| Porcine circovirus typeⅡ(PCV2) is the main causative pathogen of post-weaning multisystemic wasting syndrome (PMWS), the virus was first discovered in 1991 from Canada. To date, PMWS has spread all regions over the world which becomes a major economic disease seriously affected the world's pork production. PCV2 is a small non-enveloped virus with single DNA strand, the entire genome is about 1.76Kb, including two open reading frames, ORF1 encoded the Rep protein which is relevant to the replication of the virus. ORF2 encoded a 28kDa capsid protein, which is the only structural protein of the PCV2.This study used the PCR protocol to detect the PCV2 from the organizations from Changchun pigs.PCV1-free PK-15 cells was used to virus isolation. Virus-specific fluorescence was detected by indirect immunofluorescence assay (IFA). The whole genome DNA was extracted from the virus cultured by PK-15 cells as the template to amplify the virus by two steps, the full-long DNA was cloned and sequenced , then take comparison with the sequences published in Gene Bank and take the analysis of the phylogenetic tree. Application of software DNA Star shows that, the nucleotide homology and similarity between the PCV2 Changchun isolation and other strains is arrange from 94.2 percent to 99.5 percent, the PCV2 Changchun isolation is showed the highest homology with Britain and France strains.As the matter of the potential influence induced by the nuclear localization signal, complete ORF2 protein is difficult to express in E. coli. This study used PCR protocol to knock out the whole NLS sequence, and clone the NSL free ORF2 to the expression vector pGEX-6P-1, transmit the recombinant plasmid to the E.coli BL21 (DE3) receptor. Though optimization of the IPTG density and the induction time, the optimized the condition was set to induce 4h in 37℃by IPTG concentration of 1.0mmol / L. The structural protein encoded by ORF2 gene was expressed successfully. The molecular weight of the recombinant fusion protein GST-ORF2-N is about 47kDa, Western-blot analysis showed that the protein of the whole bacteria expression can be identified by PCV2-specific antibodies.The recombinant expression protein is present in the form of inclusion, the experiment use the urea gradient method to elute inclusion, most bacteria protein was removed by urea gradient elution, the inclusion which contains the recombinant protein is dissolved by 8M urea. The recombinant protein was recovered by protein electrophoresis, and resolve in protein extract. The SDS-PAGE electrophoresis showed that recombinant protein is apparent and but no other protein, it reached the goal to protein purification.The dialysis renaturation recombinant protein mingle with Freund's complete adjuvant to vaccinate the rabbits in the amount of 500ug each. Twice enhanced vaccinations come after the first. The ELISA assay was preformed to test the serum antibody titer . The results showed that the preparation of rabbit anti-PCV2 capsid protein serum titer is up to 1:12800. It proved that the protein is of good antigen and immunogenicity. The serum antibody has a very good prospect for PCV2-related experiments no matter on the basic research and epidemiological studies.
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