Zhang Qingyuan, Zhu Jinshun, Wang Lude, Wang Qi, Zhu Shanli, Chen Shao, Zhang Lifang, Jiang Pengfei
Institute of Molecular Virology and Immunology, Department of Medical Microbiology and Immunology, Wenzhou Medical University, Wenzhou 325000, China.
Institute of Molecular Virology and Immunology, Department of Medical Microbiology and Immunology, Wenzhou Medical University, Wenzhou 325000, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2020 Mar;36(3):264-270.
Objective To express E6 protein of human papillomavirus (HPV) type 16 in prokaryotic expression system and prepare its polyclonal antibody. Methods HPV16 E6 gene was obtained from Siha cells by PCR and cloned into pET21a(+) vector to construct the recombinant plasmid pET21a(+)/HPV16 E6 that was confirmed by sequencing. The recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3). The HPV16 E6-His tag recombinant protein was expressed after the induction of isopropyl beta-D-1-thiogalactopyranoside (IPTG), purified by Ni-NTA affinity chromatography, and then analyzed by Western blot analysis. The purified HPV16 E6 recombinant protein was used to immunize Japanese white rabbits to prepare polyclonal antibody. The titer of the serum polyclonal antibody was determined by ELISA. The specificity of the polyclonal antibody was analyzed by Western blotting and immunofluorescence. Results The recombinant plasmid pET21a(+)/HPV16 E6 was successfully constructed and confirmed by sequencing. After the recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3), the recombinant HPV16 E6 protein was expressed and purified by affinity chromatography. The polyclonal antibody at a titer of 1:40 000 was obtained by immunizing Japanese big-ear white rabbit with the purified recombinant HPV16 E6 protein, and its specificity was confirmed by Western blotting and immunofluorescence assay. Conclusion HPV16 E6 recombinant protein was successfully expressed and the rabbit polyclonal antibody against HPV16 E6 recombinant protein was prepared.
目的 在原核表达系统中表达人乳头瘤病毒16型（HPV16）E6蛋白并制备其多克隆抗体。方法 通过PCR从Siha细胞中获取HPV16 E6基因，并克隆至pET21a(+)载体，构建重组质粒pET21a(+)/HPV16 E6，经测序鉴定。将重组质粒pET21a(+)/HPV16 E6转化至大肠杆菌BL21(DE3)。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达HPV16 E6-His标签重组蛋白，采用Ni-NTA亲和层析法纯化，随后进行蛋白质免疫印迹分析。用纯化的HPV16 E6重组蛋白免疫日本大耳白兔制备多克隆抗体。采用酶联免疫吸附测定（ELISA）法测定血清多克隆抗体效价。通过蛋白质免疫印迹和免疫荧光分析多克隆抗体的特异性。结果 成功构建重组质粒pET21a(+)/HPV16 E6并经测序鉴定。将重组质粒pET21a(+)/HPV16 E6转化至大肠杆菌BL21(DE3)后，表达重组HPV16 E6蛋白并通过亲和层析法纯化。用纯化的重组HPV16 E6蛋白免疫日本大耳白兔获得效价为1:40 000的多克隆抗体，经蛋白质免疫印迹和免疫荧光分析证实其特异性。结论 成功表达HPV16 E6重组蛋白并制备了抗HPV16 E6重组蛋白的兔多克隆抗体。
