HPV16型E7重组蛋白的表达及其多克隆抗体的制备

Expression of HPV16 E7 protein and preparation of its polyclonal antibody

目的制备人乳头瘤病毒(HPV)16型E7原核表达蛋白及其多克隆抗体。方法用PCR方法从宫颈癌组织中扩增HPV16 E7基因,克隆至pET21a(+)载体并构建pET21a(+)/HPV16 E7重组质粒,测序鉴定;将重组质粒转化至大肠杆菌BL21(DE3),经异丙基硫代-β-D-硫代吡喃半乳糖苷(IPTG)诱导后表达重组蛋白,用Ni-NTA亲和层析法纯化并经SDS-PAGE及Western blot分析鉴定;纯化的HPV16 E7重组蛋白免疫日本大耳白兔制备多克隆抗体,用ELISA检测多克隆抗体的效价,并用Western blot法及免疫荧光技术分析多克隆抗体的特异性。结果 HPV16 E7重组蛋白可通过原核表达系统进行表达和纯化;通过兔免疫后可制备特异性的多克隆IgG抗体,效价达1∶30 000;经Western blot法和免疫荧光染色结果证实兔多克隆抗体可特异性识别HPV16 E7蛋白。结论成功进行了HPV16 E7原核表达并制备了HPV16 E7兔多克隆抗体。

Objective To prepare a prokaryotic expression vector carrying E7 protein of human papillomavirus （HPV） type 16 and a polyclonal antibody against it. Methods The HPV16 E7 gene was amplified by PCR from the tissue samples of cervical cancer and cloned into the pET21a （＋） prokaryotic expression vector. The constructed pET21a （＋）/HPV16 E7 recombinant plasmid was transformed into E. coli BL21 （ DE3 ） and induced by isopropyl beta-D-l-thiogalactopyranoside （IPTG）. The recombinant protein of HPVI6 E？ was purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis. To prepare the polyclonal antibody, the rabbits were immunized with the purified recombinant protein HPVl6 E7. The titers of specific antibodies were detected by ELISA, The specificity activity was further detected by Western blotting and immunofluorescence analysis. Results HPV16 157 recombinant protein was expressed and purified in the prokaryotic expression system, The polyclonal antibodies were produced in the rabbits immunized with the recombinant protein. The titer of specific antibodies was up to 1：30 000. Western blotting and immunofluorescence analysis indicated that the polyclonal antibody could specifically recognize the HPV16 E7 protein. Conclusion HPV16 E7 recombinant protein was successfully expressed and its polyclonal antibody was prepared.