Cloning And Expression Of ORF2 Gene Of Porcine Circovirus Type 2 And The Research Of Its Immunogenicity

Procine circovirus type 2(PCV2), the causative agent of postweaning multisystemicwasting syndrome (PMWS), and was considered as the etiological agent of some disease such as porcine dermatitis nephropathy syndrome (PDNS),congenital tremor AII, (CT-AII) respiratory and proliferative and necrotizing pneumonia(PNP) etc. The virus is mainly against the immune system, lead the immune function to decline. And often secondary onset and Concurrent other pathogen infection, leading to more serious consequences.It has seriously affected the healthy development of pig industry. PCV2 has two primary open reading frames,ORF2-coding product is the primary structural protein,can stimulate the body to produce certain immune protective reaction,and PCV1 and PCV2,Cap protein homology is lower, no cross-reaction of antigenicity is observed between them.Therefore,ORF2 protein has important significance in serology diagnosis and genetic engineering subunit vaccine research.In this study, using prokaryocyte and Bac-to-bac baculovirus expression system to express the ORF2 gene encoding Cap protein of PCV2,through the experimental analysis on its immunogenicity.Firstly, the research of expression of capsid protein of PCV2 in prokaryocyte pression system. According to the publish of PCV2 in GenBank WH strain (FJ598044) ORF2 gene sequences design a pair of primers,using PCV2 WH strain genomic DNA as a template, the nuclear localization signal (NLS)-defected ORF2 gene was amplified.Insert it into the prokaryotic expression vector of pET-32a(+).The recombinant expression vector pET-32a-Cap579 was transformed into the BL2(DE3),It was induced by IPTG. SDS-PAGE and Western blotting analysis of the expression products of reconstructed pET-32a-Cap579.The results showed that the ORF2 gene had been expressed.the molecular weight of recominant Cap protein was 40kDa, the recombinant Cap protein existed in the form of soluble and inclusion body protein and its main form was soluble. It can react with PCV2 positive serum which by Western blotting,the concentration of the purified recombinant protein is about 200 μg/mL,The specific antiserum against Cap was produced in rabbit immunized three times by the purified fusion protein cap.Secondly, the research of expression of capsid protein of PCV2 by eukaryotic system in Sf9 insect cells, To improve the expression PCV2 Cap protein in Sf9 insect cells,we are designed and synthesized a full-length PCV2 ORF2 gene sequence in which wild-type codons were replaced with codons from highly expressed in Sf9 insect.The target gene was clone into Bac-to-bac expression baculovirus system pFastBacTMHTA transfer vevtor of Bac-to-bac expression baculovirus system. The constructed pFastHTA-Cap2 was transformed into DH10Bac, resulting the recombinant baculovirus plasmid (rBacmid-Cap2) which was confirmed by blue-white plaque assayand antibotic selection.The rBacmid-Cap2 was then transfected into Sf 9 insect cells in logarithmic growth phase by Cellfection transfection reagent for harvesting recombinant baculovirus rBac-cap2 and amplify the recombinant virus.two methods for titering recombinant baculovirus including the limited dilution method,the indirect immunofluorecent assay.The results indicated, two methods of P3 generation of titering recombinant baculovirus were 1.64×108 pfu/mL,1.01×108 pfu/mL.Shows that both methods can be used for titering recombinant baculovirus.SDS-PAGE,Western blotting and IFA analysis of the expression products.The results showed that the optimized synthesis of PCV2 ORF2 gene had been expressed,the molecular weight of recominant Cap protein was 32.5 kDa and could be recognized by the anti-serum against PCV2.The optimal conditions for expressing the recombinant protein were that the recombinant baculovirus was inoculated at multicity of infection of 2.Lastly, Anti-Cap serum antibody was dynamic monitoring in mice,Mice immune experiments indicated the recombinant protein could activate fairly strong immune response.Two weeks after the first immunization, specific antibody was tested, three weeks after the booster immunization,antibody level continue to rise. Neutralizing activity was observed in serum neutralization test, The results showed that the neutralization antibody titer was up to 1:7.17 in the third week, After the booster immunization, the neutralization antibody titer continued to inerease in the third week,the neutralization antibody titer was up to 1:12.6.In conclusion, using prokaryocyte and Bac-to-bac baculovirus expression system to express the ORF2 gene of PCV2 and it have better immunogenicity, it provided a substance foundation to for developing PCV2 subunit vaccine and diagnosis.