Expression Of Nucleocapsid Gene Of Porcine Circovirus Type 2 In Prokaryote And Eukaryote System

Some kinds of diseases are related with porcine circovirus type 2 in porcine groups including PMWS, PDNS, CT, PRDC et al. Especially, PMWS is the most serious and has made great economic losses in the world. China can not escape by sheer luck. At present, there is no efficient diagnostic kit in the domestic market. The rapid and specific diagnostic method is much important to control these diseases .The nucleocapsid protein of PCV-2 encoded by ORF2 is a major structural protein of virus and has specif immunogenicity .The Cap gene is a conserved sequence in the viral genome .The expressed product would be used in diagnosis .So two aspects as follows in this research are to set up the diagnostic method on the basis of Cap protein and protect against the diseases associated with PCV-2.1 Expression in prokaryote systemPCV-2 was inoculated in PK-15 cells. The Cap gene was amplified by polymerase chain reaction (PCR) from the extracted virus DNA and cloned into pET32-a.The recombinant piasmid pETORF2 was constructed and transformed into the BL21. A fusion protein was induced with IPTG and expressed, but the molecular weight of the fusion protein was not accord with the expected. In the same way, pETRF2 was constructed and the part of Cap gene was included. The result indicated the same situation. After the analysis of Cap gene, there existed many Arg rare codons (AGG/AGA). It led to translation obstruct and the Cap protein could not be expressed. So the Cap gene was transformed. On the basis of not changing the ammonia, the sequence of codons was replaced by the major codons of E.coli. According to the changed sequence and the principle of gene SOEing, four primers were designed and the gene including the epitopes of Cap was amplified by touchdown PCR. Then it was cloned into pET32-a, the recombinant piasmid pETP!P4 was constructed. The result of SDS-PAGE indicated the correct molecular weights with the expected.2 Expression in eukaryote systemThe virus DNA was extracted from the PK-15 cells inoculated with PCV-2. Cap gene was amplified bv PCR and cloned into the pSecTag2 vector. It was named pSecORF2. Cap gene including vector signal peptide sequence was amplified from vectors pSecORF2. Then it was cloned into pIREShyg vector. The recombinantexpression vector pIRESiORF2 was constructed. CHO cells were transfected in vitro with calcium phosphate. After Hygromycine B selection, a stable cell strain that can express the Cap gene judged by. PCR and Immunofluorescence Assay (IFA) .It lays the groundwork of diagnostic method on the basis of Cap gene.