论文部分内容阅读
目的 构建人β防御素与人乳头瘤病毒 HPV16 E6羧基端基因的真核表达质粒 ,并检测其在真核细胞中的表达情况 ,探讨其作为 DNA疫苗治疗宫颈癌的可行性。方法 利用分子克隆技术 ,将 HBD2基因片段与 HPV16 E6羧基端基因片段连接 ,插入真核表达质粒 pc DNA3.1,经测序鉴定后 ,用阳离子脂质体法转染真核细胞 COS7,RT- PCR及免疫组化法检测其表达。结果 重组质粒 pc DNA3.1/ HBD2 - HPV16 E6 C转染 COS7细胞 4 8小时后 ,RT- PCR扩增出插入的 HBD2 - HPV16 E6 C片段 ,免疫组化染色呈棕黄色阳性反应。结论 人 β防御素与人乳头瘤病毒 HPV16 E6羧基端融合基因能在真核细胞中有效表达 ,为今后进行整体动物 DNA免疫试验奠定了基础。
Objective To construct eukaryotic expression plasmid of human beta-defensin and human papillomavirus HPV16 E6 carboxyl terminal gene and test its expression in eukaryotic cells and to explore its feasibility as a DNA vaccine for the treatment of cervical cancer. Methods The molecular cloning technique was used to connect the HBD2 gene fragment with the HPV16 E6 carboxyl terminal gene fragment. The recombinant plasmid pcDNA3.1 was inserted into the eukaryotic expression vector pcDNA3.1. After sequencing, the recombinant plasmid was transfected into COS7 cells by cationic liposome. And immunohistochemistry to detect its expression. Results The recombinant plasmid pcDNA3.1 / HBD2 - HPV16 E6 C was transfected into COS7 cells. After 48 hours, the inserted HBD2 - HPV16 E6 C fragment was amplified by RT - PCR and the positive staining was positive by immunohistochemistry. Conclusion The fusion gene of human β-defensin and human papillomavirus HPV16 E6 carboxy-terminal can be efficiently expressed in eukaryotic cells, which lays the foundation for the DNA immunoassay of whole animal in the future.