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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 a pGEM T S vector containing conserved regions of the hMSH2 gene cDNA containing restriction endonucleases PstI and KpnI and compare the sequence changes to provide experimental materials for future toxicological studies. Methods Total RNA was extracted from human embryonic lung fibroblast HLF and amplified by RT PCR. The recombinant plasmid was directly linked to T vector and transformed into E. coli DH5α. Positive clones were screened by blue and white spot assay. Plasmids were extracted and identified by enzyme digestion. analysis. RESULTS: A positive PCR product of 6 31 bp containing restriction enzyme sites was obtained by RT PCR. The T-vector clone, restriction enzyme digestion and sequence analysis confirmed that the sequence homology of the cloned fragment to the gene in genbank was 99.5%. Conclusion This study successfully constructed a T-vector clone containing the cDNA conserved region of the hMSH2 gene. This clone provides a tool for the study of DNA mismatch repair defects and carcinogenesis.