Zheng Qiaoli, Wang Tuan, Jiang Shaojie, Han Rui, Jin Na, Zhu Jiang, Zhou Qiang, Wang Hui, Chen Xianzhen, Cheng Hao
Department of Dermatology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China.
Department of Radiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China.
PLoS One. 2016 Dec 29;11(12):e0169138. doi: 10.1371/journal.pone.0169138. eCollection 2016.
The persistent infection of high-risk human papillomavirus (HPV) is one of the most common causes of cervical cancer worldwide, and HPV type 58 is the third most common HPV type in eastern Asia. The E7 oncoprotein is constitutively expressed in HPV58-associated cervical cancer cells and plays a key role during tumorigenesis. To study the biological function of HPV58 E7 and to characterize E7 protein-host cell interactions, we cloned the human HPV58 E7 gene and produced specific E7 antibodies. The HPV58 E7 gene was cloned into a prokaryotic expression vector, pGEX-4T2. The recombinant plasmid pGEX-4T2-(HPV58-E7) was transformed into Escherichia coli DH5α and expressed as a fusion protein containing a GST tag. After purification and removal of the GST affinity tag, the E7 protein was used as an antigen for the production of antiserum in rabbits. The specificity of the purified HPV58 E7 antibody was detected by western blotting, immunofluorescence and immunohistochemistry analysis. These methods demonstrated that the polyclonal antibody could specifically recognize the endogenous and the recombinant HPV58 E7 proteins. Immunohistochemistry analysis indicated that the E7 protein was localized in the nucleus of cervical cancer cells.
高危型人乳头瘤病毒（HPV）的持续感染是全球宫颈癌最常见的病因之一，而HPV 58型是东亚地区第三常见的HPV类型。E7癌蛋白在与HPV58相关的宫颈癌细胞中持续表达，并在肿瘤发生过程中起关键作用。为了研究HPV58 E7的生物学功能并表征E7蛋白与宿主细胞的相互作用，我们克隆了人HPV58 E7基因并制备了特异性E7抗体。将HPV58 E7基因克隆到原核表达载体pGEX-4T2中。重组质粒pGEX-4T2-(HPV58-E7)转化到大肠杆菌DH5α中，并表达为含有GST标签的融合蛋白。纯化并去除GST亲和标签后，将E7蛋白用作抗原在兔体内制备抗血清。通过蛋白质免疫印迹、免疫荧光和免疫组织化学分析检测纯化的HPV58 E7抗体的特异性。这些方法表明多克隆抗体能够特异性识别内源性和重组HPV58 E7蛋白。免疫组织化学分析表明E7蛋白定位于宫颈癌细胞的细胞核中。
