Optimization Of Anti-porcine Circovirus Type2Virus Gene And Testing Its Antiviral Effect

Porcine circovirus type2(PCV2) is the main pathogen，which can cause PMWS，and is alsoclosely related with PNP，CT-AII，PDNS, Breeding Disorder and so on. The disease associated withPCV2had been a kind of disease which causes huge economic loss and seriously harmed swine industryCapsid targeted viral inactivation (CTVI) is a new antiviral strategy, which is based on protein.Compared with other antiviral strategies based on nucleic acid, CTVI possesses five advantages, such ashigh stability, good specificity and tolerance to the mutation of virus genome. The emergence of CTVIprovided a new idea in screening anti-PCV2virus.In previous research, our laboratory had constructed the eukaryotic expression structures forJNPC(staphylococcal nuclease gene at amino terminal)and PNJC(staphylococcal nuclease gene atcarboxyl terminal) gene,using pIRESNeo as the vector. By cell transfection, the cell lines withpIRESNeo vectors had been obtained, which could express one of the two genes mentioned abovesteadily. The transgenic cell lines and the protein products have been identified by PCR, Northern Blot,RT-PCR, Western Blot etc.Meanwhile, the anti-viral effect of the cell line had been masured by the viruspassage experiment. The result showed that the fusion gene JNPC had some extent anti-virus ablity. Butcompared with other applications of CTVI in antiviral study， the antiviral efficiency of this gene waslower. Analyzing the transgenic cell lines through RT-PCR and sequencing shows that the loweranti-viral effect is due to the abnormal splicing site of the fusion genes and perhaps codon preference.To get more effective antiviral gene of PCV2, the gene JNPC will been redesigned and synthesized andthe structure of the vector need be reconstructed.In this research, the eukaryotic expression structures is reconstructed by inserting the redesigningand synthesizing gene JNPC into the new modified vector. Transgenic cell lines are obtained，which canexpress modified JNPC and Cap genes stably, and its antiviral ability is detected.The eukaryotic expression structures for JNPC and Cap gene had been constructed, usingpattb-INS-CUBC-IRES-Neo-GHPA as the vector. By cell transfection and G418screening, thetransgenic cell lines expressing modified JNPC and Cap stably had been obtained. Then, by PCR andRT-PCR, the transgenic cell lines were identified,by DNA digestion test, the activity of expressedprotein products were examined. The results showed that the exogenous genes had been cloned into thetransgenic cell lines, the abnormal splicing phenomenon disappeared, and the fusion protion isexpressedstably possesses better nuclease activity.The three transformed cell lines and the untransformed cells all had the virus pass-generationexperiment. To analyse the anti-viral effect，the PCV2relative titer had been determined, bysemi-quantitative PCR and fluorescence quantitative PCR. Contiunuous passage，after the eighthpassage，the viral titer in JNPC transgenic cell line fell to0, while the viral titer in other control groupsis very contiguous. the fusion gene JNPC with a better anti-viral effect is gotten, by redesigning andsynthesized gene JNPC,and reforming the structure of the vector，which lay the foundation for breeding anti-PCV2transgenic pigs.