Study On Clone And Expression Of Porcine Circovirus2Capsid Protein Gene

Porcine circovirus2was isolated from pigletsin by Clark et al. in1997. The pigletsin was from the Western Canada and had been diagnosed to suffer from postweaning multisystemic wasting syndrome. The virus is a small pathogenic virus with a single-stranded circular DNA genome, and was suggested to be associated with some diseases such as porcine dermatitis and nephropathy syndrome, congenital tremor, late-term abortions and exudative epidermitis, and caused serious economic loss in the global pig breeding industry. Vaccine is an important means of prevalence control, and reducing the loss of pig breeding industry. The first PCV2vaccine was licensed in North America in2006. A PCV2subunit vaccine, which had been expressed by baculovirus, had been developed by Blinger Company and registered for sale in China in2009. Some PCV2vaccine had been registered in China recently. The whole inactivated virus vaccine was developed successfully by the Harbin Veterinary Research Institute with LG strains of porcine circovirus2and Nanjing Agricultural University with SH strain of porcine circovirus2, respectively. The antigen value of these vaccines was lower; the vaccination needs to be enhanced many times to ensure the effect. There is no subunit vaccine of PCV2developed by prokaryotic expression at home and abroad for the present. The capsid protein was considered to be the main immunogenic protein of PCV2, and there was no Cross protection between the capsid protein of PCV1and PCV2. The aim of the study was to research prokaryotic expression of cap protein of PCV2, which will lay the foundation for the research of subunit vaccine.The main aspects of our study were as follows:1. The cloning research of porcine circovirus2capsid gene.The pathologic tissue was from a PCV2infected pig, which was come from a farm in Jingzhou. The inguinal lymph node was homogenized, the total DNA was abstracted from it. The virus content was very low in pathologic tissue; the target gene was invisible in agarose gel electrophoresis, after general PCR. The gene of PCV2capsid was amplified by nested PCR and sequenced. We found that there are nine rare codons and two series of rare codons in the N-terminal of the gene, which will seriously affect the expression in E.coli. BL21(DE3) or terminate the expression. In the view of this, we designed4pairs of primers to splice and modify the gene of PCV2capsid, and ligated them to pMD18-T vector.2. Construction of expression vector and expression of soluble target proteinFive recombinant pMD18-T vectors containing different PCV2cap gene were digested by Bam ⅡⅠ and Hind Ⅲ. fragments of cap gene were subcloned into the Bam H Ⅰ and Hind Ⅲ site of pET28a. The recombine expression vectors were identified by PCR. restriction enzyme and SDS-PAGE. The results showed that the full-length gene of capsid failed to express the target protein in E.coli.BL21.The capsid genes of N-termination deleted, MNdwPCV2cap and MNdxPCV2cap, could be expressed in E.coli.BL21,but most of the expression products were in the form of inclusion bodies. Another two capsid genes MNdyPCV2cap and MNdzPCV2cap, which were the modification genes of capsid, could express the target proteins, most of them were soluble, and the molecular weight is about27KD.3.Antigenicity detectionThe antigen titers of capsid protein expressed by E.coil.BL21/pET28a-MNdz PCV2cap was detected by AGP. The content of protein was1.149mg/ml.The theory of AGP was that antigens and corresponding antibodies could react in the present of electrolytes at a suitable temperature.They would form a visible precipitate after a certain time.In the experiment, swine anti-PCV2cap serum, which was diluted at the ratio of1:8,was added into the center well of the plate was, while antigen, diluted from the original to1/16, and the negative control were added into the wells of circumjacent respectively. Then we put them at37℃with24-48h. The result was that the titer of antigen was1/8.We also detected the antigenicity of expressed protein by Western blotting. SDS-PAGE was done at first, and then proteins in the gel were transferred onto nitrocellulose membranes. They were absorbed by noncovalent. Swine anti-PCV2serum reacted with the antigen at first and then with HRP-conjugated goat anti-swine Ig G. Finally under the condition of hydrogen peroxide, added the DAB and immune band turned brown. The molecular weight is about27KD. Proved that antigen has antigenicity.4. Purification of expression proteinThe expression of soluble target protein was purified by Ni column affinity chromatography. Commercial expression vector pET28a could express six histidine residues in N-terminal fusing with target protein. The protein itself contains few histidines, and they were folded and wrapped in the protein interior, while the exterior almost no histidine. The histidine residue, which fusion express with the target protein, could form complexes chelated with many metal ions. We separated the different proteins by different concentrations of imidazole. Because imidazole and protein competitive binding in the Ni column and the different protein binding capacity was different. Like this we separated the target proteins from others. And we got a purified27KD target protein.5. Correlation of protein content and antigen titer The preliminary purification expression protein was diluted with doubling diluted method, and the protein content was detected with Brodford method, the antigen titers was detected with AGP. The results showed that the minimum detectable value of AGP was0.255mg (protein)/ml.Correlation analysis showed that there was a remarkable positive correlation between total protein content of expression product, in the range of0.255～0.857mg/ml, and the titer of log0.5-AGP, the correlation coefficient (r)=0.9938. The antigen titer was no longer changed correspondingly when the total protein content was more than0.857mg/ml.Conclusion: we had completed the expression of PCV2capsid protein in prokaryotic cells successfully; most of expression products were solubility. The antigenicity of soluble expression products was detected by AGP and western blot. The research achievement had laid a good foundation for the development of PCV2subunit vaccine and antibody detection kit in the future.