DSB修复基因hKu70反义RNA真核表达载体构建

Construction of eukaryotic expression vector of DNA double-strand break repair gene hKu70 antisense RNA

目的 构建人DNA双链断裂 (DSB)修复基因hKu70反义RNA真核表达载体pEGFP C1 K ,为以后的hKu70基因功能和毒理学研究提供实验材料。方法 提取人胚肺成纤维细胞 (HLF)总RNA ,逆转录酶 多聚酶链式反应 (RT PCR)扩增hKu70基因cDNA保守序列 ,经与pGEM T载体连接、筛选、克隆、抽提质粒和双酶切后 ,将纯化的hKu70基因cDNA保守序列反向插入绿色荧光蛋白表达载体pEGFP C1中 ,筛选、克隆、抽提质粒 ,从而构建hKu70基因反义RNA真核表达载体pEGFP C1 K。结果 经RT PCR获得 467bp含限制性内切酶位点的DNA片段 ,T载体克隆后经双酶切、测序 ,确定该片段为hKu70基因cDNA ,进而构建反义RNA真核表达载体pEGFP C1 K ,并双酶切 ,测序确证。结论 成功构建hKu70基因反义RNA真核表达载体pEGFP C1 K ,为建立该基因低表达细胞株。

Objective To construct the pEGFP\|C1\|K vector,an eukaryotic expression plasmid of DNA double\|strand break(DSB) repair gene hKu 70 antisense RNA and provide experimental material for the studies of hKu 70 gene function and toxicology.Methods The conservative region of hKu 70 gene was amplified by reverse transcriptage\|polymerase chain reaction(RT\|PCR) after total RNA being extracted from human embryo lung fibroblast(HLF) and then cloned into pGEM\|T vector.The recombinant plasmids were screened and extracted.The pEGFP\|C1\|K vector,an eukaryotic expression plasmid of hKu 70 gene antisense RNA was constructed after the object hKu 70 cDNA fragment was inserted into pEGFP\|C1 vector reversedly and the recombinants were screened,cloned and extracted.Results 467 bp DNA fragment containing conservative region of hKu 70 gene and restriction sites were obtained by RT\|PCR.After cloned by pGEM\|T vector and certified by restriction analysis and sequencing,pEGFP\|C1\|K vector was successfully constructed by means of recombinant DNA technology.Conclusion The efficient expression vector of hKu 70 gene antisense RNA,pEGFP\|C1\|K has been constructed successfully,it can be used to establish the human cell line which express lower hKu 70 protein and in researches about DSB repair deficiency and toxicology.