HPV病毒E1蛋白多克隆抗体制备及在宫颈上皮病变检测的运用  

Development of polyclonal antibody against E1 protein of HPV and application in detection of cervic lesions

作　　者：张爱英[1] 沈棋[1] 龚一蕾[1] 侯琳[1] 张波[1] 

机构地区：[1]北京大学医学部病理学系,北京100083

出　　处：《现代生物医学进展》2006年第9期12-14,20,共4页Progress in Modern Biomedicine

摘　　要：目的：研制人乳头瘤病毒(HPV)E1蛋白抗体,并运用于组织病理学免疫组织化学检测。方法：通过基因重组、原核表达并纯化阳HPV E1蛋白片段；经免疫兔制备多克隆抗体,并经免疫印迹及基因转染鉴定特异性；免疫组织化学法检测13例宫颈病变中E1蛋白,经原位杂交验证。结果：将PCR扩增的E1蛋白保守功能域(第362～456aa)基因片断重组于原核表达载体pEK- 318的6×His标签下游,经诱导表达和亲合层析,纯化出14kD His-E1蛋白。制备的多克隆血清经免疫印迹法检测显示不仅可与His—El蛋白结合,也与HeLa细胞内源性及基因转染表达的天然E1蛋白反应。运用免疫血清检测7例宫颈尖锐湿疣和6例宫颈上皮内瘤病(CIN)Ⅲ级病例显示：7例宫颈尖锐湿疣6例为阳性染色,阳性反应主要分布于病灶中挖空细胞的胞核。同时6例CINⅢ级病变全部阳性,着色主要为非典型增生鳞状上皮细胞核。为了确定上述病例中E1蛋白免疫组化检测的准确性,运用原位杂交法进行了验证。结果显示：7例宫颈尖锐湿疣均为HPV/11型杂交阳性,6例CINⅢ级病变全部为HPV16型杂交阳性,显色部位与免疫组化基本一致。结论：本研究结果显示研制的HPV E1蛋白抗体可以用于病理组织学HPV病毒检测。Objective： To develop polyclonal antibody against E1 protein of HPV （human papilloma virus） and apply to immunohistochemical analysis in histopathology. Methods： A portion of E1 protein of HPV was purified by DNA recombinant and prokaryotic expression. Polyclonal antibody was generated by immunization of rabbits and its specificity was identified by immunoblotting and gene transferring. The expression of E1 in 13 cases of cervic lesions was analyzed by immunohistochemistry and existence of HPV genome was identified by in situ hybridization. Results： A DNA fragment encoding conservative domain of E1 amplified by PCR was inserted into the downstream of 6 × His tag of pEK - 318 vector. His - E1 （14kD） was purified by affinity chromatography after induced expression in E. coli.The prepared polyelonal antibody from immunized rabbit could recognize His- E1 and native E1 expressed in both original and HPV- transfected HeLa cells by immunoblotting. The immunohistochemical staining in using the prepared anfisera showed that 6/7 of condyloma acuminatum were positive and all 6 cases of cervical intraepithelial neoplasia Ⅲ （CIN Ⅲ） were positive. The positive distribution in condyloma acununatum was mainly in nuelei of atypical hyperplasia cells in CIN Ⅲ. The results of immunohistochemical staining were verified by in situ hybridization, proving that all eases were positive in detection of HPV genome and the distribution was consistent with those of immunohistochemistry. Conclusions： The prepared polyclonal antibody against E1 protein of HPV could be used in the detection of HPV in histopatholgy.

关 键 词：人乳头瘤病毒 抗体 免疫组化 原住杂交 

分 类 号：R737.3[医药卫生—肿瘤]