人乳头瘤病毒16型E7蛋白的表达、纯化及抗血清制备  

Expression and purification of human papiilomavirus 16 E7 protein and preparation of its antiserum

作　　者：颜学勤[1] 王莹莹[1] 嵩钰佳[1] 王鑫[1] 陈思佳[1] 魏兰兰[1] 钟照华[1] 商庆龙[1] Yan Xueqin;Wang Yingying;Song Yujia;Wang Xin;Chen Sijia;Wei Lanlan;Zhong Zhaohua;Shang Qinglong(Department of Microbiology,Harbin Medical University .Harbin150081)

机构地区：[1]哈尔滨医科大学微生物学教研室,150081

出　　处：《国际免疫学杂志》2018年第5期514-518,共5页International Journal of Immunology

基　　金：黑龙江省教育厅面上项目(12541327)。

摘　　要：目的建立人乳头瘤病毒(human papillomavims,HPV)16型E7蛋白的原核表达系统,表达HPv16E7蛋白,制备小鼠抗HPV16E7血清。方法采用PCR方法扩增HPV16E7基因,构建人pET21b质粒,重组表达载体经鉴定后转化大肠埃希菌BL21(DE3)。诱导表达后经十二烷基硫酸钠(sodium dodecyl sulphate,SDS)-聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)和westemblot鉴定表达产物。提取包涵体进行变性复性处理后纯化,纯化后的可溶性蛋白免疫Balb/C小鼠,检测小鼠体内γ-干扰素(interferon-γ,IFN-γ)水平、CD4/CD8比值和抗血清滴度变化。结果PcR扩增片段为0.3Kb,酶切和序列测定证实重组质粒构建正确。SDS—PAGE在11KD处出现蛋白条带。蛋白表达以包涵体为主。免疫后小鼠抗体效价升高,CD4/CD8比值无升高,IFN-γ无反应。结论成功制备可溶性HPV16E7蛋白和小鼠抗HPvl6E7高效价抗血清。Objective To express and purify human papillomavirus type 16(HPV16) E7 protein from bacteria and to prepare the antiserum of HPV16 E7 protein. Methods HPV16 E7 amplified by PCR was inserted into pET21b. The recombinant pET21b-HPV 16E7 vector was transferred into BL21 ( DE3). The expression product was identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis ( SDS-PAGE ) and Western blotting. BALB/c mice were immunized with HPV16E7 soluble protein which was obtained by purification, denaturation and renaturation of the inclusion bodies. IFN-γ,CD4/CD8 ratio,and antiserum titer were examined. Results PCR fragment of HPV16 E7 was 0. 3 Kb. Restriction digestion and DNA sequencing showed that pET21b-HPV 16E7 was constructed successfully. Relative molecular mass( Mr) of HPV16 E7 was 11000 in SDS-PAGE. In the immunized Balb/c mice,high antiserum titers were detected,while IFN-γ and CD4/CD8 rati-o did not change obviously. Conclusion Soluble HPV16 E7 protein and the antiserum against HPV16 E7 were obtained successfully.

关 键 词：人乳头瘤病毒16型 E7蛋白 抗血清 

分 类 号：R392[医药卫生—免疫学]