C型口蹄疫病毒VP1基因的串联与原核表达

Tandem and Prokaryotic Expression of VP1 Gene of Foot-and-Mouth Disease Virus Type C

目的构建C型口蹄疫病毒VP1基因原核表达系统,并鉴定其表达产物的免疫反应性。方法首先合成C型口蹄疫病毒C-S8C1株VP1基因的3个抗原表位,并克隆到pMD18-T载体上,再按设计的酶切位点酶切后连接,构建出C型VP1双拷贝基因片段(VP1C)。将VP1C基因连接到原核表达载体pET-CKS上,构建重组质粒pETCKS-VP1C,转入BL21菌中进行原核表达。采用SDS-PAGE和Western blot检测其表达产物,并利用包涵体洗涤法对表达产物进行初步纯化。结果VP1C基因在大肠杆菌中获得表达,产量占沉淀蛋白的45%,且表达产物可以被C型口蹄疫病毒标准血清所识别。VP1C重组蛋白纯度达84.1%。结论已成功构建了C型口蹄疫病毒VP1基因原核表达系统,所表达的VP1C有良好的免疫反应性,可以作为检测C型口蹄疫病毒的候选基因。

Objective To construct a prokaryotic expression system for the VP1 gene of foot-and-mouth disease virus（FMDV） type C and study the immunoreactivity of expressed product. Methods Synthesize three epitopes of VP1 gene of C-S8C1 strain of FMDV and clone into pMD18-T vector. Digest the recombinant plasmid with restriction endonuclease and llnk the digested fragments to construct a double copy gene fragment VP1C. A recombinant plasmid pETCKS-VP1C was constructed by inserting VP1C gene fragment into prokaryofic expression vector pET-CKS and transformed to E. coli BL21 for expression. Identify the expressed product by SDS-PAGE and analyze its immunoreactivity by Western blot. Purify the expressed protein, in a form of inclusion body, by washing. Results VP1C gene was expressed in E.coli. The expressed product was recognized by the standard antiserum against FMDV type C and contained 45% of total somatic protein. The purity of recombinant VP1C protein after purification by the washing of inclusion body reached 84.1%. Conclusion A prokaryotic expression system for the VP1 gene of FMDV type C was successfully constructed. The expressed VP1C showed good immunoreactivity and might be used as a candidate gene for detecting FDMV type C.