Prokaryotic Expression Of P27 Gene Of Avian Leucosis Virus And Production Of The Rabbit Anti-p27 Antibody

P27 gene is one of rather conservative genes among different subgroups of avian myeloblastosis virus. The capsid p27 protein is the major component of the core shell of avian avian myeloblastosis virus and the major group specific antigen. So p27 protein is the optimum antigen used to develop group specific antibody. In the experiment, a pair of primer which specifies p27 gene was designed according to AMV BAI-A strain sequence reported previously. P27 gene was obtained by PCR with the recombinant plasmid PGEM-gag as the template. The PCR products were cloned into PGEM-T Easy vector. Plasmid PGEM-p27 and Expression Vector pET-28a were digested respectively with the restriction enzyme, and then ligated to construct the recombinant expression plasmid. Correct recombinant expression plasmids pET28a-p27 were obtained after transformants were identified by PCR assay and the BamH I-Sal I digestion assay. The prokaryotic expression construct for p27 was transformed into BL21 (DE3), and then recombinant engineering bacteria were selected and identified. Culture engineering bacteria in the gross and add 3mM IPTG to induce foreign protein expression. The cells were harvested by centrifugation and lysed by sonication. Then collected the soluble protein and purified the protein with Ni-NTA affinity chromatography. Purified recombinant protein was analyzed by SDS-PAGE and Western-blotting. In order to get antibody, the rabbits were immunized with p27 protein according to the routine method and then collected the serum. The titer of the rabbit anti-p27 antibody was measured by agar diffusion assay and ELISA assay.The results showed that p27 gene of avian myeloblastosis virus was successfully subcloned from pGEM-gag. BL21(DE3)strains with pET28a-p27 constructs successfully expressed the recombinant protein bearing an N-terminal poly-His tag with molecular weight of 30KDa, which is consistent with the predicted putative molecular weights of p27. ID muli-analysis showed that the recombinant protein was accounted for about 25.3% in the total bacterial proteins before purified and above 97% after purification. The recombinant protein was proved to be the specific p27 protein with western-blotting using the Rabbit anti-p27 IgG HPR conjugated as the antibody. The agar diffusion assay showed that the anti-serum titer of immune rabbit was 1: 32. The anti-serum was proved to be the specific anti-p27 with the Western-blotting using the anti-serum as the first antibody. ELISA in which the plant was coated with 1: 200~ 1: 32000 diluted serum showed that the OD is above 1.0 when the serum was diluted 4000.In conclusion, the experiment laid a good foundation for the research of the structure of p27 protein, the preparation of the monoclonal antibody against p27 protein and the development of ELISA antigen diagnosis Kit.