Ding Yang, Jiang Shaojie, Chen Xianzhen, Chen Lihong, Zhang Xing, Cheng Hao
Department of Dermatology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China.
Center of Biomedicine Research, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Jun;30(6):618-22.
To express human papillomavirus type 11, E7 protein (HPV11E7) via a prokaryotic expression vector and produce anti-HPV11E7 polyclonal antibody.
A prokaryotic expression vector pGEX-4T2-HPV11E7 was constructed and soluble GST-HPV11E7 fusion protein was expressed in E.coli by IPTG induction and purified. The purified HPV11E7 protein was used to immunize New Zealand rabbits to prepare the anti-HPV11E7 polyclonal antibody followed by protein G agarose purification to obtain the IgG type polyclonal antibody. Western blotting and immunofluorescence analysis were used to test the specificity and titer of the antibody.
SDS-PAGE analysis demonstrated that large amounts of soluble GST-HPV11E7 fusion protein was expressed in E.coli after 6 hours of IPTG induction. Western blotting and immunofluorescence confirmed that the purified anti-HPV11E7 polyclonal IgG antibody obtained from immunized rabbits had a high titer and specificity.
The prokaryotic expression system could express a great deal of soluble HPV11E7 protein, and anti-HPV11E7 polyclonal IgG antibody from HPV11E7-immunized rabbits was proved to have a high titer and specificity.
通过原核表达载体表达人乳头瘤病毒11型E7蛋白（HPV11E7）并制备抗HPV11E7多克隆抗体。
构建原核表达载体pGEX-4T2-HPV11E7，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导在大肠杆菌中表达可溶性谷胱甘肽S-转移酶（GST）-HPV11E7融合蛋白并进行纯化。用纯化的HPV11E7蛋白免疫新西兰兔制备抗HPV11E7多克隆抗体，经蛋白G琼脂糖纯化获得IgG型多克隆抗体。采用蛋白质免疫印迹法和免疫荧光分析法检测抗体的特异性和效价。
十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析显示，IPTG诱导6小时后，大肠杆菌中大量表达可溶性GST-HPV11E7融合蛋白。蛋白质免疫印迹法和免疫荧光分析证实，从免疫兔获得的纯化抗HPV11E7多克隆IgG抗体具有高效价和特异性。
原核表达系统可大量表达可溶性HPV11E7蛋白，且经HPV11E7免疫的兔所产生的抗HPV11E7多克隆IgG抗体具有高效价和特异性。
