人乳头瘤病毒16型E2蛋白表达、纯化及抗血清制备  

Expression and purification of HPV16 E2 protein and preparation of its antiserum

作　　者：孙宇辉[1,2] 张沿君[1] 刘明明[1] 唐丽萍[3] 张光虹[1] 魏兰兰[1] 谷鸿喜[1] 商庆龙[1] 

机构地区：[1]哈尔滨医科大学微生物学教研室,黑龙江省感染与免疫重点实验室,黑龙江哈尔滨150081 [2]哈尔滨医科大学附属第一医学院妇科,黑龙江哈尔滨150040 [3]哈尔滨医科大学附属肿瘤医院妇科,黑龙江哈尔滨150086

出　　处：《细胞与分子免疫学杂志》2013年第9期940-944,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81000726,30901706);黑龙江省留学归国基金(LC2009C26)

摘　　要：目的表达人乳头瘤病毒16型(HPV16)E2蛋白,并制备小鼠抗HPV16 E2血清。方法采用PCR技术扩增HPV16E2基因,构建入pET21b载体,重组表达载体pET21b-HPV16E2经鉴定后转化大肠埃希菌BL21(DE3),诱导表达并鉴定表达产物。经纯化、变性和复性方法,制备可溶性HPV16 E2蛋白。免疫BALB/c小鼠制备抗血清,检测小鼠IFN-γ、CD4^+T细胞、CD8^+T细胞、CD4/CD8比值和抗血清滴度变化。结果酶切和测序结果表明pET21b-HPV16 E2构建成功。表达蛋白相对分子质量(M_r)为42 000,Western blot法证明具有较高特异性。小鼠抗血清效价升高,CD4^+T细胞数量和CD4/CD8比值升高,小鼠IFN-γ无升高。结论成功制备可溶性HPV16 E2蛋白和小鼠抗HPV16 E2高效价的抗血清。Objective To express and purify the human papillomavirus type 16 （HPV16） E2 protein in prokaryotic bacteria and prepare the antiserum of HPVl6 E2. Methods After amplified by PCR, HPV16 E2 was inserted into pET21b vector. The recombinant pET21b-HPV16E2 vector was transfected into E. coli BL21 （DE3）. Expression product was identified after induction. Through purification, denaturation and renaturation, soluble protein was obtained. With the HPV16 E2 protein, we immunized BALB/c mice and examined mouse IFN-y, CD4~ T ceils, CD8~ T cells, CD4/CD8 ratio and antiserum titer. Results Restriction digestion and DNA sequencing showed pET21b-HPV16E2 was constructed successfully. Relative molecular mass （M,） of HPVl6 E2 was 42 000 in SDS-PAGE and the specificity of the protein was confirmed with Western blotting. The antiserum could specifically bind with HPVl6 E2 protein. In the immunized BALB/c mice, antiserum titre, CD4＋ T cell count and CD4/CD8 ratio increased, while mouse IFN-γ did not change obviously. Conclusion Soluble HPV16 E2 protein was obtained successfully. The antiserum of high titer against HPVl6 E2 was prepared in mice.

关 键 词：人乳头瘤病毒16型 E2蛋白 表达 抗血清 

分 类 号：R373.9[医药卫生—病原生物学] R392-33[医药卫生—基础医学]