【摘要】 目的原核表达、纯化狂犬病病毒(Rabies virus,RV)核蛋白(Nucleoprotein,NP),并检测其免疫原性。方法 RT-PCR扩增RV CVS-11株NP全长基因序列,克隆至原核表达载体pET-30a(+),构建重组原核表达质粒pET-30a-N,转化E.coliBL21(DE3),IPTG诱导表达,表达的重组蛋白经Ni2+柱亲和层析纯化后,进行Western blot分析,并分别以A(lOH)3和CpG1826作为佐剂经腹腔免疫BALB/c小鼠,ELISA法检测小鼠的血清特异性抗体水平。结果重组原核表达质粒pET-30a-N经双酶切和测序证实构建正确;表达的重组蛋白相对分子质量约51 800,表达量约占菌体总蛋白的35%,主要以包涵体形式存在;纯化的重组蛋白纯度达90%以上,可与抗RV小鼠血清特异性结合,分别以A(lOH)3和CpG1826作为佐剂免疫小鼠的血清特异性抗体滴度对数的GMT值(3.81±0.22和3.68±0.20)明显高于阴性对照组(1.25±0.22),且差异均有统计学意义(P<0.01)。结论已成功表达并纯化了RV NP,其免疫原性良好,为进一步研究其功能奠定了基础。更多还原

【Abstract】 Objective To express the minor capsid protein L2 derived from human papillomavirus(HPV) 58 which caused majority of cervical cancer in China in prokaryotic cells,purify the expressed product and prepare its monoclonal antibody(McAb).Methods L2 gene was amplified from plasmid pHPV58 containing the genome of HPV-58 and inserted into modified prokaryotic expression vector pGEX-KGV.The constructed recombinant plasmid pGEX-KGV-HPV58 L2 was transformed to E.coli BL21(DE3) for expression under induction of lactose.The expressed protein was re-naturalized by dialysis and purified by affinity chromatography,based on which McAb was prepared by hybridoma technique and identified.Results Restriction analysis and sequencing proved that recombinant plasmid pGEX-KGV-HPV58 L2 was constructed correctly.The expressed protein,mainly existing in a form of inclusion body,contained 15.5% of total somatic protein and reached a purity of 95% after purification.A total of cell strains secreting high titer McAbs were obtained.Conclusion HPV-58 L2 protein was successfully expressed in prokaryotic cells and purified,and its McAb was prepared,which laid a foundation of further study on the antigenic epitope of L2 protein as well as development of relevant prophylactic antibody drug and broad spectrum recombinant vaccine.更多还原