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目的克隆并表达重组肠道病毒71(EV71)VP1基因,进行抗血清的制备并检测抗血清效价。方法利用原核表达系统将PCR获得的VP1片段构建成原核表达载体pET32a(+)-VP1,诱导其表达VP1融合蛋白并利用包涵体纯化方法进行重组蛋白的纯化,将纯化的重组蛋白免疫新西兰兔制备VP1抗血清,ELISA检测抗体效价。同时构建真核表达载体,利用其在真核细胞中的瞬时表达检测抗血清的特异性。结果通过克隆获得VP1原核表达载体,IPTG诱导VP1蛋白表达并纯化,SDS-PAGE结果显示重组蛋白表达且纯化浓度较高,将重组蛋白乳化后免疫新西兰兔3次,取血检测抗血清的效价,ELISA结果显示抗体效价为1∶64 000,利用所构建真核表达载体pcDNA3.1(+)-VP1表达VP1对抗血清进行检测,Western blot结果表明抗血清可较好的与VP1特异性结合。结论制备具有免疫原性的VP1蛋白及其效价较高的抗血清,为进一步研究抗EV71诊断方法、血清学诊断试剂盒及抗病毒疫苗的研制奠定基础。
Objective To clone and express VP1 gene of recombinant enterovirus 71 (EV71), prepare antiserum and test antiserum titer. Methods The prokaryotic expression system was used to construct the prokaryotic expression vector pET32a (+) - VP1. The VP1 fusion protein was expressed and the recombinant protein was purified by inclusion body purification. The purified recombinant protein was immunized into New Zealand rabbits VP1 antiserum, ELISA detection of antibody titer. At the same time, eukaryotic expression vector was constructed and the specificity of antiserum was tested by its transient expression in eukaryotic cells. Results VP1 prokaryotic expression vector was obtained by cloning. VP1 protein was induced and purified by IPTG. The recombinant protein was expressed and purified by SDS-PAGE. The recombinant protein was emulsified and immunized New Zealand rabbits three times. , The result of ELISA showed that the titer of antibody was 1:64 000. The antiserum was detected by using the eukaryotic expression vector pcDNA3.1 (+) - VP1 to express VP1. Western blot results showed that the antiserum could specifically bind to VP1 . Conclusion The VP1 protein with high immunogenicity and antiserum with high titer were prepared, which lays the foundation for further research on the diagnostic method of EV71, serological diagnostic kit and antiviral vaccine.