鲤春病毒血症病毒基质蛋白基因的原核表达及多克隆抗体的制备

根据鲤春病毒血症病毒(SVCV)基因组序列(GenBank:NC_002803),人工合成M基因开放阅读框序列,克隆至原核表达载体pET-32a(+),转化工程菌株BL21 (DE3),在0.1 mmol·L-1 IPTG、37℃下诱导5h,获得高效表达的M蛋白,但其主要以包涵体的形式存在,经超声破碎、包涵体纯化后,再...

Prokaryotic expression of Spring viremia of carp virus matrix protein and preparation of polyclonal antibody

The open reading frame of Spring viremia of carp virus(SVCV) matrix(M) protein gene was artificially synthesized according to the genome sequence of SVCV available in GenBank(NC_002803),and then cloned into the pMD19-T vector.After restriction digestion and sequence verification,the fragment of M protein gene was cloned into the prokaryotic expression vector pET-32a(+),and then transformed into Escherichia coli BL21(DE3) strain.The M protein was highly expressed in E.coli under the induction of 0.1 mmol·L-1 IPTG at 37 ℃ for 5 hours,and accumulated in inclusion bodies.The pure M protein was obtained after E.coli ultrasonication,purification of inclusion bodies and Ni-NTA His-bind Resin purification.Purified M protein was used to immunize rabbits to produce rabbit anti-M serum.The anti-M serum was titrated by ELISA,showing positive signal down to a 1∶ 128000 dilution.Western blot analysis indicated that the serum could specifically recognize the natural virus M antigen.In conclusion,the experiments provide important research tools for further study of SVCV diagnosis and M protein gene function.