58型人乳头瘤病毒L2蛋白的原核表达、纯化及单克隆抗体的制备

目的原核表达并纯化我国高发的致宫颈癌58型人乳头瘤病毒(Human papillomavirus,HPV)次要衣壳蛋白L2,并制备其单克隆抗体。方法从含有HPV-58基因组的质粒pHPV58中扩增L2基因,插入改构的原核表达载体pGEX-KGV中,转化大肠杆菌BL21(DE3),乳糖诱导表达,透析复性并亲和层析纯化重组蛋白,采用杂交瘤技术制备单克隆抗体,并对其进行鉴定。结果重组表达质粒pGEX-KGV-HPV58 L2经双酶切和测序证明构建正确;表达的重组蛋白主要以包涵体形式存在,表达量可占菌体总蛋白的15.5%;纯化的重组蛋白纯度可达95%;共获得10株抗HPV-58 L2的高效价单克隆抗体。结论已成功原核表达、纯化了HPV-58 L2重组蛋白,并制备了单克隆抗体,为下一步HPV-58 L2蛋白抗原表位的研究以及相关预防性抗体药物和广谱重组疫苗的开发奠定了基础。

Prokaryotic Expression and Purification of Minor Capsid Protein L2 Derived from Human Papillomavirus 58 and Preparation of Monoclonal Antibody

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.