Research in Veterinary Science 98 (2015) 39–41

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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c

Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope Limeng Zhang a,1, Hua Zhang a,1, Ziyao Fan a, Xue Zhou a, Liquan Yu a, Hunan Sun a, Zhijun Wu a, Yongzhong Yu a, Baifen Song a, Jinzhu Ma a, Chunyu Tong a, Zhanbo Zhu b, Yudong Cui a,b,* a b

College of Life Science and Technology, HeiLongJiang BaYi Agricultural University, Daqing 163319, China College of Animal Science and Veterinary Medicine, HeiLongJiang BaYi Agricultural University, Daqing 163319, China



Article history: Received 15 August 2014 Accepted 6 November 2014 Keywords: Streptococcus dysgalactiae GapC Phage display Epitope Homology


Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97–103 of the S. dysgalactiae GapC. In addition, the epitope 97TGFFAKK103 showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of 97TGFFAKK103, and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine. © 2014 Elsevier Ltd. All rights reserved.

1. Short communication The Streptococcus dysgalactiae (S. dysgalactiae) is responsible for a significant proportion of cases of mastitis in lactating and nonlactating cows (Barkema et al., 1999; Wang et al., 1999). S. dysgalactiae produces a number of surface proteins that have been associated with its virulent properties (Calvinho et al., 1998; Oliver et al., 1998). It is reported that surface proteins could confer protection against infection of S. dysgalactiae (Song et al., 2002). These events make these surface proteins to be good candidate for vaccine study. Currently, several genes encoding surface proteins have been isolated and their proteins showed significant protection of immunized animals from challenge with S. dysgalactiae (Song et al., 2002). One of the surface proteins is the streptococcal surface dehydrogenase (GapC), possessing glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity. GAPDH is a key glycolysis enzyme that reversibly catalyses the conversion of glyceraldehyde 3-phosphate to 1,3 bis-phosphoglycerate (Pancholi and Fischetti, 1992). Besides its role in glycolysis, GapC has been found to be a multifunctional protein that serves as a potential virulent factor (Fontaine et al.,

* Corresponding author. Tel.: +86 0459 6819290; fax: +86 0459 6819290. E-mail address: [email protected] (Y. Cui). 1 Both authors equally contributed to this work. 0034-5288/© 2014 Elsevier Ltd. All rights reserved.

2002). Immunization with recombinant GapC protein appeared to confer protection following challenge with the S. dysgalactiae (Song et al., 2001). Similarly, our previous study suggested that GapC protein functions as the immunodominant protein and is responsible for eliciting antibodies against S. dysgalactiae. It is well known that antigen elicits immune responses mainly through antigen epitopes. B-cell epitope is defined as regions on the surface of the native antigen that is recognized by binding to B-cell receptors or specific antibodies. However, the B-cell epitope in S. dysgalactiae GapC protein has not been finely mapped, and the core sequence of the epitope needs to be determined. Epitope is the focus of immunological research as well as the target of developing epitope-based vaccines (Li et al., 2007, 2008). The objective of the present study was to produce the mouse monoclonal antibody against S. dysgalactiae GapC, and further to map its conserved B-cell epitope. We firstly used two online software programs (http://tools and BepiPred/) to predict the B-cell epitopes of the S. dysgalactiae GapC protein. The results showed that the 30–50 aa fragment in the N-terminus of GapC received the highest scores and was determined as the continuous-epitope fragments (data not shown). Fragment encoding 1–150 aa of S. dysgalactiae GapC was cloned into pET-32a(+) plasmid resulting in the corresponding His-TrxA fusion protein. The recombinant GapC1–150 was purified using Ni-NTA purification system (Merck). The purified GapC 1–150 protein was confirmed by SDS-PAGE and Western blot (Fig. 1A).


L. Zhang et al./Research in Veterinary Science 98 (2015) 39–41

Fig. 1. (A) The purified recombinant GapC1–150 protein was detected by SDS-PAGE and Western blot. (B) The purified mAb1E11 was determined by SDS-PAGE. (C) The reactivity of mAb1E11 with the recombinant GapC of S. dysgalactiae, S. uberis, S. agalactiae and S. aureus was determined by Western blot. (D) The reactivity of mAb1E11 with the whole bacteria of inactivated S. dysgalactiae, S. agalactiae and S. uberis was confirmed by indirect ELISA (* p < 0.05; ** p < 0.01). (E) Detection of the binding activities of 11 positive phage clones to mAb1E11 using sandwich ELISA.

Splenic cells of the GapC1–150-immunized mice were fused with SP2/0 mice myeloma cells. The hybridoma lines secreting anti-GapC1–150 monoclonal antibodies (mAbs) were established by hybridoma technique (Kohler and Milstein, 1975; Mazzarotto et al., 2009). The mAbs to the recombinant GapC1–150 were successfully purified from mouse ascites (Fig. 1B) (animal experiments were approved by Ethics Committee of Experimental Animal Center, Heilongjiang Bayi Agricultural University). The classes of the mAbs were determined to be IgG1 and κ chains (55 kDa and 26 kDa) using Mouse Monoclonal Antibody Isotyping Kit (Promega, USA). One of the cell lines, referred to as mAb1E11, could stably secrete antiGapC antibody at high titer for more than 10 passages. Antibody titers of the culture supernatant and ascites fluid were 1:6400 and 1:1.28 × 105. To characterize the mAb1E11 specificity for binding GapC, fulllength S. dysgalactiae gapC gene was cloned into pET-32a(+) plasmid resulting in the corresponding His-TrxA fusion proteins. Other S. uberis, S. agalactiae and Staphylococcus aureus (S. aureus) gapC genes were cloned into pET-30a(+) plasmid resulting in the Hisfusion proteins, respectively. Western blot analysis showed that, besides truncated GapC1–150 of S. dysgalactiae (Lane 2), mAb1E11 also recognized full-length recombinant GapC of S. dysgalactiae (Lane 1), S. agalactiae (Lane 3) and S. uberis (Lane 4). However, mAb1E11

did not recognize recombinant full-length GapC protein of S. aureus (Lane 5) (Fig. 1C). The reactivity of mAb1E11 with the whole cells of the three streptococcus species was determined by indirect ELISA. The results showed that both positive GapC serum and the mAb1E11 could significantly react with the whole cells of S. dysgalactiae, S. agalactiae and S. uberis (Fig. 1D), but the negative control antibody (mAb Trap) failed to react with these Streptococcus. These results imply that the epitope recognized by mAb1E11 lies in GapC of the three Streptococcus species and is exposed on bacterial surface. Subsequently, the Ph.D.-12 Phage Display Peptide Library (New England Biolabs, Beverly, MA, USA) containing random 12-mer peptides was screened with mAb1E11 according to the previous methods (Li et al., 2007, 2008). After three successive rounds of biopanning, the screened phage bound to the mAb1E11 was well enriched and the yield of positive phage clones significantly increased. Twentyfive clones selected by mAb1E11 were analyzed by sandwich ELISA, and 11 clones among them showed specific reactivity to mAb1E11 (Fig. 1E). The single-strand phage DNAs extracted from the 11 ELISAconfirmed positive clones was sequenced and the amino acid sequences were deduced. The amino acid sequences of the 11 clones showed a consensus motif TGFFAKK (Fig. 2A). The motif exactly matched with amino acids 97–103 of the S. dysgalactiae GapC, which

L. Zhang et al./Research in Veterinary Science 98 (2015) 39–41


the motif represents the minimal reactivity unit of the continuous epitope recognized by mAb1E11. Lastly, the epitope sequences of 7 Streptococcus species and 11 other microbial species were aligned to further analyze whether the characterized epitope is conserved. It is shown that all amino acids in the motif are identical among all 7 Streptococcus species, indicating that the motif represented a conserved epitope on the GapC protein of Streptococcus. However, the epitope motif of Streptococcus is different from the motif in other microbial species (Fig. 2C). This motif 97TGFFAKK103 represents a highly conserved epitope in the GapC proteins of Streptococcus species, which might be available for the further study of epitope-based vaccines. Acknowledgments This work was supported by National Natural Science Foundation of China (NSFC, Grant Nos. 31072120), Science and Technology Project for Heilongjiang Agricultural Reclamation Administration (HNK11A-08-01-04), and National “Twelfth Five-Year” Plan for Science & Technology Support (Grant Nos. 2012BAD12B03 and Nos. 2012BA12B05). References

Fig. 2. (A) Displaying peptide sequences from the positive phage clones through biopanning in Ph.D.-12 library. Serine (S) could substitute for threonine (T), and arginine (R) could substitute for lysine (K) because of their similar structure. Conservative amino acid motifs are blue-indicated and underlined. (B) The reactivity of various recombinant GST-fusion proteins with mAb1E11 was confirmed using Western blot. The mutant amino acids are red-indicated and underlined. (C) Alignment of sequences containing the epitope on GapC from different microbial species. The homologous amino acids are blue-indicated and underlined. For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.

suggested that the motif 97 TGFFAKK 103 is an epitope of the S. dysgalactiae GapC protein. DNA fragments encoding the motif 97TGFFAKK103 were cloned and a series of mutated recombinant GST-fusion proteins were purified, respectively. Western blot results showed that G98A, F99A, F100A, and K103A mutations completely destroyed the reactivity of the epitope with mAb1E11. However, the mutants T97A and K102A showed slightly reduced reactivity of the epitope with mAb1E11 (Fig. 2B). These results confirm that the motif 97TGFFAKK103 is an authentic epitope in the GapC protein of S. dysgalactiae and

Barkema, H.W., Schukken, Y.H., Lam, T.J., Beiboer, M.L., Benedictus, G., Brand, A., 1999. Management practices associated with the incidence rate of clinical mastitis. Journal of Dairy Science 82, 1643–1654. Calvinho, L.F., Almeida, R.A., Oliver, S.P., 1998. Potential virulence factors of Streptococcus dysgalactiae associated with bovine mastitis. Veterinary Microbiology 61, 93–110. Fontaine, M.C., Perez-Casal, J., Song, X.M., Shelford, J., Willson, P.J., Potter, A.A., 2002. Immunisation of dairy cattle with recombinant Streptococcus uberis GapC or a chimeric CAMP antigen confers protection against heterologous bacterial challenge. Vaccine 20, 2278–2286. Kohler, G., Milstein, C., 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497. Li, Y., Ning, Y.S., Wang, Y.D., Hong, Y.H., Luo, J., Dong, W.Q., et al., 2007. Production of mouse monoclonal antibodies against Helicobacter pylori Lpp20 and mapping the antigenic epitope by phage display library. Journal of Immunological Methods 325, 1–8. Li, Y., Ning, Y.S., Wang, Y.D., Luo, J., Wang, W., Dong, W.Q., et al., 2008. Production of mouse monoclonal antibodies against Helicobacter pylori catalase and mapping the antigenic epitope by phage display library. Vaccine 26, 1263–1269. Mazzarotto, G.A., Raboni, S.M., Stella, V., Carstensen, S., de Noronha, L., Levis, S., et al., 2009. Production and characterization of monoclonal antibodies against the recombinant nucleoprotein of Araucaria hantavirus. Journal of Virological Methods 162, 96–100. Oliver, S.P., Almeida, R.A., Calvinho, L.F., 1998. Virulence factors of Streptococcus uberis isolated from cows with mastitis. Journal of Veterinary Medicine Series B-Infectious Diseases and Veterinary Public Health 45, 461–471. Pancholi, V., Fischetti, V.A., 1992. A major surface protein on group-a Streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding-activity. Journal of Experimental Medicine 176, 415–426. Song, X.M., Perez-Casal, J., Bolton, A., Potter, A.A., 2001. Surface-expressed mig protein protects Streptococcus dysgalactiae against phagocytosis by bovine neutrophils. Infection and Immunity 69, 6030–6037. Song, X.M., Perez-Casal, J., Fontaine, M.C., Potter, A.A., 2002. Bovine immunoglobulin A (IgA)-binding activities of the surface-expressed Mig protein of Streptococcus dysgalactiae. Microbiology (SGM) 148, 2055–2064. Wang, S.M., Deighton, M.A., Capstick, J.A., Gerraty, N., 1999. Epidemiological typing of bovine streptococci by pulsed-field gel electrophoresis. Epidemiology and Infection 123, 317–324.

Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope.

Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in a...
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