hMSH2基因cDNA保守区域的T载体克隆及序列分析  

The conservative region of hMSH2 gene cDNA cloning by T-vector and sequencing

作　　者：何云[1] 庄志雄[2] 杨杏芬[1] 陈铁江[1] 陈雯[1] 

机构地区：[1]中山医科大学公共卫生学院卫生毒理学教研室,广州510080 [2]中国预防医学科学院深圳研究中心

出　　处：《卫生毒理学杂志》2001年第1期8-10,共3页Journal of Health Toxicology

摘　　要：目的　构建含限制性内切酶位点PstⅠ、KpnⅠ的hMSH2基因cDNA保守区域的pGEM T S载体 ,比较分析序列的变化 ,为以后的毒理学研究提供实验材料。方法　从人胚肺成纤维细胞HLF中抽提总RNA进行RT PCR扩增 ,直接与T载体连接后转化大肠杆菌DH5α ,用蓝 白斑试验筛选阳性克隆 ,抽提质粒进行酶切鉴定 ,再行序列分析。结果　经RT PCR获得 6 31bp含限制性内切酶位点的阳性产物 ,T载体克隆、酶切鉴定及序列分析后证实 ,克隆片段与genbank中该基因的序列同源性为 99 5 %。结论　本文成功地构建了含hMSH2基因cDNA保守区域的T载体克隆 ,该克隆可为DNA错配修复缺陷与致癌关系研究提供工具。Objective To construct the pGEM\|T\|S vector containing the conservative region of hMSH2 gene cDNA with two recognition sites for the enzymes Pst Ⅰ and Kpn Ⅰ,to compare its sequence with Genbank and provide experimental material for toxicological study.Methods Following total RNA being extracted from human embryo lung fibroblast HLF and RT\|PCR being carried out,T\|vector was linked with PCR products.After \%E.coli\% DH5α was transformed with recombinants and screened with blue/white blot test for positive clones from which plasmids were then extracted,the recombinants were identified by restriction analysis and sequencing.Results 631 base pairs fragment containing restriction sites was obtained through RT\|PCR.After clonging by T\|vector and being identified by restriction analysis and sequencing,it is confirmed that the object fragment has 99\^5% homology with hMSH2 sequence in Genbank.Conclusion The T\|vector clone with the conservative region of hMSH2 gene cDNA has been constructed successfully.This clone can be used in research about the role of mismatch repair deficiency in carcinogenesis.

关 键 词：HMSH2 RT-PCR T载体 序列分析 毒理学 

分 类 号：R994.6[医药卫生—毒理学]