【摘要】 目的利用突变修饰后的中国山东地方株人乳头瘤病毒16型(humanpapillomavirustype16,HPV16)E6E7融合基因(fmE6E7),研究治疗HPV16感染相关疾病的DNA疫苗,并进一步探索利用共激活分子B7-1基因,研究更加活化细胞免疫的加强疫苗。方法将用PCR法扩增获得fmE6E7基因插入真核表达质粒pVR1012中获得pVR1012-fmE6E7,瞬时转染Cos-7细胞,免疫荧光组织化学法检测证实其表达后,在C57BL/6小鼠肌肉内进行pVR1012-fmE6E7单独免疫,或与小鼠共激活分子B7-1基因真核表达质粒(pcDNA3.1-B7-1)联合免疫。51Cr释放法分析免疫小鼠的细胞毒性T淋巴细胞(cytotoxicTlymphocytes,CTL)活性,间接ELISA法检测小鼠血清中E7特异性抗体。用5×105个C3细胞皮下接种C57BL/6小鼠,分析小鼠体内诱发的特异性抗瘤免疫水平。结果修饰后的E6E7基因免疫可诱导机体产生特异的抗体反应和CTL反应,小鼠B7-1基因与fmE6E7联合免疫可显著提高特异性CTL活性,并可保护33%(2/6)的小鼠免受C3肿瘤细胞的攻击,而单独fmE6E7基因免疫则不能抑制C3瘤细胞的生长,联合B7-1基因免疫对诱发的抗体水平无加强作用。结论中国山东地方株E6E7融合基因可用于DNA疫苗的构建,B7-1基因协同免疫可提高疫苗的细胞免疫水平,利用B7-1基因作为HPV16DNA疫苗的协同因子具有重要价值。更多还原

【Abstract】 Objective To develop a therapeutic vaccine against human tumors associated with human papillo-mavirus type16E6E7(HPV16E6E7)which is modified from a Chinese patient of the cervical cancer which possessing the antigenicity and no transforming activity,and explore more active vaccine for inducing cellular immunity with mouse co-stimulatory molecular B7-1gene.Methods The modified E6E7gene expression plasmid pVR1012-fmE6E7was constructed and transfected Cos-7cells,and the E7protein specific expression was testified by immunofluorescence assay.C57BL/6mice were immunized intramuscularly with pVR1012-fmE6E7alone or in combination with B7-1gene expression plasmid(pcDNA3.1-B7-1).The activity of cytotoxic T lymphocytes(CTLs)was analyzed with 51 Cr specific release assay and the specific antibody in sera was analyzed by indirect ELISA.HPV16positive C57BL/6tumor cells C3were inoculated subcutaneously in the vaccinated mice to assay the growth of transplanted tumors.Results The specific CTLs and antibody from immunized mice were induced efficaciously by the E6E7gene immunization,and co-administration of B7-1gene could significantly enhanced the CTLs immune responses of fmE6E7,and protected33%immunized mice against C3tumor cells challenge.In contrast,all the mice immunized only with fmE6E7gene developed transplanted tumors after C3cells challenge.There was no difference in E7specific antibody responses between mice immunized with the E6E7gene only and co-administration with B7-1gene.Conclusions The modified E6E7gene can be used as target gene for developing DNA vaccine,and B7-1gene may represent an attractive adjuvant for enhancement of the specific cellular immune responses.更多还原