HSV1-TK逆转录病毒载体构建及表达

目的　构建含ＴＫ基因逆转录病毒载体并检测其在转染细胞中的表达，为肺癌基因治疗积累实验资料。方法从 ＰＨＳＶ１０６质粒中切下约２．４ｋｂ ＴＫ基因片段，插入逆转录病毒载体ＰＬＸＳＮ多克隆位点，酶切筛选正、反向连接质粒。正 向连接子电穿孔法转化肺癌细胞Ａ５４９，用ＰＣＲ、细胞原位杂交法分别检测ＴＫ基因整合和表达。结果　经酶切鉴定得到 正向连接子，电穿孔法转导Ａ５４９细胞，经Ｇ４１８筛选出了转基因细胞，ＴＫ基因已整合在转染细胞中并阳性表达。结论 成功构建了含ＴＫ基因逆转录病毒载体，转导该载体的肺癌细胞可表达ＴＫ基因。

Construction of retroviral vector containing HSV1-TK gene and its expression

Objective: To construct a retroviral vector containing TK gene and examine its expression in the lung cancer cells transfected with this gene for facilitating the study of gene therapy for lung cancer. Methods A approximately 2.4 kb TK gene segment was excised from plasmid PHSV106,and inserted into the polyclonal site of retroviral vector PLXSN. A positive linking plasmid was selected by restriction endonuclease analysis and used to transduce pulmonary cancer cell line A549 by electroporation. in which the recombination and expression of TK gene were examined by PCR and in situ hybridization. Results A positive connective plasmid was selected and verified by restriction endonuclease analysis, and the transgene-bearing cells were screened by G418, in which TK gene expression was observed. Conclusion A retroviral vector containing TK gene was sucessfully constructed, and the cells transfected with the vector are capable of expressing TK gene.