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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 ;基因免疫仅需少量抗原即可诱导机体产生较强的免疫应答
Objective To construct eukaryotic expression plasmid of human papillomavirus type 16 (HPV16) E7 Hubei strain (HB) gene and investigate its expression in mammals in vitro and in vivo, and to provide basis for gene therapy of HPV related tumors in this region.Methods Molecular cloning (HPV16 E7-HB) was introduced into NIH3T3 cells, large and small mice, and PCR, RT-PCR and RT-PCR were used to detect the expression of HPV16 E7- - PCR and immunofluorescence staining were used to detect the exogenous gene and its product (m R-NA, protein). ELISA was used to detect the antiserum of the immunized animals.Results The size of the exogenous gene and the size, The insertion sites were correct. RT-PCR showed specific amplification bands after transfected cells for 2 days. The specific E7 protein fluorescent particles were found in the cytoplasm and a few in the nucleus after smear. The results showed that E7-HBDNA The positive rate of RT-PCR was 3/3 in rat and 1/10 in mice, and the specific anti-E7 antibody was detected in sera of the 2nd week after the gene immunization, although the E7 protein was not detected in the tissue section. HPV 16 E7-HB eukaryotic expression plasmid was constructed successfully and can be effectively expressed in mammals in vitro and in vivo. Gene immunization can induce the body to produce stronger immune response with only a small amount of antigen