Construction Of Fusion Expression Vector's And Expression Of Classical Swine Fever Virus's Glycoprotein E₂ Gene

The main protective antigen E2 of classical swine fever virus can induce to produce neutralizing antibody, and the protective immunity.Prokaryotic expression plasmid pGEX-4T-E2 is constructed by the connection between GST fusion prokaryotic expression vector pGEX-4T-l and target gene E2 And the test to recombinant plasmid is made by means of enzyme digest and sequencing technique. IPTG inducing expression with host bacterium of BL21 is done.Eukaryotic recombinant transfer plasmid pAcsec-E2 with GST is constructed by the connection between AcMNPV baculovirus transfer vector pAcSecG2T and target gene E2. The assays of enzymolysis and PCR were carried out to confirm the correctness. The pAcsecG2T-E2 plasmid and liner Bombyx mori's baculovirus are cotransfected into Bombyx mori's nurturing cell. And the recombinant virus with target E2 gene is screened by plaque assay. PCR amplification is made to measure recombinant virus. And then recombinant virus Bm-BacPAK-E2 is screened and purified further by plaque assay. The recombinant virus is respectively expressed in Bm-N, and Bombyx mari's larva and pupa.SDS-PAGE electrophoresis is used to test the effect of both expression of prokaryotic fusion and baculovims GST's eukaryotic. The result shows there's aspecial band at about 63kD which is accordant to the protein molecular weight deduced by the target gene.In our research, prokaryotic expression plasmid pGEX-4T-E2 and eukaryotic transfer plasmid have been constructed. And according to the result, the conclusion that E2 has got its fusion expression in BL21 can be implied, which will be the molecular foundation work of purification of antigen protein prokaryoticlly expressed and exploitation of CSFV diagnose reagents. And, also, CSFV F114E2 gene's fusion protein has been preliminarily expressed by the baculovirus expression system in this research. Perhaps, in some aspects, this will contribute to the development of CSFV's new type of subunit vaccine.