Soluble Prokaryotic Expression,purification And Immunogenicity Analysis Of VP2 Protein Of Infectious Bursal Disease Virus

Infectious bursal disease(IBD),which usually occurs in 3~12 weeks old chickens and young chicken,is an acute and highly contagious disease caused by Infectious bursal disease virus(IBDV).The virus mainly destroys bursal B lymphocyte,leading to immune suppression of sick chickens as well as great economic losses.In view of the VP2 is the major structural protein and host-protective antigen of IBDV and is a hot gene nowadays.In this experiment,we constructed the VP2-LS3 recombinant prokaryotic expression vectors with seven kinds different of fusion tags,and hoping can obtain highly soluble VP2-LS3 recombinant protein in E.coli BL21(DE3)strain,The detection result showed that VP2-LS3 recombinant protein had good immune activity and formed LS3 protein cage by Microscopic examination.The present study layed the foundation for the development of IBDV genetic engineering subunit vaccine.Firstly,Nde I and Bam HI restriction sites were designed for the amplification of nucleotide sequence of seven kinds of fusion tags by the comparison of nucleotide sequences(His6-Grifin-TEV,His6-GST-TEV,His6-MBP-TEV,His6-Nus A-TEV,His6-?-crystallin-TEV,His6-Ars C-TEV and His6-Ppi B-TEV).The product of amplifiction from seven tags were connected with p MD19-T vector and transformed,and determined correct by bacteria liquid PCR and sequencing analysis.Target fragment of the correct seven labels and VP2-LS3 gene were connected with the p ET-21 b expression vector respectively.The constructed recombinant expression vectors were transformed into the E.coli BL21(DE3)strain,recombinant proteins expression were induced by IPTG,and analyzed the expression level and soluble of VP2-LS3 protein with different fusion tags by running SDS-PAGE electrophoresis.Screening the most suitable fusion tags for VP2-LS3 protein and abundantly inducible expression.The results showed that in addition to Ppi B labels,other labels promoted the soluble expression of VP2-LS3 protein in varying degrees,which MBP tag was the best and soluble expression level reached 69.5 %.Secondly,The VP2-LS3 recombinant protein was purified by Ni-NTA Agarose and VP2-LS3 target protein was obtained by TEV enzyme digestion.Electron microscopy observed and analyzed whether VP2-LS3 recombinant protein formed protein cage nanoparticles.The test results indicated that the TEV protease could separate the His6-MBP tag protein and VP2-LS3 protein,and obtained high-purity VP2-LS3 protein.Microscopic examination showed that LS3 could form nanoparticles cage.Finally,The purified VP2-LS3 recombinant protein emulsified with equal volume adjuvant to prepare gene engineering subunit vaccine,then rabbit antisera was gained from injected rabbits by subcutaneous injection.Analysising by the Western blot and indirect ELISA.Western blot analysis results indicated that VP2 protein could react with positive serum of IBDV,but not react with the negative control sera.The indirect ELISA test showed that rabbit-anti VP2 serum titer was up to 1:1 2800.The above experiment results suggested that the VP2 protein could be wrapped by LS3 protein cage,and the synthetic VP2 protein had a satisfactory immunogenicity,which will lay the foundation for the development of IBDV gene engineering subunit vaccine.