人乳头瘤病毒16型(湖北株)E7基因在体内和体外的表达

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ＨＰＶ１６Ｅ７－ＨＢ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（ＨＰＶ１６Ｅ７－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（ｍＲ－ＮＡ，蛋白质。

Expression of HPV16E7-HB gene in vitro and in vivo

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.