Cancer Letters, 65 (1992) 201- 207
Elsevier Scientific Publishers Ireland Ltd.
Polyclonal and monoclonal antibodies monospecific to MMTV LTR orf protein produced in E. co/i Satomi Hagaa, Katsuya Shigesadab, Yujiro Nambab, Harutaka Tanakab, Shunsuke Junji Morimotoc, Shingo Hiroishic, Hiroshi Yamamotoa, Nurul H. Sarkard and Jo Hilgerse
“The Second Deportment of Anatomy and The Second Department of Pathology, JVara Medical University, bThe Institute for Virus Research, Kyoto Uniuersity (Japan) dDepartment of Immunology and Microbiology, Medical College of Georgia (USA) and ‘Research Laboratory for Oncological Gynecology, Vrije Unioersiteit (The Netherlands) (Received (Accepted
11 May 1992) 18 May 1992)
Summary Monoclonal and polyclonal antibodies specific to an open reading frame of the mouse mammary tumor uirus long terminal repeat were generated using an open reading frame,!I-galactosidase fusion protein produced in E. coli. Both antibodies reacted with the open reading frame-fi-galactosidase fusion protein but not with &galactosidase alone using an immunoblotting technique. It is concluded that these antibodies were specific for the protein encoded by the open reading frame of the mouse mammary tumor virus long terminal repeat.
Keywords: mouse mammary tumor virus; long terminal repeat; open reading frame; colloidal gold; monoclonal
Introduction The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) has an open
reading frame (orf) in its U3 region [l] and this is a unique feature in MMTV compared with other retroviruses. The orf was discovered by
DNA sequencing of the proviral LTR  and by in vitro translation of the 3 ’ end of genomic RNA of MMTV [3 - 61. The orf is well conserved in all MMTV isolates tested so far by molecular techniques [2,7 - lo]. From the sequence, the orf is sufficient to code for a 40 000 M, protein  and many studies have been performed to test whether the orf gene expression occurs in vitro. It has been found that 1.6 - 1.7 kb transcripts of the orf are expressed in the lactating mammary gland [11,12], a T-cell lymphoma [ll], and normal spleen cells . The protein products of the orf [3,11,14] were reported to be glycoproteins  by immunoprecipitation technique. However, antibodies in these experiments were made against the synthetic peptide antigen and showed a high non-specific cross reaction in histochemical experiments. We tried to make an antibody against the almost complete orf protein using molecular biological techniques. Materials and Methods
Correspondence Anatomy, Nara, 634,
Nara Medical University, Japan.
Printed and Published in Ireland
Bacterial strain We prepared
Elsevier Scientific Publishers Ireland Ltd
CSH50 (F’gal iq) carrying lac Z, orf (C3H virus origin), [email protected]
Orf transformed and control (vector only) E. coli were cultured in L-broth medium containing 50 pg/ml of ampicillin at 37OC for 8 h. Purification of [email protected]
(orf-/?-gal) fusion protein A 4.5-g quantity of E. co/i was harvested by centrifugation from 1.8 1 of culture medium. The cells were suspended in 30 ml of 20 mM Tris-HC! (pH 7.5), 20% glycerol, 10 mM MgCle, 10 mM mercaptoethanol (buffered glycerol, BG) containing 4.95 mg of PMSF (phenylmethanesulfonyl fluoride) and 10.95 mg of TLCK (W-tosyl-L-lysyl chloromethyl ketone) and were disrupted by sonication. The cell extract was centrifuged in a Sorval centrifuge at 40 000 x g for 30 min and the supernatant was discarded. The pellet was suspended in 8 ml of 2 M guanidine- HC!, 1 mM dithiothreitol (DTT) containing buffer BG with a Potter homogenizer and the fusion protein was recovered by centrifugation for 40 000 x g for 30 min at OOC. The pellet was then washed with distilled water until the guanidine was completely removed, subsequently the pellet was suspended in 5 ml of 0.1 M phosphate buffer (pH 7.0), containing 10 mM DTT, 3.3% SDS and stored at 32OC overnight. A l-ml quantity of protein solution was loaded onto the first CL-6B column (bed vol., 70 ml). A 0.1-M phosphate buffer (pH 7.0) containing 0.1% SDS and 1 mM DTT was applied to the column and the peak protein fractions were pooled. All peak fractions were concentrated with Lyphogel (ATTO, Osaka, Japan) to 4 ml loaded onto the second CL-6B column (bed vol., 670 ml) and eluted with the same buffer. The peak protein fractions were pooled. Rabbit antiserum against the or-f-/3-gai fusion protein Anti orf-P-gal serum was raised in rabbits by six weekly l-ml injections of protein solution (50 pg of protein each) with the same volume of complete Freund’s adjuvant.
Preparation of the specific antiserum against the orf from anti orf-&gal rabbit serum CNBr activated Sepharose 4B gel was coupled with o&&gal or P-gal according to the manufacturer (Pharmacia Fine Chemicals). Anti orf-&gal serum was loaded onto P-galCNBr activated Sepharose 4B first. The flowthrough fractions were loaded onto orf-P-galCNBr activated Sepharose 4B and were eluted with 1 N CH,COOH. Preparation of the monoclonal antibody against the orf-P-gal fusion pritein Monoclonal antibody against the orf-p-gal fusion protein was prepared according to the methods of Kohler and Milstein [ 151. The titer of the monoclonal antibody was determined using Multi Micro Filters ‘Dot Plate’ (AD-
VANTEC). Preparation of immunocolloidal gold Colloidal gold was prepared by reducing tetra-chloroaoric acid with tannic acid [ 161 and then coating with Protein A [17,18]. Protein A-gold was centrifuged at 55 000 x g for 40 min on a 3-ml cushion of 5% glycerol containing 0.05% polyethylene glycol and 0.01% sodium merthiolate. At the bottom of the tube, approximately 2 ml of condensed sol and small, densely packed pellets of protein A-gold were found from 100 ml of the original colloidal gold. Immediately before use, the sol was diluted lo-fold with 1% BSA in PBS and centrifuged at 6500 x g for 10 min to remove aggregates. The supernatant was diluted lOOfold for materials embedded in Lowicryl K4M [19,20]. Thin section immunocolloidal
E.coli, strain PSI3104 was fixed with 0.5% glutaraldehyde and embedded in Lowicryl K4M at - 20°C. Thin sections were picked up on coltodion-carbon-coated grids. These were floated thin sections downwards on a drop of the following reagents at room temperature in the order; (1) 1% BSA in PBS for 5 min, (2) antiserum on monoclonal antibody for 30 min, (3) 1% BSA in PBS 3 times, each for 5 min,
(4) protein A-gold sol for 30 min, (5) 1% BSA in PBS, 3 times each for 5 min, (6) distilled water, 3 times each for 5 min, (7) saturated aqueous solution of uranyl acetate for 10 min and finally (8) distilled water, 3 times each for 5 min. Electrophoresis of proteins Protein was separated by electrophoresis in 7.5% or 12% polyacrylamide gels in 0.1% SDS according to Laemmli [Zl]. Proteins (approx. 50 pg) were mixed with 10 ~1 of sample buffer (125 mM Tris - HCI, pH 8.8, 2% SDS, 20 mM DTT, 0.02 mg/ml bromophenol blue and 10% glycerol). The samples were heated at 70°C for 3 min and loaded on the gel. After electrophoresis, proteins were stained with Coomassie Brilliant Blue.
electrophoresis (Fig. 2). In this strain P-gal or orf-P-gal fusion protein was recognized by the appearance of polypeptides of the expected molecular size. Figure 3 shows the 2 M guanidine - HCI fraction and the peak fractions of the eluent from the second CL-6B column. In CL-6B eluent, two bands were seen which were the products translated at the different initiation (ATG) codes of the orf gene (Fig. 1). Absorption of the rabbit antiserum with @gal Figure 4 shows the specificity of the rabbit antiserum using a Dot Plate, after absorption by fl-gal-CNBr activated Sepharose 4B affinity column. The original antiserum reacted with both orf-P-gal fusion protein and P-gal, but
R43SdtSi Purification of the fusion proteins E. coli (strain pSI3104) were disrupted by sonication and analysed by polyacrylamide gel
Construction of recombinant plasmids containing MMTV LTR orf gene and &gal gene.
Fis. 2. Analysis of products derived from E. coli which consists of &gal plasmid (&gal) or o&&gal plasmid (orf) The molecular size is described at the left side.
Fig. 3. The PAGE analysis of 2 M guanidine - HCI fraction (lane a), CL-6B gel filtration peak fraction molecular marker (d). The molecular size is described at the right side.
Fie. 4. The titer of the monospecific rabbit antiserum. The antigen used is indicated at the right side and antiserum is described at the left side. Dilution used is lOO-fold, 3 x lOO-fold, etc. Ori, indicates an original antiserum and an orf-lac z indicated after absorbed antiserum by affinity column.
Fig. 5. treated
The titer of the monoclonal antibodies. The antigen P-gal, orf-lac z indicates purified fusion protein.
used is indicated
at the top. P-gal-SDS
Fis. 6. lmmunoelectron microscopy of orf plasmid containing E. co/i (A) and orf plasmid absent E. coli (B). Monoclonal antibody c 1.40 (original dilution) was used. Gold particles, although few, are seen in the cytoplasm of orf plasmid containing E. coli ( x 21000).
Table 1. Number
of gold particles per pm* of:
after absorption over a P-gal column, the antiserum reacted only with orf-P-gal fusion protein. Antibodies against P-gal were removed using these methods and the antiserum was concluded to be the monospecific antibody against the LTR orf protein. Monoclonal antibody Figure 5 shows the specific reaction of the monoclonal antibodies. The ~1.40, ~1.62, cl.195 reacted with the orf-&gal fusion protein, but not with P-gal alone. It is therefore concluded that these clones secrete monoclonal antibodies against LTR orf protein. Immunocolloidal gold method Table I and Fig. 6 show the results of the immunocolloidal gold electron microscopy. The gold particles were mainly seen in the cytoplasm of orf-&gal plasmid containing E. coli (Fig. 6A, arrow), but not seen in only pBR322 plasmid containing E. co/i (Fig. 6B) or non-cellular space (Fig. 6A and 6B). Table I quantifies the results seen in Fig. 6. In the cytoplasm of [email protected]
plasmid containing E. co/i, 6.23 gold particles per pm* were seen, whereas 0.6-0.7 gold particles per pm* were seen in the extracellular space or cytoplasm of E. co/i containing the plasmid pBR322. Discussion
LTR orf is believed to play an important role in oncogenesis of the MMTV, thus, it is very important to know the function and the expression of LTR orf products. While the proteins or transcripts of LTR orf were found in cell lines [3,14] and tissues many [3,11,12,14], the orf proteins could not be
found in mammary tumors themselves. It is, therefore uncertain whether the orf was related to the oncogenes is of mammary tumor caused by MMTV. The presence of the proteins or the transcripts in lymphoid cells [l 1,141may suggest that the mammary tumor or MMTV might have some role in causing leukemia [22,23]. But, Carr et al.  suggested that orf may play a role to the oncogenesis in MMTV although perhaps not directly. An antibody specific to LTR orf should facilitate the analysis. However, antibodies against MMTV LTR orf have not been made directly from the proviral LTR orf producing protein so far. In this communication, we report on specific antibodies produced against MMTV LTR orf protein synthesized in E. co/i. In regard to specific reactivity against the orf, immunoelectron microscopy showed no differences between a monoclonal antibody and absorbed polyclonal rabbit antiserum by immunoelectron microscopy (data not shown). By using monoclonal antibodies against LTR orf, the cytoplasm of the orf-P-gal plasmid containing E. co/i showed 8.8 - 10.4-times more gold particles comparing with control E. co/i or no cellular space. It is concluded that the polyclonal and the monoclonal antibodies will be useful for further studies of MMTV orf protein. Acknowledgments
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