摘要
为了分离、克隆单纯疱疹病毒I型(HSV-I) SM_(44)株胸苷激酶基因(TK),采用原代兔婴肾细胞培养HSV-ISM_(44) 株病毒,提取病毒核酸作为模板,设计了 HSV-ITK基因的上、下游引物,在引物的 5’端分别引入 BamHI和 EcoR I 限制性内切酶位点,通过PCR方法扩增出 TK基因,经BamHI/EcoRI双酶切与 PUC19质粒载体相连接,构建重组 体转化受体菌JM109,通过筛选和酶切鉴定,获得HSV-ISM_(44) TK基因阳性克隆,行全序列测定分析,与HSV-ICL101 株 TK基因同源性为 99.02%,存在 11个核苷酸和 7个氨基酸的差异。 HSV-ITK基因的克隆成功,为该基因应用于恶 性肿瘤的自杀基因治疗奠定了基础。
In order to isolating and cloning the HSV-I TK gene, HSV-I strain SM_44 virus were proliferated by culturing primary rabbit fetal kidney cells, and the virus nucleic acid was extracted as template. The 5'terminals of the primers to amplify TK gene were incorporated restriction endonuclease sites of BamH I and EcoR I. The PCR products of TK gene fragment were inserted into PUC 19 plasmid vector at corresponding sites, then transformed into E.coli JM109.By restriction enzyme cleavage characterization, the recombined clone of TK gene was obtained and named PTKs. The nucleotide sequences were deterimined. Comparing with the TK gene of HSV-I strain CL101, HSV-I SM_44 TK gene have homology of 99.2% for nucleotide sequence, and 98. 12% for amino acid sequence. The clone of HSV-I SM_44 TK gene might contribute to using in the suicide gene therapy of cancer.
出处
《基础医学与临床》
CSCD
2000年第2期34-36,40,共4页
Basic and Clinical Medicine
基金
湖北省科委资助!SK-971P1603
关键词
单纯疱疹病毒
胸苷激酶基因
克隆
序列测定
herpes simplex virus type I
thymidine kinase gene
PCR
clone
sequence