Tang Yi, Zhou Qiang, Wang Qi, Cheng Hao
Department of Dermatology and Venereology, School of Medicine, Sun Run Run Shaw Hospital, Zhejiang University, Hangzhou 310016, China.
Bing Du Xue Bao. 2011 Sep;27(5):416-20.
To express and prepare polyclonal antibody of Human papillomavirus type 6b (HPV6b) E7 protein. a prokaryotic expression vector pGEX-4T-2/HPV6b E7 was constructed and GST-HPV6b E7 fusion protein was expressed as a soluble protein in E. coli. The expressed fusion protein was purified via Glutathione-Sepharose 4B column and thrombin cleavage in order to obtain HPV6b E7 protein. Polyclonal IgG antibody was prepared by immunizing New-Zealand rabbits with HPV6b E7 protein. Western-Blot and immunofluorescence analysis showed that the polyclonal IgG antibody could specifically recognize HPV6b E7 protein and its titer was identified. SDS-PAGE analysis demonstrated that large amounts of soluble GST-HPV6b E7 fusion protein was expressed in E. coli after 3.0-6.0 hours of IPTG induction. Polyclonal IgG antibody successfully prepared from immunized rabbits showed high titer and high specificity as confirmed by Western-Blot and immunofluorescence. The preparation of anti-HPV6b E7 polyclonal antibody will facilitate further research on the biological and immunological functions of HPV6b E7 protein.
为表达和制备人乳头瘤病毒6b型（HPV6b）E7蛋白的多克隆抗体，构建了原核表达载体pGEX-4T-2/HPV6b E7，并使GST-HPV6b E7融合蛋白在大肠杆菌中作为可溶性蛋白表达。表达的融合蛋白通过谷胱甘肽-琼脂糖4B柱纯化和凝血酶切割以获得HPV6b E7蛋白。用HPV6b E7蛋白免疫新西兰兔制备多克隆IgG抗体。Western-Blot和免疫荧光分析表明，多克隆IgG抗体可特异性识别HPV6b E7蛋白并测定其效价。SDS-PAGE分析表明，IPTG诱导3.0-6.0小时后，大肠杆菌中表达了大量可溶性GST-HPV6b E7融合蛋白。经Western-Blot和免疫荧光证实，从免疫兔成功制备的多克隆IgG抗体具有高效价和高特异性。抗HPV6b E7多克隆抗体的制备将有助于进一步研究HPV6b E7蛋白的生物学和免疫学功能。
