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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 HPV16 DNA vaccine using the Chinese Shandong local isolate HPV16 E6E7 gene, which has been modified by mutation to eliminate the transformation activity and retain the antigenicity. Methods Site-directed mutagenesis of the E6 stop codon and ensuring that the E7 reading frame was unchanged; site-directed mutagenesis of the Rb binding region of E7 protein was found to be critical for its conversion activity. The mutated gene was named fmE6E7. PCR amplification of fmE6E7, recombinant pLNCX vector, liposome transfected 3T3 cells, immunofluorescence histochemistry and Western blot detection of transfected cell protein expression. The transformation activity of fmE6E7 was detected by soft agar colony culture and BALB mice subcutaneously inoculation. Then, fmE6E7 was amplified by PCR and eukaryotic expression plasmid pVR10 12 fmE6E7 was constructed. The naked DNA was directly immunized in the muscle of C5 7BL 6 mice. The cytotoxic T lymphocyte activity of immunized mice was analyzed by 51 Cr release method. E7 specific antibody in murine serum. Results Sequencing confirmed the expected mutation results. No colony formation was observed in soft agar medium of pLNCX fmE6E7 transfected cells in vitro for 3 weeks. No xenograft tumor was formed in nude mice after 2 days of subcutaneous inoculation (0 3). The immunized mice obtained better E7-specific antibody E and antigen-specific CTL. CONCLUSION: The modified E6E7 gene can be fused and expressed, and the specific cellular immunity and humoral immunity can be induced simultaneously with the elimination of transformation activity. This indicates that the E6E7 gene of Shandong local strain can be used as the target gene of HPV16 therapeutic DNA vaccine.