Research On Production Of Virus Like Particle Via Escherichia Coli System Of Mink Viral Enteritis

Mink viral enteritis caused by Mink Enteritis Virus(MEV)has brought great losses to the mink industries.VP2 protein is the main capsid protein of MEV and has important antigenic sites.Therefore,VP2 protein was an important candidate of study of gene-engineering vaccines.Firstly,sequence of MEV VP2 was optimized by the codon optimization software online.Then,it was cloned into the p ET-30a vector to construct the recombinant plasmid and named p ET-30a-VP2.p ET-30a-VP2 was expressed alone and with p Tf16 in ER2566,respectively.Cells were ultrasonic broken,and analysed by SDS-PAGE.The results showed that when co-expressed with p Tf16,VP2 protein existed in a soluble form was about 65 ku.The soluble VP2protein was analyzed by Westetn-blotting,and the result showed that it had good reactivity.Secondly,in order to express soluble VP2 protein effectively,the induction conditions for co-expression were optimized.When cells were induced with 2 g/L L-arabinose,0.2 mmol/L IPTG,25? for 16 h,protein expression was better.Then protein was purified in two steps by ammonium sulfate precipitation and sucrose density centrifugationmethod,and the purity was as high as 90%.After that,the process conditions of sucrose density gradientcentrifugation were optimized to improve the purification efficiency and provide the possibility for large-scale industrial production of protein in the later stage.Finally,the purified VP2 protein was self-assembled,and the size of VLPs were identified by DLS.The results showed that when p H was 8.0 and Na Cl concentration was 150 mmol/L,the size of VLPs was most similar to that of natural MEV.The results of TME showed that a lot of uniform VLPs were formed under this condition.The results of hemagglutination also showed that the VLPs could cause hemagglutination of porcine red blood cells,up to 2¹⁴.The vaccine prepared with these VLPs was used to immunize minks.The HI titer was measured up to 2¹¹,indicating that the VLPs vaccine had no less immunogenicity than the inactivated vaccine,laying a foundation for further research and development of the MEV genetic engineering subunit vaccine.