HSV-TK基因逆转录病毒重组体的构建和鉴定

THE CONSTRUCTION AND IDENTIFICATION OF HSV TK GENE RETROVIRVAL VECTOR

利用逆转录病毒载体ｐＬＮＳＸ构建了带ＨＳＶＴＫ基因的逆转录病毒重组体ｐＬＮＳＴＫ，用磷酸钙沉淀法转染ＰＡ３１７包装细胞，经Ｇ４１８筛选抗性克隆，建立了产生重组病毒的载体产生细胞系ＰＡ３１７／ＴＫ，用ＮＩＨ３Ｔ３细胞测定病毒（ＣＦＵ）滴度为３×１０８Ｌ－１。提取ＰＡ３１７／ＴＫ细胞的ＤＮＡ和细胞上清液的ＲＮＡ，在特异性引物引导下，分别用ＰＣＲ和ＲＴＰＣＲ检测证实ＰＡ３１７／ＴＫ细胞整合了ＨＳＶＴＫ基因并产生重组病毒，为ＨＳＶＴＫ基因治疗恶性肿瘤的实验研究打下了基础。

he recombinant retrovirus expressive vector pLNS TK was constructed with HSV TK cDNA and retrovirus vector pLNSX. The pLNS TK was certified by restriction endonuclease digestion and transfected into PA317 packing cell by calcium phosphate transfection method. The HSV TK vector producer cell line PA317/TK was selected in G418 medium. The virus titer was about 3×10 8 CFU/L with NIH3T3 cells. The integration of HSV TK cDNA into PA317 producing cells was identified by PCR method in the genomic DNA and by RT PCR method in RNA of supernatant of PA317/TK cells. The experiment makes it possible to carry out tumor gene therapy with HSV TK gene.