Cloning And Expressing Of VP1 And VP2 Fusion Genes Of Chicken Anaemia Virus In E.coli

Chicken anaemia virus is a virus causing chicken anaemia complicated by aplasia of the bone marrow and atrophy of the lymphoid organs in young chickens and a transient severe immunodeficiency in older chickens. The VP1 protein of CAV is not only a main structural protein, but also the only protein in a CAV particle. The VP2 may play a certain role in assisting VP1 to assemble the capsids of virus as the companion or framework protein (Scaffold protein).It is reported that the chicken immunized by the fracted sect cells that can simultaneously express VP1 and VP2 produced the antibody against CAV, but their expression deal is low and it doesn't fit to the demand of the large-scale usage in production farm. If the fusion protein of VP1 and VP2 that was expressed in E.coli can form neutralizing antigen epitopes, this problem is resolved. Is it true? This study is a basic study about it by cloning and expressing of VP1 and VP2 fusion genes of chicken anaemia virus in E.coli.In this study, the VP1 and VP2 gene of CAV were magnified by polymerase chain reaction using designed primers and were cloned and sequenced. Their sequences of DNA and amino acids were analyzed with computer software on molecular biology. Based on the analysis results, three partition of VP1 gene of CAV were cloned and inserted into the bacterial plasmids pGEX-4T-1. The recombinant plasmids containing VP1 partiton gene of CAV were identified by restriction enzyme analysis and PCR method. Then the whole VP2 gene was inserted into the recombinant plasmids. The new recombinant plasmids containing VP1 and VP2 fusion gene of CAV were identified.The recombinant fusion protein was highly expressed in the form of inclusion bodies in E.coli BL21 induced by 1mmol/L IPTG. The molecular weight of the fusion protein is 95kD, 80kd and 66kD. Western-blot analysis with sera against CAV showed the fusion protein had good specificity. After the inclusion bodies of fusion proteins were purified with washing buffer consisting of 4mol/L urea for several times, they were dissolved by the denaturing buffer containing 1% SDS as denaturant. Then the purification of the fusion proteins by electrodialysis method also attained perfect result.A good renaturation result was obtained using gradient dialysis with lower protein concentration. The specificity of the fusion proteins was identified using ELISA method. One fusion protein named F exhibited specific binding to antibodies against CAV. The result shows that this purified protein has excellent specificity and it can be adjusted for CAV antibody test kit or be tested for its immunogenic feature.