人乳头瘤病毒HPV33型E6E7蛋白的表达及多克隆抗体的制备  

Preparation of the polyclonal antibody of human papillomavirus type HPV33 for E6E7

作　　者：卢钧雄[1] 段炼[1] 王若伦[1] 易俊波[1] 

机构地区：[1]广州医科大学附属第二医院,518000

出　　处：《中华实验和临床病毒学杂志》2016年第1期80-83,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金项目（81403031）

摘　　要：目的表达人乳头瘤HPV33型病毒癌蛋白E6E7，并免疫小鼠制备抗体。方法从HPV33型基因组中扩增出E6E7的基因片段，通过基因测序加以证实；将其克隆至原核表达载体pET28a中，在大肠埃希菌中经IPTG诱导表达，经HIS亲核层析对重组蛋白进行纯化，收集重组蛋向免疫的小鼠阳性血清，经ProteinA／G亲核层析柱纯化得到多克隆抗体，同时将E6E7克隆至真核表达载体pCDNA3．1／HisC中，转染293细胞，分别采用免疫荧光法和Western Blot检测抗体的特异性．、结果用得到高纯度的重组蛋白E6、E7制备的多克隆抗体，具有良好的特异性。结论这种高纯度重组E6、E7蛋白和抗体有望用于HPV33型人乳头瘤疾病的临床检测及发生机制的研究。Objective To clone and efficiently express E6E7 proteins of human papilloma virus HPV33 in E. coli. The recombinant proteins were purified and immunized to mice, and polyclonal antibodies were purified. Methods E6 and E7 genes of HPV33 were amplified by PCR using HPV33 human papilloma virus as template, and were sub-cloned into pET28a and pCDNA3.1 / His C vectors. The recombinant plasmids pET28a-HPV33 E6 and pET28a-HPV33 E7 were transformed into Escherichia coil BL21 （DE3） strain. We purified the recombinant proteins and immunized mice for preparing antibodies, and the polyclonal antibodies were purified. Moreover the plasmids pCDNA3. 1 / His C-HPV33 E6 and pCDNA3. 1 / His C-HPV33 E7 were transfected into 293 cells. The cells were used in immunoflnorescence and Western-Blot to detect antibody specificity. Results The recombinant proteins were successfully obtained, and the polyelonal antibody was prepared, which had good specificity. Conclusion The recombinant proteins and antibodies may be used in the clinical detection and pathogenesis research of HPV33 type human papilloma disease.

关 键 词：宫颈肿瘤 人乳头瘤HPV33 E6 E7蛋白 多克隆抗体 

分 类 号：R392-33[医药卫生—免疫学]