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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。
Objective To express and purify the secondary capsid protein L2 of human papillomavirus (HPV) 58, which is highly expressed in our country, and to prepare its monoclonal antibody. Methods The L2 gene was amplified from the plasmid pHPV58 containing HPV58 and inserted into the prokaryotic expression vector pGEX-KGV. The recombinant plasmid was transformed into E. coli BL21 (DE3), induced by lactose, purified by dialysis and purified by affinity chromatography Protein, hybridoma technology to prepare monoclonal antibodies, and its identification. Results The recombinant plasmid pGEX-KGV-HPV58 L2 was proved to be correct by double enzyme digestion and sequencing. The recombinant protein was mainly expressed in inclusion bodies and accounted for 15.5% of the total bacterial proteins. The purity of the purified recombinant protein was up to 95%. A total of 10 high titer anti-HPV-58 L2 monoclonal antibodies were obtained. Conclusion The prokaryotic expression of HPV-58 L2 recombinant protein has been successfully purified and the monoclonal antibody has been prepared, which laid the foundation for the further study on HPV-58 L2 epitopes and the development of related preventive antibodies and broad-spectrum recombinant vaccines basis.