HPV-6/11 L1/E6、HPV-6/11 L1/E7嵌合DNA疫苗质粒的构建

目的　构建HPV 6 / 11L1/E6、HPV 6 / 11L1/E7预防、治疗性DNA疫苗质粒。方法　运用PCR扩增HPV 6 /11L1、E6、E7序列 ,PCR产物连接至pGEMT Easy质粒 ,测序鉴定正确后 ,分别连接至真核表达载体pVAX ,再用酶切、连接而构建成HPV 6 / 11L1 E6 /E7 pVAX质粒。运用电穿孔法将所获得质粒转染COS 7细胞 ,免疫组化检测蛋白表达情况。结果　所构建质粒的插入目的片段测序正确 ;免疫组化检测在胞浆胞核可见棕黄色点状阳性产物沉积。结论　所构建的HPV 6 / 11L1 E6 /E7 pVAX融合蛋白表达质粒可在体外表达L1 E6 /L1 E7蛋白 ,为今后进行DNA疫苗的动物实验及临床实验研究做好准备

The construction of chimerical DNA vaccine plasmid of HPV-6/11 L1/E6 and HPV-6/11 L1/E7

Objective To construct HPV-6/11 L1/E6 and HPV-6/11 L1/E7 prophylactic and therapeutic DNA vaccine plasmid. Methods HPV-6/11 L1, E6 and E7 sequences were amplified by PCR, and PCR products were inserted into the pGEM T-Easy vector and identified by sequencing. Then they were cloned into eukaryotic expression vector pVAX, cleaved by restriction enzymes and ligased to construct HPV-6/11 L1-E6/E7-pVAX plasmid. The constructed plasmid was transfected into COS-7 cell to detect the protein expression by immunohistochemistry. Results Sequences in the constructed plasmids were correct; in cytoplasma, sometimes in nucleus, yellow positive products were found with immunohistochemistry. Conclusion The constructed fusion protein expression plasmid HPV-6/11 L1-E6/E7-pVAXcan express L1-E6/L1-E7 protein in vitro, which has paved a way for animal experiment and clinical study in the future.