人乳头瘤病毒16型(湖北株)E7基因在体内和体外的表达  

Expression of HPV16E7-HB gene in vitro and in vivo

作　　者：伍欣星[1] 盛德乔[1] 王宇哲[1] 赵旻[1] 丁晓华[1] 邱小萍[1] 谭云[1] 戴天力[1] 赵文先[1] 

机构地区：[1]湖北医科大学病毒所分子生物学研究室,湖北武汉430071

出　　处：《第四军医大学学报》2000年第3期295-299,共5页Journal of the Fourth Military Medical University

基　　金：湖北省科委重大科研项目!(931P1801);湖北省自然科学基金资助项目!(97J077)

摘　　要：目的　构建人乳头瘤病毒 16型 (HPV16 ) E7湖北株(HB)基因真核表达质粒 ,探讨其在哺乳动物体外、体内表达状况 ,为本地区 HPV相关肿瘤的基因治疗提供依据 .方法　采用分子克隆技术 ,构建 HPV16 E7- HB(已作测序 )重组表达质粒 ;磷酸钙 - DNA沉淀技术及基因免疫技术将外源目的基因 (HPV16 E7- HB)导入 NIH3T3细胞及大、小鼠体内 ;PCR,RT- PCR,免疫荧光染色技术对外源目的基因及其产物 (m R-NA,蛋白质 )进行检测 ;EL ISA法用于检测免疫动物抗血清 .结果　重组后外源目的基因及载体 DNA大小、方向、插入位点均正确 ;转染细胞 2 d后 RT- PCR出现特异扩增带 ;细胞涂片可见特异性 E7蛋白荧光颗粒分布于胞质中 ,少数在胞核 ;基因免疫后肌组织 E7- HBDNA持续阳性 ;RT- PCR阳性率大鼠 3/3、小鼠 1/10 ;虽然组织切片未能检出 E7蛋白 ,但基因免疫后第 2周小鼠血清检出了特异性抗 E7抗体 .结论　HPV16 E7- HB真核表达质粒构建成功 ,并能够在哺乳动物体外、体内有效表达 ;AIM To construct the recombined expressionplasmid of HPV16E7 HB (sequenced) and to explore its expression status in vitro and in vivo so as to provide gene therapy basis for HPV related tumors. METHODS Using molecular cloning, the expression plasmid (pLE7 HBSN) was constructed, which was transfected into NIH3T3 cell and injected into BALB/c mice and Wistar rat by calcium phosphate DNA coprecipitation and genetic immunization technique respectively. E7 HBDNA, E7 HBmRNA and E7 HB protein were detected by PCR, RT PCR and fluorescent immunoassay (FI). The sera of inoculated mice was detected ELISA. RESULTS The inserted gene fragment and vector DNA were confirmed to be correct in size, direction and seat after the recombination. Specific band and specific fluorescence could be seen in 2 day transfecion cells. E7 protein was mostly in cytoplasm and the nuclear was faintly stained. pLE7 HBSN was injected into the muscles of mice and rat through the genetic immunization. HPV16E7 HBDNA could be detected in muscle at least at week 4, and the positive rates of RT PCR of Wistar and BALB/c were 3/3 and 1/10. Though we did not observe the specific fluorescence of E7 protein in muscle section; we detected against E7 antibody after inoculation at week 2, 3, 5 and 6. CONCLUSION HPV16E7 HB is successfully constructed, which can be expressed in vitro and in vivo . As for genetic immunization, a strong immune reaction can be induced by only a small amount of antigen.

关 键 词：乳头瘤病毒 基因表达 基因免疫 

分 类 号：R373.9[医药卫生—病原生物学]