Accepted Manuscript Title: Construction of Eimeria tenella multi-epitope DNA vaccines and their protective efficacies against experimental infection Author: Xiaokai Song Lixin Xu Ruofeng Yan Xinmei Huang Xiangrui Li PII: DOI: Reference:
S0165-2427(15)00124-5 http://dx.doi.org/doi:10.1016/j.vetimm.2015.05.005 VETIMM 9346
To appear in:
VETIMM
Received date: Revised date: Accepted date:
24-11-2014 13-4-2015 26-5-2015
Please cite this article as: Song, X., Xu, L., Yan, R., Huang, X., Li, X.,Construction of Eimeria tenella multi-epitope DNA vaccines and their protective efficacies against experimental infection, Veterinary Immunology and Immunopathology (2015), http://dx.doi.org/10.1016/j.vetimm.2015.05.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript Click here to view linked References
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Construction of Eimeria tenella multi-epitope DNA
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vaccines and their protective efficacies against experimental
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infection
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Xiaokai Songa, Lixin Xua, Ruofeng Yana, Xinmei Huanga,b, Xiangrui Lia* a
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College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, P.R. China
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Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Science, Nanjing, Jiangsu 210014, P.R. China
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* Corresponding author. Tel：+86 25 8439 9000
Fax: +86 25 8439 9000
E-mail address: [email protected] (X. Li)
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Abstract: The search for effective vaccines against chicken coccidiosis remains a
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challenge because of the complex organisms with multiple life cycle stages of
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Eimeria. Combination of T-cell epitopes from different stages of Eimeria life cycle
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could be an optimal strategy to overcome the antigen complexity of the parasite. In
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this study, 4 fragments with concentrated T-cell epitopes from the sporozoite antigen
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SO7 and the merozoite antigen MZ5-7 of Eimeria tenella were cloned into eukaryotic
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expression vector pVAX1 in different forms, with or without chicken cytokines IL-2
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or IFN-γ genes as genetic adjuvants, to construct multistage, multi-epitope DNA
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vaccines against Eimeria tenella. Transcription and expression of the multi-epitope
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DNA vaccines in vivo were detected by reverse transcription-PCR (RT-PCR) and
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Western blot. On the basis of survival rate, lesion score, body weight gain, oocyst
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decrease ratio and the anti-coccidial index (ACI), Animal experiments were carried
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out to evaluate the protective efficacy against Eimeria tenella. Results showed the
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constructed DNA vaccines were transcribed and translated successfully in vivo.
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Animal experiment showed that the multi-epitopes DNA vaccines were more effective
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to stimulate immune response than single fragment. Compared with the DNA
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vaccines composed with less T-cell epitopes, DNA vaccine pVAX1-m1-m2-s1-s2
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containing 4 fragments with concentrated T-epitopes provided the highest ACI of
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180.39. DNA vaccines composed of antigens from two developmental stages were
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more
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pVAX1-m1-m2-s1-s2 provided the most effective protection with the ACI of 180.39.
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Furthermore, cytokines IL-2 or IFN-γ could improve the efficacy of the multi-epitope
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effective
than
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Especially
DNA
vaccine
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DNA vaccines significantly. Overall, pVAX1-m1-m2-s1-s2-IFN-γ provided the most
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effective protection with the ACI of 189.92. The multi-epitope DNA vaccines
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revealed in this study provide new candidates for Eimeria vaccine development.
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Key words: Eimeria tenella; T-cell epitopes; DNA vaccine; cytokine
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1. Introduction
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Avian coccidiosis, caused by parasites of the genus Eimeria, is a worldwide
51
problem with economic losses to the world’s poultry industry. (Dalloul and Lillehoj,
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2006; Reid et al., 2014). Eimeria tenella (E. tenella) is one of the seven Eimeria
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species that infects domestic chickens and contributes significantly to the economic
54
losses induced by the chicken coccidiosis (Reid et al., 2014; Williams et al., 1999).
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Control of Eimeria in poultry is presently accomplished by prophylactic
56
chemotherapy and vaccination with either virulent or live attenuated parasites (Blake
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and Tomley, 2014; Dalloul and Lillehoj, 2006). But the rapid emergence of drug
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resistant parasites, relatively high production cost and potential reversion to virulence
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of live vaccines have led to search for new approaches to coccidiosis control (Du et al.,
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2005; Ivory and Chadee, 2004; Song et al., 2013).
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Recent studies suggested that DNA-based vaccines maybe effective in eliciting
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adequate immunity against coccidian infection without the disadvantages associated
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with chemoprophylaxis and live vaccines (Ding et al., 2012; Hoan et al., 2014; Ivory
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and Chadee, 2004). It was reported that cell-mediated immunity played main roles in
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the protection against chicken coccidiosis (Chapman, 2014; Dalloul and Lillehoj,
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2006; Jenkins, 1998). T-cell epitope, the minimal antigenic units, is a set of amino
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acid residues presented by histocompatibility complex (MHC) molecules and
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recognized by the T cells of the host system. (Desai and Kulkarni-Kale, 2014; Cho
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and Celis, 2012). Since T-cell epitope, a relatively tiny, but immunologically relevant,
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sequence is often capable of inducing cellular immune response to a large and
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complex pathogen (Desai and Kulkarni-Kale, 2014; Suhrbier, 1997), multi-epitope
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DNA vaccines have been constructed and showed effective efficacies in HIV,
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hantavirus, Hepatitis C virus (HCV), Plasmodium falciparum, Toxoplasma gondii and
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Melanoma (Cho and Celis, 2012; Cong et al., 2014; Hanke et al., 1999; Pishraft Sabet
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et al., 2014; Suhrbier, 1997; Zhao et al., 2012). Since the complex life cycle of the
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Eimeria parasite involves both asexual and sexual developmental stages, vaccine
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based on a single antigen from one developmental stage of the parasite could not
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provide effective protection (Badran and Lukešová, 2006; Jenkins, 1998). Thus,
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developing T-cell epitopes DNA vaccines with antigens from different stages of
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Eimeria life cycle could be a new alternative control strategy (Blake and Tomley,
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2014; Ding et al., 2012; Ivory and Chadee, 2004; Jenkins, 1998; Olszewska and
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Steward, 2001).
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Sporozoite antigen SO7 of E. tenella is associated with the refractile body (RB)
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and plays a potential role in cellular invasion (de Venevelles et al., 2006; Fetterer et al.,
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2007). Some subsequent researches proved that SO7 could induce protection against
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E. tenella (Crane et al., 1991; Yang et al., 2010). The surface antigen MZP5-7
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(encoded by MZ5-7 gene) from the second generation merozoite of E. tenella was
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first identified by Binger et al. (1993) and could produce partial protection against E.
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tenella challenge infection (Geriletu et al., 2011). In previous studies, SO7 and MZ5-7
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genes were cloned and the immunization with the DNA vaccines based on these two
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genes could provide effective protection against E. tenella (Geriletu et al., 2011; Song
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et al., 2013). These results suggested that SO7 and MZ5-7 genes might contain
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protective epitopes to the challenge of E. tenella.
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Cytokines are key regulators of the immune system. They are essential to shape
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the innate and adaptive immune responses, as well as for the establishment and
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maintenance of immunological memory (Chabalgoity et al., 2007). The use of
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cytokines as adjuvants in poultry is attracting considerable attention (Asif et al., 2004).
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It was reported that immune responses induced by DNA vaccination could be
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enhanced by co-delivery of recombinant cytokines or plasmids encoding these
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cytokines (Chabalgoity et al., 2007; Min et al., 2002; Shah et al., 2011). Our previous
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researches suggested that IL-2 or IFN-γ could effectively improve protective
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immunity of DNA vaccines against coccidiosis (Xu et al. 2008; Song et al., 2010；
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Shah et al. 2011).
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Here we reported the selections of effective epitopes from SO7 and MZ5-7
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antigens and the construction of multi-epitope DNA vaccines of E. tenella and their
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protective immunity against experimental E. tenella infection.
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2. Materials and methods
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2.1. Plasmids, parasites and animals
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Plasmids
pMD18-T-SO7,
pMD18-T-MZ5-7,
pMD18-T-IFN-γ
and
pMD18-T-IL-2 were provided by the Laboratory of Veterinary Molecular and
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Immunological Parasitology, Nanjing Agricultural University, China. Sporulated
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oocysts of E. tenella isolated from Jiangsu Province of China (JS) were passed
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through chickens at least every 3 months. New-hatched Hy-Line layer chickens
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(commercial breed W-36) were raised in a sterilized room under coccidia-free
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conditions until the end of the experiment. Food and water without anti-coccidia
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drugs were available ad libitum. All experiments were approved according to the
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Animal Care and Use Committee of the Jiangsu Province Animal Care Ethics
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Committee.
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2.2 Analysis and selection of T cell multi-epitope fragments from SO7 and MZ5-7
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T-cell epitopes of SO7 and MZ5-7 were analyzed with Rothbard-Taylor method
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and AMPHI method of DNAStar software. Rothbard-Taylor method locates a
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common sequence motif which is of 3 to 4 residues consisting of glycine followed by
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hydrophobic residues (Rothbard and Taylor, 1988). The AMPHI method assumes
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T-cell antigenic sites are composed of amphipathic helices (Margalit et al., 1987).
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Subsequently 4 fragments with concentrated T-cell epitopes were selected from the 2
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antigens. The fragments selected from SO7 were named as s1 and s2. The fragments
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selected from MZ5-7 were named as m1 and m2. Characteristics of the selected four
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fragments were shown in supplementary Table 1. The m1 is the 107 amino acids
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residues located in the region from 39 to 145 amino acids of MZ5-7. The m2 is the
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117 amino acids residues located in the region from 183 to 299 amino acids of MZ5-7.
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The s1 is the 95 amino acids located in the region from 3 to 97 amino acids of SO7.
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The s2 is the 80 amino acids located in the region from 124 to 203 amino acids of
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SO7. The m1 contains 4 T-cell epitopes, RLAK, RLAAG, DFVG, and EVLIQ which
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were located in the regions of 49-52, 88-92, 125-128 and 141-145 amino acids of
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MZ5-7 respectively. The m2 contains 6 T-cell epitopes namely, GYLG, KTAK,
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GYVVEVLMK, DMMTS, GVLMD and GMLTQ located in the regions of 187-190,
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199-202, 243-251, 259-263, 272-276 and 295-299 amino acids of MZ5-7 respectively.
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The s1 contains 2 T-cell epitopes namely LPAEGERAPRPAPGT and EQLA located
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in the regions of 21-35 and 74-77 amino acids of SO7 respectively.
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4 T-cell epitopes namely VESSKDTVL, LRGYQ, LGYG and QPSSY located in the
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regions of 145-153, 171-175, 183-186 and 197-201 amino acids of SO7 respectively.
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2. 3. Cloning of T cell multi-epitope fragments
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To clone the 4 fragments with concentrated T-cell epitopes, PCR amplifications
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were performed using the corresponding primers (supplementary Table 1) with
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pMD18-T-SO7 or pMD18-T-MZ5-7 as templates. The PCR products were cloned into
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pMD18-T vector producing recombinant plasmids pMD18-T-m1, pMD18-T-m2,
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pMD18-T-s1 and pMD18-T-s2. Subsequently, the recombinant plasmids were
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confirmed by endonuclease digestion assay and DNA sequencing.
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2. 4. Constructions of DNA vaccines containing one fragment
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DNA vaccines pVAX1-m1, pVAX1-m2, pVAX1-s1 and pVAX1-s2, containing
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one fragment were constructed separately. Recombinant plasmids pMD18-T-s1 and
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eukaryotic expression vector pVAX1 (Invitrogen) were digested with EcoRI and XbaI.
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The m1 and pVAX1 fragments were purified and ligated together to produce DNA
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vaccine pVAX1-s1. Hereafter pVAX1-s1 was confirmed by endonuclease digestion
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assay and DNA sequencing. Similarly, DNA vaccines pVAX1-s2, pVAX1-m1 and
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pVAX1-m2 were constructed.
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2. 5. Constructions of DNA vaccines containing two fragments with and without
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cytokines
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To construct DNA vaccines containing two fragments with cytokines,
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pMD18-T-m1 and pVAX1 were digested with HindⅢ and EcoRI and the fragments
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m1 and pVAX1 were ligated together producing pVAX1-m1. Subsequently,
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pVAX1-m1 and pMD18-T-s1 were digested with EcoRI and XhoI, and the fragments
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s1 and pVAX1-m1 were ligated together producing pVAX1-m1-s1. And then
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pVAX1-m1-s1, pMD18-T-IFN-γ and pMD18-T-IL-2 were digested with XhoI and
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XbaI. The fragments IFN-γ, IL-2 and pVAX1-m1-s1 were ligated together,
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respectively, producing DNA vaccines pVAX1-m1-s1-IFN-γ and pVAX1-m1-s1-IL-2.
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Using the same method above, DNA vaccines pVAX1-m1-s2-IFN-γ,
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pVAX1-m1-s2-IL-2,
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pVAX1-m2-s2-IFN-γ and pVAX1-m2-s2-IL-2 were also constructed.
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pVAX1-m2-s1-IFN-γ,
pVAX1-m2-s1-IL-2,
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To construct DNA vaccines composed of two fragments without cytokines,
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pVAX1-s1 and pVAX1-s2 constructed in procedure 2.4 were digested with EcoRI and
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XbaI to get the fragments s1 and s2 with termination code. At the same time,
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pVAX1-m1-s1-IL-2 and pVAX1-m2-s1-IL-2 constructed in 2.5 were digested with
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EcoRI and XbaI to get vector fragments pVAX1-m1 and pVAX1-m2. Hereafter, the
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antigen fragments s1 and s2 with termination code were ligated with vector fragments
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pVAX1-m1 and pVAX1-m2 respectively to produce DNA vaccines pVAX1-m1-s1,
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pVAX1-m1-s2, pVAX1-m2-s1 and pVAX1-m2-s2.
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2.6. Constructions of DNA vaccines containing four fragments with and without
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cytokines To construct pVAX1-m1-m2-s1-s2-IFN-γ and pVAX1-m1-m2-s1-s2-IL-2,
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pMD18-T-m1 and pVAX1 vector were digested with NheI and HindⅢ and the
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fragments m1 and pVAX1 were ligated together producing pVAX1-m1. Subsequently,
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recombinant plasmid pMD18-T-m2 and pVAX1-m1 vector were digested with KpnI
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and EcoRI and the fragments m2 and pVAX1-m1 were ligated together producing
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pVAX1-m1-m2. Recombinant plasmids pMD18-T-s1 and pVAX1-m1-m2 vector were
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digested with KpnI and EcoRI and the fragments s1 and pVAX1-m1-m2 were ligated
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together producing pVAX1-m1-m2-s1. Recombinant plasmids pMD18-T-s2 and
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pVAX1-m1-m2-s1 vector were digested with EcoRI and NotI and the fragments s2
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and pVAX1-m1-m2-s1 were ligated together to producing pVAX1-m1-m2-s1-s2.
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Recombinant plasmids pMD18-T-IFN-γ, pMD18-T-IL-2 and pVAX1-m1-m2-s1-s2
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vector were digested with NotI and XbaI and the fragments IFN-γ, IL-2 and
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pVAX1-m1-m2-s1-s2 were ligated together, respectively, producing DNA vaccines
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pVAX1-m1-m2-s1-s2-IFN-γ and pVAX1-m1-m2-s1-s2-IL-2.
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To construct pVAX1-m1-m2-s1-s2, recombinant plasmid pVAX1-s2 constructed
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in procedure 2.4 and pVAX1-m1-m2-s1-s2-IL-2 constructed above was digested with
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EcoRI and XbaI. Subsequently, the target fragments s2 and vector pVAX1-m1-m2-s1
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were ligated together producing DNA vaccine pVAX1-m1-m2-s1-s2.
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2.7. Transcription detection of constructed DNA vaccines in vivo by RT-PCR
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Chickens were vaccinated with the constructed DNA vaccines by intramuscular
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injection. Seven days later, about 0.5 g of the injected muscle of each chicken was cut
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for total mRNA extraction. After digestion of plasmid DNA vaccines by adding
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DNaseI (TaKaRa) into the mRNA, reverse transcription-polymerase chain reaction
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(RT-PCR) were performed with the cloning primers of m1, m2, s1, s2, chIFN-γ and
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chIL-2 genes (Supplementary Table 1), respectively. Electrophoresis in 1% agarose
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gel was performed to examine the transcription of the target genes. Meanwhile the
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same site muscle from non-injected chickens was selected as control.
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2.8. Expression detection of target genes in vivo by Western blot
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2.8.1. Preparation of rat antiserum to sporozoites and merozoites
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Soluble sporozoite and second-generation merozoite antigens of E. tenella were
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prepared with the reported methods (Liu, et al., 2009; Khalafalla et al., 2011). Rats
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were vaccinated subcutaneously on the back with 100 μg antigen emulsified in
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Freund's Adjuvant Complete (Sigma) at 30 days of age, 14 days later, the second dose
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was given with Freund's Adjuvant Incomplete (Sigma). 14 days later, a third dose
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without adjuvant was given. Blood were obtained from tails of the rats for titer
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determination of antiserum by enzyme linked immunosorbent assay 7 days post the
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third dose. A fourth even fifth dose would be given unless the titers of antiserum were
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satisfactory. The antiserum was collected and stored at -20 ℃ for further use when
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the titers of antiserum were satisfactory.
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2.8.2. Western blot assay
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Chickens were vaccinated with the recombinant DNA vaccines and seven days
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post-vaccination, the injected muscle was grinded and treated with RIPA solution (0.1
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mol/L phenylmethylsulfonyl fluoride (PMSF), 50 mmol/L Tris-HCl, 150 mmol/L
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NaCl, 1% Nonnidet P-40, 0.1% SDS) for 3 h. After centrifugation at 13000 rpm, the
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supernatant was collected. Meanwhile the muscle from the same site of non-injection
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was selected as control. Then Western blot was done to detect the target proteins with
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rat antiserum to sporozoites or merozoites as first antibody (Song et al., 2013; Xu et
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al., 2008). Briefly, proteins collected from the injected muscle were separated by
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sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then
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transferred to a nitrocellulose membrane (Bio-Rad). The membrane was incubated
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with rat antiserum against sporozoites or second-generation merozoites and
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horseradish peroxidase (HRP)-conjugated rabbit anti-rat IgG (Sigma) as secondary
232
antibody. The bound antibody was detected using 3, 30-diaminobenzidine (DAB).
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2.9. Evaluation of protection
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Efficacy of protection was evaluated on the basis of survival rate, lesion score,
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body weight gain, oocyst decrease ratio and ACI. Survival rate was estimated by the
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number of surviving chickens divided by the number of initial chickens. Body weight
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gain of the chickens in each group was determined by the body weight of the chickens
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at the end of the experiments subtracting the body weight at the time of challenge.
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Lesion score of the chickens from each group was investigated according to the
240
method of Johnson and Reid (1970). Enteric content of the chickens and daily fecal
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samples between days 5 and 10 post-infection were collected for oocyst counting
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using McMaster’s counting technique as described (Ding et al., 2004; Jang et al.,
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2011). Oocyst decrease ratio was calculated as follows: the number of oocysts from
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positive control chickens-vaccinated chickens/positive control chickens×100%. Body
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weight
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mean±S.D.(standard deviation). ACI is a synthetic criterion for assessing the
247
protective effect of a medicine or vaccine and calculated as follows: (relative rate of
248
weight gain + survival rate) - (lesion value + oocyst value). ACI is considered "good"
249
when 180 or more, "moderate" when 160-179, and "poor" when below 160
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(McManus et al., 1968).
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2.10. Immunization of chickens
oocyst
output
and
lesion
scores
were
expressed
as
the
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At 14 days of age, chickens were weighed and randomly distributed to 4
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vaccinated groups and 3 control groups. As shown in Table 1, the 4 vaccinated groups
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were DNA vaccines containing four fragments vaccinated group (4f), DNA vaccines
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containing two fragments with cytokine vaccinated group (2f+c), DNA vaccines
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containing two fragments vaccinated group (2f) and DNA vaccines containing one
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fragments vaccinated group (1f). The 3 control groups were vector control (vc),
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unchallenged control (uc) and challenged control (cc). The vaccinated groups 4f, 2f+c,
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2f and 1f contained 3, 8, 4 and 4 treatments with DNA vaccine (30 chickens/treatment)
260
respectively, and each control group had 30 chickens. Vaccinated group chickens were
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immunized with 100 µg the corresponding DNA vaccines by leg intramuscular
262
injection, respectively. The challenged control group and unchallenged control
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chickens were injected with sterile TE and empty vector control group was
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intramuscularly immunized with 100 µg pVAX1 at the same injection site. A booster
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immunization was given 7 days post the first immunization. At 28 days of age, the
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chickens were challenged with 5×104 sporulated oocysts of E. tenella JS except the
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unchallenged control group. Seven days post challenge, all the chickens were
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weighted. Half chickens of each group were slaughtered for lesion score and the rest
269
half was left for oocyst counting until 10 days post infection. Average body weight
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gain, oocyst decrease ratio, lesion score, and ACI were calculated as described above.
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2.11. Statistical analysis
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Statistical analysis was performed using R statistical package (R Core Team,
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2013). For body weight gain, differences of means between pairwise groups were
274
tested using Turkey-Kramer method and considered significant at p < 0.05. Since
275
lesion scores and oocyst output don’t follow normal distribution, differences between
276
groups were tested with pairwise comparisons using Wilcoxon rank sum test and
277
considered significant at p < 0.05. The differences between treatments of each
278
vaccinated group were also tested.
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3. Results
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3.1. Identification of the DNA vaccines
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The constructed recombinant DNA vaccines were confirmed by endonuclease
282
digestion. The aim fragments were produced after digestion with the corresponding
283
endonuclease. As shown in Figure 1A, digestion of recombinant plasmid
284
pVAX1-m1-m2-s1-s2-IFN-γ and pVAX1-m1-m2-s1-s2-IL-2 with NheI and HindⅢ
285
produced fragments of approximate 336 bp (Fig.1A, lane 5 and 10) which were equal
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to the molecular mass of m1, while digestion with NheI and KpnI produced fragments
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of approximate 702 bp of m1-m2 (Fig. 1A, lane 4 and 9), digestion with NheI and
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EcoRI produced fragments of approximate 1011 bp of m1-m2-s1 (Fig. 1A, lane 3 and
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8), digestion with NheI and NotI produced fragments of approximate 1275 bp of
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m1-m2-s1-s2 (Fig. 1 A, lane 2 and 7), digestion with NheI and XbaI produced
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fragments of approximate 1790 bp of m1-m2-s1-s2-IFN-γ (Fig. 1A, lane 1) and 1727
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bp of m1-m2-s1-s2-IL-2 (Fig. 1A, lane 6), respectively.
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3.2. Transcription and expression of constructed DNA vaccines in vivo
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Transcription and expression of the DNA vaccines in vivo were detected by
295
RT-PCR and Western blot, separately. RT-PCR produced specific DNA bands of m1,
296
m2, s1, s2, IFN-γ and IL-2 from muscles injected with the corresponding DNA
297
vaccines, respectively (Fig. 1B). No amplification products were generated in the
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non-injection controls (Fig. 1B, lane 6 and 12). Western blot detection of
299
pVAX1-m1-m2-s1-s2-IL-2 or pVAX1-m1-m2-s1-s2-IFN-γ injected muscles revealed
300
two bands about 60.7 kDa and 63 kDa (Fig. 1C, lane 1-2). Eight bands with the
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molecular masses of about 41.2 kDa, 42.3 kDa, 40.8 kDa, 39.6 kDa, 39 kDa, 37.3
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kDa, 40.5 kDa and 38.5 kDa were detected from the pVAX1-m1-s1-IFN-γ,
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pVAX1-m2-s1-IFN-γ,
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pVAX1-m1-s1-IL-2, pVAX1-m1-s2-IL-2, pVAX1-m2-s1-IL-2, pVAX1-m2-s2-IL-2
305
injected muscles, respectively(Fig. 1C, lane 4-12), and no protein was detected in the
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non-injected muscle (Fig. 1C, lane 3-4).
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3. 3. Protective efficacies of multistage, multi-epitope DNA vaccination against E.
308
tenella challenge
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pVAX1-m2-s2-IFN-γ,
pVAX1-m1-s2-IFN-γ,
Page 14 of 41
309
There were no deaths in any group following coccidial infection. Comparison of
310
protective efficacy on the four types of DNA vaccines (1f, 2f, 2f+c and 4f) was
311
showed in Table 2. The protective efficacies of each type of
312
showed in Table 3 to Table 6 separately.
313
3.3.1. Protective efficacies of the DNA vaccines containing one fragment (1f)
ip t
DNA vaccine were
The immunization efficacies of DNA vaccines containing one fragment (1f) are
315
described in in Table 3. Challenged control and pVAX1 vector control groups
316
exhibited significantly reduced weight gain indicative of active intestinal disease
317
compared with the unchallenged control group (P < 0.05).
an
us
cr
314
The body weight gain of chickens immunized did not significantly differ with
319
that of unchallenged control group (p>0.05), but was significantly higher than that of
320
challenged control group and pVAX1 vector control group (p0.05).
ce pt
ed
M
318
Immunization with the constructed DNA vaccines resulted in lower oocyst
324
output and higher oocyst decrease ratio as compared with challenged control group
325
and pVAX1 vector control group (p0.05). DNA vaccines
327
immunized groups presented significant lower lesion scores compared with
328
challenged control group and pVAX1 vector control groups (p 0.05) between numbers with the same letter.
Ac
698
ce pt
695
699
Table 3 Protective efficacy of the multi-epitope DNA vaccines containing one
700
fragment (group 1f) against E. tenella challenge
701
Note: significant difference (P < 0.05) between numbers with different letters. No
702
significant difference (P > 0.05) between numbers with the same letter.
703 704
Table 4 Protective efficacy of the multi-epitope DNA vaccines containing two
Page 32 of 41
705
fragments (group 2f) against E. tenella challenge
706
Note: significant difference (P < 0.05) between numbers with different letters. No
707
significant difference (P > 0.05) between numbers with the same letter.
ip t
708
Table 5 Protective efficacy of the multi-epitope DNA vaccines containing two
710
fragments with cytokines (group 2f+c) against E. tenella challenge
711
Note: significant difference (P < 0.05) between numbers with different letters. No
712
significant difference (P > 0.05) between numbers with the same letter.
us
cr
709
an
713
Table 6 Protective efficacy of the multi-epitope DNA vaccines containing four
715
fragments with and without cytokines (group 4f) against E. tenella challenge
716
Note: significant difference (P < 0.05) between numbers with different letters. No
717
significant difference (P > 0.05) between numbers with the same letter.
ed
ce pt
718
M
714
Supplementary Table 1 Characteristics of the selected four fragments and the primers
720
of chicken cytokines (aa= amino acids)
Ac
719
Page 33 of 41
Ac ce p
te
d
M
an
us
cr
ip t
Fig.1 Click here to download high resolution image
Page 34 of 41
Table 1
Second
Groups
First vaccination vaccination (14 days of age)
Treatments
(21 days of age)
Challenge (28 days of age)
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-s1
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-s2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s1
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s2
100 µg DNA
100 µg DNA
pVAX1-m2-s1
100 µg DNA
100 µg DNA
pVAX1-m2-s2
100 µg DNA
100 µg DNA
pVAX1-m1-s1-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s1-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s2-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s2-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2-s1-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2-s1-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2-s2-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2-s2-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-m2-s1-s2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-m2-s1-s2-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-m2-s1-s2-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
vc
pVAX1
100 µg pVAX1
100 µg pVAX1
5×104 E. tenella
uc
TE
100 µl TE
100 µl TE
PBS
cc
TE
100 µl TE
100 µl TE
5×104 E. tenella
4f
5×104 E. tenella 5×104 E. tenella 5×104 E. tenella
cr
us
an
M
ed
2f+c
ce pt
2f
Ac
1f
ip t
pVAX1-m1
Page 35 of 41
Table 2
Groups
Average body weight gain
Mean lesion scores
Lesion decrease ratio (%)
Oocyst output (×106)
Oocyst decrease ratio (%)
ACI
1f
82±15.05b
1.6±1.02c
52.27
4.25±4.39bc
42.67
167.79
2f
85.86±14.17b
1.18±0.94b
62.35
3.62±3.72bc
92.19±10.92c
1.37±0.85
4f
94.94±11.87c
1.22±0.87b
vc
53.72±8.25a
3.10±0.61
uc
88.40±7.73bc
0a 3.40±0.77
3.51±3.66
50.24
179.73
64.05
2.93±3.86b
60.55
186.10
7.13
110.24
100
200
0
100.13
8.82
6.89±7.55
100
0a c
0
7.42±6.7
bc
ce pt
ed
M
an
us
47.48±11.49a
d
58.82
Ac
cc
d
175.70
ip t
2f+c
52.47
b
cr
bc
Page 36 of 41
81.13±16.95bc
Lesion decrease ratio (%)
Oocyst output (×106)
Oocyst decrease ratio (%)
ACI
1.63±1.16c
51.96
4.25±3.48b
42.73
166.04
50.98
4.41±3.91
b
40.57
162.48
55.88
4.14±5.57b
44.25
170.41
52.94
4.22±4.54
b
43.19
170.49
8.82
6.89±7.55c
7.13
110.24
100
200
0
100.13
c
pVAX1-m2
78.20±12.04b
1.67±1.03
pVAX1-s1
83.87±13.60bc
1.50±0.90bc c
pVAX1-s2
84.8±16.87bc
1.60±1.04
pVAX1
53.72±8.25a
3.10±0.61d
a
0
100
0
3.40±0.77e
0
7.42±6.7c
Ac
ce pt
ed
M
an
Unchallenged control 88.40±7.73c Challenged control 47.48±11.49a
a
ip t
pVAX1-m1
Mean lesion scores
us
Treatments
Average body weight gain
cr
Table 3
Page 37 of 41
Table 4
Average body weight gain
Treatments
Mean lesion scores
Lesion decrease ratio (%)
Oocyst output (×106)
Oocyst decrease ratio (%)
ACI
83.15±17.25b
0.87±0.86b
74.51
3.88±3.61b
47.66
175.19
pVAX1-m1-s2
87.81±11.66b
1.13±0.97bc
66.67
3.72±5.57b
49.84
177.97
55.88
2.79±1.61
b
64.50
4.07±3.03b
87.73±14.26b
1.50±1.04
pVAX1-m2-s2
84.82±13.06b
1.21±0.80bc
pVAX1
53.72±8.25a
3.10±0.61
Unchallenged control
88.40±7.73b
0a
47.48±11.49a
3.40±0.77
e
8.82
6.89±7.55
100
0a
0
c
175.22
45.17
173.92
7.13
110.24
c
100
200
7.42±6.7
0
100.13
Ac
ce pt
ed
M
an
Challenged control
d
62.40
cr
pVAX1-m2-s1
us
c
ip t
pVAX1-m1-s1
Page 38 of 41
Table 5
92.7±12.23b
pVAX1-m1-s2-IL2
92.5±9.66b
pVAX1-m1-s2-IFNγ
94.97±11.40b
pVAX1-m2-s1-IL2
91.1±12.97b
pVAX1-m2-s1-IFNγ
89.65±10.19b
pVAX1-m2-s2-IL2
91.82±11.07b
pVAX1-m2-s2-IFNγ
93.22±11.54b
Oocyst decrease ratio(%)
ACI
1.37±0.89b
59.80
3.7±5.2b
50.12
180.36
53.61
181.63
1.37±1.19
b
1.27±1.08
b
1.23±0.90
b
1.53±0.97
b
1.23±0.82
b
1.47±0.86
b
1.50±0.86
b c
pVAX1
53.72±8.25a
3.10±0.61
Unchallenged control
88.40±7.73b
0a
47.48±11.49a
62.75 63.73 54.90 63.73 56.86 55.88 8.82 100
3.40±0.77
d
3.44±4.2
3.04±3.22
b
2.81±2.84
b
4.02±5.23
b
0
59.01
183.1
62.12
185.86
45.76
178.46
b
58.12
179.02
4.15±2.76
b
44.00
179.25
3.81±4.58
b
48.59
180.91
6.89±7.55
c
7.13
110.24
100
200
0
100.13
3.11±3.45
0a
c
7.42±6.7
Ac
ce pt
ed
M
Challenged control
59.80
b
ip t
pVAX1-m1-s1-IFNγ
Oocyst output (×106)
cr
91.58±13.66b
Lesion decrease ratio (%)
us
pVAX1-m1-s1-IL2
Mean lesion scores
an
Average body weight gain
Treatments
Page 39 of 41
Table 6
Mean lesion scores
Lesion decrease ratio (%)
Oocyst output (×106)
92.37±13.72bc
1.53±1.11b
54.90
3.93±4.9b
96.02±13.21bc
1.10±0.80
b
1.03±0.56
b
3.10±0.61
c
pVAX1-m1-m2-s1-s2 pVAX1-m1-m2-s1-s2-IL2 pVAX1-m1-m2-s1-s2-IFNγ pVAX1
96.45±7.69c 53.72±8.25a
Unchallenged control Challenged control
88.40±7.73b
0
69.61 8.82 100
3.40±0.77
d
46.99
180.39
2.54±3.42
b
65.74
187.98
2.31±2.92
b
68.93
189.92
6.89±7.55
c
7.13
110.24
100
200
0
100.13
a
0
c
0
ACI
7.42±6.7
Ac
ce pt
ed
M
an
us
cr
47.48±11.49a
a
67.65
Oocyst decrease ratio(%)
ip t
Average body weight gain
Treatments
Page 40 of 41
Supplementary material for online publication only
Supplementary Table 1 Characteristics of the selected four fragments and the primers of chicken cytokines (aa=amino acids)
chIL-2
Reverse: 5’-AAAGAATTCCTACAGCGCGCAGC-3’ Forward: 5’-GCAGAATTCATGAAACTGGC-3’ Reverse: 5’ –AAATCTAGACTACGCCCCGT- 3’ Forward: 5’- TTACGCGGCGCAATGACTTGCCA -3’
117
6
95
80
2
4
ntal stages Merozoite, Sporozoite
ip t
MZ5-7
MZ5-7
cr
Forward: 5’-CCCGGATCCATGGACCTCTT-3’
4
us
chIFN-γ
Reverse: 5’ -CCCTCTAGACTATTGAGTTA -3’
107
Developme
Merozoite, Sporozoite
SO7
Sporozoite
SO7
Sporozoite
an
s2
Forward: 5’- AAAGAATTCATGCAAACACT-3’
Source
Reverse: 5’ – CCCTCTAGATTAGCAATTGCATCTCCTC - 3’ Forward: 5’- AAAGCGGCCGCAATGATGTGCAAAGTA -3’
Reverse: 5’ – CCCTCTAGATTATTTTTGCAGATATCTCAC - 3’
M
s1
Reverse: 5’-CCCGAATTCCTATTGGATAAGG-3’
T cell motifs
ed
m2
Forward: 5’-AAAGGATCCATGCAGGAAGT-3’
Size(aa)
ce pt
m1
Primers
Ac
Fragments
Page 41 of 41
S0165-2427(15)00124-5 http://dx.doi.org/doi:10.1016/j.vetimm.2015.05.005 VETIMM 9346
To appear in:
VETIMM
Received date: Revised date: Accepted date:
24-11-2014 13-4-2015 26-5-2015
Please cite this article as: Song, X., Xu, L., Yan, R., Huang, X., Li, X.,Construction of Eimeria tenella multi-epitope DNA vaccines and their protective efficacies against experimental infection, Veterinary Immunology and Immunopathology (2015), http://dx.doi.org/10.1016/j.vetimm.2015.05.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript Click here to view linked References
1
Construction of Eimeria tenella multi-epitope DNA
2
vaccines and their protective efficacies against experimental
3
infection
ip t
4
Xiaokai Songa, Lixin Xua, Ruofeng Yana, Xinmei Huanga,b, Xiangrui Lia* a
6
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, P.R. China
b
us
7 8
cr
5
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Science, Nanjing, Jiangsu 210014, P.R. China
an
9
M
10 11
ed
12
15 16 17 18
Ac
14
ce pt
13
* Corresponding author. Tel：+86 25 8439 9000
Fax: +86 25 8439 9000
E-mail address: [email protected] (X. Li)
19 20 21 22
Page 1 of 41
Abstract: The search for effective vaccines against chicken coccidiosis remains a
24
challenge because of the complex organisms with multiple life cycle stages of
25
Eimeria. Combination of T-cell epitopes from different stages of Eimeria life cycle
26
could be an optimal strategy to overcome the antigen complexity of the parasite. In
27
this study, 4 fragments with concentrated T-cell epitopes from the sporozoite antigen
28
SO7 and the merozoite antigen MZ5-7 of Eimeria tenella were cloned into eukaryotic
29
expression vector pVAX1 in different forms, with or without chicken cytokines IL-2
30
or IFN-γ genes as genetic adjuvants, to construct multistage, multi-epitope DNA
31
vaccines against Eimeria tenella. Transcription and expression of the multi-epitope
32
DNA vaccines in vivo were detected by reverse transcription-PCR (RT-PCR) and
33
Western blot. On the basis of survival rate, lesion score, body weight gain, oocyst
34
decrease ratio and the anti-coccidial index (ACI), Animal experiments were carried
35
out to evaluate the protective efficacy against Eimeria tenella. Results showed the
36
constructed DNA vaccines were transcribed and translated successfully in vivo.
37
Animal experiment showed that the multi-epitopes DNA vaccines were more effective
38
to stimulate immune response than single fragment. Compared with the DNA
39
vaccines composed with less T-cell epitopes, DNA vaccine pVAX1-m1-m2-s1-s2
40
containing 4 fragments with concentrated T-epitopes provided the highest ACI of
41
180.39. DNA vaccines composed of antigens from two developmental stages were
42
more
43
pVAX1-m1-m2-s1-s2 provided the most effective protection with the ACI of 180.39.
44
Furthermore, cytokines IL-2 or IFN-γ could improve the efficacy of the multi-epitope
Ac
ce pt
ed
M
an
us
cr
ip t
23
effective
than
the
single-stage
ones.
Especially
DNA
vaccine
Page 2 of 41
DNA vaccines significantly. Overall, pVAX1-m1-m2-s1-s2-IFN-γ provided the most
46
effective protection with the ACI of 189.92. The multi-epitope DNA vaccines
47
revealed in this study provide new candidates for Eimeria vaccine development.
48
Key words: Eimeria tenella; T-cell epitopes; DNA vaccine; cytokine
49
1. Introduction
ip t
45
Avian coccidiosis, caused by parasites of the genus Eimeria, is a worldwide
51
problem with economic losses to the world’s poultry industry. (Dalloul and Lillehoj,
52
2006; Reid et al., 2014). Eimeria tenella (E. tenella) is one of the seven Eimeria
53
species that infects domestic chickens and contributes significantly to the economic
54
losses induced by the chicken coccidiosis (Reid et al., 2014; Williams et al., 1999).
55
Control of Eimeria in poultry is presently accomplished by prophylactic
56
chemotherapy and vaccination with either virulent or live attenuated parasites (Blake
57
and Tomley, 2014; Dalloul and Lillehoj, 2006). But the rapid emergence of drug
58
resistant parasites, relatively high production cost and potential reversion to virulence
59
of live vaccines have led to search for new approaches to coccidiosis control (Du et al.,
60
2005; Ivory and Chadee, 2004; Song et al., 2013).
us
an
M
ed
ce pt
Ac
61
cr
50
Recent studies suggested that DNA-based vaccines maybe effective in eliciting
62
adequate immunity against coccidian infection without the disadvantages associated
63
with chemoprophylaxis and live vaccines (Ding et al., 2012; Hoan et al., 2014; Ivory
64
and Chadee, 2004). It was reported that cell-mediated immunity played main roles in
65
the protection against chicken coccidiosis (Chapman, 2014; Dalloul and Lillehoj,
66
2006; Jenkins, 1998). T-cell epitope, the minimal antigenic units, is a set of amino
Page 3 of 41
acid residues presented by histocompatibility complex (MHC) molecules and
68
recognized by the T cells of the host system. (Desai and Kulkarni-Kale, 2014; Cho
69
and Celis, 2012). Since T-cell epitope, a relatively tiny, but immunologically relevant,
70
sequence is often capable of inducing cellular immune response to a large and
71
complex pathogen (Desai and Kulkarni-Kale, 2014; Suhrbier, 1997), multi-epitope
72
DNA vaccines have been constructed and showed effective efficacies in HIV,
73
hantavirus, Hepatitis C virus (HCV), Plasmodium falciparum, Toxoplasma gondii and
74
Melanoma (Cho and Celis, 2012; Cong et al., 2014; Hanke et al., 1999; Pishraft Sabet
75
et al., 2014; Suhrbier, 1997; Zhao et al., 2012). Since the complex life cycle of the
76
Eimeria parasite involves both asexual and sexual developmental stages, vaccine
77
based on a single antigen from one developmental stage of the parasite could not
78
provide effective protection (Badran and Lukešová, 2006; Jenkins, 1998). Thus,
79
developing T-cell epitopes DNA vaccines with antigens from different stages of
80
Eimeria life cycle could be a new alternative control strategy (Blake and Tomley,
81
2014; Ding et al., 2012; Ivory and Chadee, 2004; Jenkins, 1998; Olszewska and
82
Steward, 2001).
cr
us
an
M
ed
ce pt
Ac
83
ip t
67
Sporozoite antigen SO7 of E. tenella is associated with the refractile body (RB)
84
and plays a potential role in cellular invasion (de Venevelles et al., 2006; Fetterer et al.,
85
2007). Some subsequent researches proved that SO7 could induce protection against
86
E. tenella (Crane et al., 1991; Yang et al., 2010). The surface antigen MZP5-7
87
(encoded by MZ5-7 gene) from the second generation merozoite of E. tenella was
88
first identified by Binger et al. (1993) and could produce partial protection against E.
Page 4 of 41
tenella challenge infection (Geriletu et al., 2011). In previous studies, SO7 and MZ5-7
90
genes were cloned and the immunization with the DNA vaccines based on these two
91
genes could provide effective protection against E. tenella (Geriletu et al., 2011; Song
92
et al., 2013). These results suggested that SO7 and MZ5-7 genes might contain
93
protective epitopes to the challenge of E. tenella.
ip t
89
Cytokines are key regulators of the immune system. They are essential to shape
95
the innate and adaptive immune responses, as well as for the establishment and
96
maintenance of immunological memory (Chabalgoity et al., 2007). The use of
97
cytokines as adjuvants in poultry is attracting considerable attention (Asif et al., 2004).
98
It was reported that immune responses induced by DNA vaccination could be
99
enhanced by co-delivery of recombinant cytokines or plasmids encoding these
100
cytokines (Chabalgoity et al., 2007; Min et al., 2002; Shah et al., 2011). Our previous
101
researches suggested that IL-2 or IFN-γ could effectively improve protective
102
immunity of DNA vaccines against coccidiosis (Xu et al. 2008; Song et al., 2010；
103
Shah et al. 2011).
ce pt
ed
M
an
us
cr
94
Here we reported the selections of effective epitopes from SO7 and MZ5-7
105
antigens and the construction of multi-epitope DNA vaccines of E. tenella and their
106
protective immunity against experimental E. tenella infection.
107
2. Materials and methods
108
2.1. Plasmids, parasites and animals
109 110
Ac
104
Plasmids
pMD18-T-SO7,
pMD18-T-MZ5-7,
pMD18-T-IFN-γ
and
pMD18-T-IL-2 were provided by the Laboratory of Veterinary Molecular and
Page 5 of 41
Immunological Parasitology, Nanjing Agricultural University, China. Sporulated
112
oocysts of E. tenella isolated from Jiangsu Province of China (JS) were passed
113
through chickens at least every 3 months. New-hatched Hy-Line layer chickens
114
(commercial breed W-36) were raised in a sterilized room under coccidia-free
115
conditions until the end of the experiment. Food and water without anti-coccidia
116
drugs were available ad libitum. All experiments were approved according to the
117
Animal Care and Use Committee of the Jiangsu Province Animal Care Ethics
118
Committee.
119
2.2 Analysis and selection of T cell multi-epitope fragments from SO7 and MZ5-7
an
us
cr
ip t
111
T-cell epitopes of SO7 and MZ5-7 were analyzed with Rothbard-Taylor method
121
and AMPHI method of DNAStar software. Rothbard-Taylor method locates a
122
common sequence motif which is of 3 to 4 residues consisting of glycine followed by
123
hydrophobic residues (Rothbard and Taylor, 1988). The AMPHI method assumes
124
T-cell antigenic sites are composed of amphipathic helices (Margalit et al., 1987).
125
Subsequently 4 fragments with concentrated T-cell epitopes were selected from the 2
126
antigens. The fragments selected from SO7 were named as s1 and s2. The fragments
127
selected from MZ5-7 were named as m1 and m2. Characteristics of the selected four
128
fragments were shown in supplementary Table 1. The m1 is the 107 amino acids
129
residues located in the region from 39 to 145 amino acids of MZ5-7. The m2 is the
130
117 amino acids residues located in the region from 183 to 299 amino acids of MZ5-7.
131
The s1 is the 95 amino acids located in the region from 3 to 97 amino acids of SO7.
132
The s2 is the 80 amino acids located in the region from 124 to 203 amino acids of
Ac
ce pt
ed
M
120
Page 6 of 41
SO7. The m1 contains 4 T-cell epitopes, RLAK, RLAAG, DFVG, and EVLIQ which
134
were located in the regions of 49-52, 88-92, 125-128 and 141-145 amino acids of
135
MZ5-7 respectively. The m2 contains 6 T-cell epitopes namely, GYLG, KTAK,
136
GYVVEVLMK, DMMTS, GVLMD and GMLTQ located in the regions of 187-190,
137
199-202, 243-251, 259-263, 272-276 and 295-299 amino acids of MZ5-7 respectively.
138
The s1 contains 2 T-cell epitopes namely LPAEGERAPRPAPGT and EQLA located
139
in the regions of 21-35 and 74-77 amino acids of SO7 respectively.
140
4 T-cell epitopes namely VESSKDTVL, LRGYQ, LGYG and QPSSY located in the
141
regions of 145-153, 171-175, 183-186 and 197-201 amino acids of SO7 respectively.
142
2. 3. Cloning of T cell multi-epitope fragments
cr
ip t
133
M
an
us
The s2 contains
To clone the 4 fragments with concentrated T-cell epitopes, PCR amplifications
144
were performed using the corresponding primers (supplementary Table 1) with
145
pMD18-T-SO7 or pMD18-T-MZ5-7 as templates. The PCR products were cloned into
146
pMD18-T vector producing recombinant plasmids pMD18-T-m1, pMD18-T-m2,
147
pMD18-T-s1 and pMD18-T-s2. Subsequently, the recombinant plasmids were
148
confirmed by endonuclease digestion assay and DNA sequencing.
149
2. 4. Constructions of DNA vaccines containing one fragment
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ed
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DNA vaccines pVAX1-m1, pVAX1-m2, pVAX1-s1 and pVAX1-s2, containing
151
one fragment were constructed separately. Recombinant plasmids pMD18-T-s1 and
152
eukaryotic expression vector pVAX1 (Invitrogen) were digested with EcoRI and XbaI.
153
The m1 and pVAX1 fragments were purified and ligated together to produce DNA
154
vaccine pVAX1-s1. Hereafter pVAX1-s1 was confirmed by endonuclease digestion
Page 7 of 41
assay and DNA sequencing. Similarly, DNA vaccines pVAX1-s2, pVAX1-m1 and
156
pVAX1-m2 were constructed.
157
2. 5. Constructions of DNA vaccines containing two fragments with and without
158
cytokines
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155
To construct DNA vaccines containing two fragments with cytokines,
160
pMD18-T-m1 and pVAX1 were digested with HindⅢ and EcoRI and the fragments
161
m1 and pVAX1 were ligated together producing pVAX1-m1. Subsequently,
162
pVAX1-m1 and pMD18-T-s1 were digested with EcoRI and XhoI, and the fragments
163
s1 and pVAX1-m1 were ligated together producing pVAX1-m1-s1. And then
164
pVAX1-m1-s1, pMD18-T-IFN-γ and pMD18-T-IL-2 were digested with XhoI and
165
XbaI. The fragments IFN-γ, IL-2 and pVAX1-m1-s1 were ligated together,
166
respectively, producing DNA vaccines pVAX1-m1-s1-IFN-γ and pVAX1-m1-s1-IL-2.
167
Using the same method above, DNA vaccines pVAX1-m1-s2-IFN-γ,
ed
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pVAX1-m1-s2-IL-2,
169
pVAX1-m2-s2-IFN-γ and pVAX1-m2-s2-IL-2 were also constructed.
ce pt
pVAX1-m2-s1-IFN-γ,
pVAX1-m2-s1-IL-2,
168
To construct DNA vaccines composed of two fragments without cytokines,
171
pVAX1-s1 and pVAX1-s2 constructed in procedure 2.4 were digested with EcoRI and
172
XbaI to get the fragments s1 and s2 with termination code. At the same time,
173
pVAX1-m1-s1-IL-2 and pVAX1-m2-s1-IL-2 constructed in 2.5 were digested with
174
EcoRI and XbaI to get vector fragments pVAX1-m1 and pVAX1-m2. Hereafter, the
175
antigen fragments s1 and s2 with termination code were ligated with vector fragments
176
pVAX1-m1 and pVAX1-m2 respectively to produce DNA vaccines pVAX1-m1-s1,
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Page 8 of 41
177
pVAX1-m1-s2, pVAX1-m2-s1 and pVAX1-m2-s2.
178
2.6. Constructions of DNA vaccines containing four fragments with and without
179
cytokines To construct pVAX1-m1-m2-s1-s2-IFN-γ and pVAX1-m1-m2-s1-s2-IL-2,
181
pMD18-T-m1 and pVAX1 vector were digested with NheI and HindⅢ and the
182
fragments m1 and pVAX1 were ligated together producing pVAX1-m1. Subsequently,
183
recombinant plasmid pMD18-T-m2 and pVAX1-m1 vector were digested with KpnI
184
and EcoRI and the fragments m2 and pVAX1-m1 were ligated together producing
185
pVAX1-m1-m2. Recombinant plasmids pMD18-T-s1 and pVAX1-m1-m2 vector were
186
digested with KpnI and EcoRI and the fragments s1 and pVAX1-m1-m2 were ligated
187
together producing pVAX1-m1-m2-s1. Recombinant plasmids pMD18-T-s2 and
188
pVAX1-m1-m2-s1 vector were digested with EcoRI and NotI and the fragments s2
189
and pVAX1-m1-m2-s1 were ligated together to producing pVAX1-m1-m2-s1-s2.
190
Recombinant plasmids pMD18-T-IFN-γ, pMD18-T-IL-2 and pVAX1-m1-m2-s1-s2
191
vector were digested with NotI and XbaI and the fragments IFN-γ, IL-2 and
192
pVAX1-m1-m2-s1-s2 were ligated together, respectively, producing DNA vaccines
193
pVAX1-m1-m2-s1-s2-IFN-γ and pVAX1-m1-m2-s1-s2-IL-2.
cr
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194
ip t
180
To construct pVAX1-m1-m2-s1-s2, recombinant plasmid pVAX1-s2 constructed
195
in procedure 2.4 and pVAX1-m1-m2-s1-s2-IL-2 constructed above was digested with
196
EcoRI and XbaI. Subsequently, the target fragments s2 and vector pVAX1-m1-m2-s1
197
were ligated together producing DNA vaccine pVAX1-m1-m2-s1-s2.
198
2.7. Transcription detection of constructed DNA vaccines in vivo by RT-PCR
Page 9 of 41
Chickens were vaccinated with the constructed DNA vaccines by intramuscular
200
injection. Seven days later, about 0.5 g of the injected muscle of each chicken was cut
201
for total mRNA extraction. After digestion of plasmid DNA vaccines by adding
202
DNaseI (TaKaRa) into the mRNA, reverse transcription-polymerase chain reaction
203
(RT-PCR) were performed with the cloning primers of m1, m2, s1, s2, chIFN-γ and
204
chIL-2 genes (Supplementary Table 1), respectively. Electrophoresis in 1% agarose
205
gel was performed to examine the transcription of the target genes. Meanwhile the
206
same site muscle from non-injected chickens was selected as control.
207
2.8. Expression detection of target genes in vivo by Western blot
208
2.8.1. Preparation of rat antiserum to sporozoites and merozoites
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Soluble sporozoite and second-generation merozoite antigens of E. tenella were
210
prepared with the reported methods (Liu, et al., 2009; Khalafalla et al., 2011). Rats
211
were vaccinated subcutaneously on the back with 100 μg antigen emulsified in
212
Freund's Adjuvant Complete (Sigma) at 30 days of age, 14 days later, the second dose
213
was given with Freund's Adjuvant Incomplete (Sigma). 14 days later, a third dose
214
without adjuvant was given. Blood were obtained from tails of the rats for titer
215
determination of antiserum by enzyme linked immunosorbent assay 7 days post the
216
third dose. A fourth even fifth dose would be given unless the titers of antiserum were
217
satisfactory. The antiserum was collected and stored at -20 ℃ for further use when
218
the titers of antiserum were satisfactory.
219
2.8.2. Western blot assay
220
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Chickens were vaccinated with the recombinant DNA vaccines and seven days
Page 10 of 41
post-vaccination, the injected muscle was grinded and treated with RIPA solution (0.1
222
mol/L phenylmethylsulfonyl fluoride (PMSF), 50 mmol/L Tris-HCl, 150 mmol/L
223
NaCl, 1% Nonnidet P-40, 0.1% SDS) for 3 h. After centrifugation at 13000 rpm, the
224
supernatant was collected. Meanwhile the muscle from the same site of non-injection
225
was selected as control. Then Western blot was done to detect the target proteins with
226
rat antiserum to sporozoites or merozoites as first antibody (Song et al., 2013; Xu et
227
al., 2008). Briefly, proteins collected from the injected muscle were separated by
228
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then
229
transferred to a nitrocellulose membrane (Bio-Rad). The membrane was incubated
230
with rat antiserum against sporozoites or second-generation merozoites and
231
horseradish peroxidase (HRP)-conjugated rabbit anti-rat IgG (Sigma) as secondary
232
antibody. The bound antibody was detected using 3, 30-diaminobenzidine (DAB).
233
2.9. Evaluation of protection
ed
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Efficacy of protection was evaluated on the basis of survival rate, lesion score,
235
body weight gain, oocyst decrease ratio and ACI. Survival rate was estimated by the
236
number of surviving chickens divided by the number of initial chickens. Body weight
237
gain of the chickens in each group was determined by the body weight of the chickens
238
at the end of the experiments subtracting the body weight at the time of challenge.
239
Lesion score of the chickens from each group was investigated according to the
240
method of Johnson and Reid (1970). Enteric content of the chickens and daily fecal
241
samples between days 5 and 10 post-infection were collected for oocyst counting
242
using McMaster’s counting technique as described (Ding et al., 2004; Jang et al.,
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234
Page 11 of 41
243
2011). Oocyst decrease ratio was calculated as follows: the number of oocysts from
244
positive control chickens-vaccinated chickens/positive control chickens×100%. Body
245
weight
246
mean±S.D.(standard deviation). ACI is a synthetic criterion for assessing the
247
protective effect of a medicine or vaccine and calculated as follows: (relative rate of
248
weight gain + survival rate) - (lesion value + oocyst value). ACI is considered "good"
249
when 180 or more, "moderate" when 160-179, and "poor" when below 160
250
(McManus et al., 1968).
251
2.10. Immunization of chickens
oocyst
output
and
lesion
scores
were
expressed
as
the
an
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gain
At 14 days of age, chickens were weighed and randomly distributed to 4
253
vaccinated groups and 3 control groups. As shown in Table 1, the 4 vaccinated groups
254
were DNA vaccines containing four fragments vaccinated group (4f), DNA vaccines
255
containing two fragments with cytokine vaccinated group (2f+c), DNA vaccines
256
containing two fragments vaccinated group (2f) and DNA vaccines containing one
257
fragments vaccinated group (1f). The 3 control groups were vector control (vc),
258
unchallenged control (uc) and challenged control (cc). The vaccinated groups 4f, 2f+c,
259
2f and 1f contained 3, 8, 4 and 4 treatments with DNA vaccine (30 chickens/treatment)
260
respectively, and each control group had 30 chickens. Vaccinated group chickens were
261
immunized with 100 µg the corresponding DNA vaccines by leg intramuscular
262
injection, respectively. The challenged control group and unchallenged control
263
chickens were injected with sterile TE and empty vector control group was
264
intramuscularly immunized with 100 µg pVAX1 at the same injection site. A booster
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Page 12 of 41
immunization was given 7 days post the first immunization. At 28 days of age, the
266
chickens were challenged with 5×104 sporulated oocysts of E. tenella JS except the
267
unchallenged control group. Seven days post challenge, all the chickens were
268
weighted. Half chickens of each group were slaughtered for lesion score and the rest
269
half was left for oocyst counting until 10 days post infection. Average body weight
270
gain, oocyst decrease ratio, lesion score, and ACI were calculated as described above.
271
2.11. Statistical analysis
us
cr
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265
Statistical analysis was performed using R statistical package (R Core Team,
273
2013). For body weight gain, differences of means between pairwise groups were
274
tested using Turkey-Kramer method and considered significant at p < 0.05. Since
275
lesion scores and oocyst output don’t follow normal distribution, differences between
276
groups were tested with pairwise comparisons using Wilcoxon rank sum test and
277
considered significant at p < 0.05. The differences between treatments of each
278
vaccinated group were also tested.
279
3. Results
280
3.1. Identification of the DNA vaccines
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Ac
281
an
272
The constructed recombinant DNA vaccines were confirmed by endonuclease
282
digestion. The aim fragments were produced after digestion with the corresponding
283
endonuclease. As shown in Figure 1A, digestion of recombinant plasmid
284
pVAX1-m1-m2-s1-s2-IFN-γ and pVAX1-m1-m2-s1-s2-IL-2 with NheI and HindⅢ
285
produced fragments of approximate 336 bp (Fig.1A, lane 5 and 10) which were equal
286
to the molecular mass of m1, while digestion with NheI and KpnI produced fragments
Page 13 of 41
of approximate 702 bp of m1-m2 (Fig. 1A, lane 4 and 9), digestion with NheI and
288
EcoRI produced fragments of approximate 1011 bp of m1-m2-s1 (Fig. 1A, lane 3 and
289
8), digestion with NheI and NotI produced fragments of approximate 1275 bp of
290
m1-m2-s1-s2 (Fig. 1 A, lane 2 and 7), digestion with NheI and XbaI produced
291
fragments of approximate 1790 bp of m1-m2-s1-s2-IFN-γ (Fig. 1A, lane 1) and 1727
292
bp of m1-m2-s1-s2-IL-2 (Fig. 1A, lane 6), respectively.
293
3.2. Transcription and expression of constructed DNA vaccines in vivo
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cr
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287
Transcription and expression of the DNA vaccines in vivo were detected by
295
RT-PCR and Western blot, separately. RT-PCR produced specific DNA bands of m1,
296
m2, s1, s2, IFN-γ and IL-2 from muscles injected with the corresponding DNA
297
vaccines, respectively (Fig. 1B). No amplification products were generated in the
298
non-injection controls (Fig. 1B, lane 6 and 12). Western blot detection of
299
pVAX1-m1-m2-s1-s2-IL-2 or pVAX1-m1-m2-s1-s2-IFN-γ injected muscles revealed
300
two bands about 60.7 kDa and 63 kDa (Fig. 1C, lane 1-2). Eight bands with the
301
molecular masses of about 41.2 kDa, 42.3 kDa, 40.8 kDa, 39.6 kDa, 39 kDa, 37.3
302
kDa, 40.5 kDa and 38.5 kDa were detected from the pVAX1-m1-s1-IFN-γ,
303
pVAX1-m2-s1-IFN-γ,
304
pVAX1-m1-s1-IL-2, pVAX1-m1-s2-IL-2, pVAX1-m2-s1-IL-2, pVAX1-m2-s2-IL-2
305
injected muscles, respectively(Fig. 1C, lane 4-12), and no protein was detected in the
306
non-injected muscle (Fig. 1C, lane 3-4).
307
3. 3. Protective efficacies of multistage, multi-epitope DNA vaccination against E.
308
tenella challenge
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pVAX1-m2-s2-IFN-γ,
pVAX1-m1-s2-IFN-γ,
Page 14 of 41
309
There were no deaths in any group following coccidial infection. Comparison of
310
protective efficacy on the four types of DNA vaccines (1f, 2f, 2f+c and 4f) was
311
showed in Table 2. The protective efficacies of each type of
312
showed in Table 3 to Table 6 separately.
313
3.3.1. Protective efficacies of the DNA vaccines containing one fragment (1f)
ip t
DNA vaccine were
The immunization efficacies of DNA vaccines containing one fragment (1f) are
315
described in in Table 3. Challenged control and pVAX1 vector control groups
316
exhibited significantly reduced weight gain indicative of active intestinal disease
317
compared with the unchallenged control group (P < 0.05).
an
us
cr
314
The body weight gain of chickens immunized did not significantly differ with
319
that of unchallenged control group (p>0.05), but was significantly higher than that of
320
challenged control group and pVAX1 vector control group (p0.05).
ce pt
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318
Immunization with the constructed DNA vaccines resulted in lower oocyst
324
output and higher oocyst decrease ratio as compared with challenged control group
325
and pVAX1 vector control group (p0.05). DNA vaccines
327
immunized groups presented significant lower lesion scores compared with
328
challenged control group and pVAX1 vector control groups (p 0.05) between numbers with the same letter.
Ac
698
ce pt
695
699
Table 3 Protective efficacy of the multi-epitope DNA vaccines containing one
700
fragment (group 1f) against E. tenella challenge
701
Note: significant difference (P < 0.05) between numbers with different letters. No
702
significant difference (P > 0.05) between numbers with the same letter.
703 704
Table 4 Protective efficacy of the multi-epitope DNA vaccines containing two
Page 32 of 41
705
fragments (group 2f) against E. tenella challenge
706
Note: significant difference (P < 0.05) between numbers with different letters. No
707
significant difference (P > 0.05) between numbers with the same letter.
ip t
708
Table 5 Protective efficacy of the multi-epitope DNA vaccines containing two
710
fragments with cytokines (group 2f+c) against E. tenella challenge
711
Note: significant difference (P < 0.05) between numbers with different letters. No
712
significant difference (P > 0.05) between numbers with the same letter.
us
cr
709
an
713
Table 6 Protective efficacy of the multi-epitope DNA vaccines containing four
715
fragments with and without cytokines (group 4f) against E. tenella challenge
716
Note: significant difference (P < 0.05) between numbers with different letters. No
717
significant difference (P > 0.05) between numbers with the same letter.
ed
ce pt
718
M
714
Supplementary Table 1 Characteristics of the selected four fragments and the primers
720
of chicken cytokines (aa= amino acids)
Ac
719
Page 33 of 41
Ac ce p
te
d
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Fig.1 Click here to download high resolution image
Page 34 of 41
Table 1
Second
Groups
First vaccination vaccination (14 days of age)
Treatments
(21 days of age)
Challenge (28 days of age)
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-s1
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-s2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s1
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s2
100 µg DNA
100 µg DNA
pVAX1-m2-s1
100 µg DNA
100 µg DNA
pVAX1-m2-s2
100 µg DNA
100 µg DNA
pVAX1-m1-s1-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s1-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s2-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-s2-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2-s1-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2-s1-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2-s2-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m2-s2-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-m2-s1-s2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-m2-s1-s2-IL-2
100 µg DNA
100 µg DNA
5×104 E. tenella
pVAX1-m1-m2-s1-s2-IFN-γ
100 µg DNA
100 µg DNA
5×104 E. tenella
vc
pVAX1
100 µg pVAX1
100 µg pVAX1
5×104 E. tenella
uc
TE
100 µl TE
100 µl TE
PBS
cc
TE
100 µl TE
100 µl TE
5×104 E. tenella
4f
5×104 E. tenella 5×104 E. tenella 5×104 E. tenella
cr
us
an
M
ed
2f+c
ce pt
2f
Ac
1f
ip t
pVAX1-m1
Page 35 of 41
Table 2
Groups
Average body weight gain
Mean lesion scores
Lesion decrease ratio (%)
Oocyst output (×106)
Oocyst decrease ratio (%)
ACI
1f
82±15.05b
1.6±1.02c
52.27
4.25±4.39bc
42.67
167.79
2f
85.86±14.17b
1.18±0.94b
62.35
3.62±3.72bc
92.19±10.92c
1.37±0.85
4f
94.94±11.87c
1.22±0.87b
vc
53.72±8.25a
3.10±0.61
uc
88.40±7.73bc
0a 3.40±0.77
3.51±3.66
50.24
179.73
64.05
2.93±3.86b
60.55
186.10
7.13
110.24
100
200
0
100.13
8.82
6.89±7.55
100
0a c
0
7.42±6.7
bc
ce pt
ed
M
an
us
47.48±11.49a
d
58.82
Ac
cc
d
175.70
ip t
2f+c
52.47
b
cr
bc
Page 36 of 41
81.13±16.95bc
Lesion decrease ratio (%)
Oocyst output (×106)
Oocyst decrease ratio (%)
ACI
1.63±1.16c
51.96
4.25±3.48b
42.73
166.04
50.98
4.41±3.91
b
40.57
162.48
55.88
4.14±5.57b
44.25
170.41
52.94
4.22±4.54
b
43.19
170.49
8.82
6.89±7.55c
7.13
110.24
100
200
0
100.13
c
pVAX1-m2
78.20±12.04b
1.67±1.03
pVAX1-s1
83.87±13.60bc
1.50±0.90bc c
pVAX1-s2
84.8±16.87bc
1.60±1.04
pVAX1
53.72±8.25a
3.10±0.61d
a
0
100
0
3.40±0.77e
0
7.42±6.7c
Ac
ce pt
ed
M
an
Unchallenged control 88.40±7.73c Challenged control 47.48±11.49a
a
ip t
pVAX1-m1
Mean lesion scores
us
Treatments
Average body weight gain
cr
Table 3
Page 37 of 41
Table 4
Average body weight gain
Treatments
Mean lesion scores
Lesion decrease ratio (%)
Oocyst output (×106)
Oocyst decrease ratio (%)
ACI
83.15±17.25b
0.87±0.86b
74.51
3.88±3.61b
47.66
175.19
pVAX1-m1-s2
87.81±11.66b
1.13±0.97bc
66.67
3.72±5.57b
49.84
177.97
55.88
2.79±1.61
b
64.50
4.07±3.03b
87.73±14.26b
1.50±1.04
pVAX1-m2-s2
84.82±13.06b
1.21±0.80bc
pVAX1
53.72±8.25a
3.10±0.61
Unchallenged control
88.40±7.73b
0a
47.48±11.49a
3.40±0.77
e
8.82
6.89±7.55
100
0a
0
c
175.22
45.17
173.92
7.13
110.24
c
100
200
7.42±6.7
0
100.13
Ac
ce pt
ed
M
an
Challenged control
d
62.40
cr
pVAX1-m2-s1
us
c
ip t
pVAX1-m1-s1
Page 38 of 41
Table 5
92.7±12.23b
pVAX1-m1-s2-IL2
92.5±9.66b
pVAX1-m1-s2-IFNγ
94.97±11.40b
pVAX1-m2-s1-IL2
91.1±12.97b
pVAX1-m2-s1-IFNγ
89.65±10.19b
pVAX1-m2-s2-IL2
91.82±11.07b
pVAX1-m2-s2-IFNγ
93.22±11.54b
Oocyst decrease ratio(%)
ACI
1.37±0.89b
59.80
3.7±5.2b
50.12
180.36
53.61
181.63
1.37±1.19
b
1.27±1.08
b
1.23±0.90
b
1.53±0.97
b
1.23±0.82
b
1.47±0.86
b
1.50±0.86
b c
pVAX1
53.72±8.25a
3.10±0.61
Unchallenged control
88.40±7.73b
0a
47.48±11.49a
62.75 63.73 54.90 63.73 56.86 55.88 8.82 100
3.40±0.77
d
3.44±4.2
3.04±3.22
b
2.81±2.84
b
4.02±5.23
b
0
59.01
183.1
62.12
185.86
45.76
178.46
b
58.12
179.02
4.15±2.76
b
44.00
179.25
3.81±4.58
b
48.59
180.91
6.89±7.55
c
7.13
110.24
100
200
0
100.13
3.11±3.45
0a
c
7.42±6.7
Ac
ce pt
ed
M
Challenged control
59.80
b
ip t
pVAX1-m1-s1-IFNγ
Oocyst output (×106)
cr
91.58±13.66b
Lesion decrease ratio (%)
us
pVAX1-m1-s1-IL2
Mean lesion scores
an
Average body weight gain
Treatments
Page 39 of 41
Table 6
Mean lesion scores
Lesion decrease ratio (%)
Oocyst output (×106)
92.37±13.72bc
1.53±1.11b
54.90
3.93±4.9b
96.02±13.21bc
1.10±0.80
b
1.03±0.56
b
3.10±0.61
c
pVAX1-m1-m2-s1-s2 pVAX1-m1-m2-s1-s2-IL2 pVAX1-m1-m2-s1-s2-IFNγ pVAX1
96.45±7.69c 53.72±8.25a
Unchallenged control Challenged control
88.40±7.73b
0
69.61 8.82 100
3.40±0.77
d
46.99
180.39
2.54±3.42
b
65.74
187.98
2.31±2.92
b
68.93
189.92
6.89±7.55
c
7.13
110.24
100
200
0
100.13
a
0
c
0
ACI
7.42±6.7
Ac
ce pt
ed
M
an
us
cr
47.48±11.49a
a
67.65
Oocyst decrease ratio(%)
ip t
Average body weight gain
Treatments
Page 40 of 41
Supplementary material for online publication only
Supplementary Table 1 Characteristics of the selected four fragments and the primers of chicken cytokines (aa=amino acids)
chIL-2
Reverse: 5’-AAAGAATTCCTACAGCGCGCAGC-3’ Forward: 5’-GCAGAATTCATGAAACTGGC-3’ Reverse: 5’ –AAATCTAGACTACGCCCCGT- 3’ Forward: 5’- TTACGCGGCGCAATGACTTGCCA -3’
117
6
95
80
2
4
ntal stages Merozoite, Sporozoite
ip t
MZ5-7
MZ5-7
cr
Forward: 5’-CCCGGATCCATGGACCTCTT-3’
4
us
chIFN-γ
Reverse: 5’ -CCCTCTAGACTATTGAGTTA -3’
107
Developme
Merozoite, Sporozoite
SO7
Sporozoite
SO7
Sporozoite
an
s2
Forward: 5’- AAAGAATTCATGCAAACACT-3’
Source
Reverse: 5’ – CCCTCTAGATTAGCAATTGCATCTCCTC - 3’ Forward: 5’- AAAGCGGCCGCAATGATGTGCAAAGTA -3’
Reverse: 5’ – CCCTCTAGATTATTTTTGCAGATATCTCAC - 3’
M
s1
Reverse: 5’-CCCGAATTCCTATTGGATAAGG-3’
T cell motifs
ed
m2
Forward: 5’-AAAGGATCCATGCAGGAAGT-3’
Size(aa)
ce pt
m1
Primers
Ac
Fragments
Page 41 of 41
