修饰后中国山东地方株HPV16 E6E7基因的转化活性检测及免疫原性研究  

Transformation activity and the immunogenicity of a human papillomavirus type 16 variant E6E7 gene from cervical carcinoma biopsy in Shangdong province

作　　者：许雪梅[1] 张明策[1] 罗利群[2] 刘晓娟[1] 朱明昭[1] 司静懿[1] 郭秀婵 宋国兴 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所生物物理室 [2]中国医学科学院中国协和医科大学肿瘤研究所免疫室 [3]中国医学科学院中国协和医科大学预防医学科学院病毒所

出　　处：《中华微生物学和免疫学杂志》2002年第4期427-432,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 ( 3970 0131)

摘　　要：目的　利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型 (HPV16 )E6E7基因 ,研制HPV16DNA疫苗。方法　定点突变E6的终止密码 ,并保证E7读码框架不变 ;定点突变E7蛋白的Rb结合区中对其转化活性维持起关键作用的第 2 4位氨基酸。突变修饰后的基因命名为fmE6E7。PCR扩增fmE6E7,重组入pLNCX载体 ,脂质体法转染 3T3细胞 ,免疫荧光组织化学及Westernblot检测转染细胞蛋白的表达。经软琼脂集落培养法和BALB c裸鼠皮下接种法检测fmE6E7的转化活性。然后PCR扩增fmE6E7,构建pVR10 12 fmE6E7真核表达质粒 ,于C5 7BL 6小鼠肌肉内直接进行裸DNA免疫 ,51 Cr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性 ,间接ELISA法检测免疫鼠血清中E7特异性抗体。结果　测序证实获得了预期的突变结果。pLNCX fmE6E7转染细胞体外软琼脂培养 3周未见集落形成 ;裸鼠皮下接种 2月后未见移植瘤形成 (0 3)。免疫鼠获得了较好的E7特异性的抗体E和抗原特异性的CTL。结论　修饰后E6E7基因可融合表达 ,转化活性消除的同时还可诱发特异的细胞免疫和体液免疫 ,表明中国山东地方株的E6E7基因可作为HPV16治疗性DNA疫苗的靶基因。Objective To develop a prophylactic and therapeutic vaccine against human papillomavirus type 16 associated cervical cancers. Methods HPV16 E6E7 genes from a Chinese were modified by site directed mutagenesis to create fused E6 and E7 coding sequences, and introduce a mutation at the Rb binding site; The fused and modified HPV16 E6E7 coding sequences named fmE6E7. HPV16-fmE6E7 genes amplified by PCR were inserted into pLNCX vector to generate pLNCX-fmE6E7 plasmids. Then transfected the immortal cell lines 3T3 with plasmids pLNCX-fmE6E7, selected with G418 to generated 3T3-fmE6E7 cell line. Transforming activity of fmE6E7 was assayed in vitro and in vivo. Results The nucleotide mutations were confirmed by nucleotide sequencing; 3T3-fmE6E7 cells showed strong nuclear and cytoplastic fluorescence. Western blotting analysis showed that an apparent molecular relative mass(M r) of 36×10 3 coordinated the predicted M r from the HPV16-fmE6E7. Tumorigenicity assay in nude mice showed that 3T3-fmE6E7 cells did not form colonies in soft agar and all of them were nontumorigenic in nude mice (0/3) for up to 2 months. The results indicated that the transforming activity of the fmE6E7 had been abolished. Intramuscular administration of pVR1012-fmE6E7 induced E7-specific antibodies. The CTLs from immunized mice were activated efficiously. Conclusion The fused and modified HPV16 E6E7 coding sequences from HPV16 variant of a Chinese were a potential target gene for the development of therapeutic DNA vaccine against HPV16, infection and associated cervical cancers.

关 键 词：免疫原性 人乳头瘤病毒16型 E6基因 E7基因 DNA疫苗 细胞毒性T淋巴细胞 宫颈癌 

分 类 号：R392[医药卫生—免疫学]