诱导型一氧化氮合酶基因真核表达载体的构建

Construction of a karyocyte expression vector carrying an inducible nitric oxide synthase gene

目的 构建含有人诱导型一氧化氮合酶基因真核表达载体pcDNA3 iNOS。方法 采用分子生物学技术 ,以HindⅢ和XbIⅠ双酶切pSPORT iNOS,电泳回收 3.96kb编码人iNOScDNA序列 ,克隆入真核表达载体pcDNA3 的HindⅢ和XbIⅠ位点。结果 重组真核表达载体pcDNA3 iNOS经酶切鉴定证明iNOS基因正向插入真核表达载体中 ,用人iNOS基因特异性引物 ,PCR分析重组质粒和进行碱基序列的测定均证明重组质粒中含有人iNOS基因序列。结论 构建的真核表达载体携有人iNOScDNA序列 ,并且含有巨细胞病毒强启动子、ploy(A)加接信号和neo标志基因 ,具有转染多种哺乳类细胞通用性 ,可用于iNOS基因的真核表达及调控的研究。

Objective To construct a karyocyte expression plasmid pcDNA 3 iNOS encoding human inducible nitric oxide synthase (iNOS). Methods The full length human iNOS cDNA (3.96kb) was isolated from pSPORT iNOS by restriction enzyme digest with Hind III and Xbl I,and was then ligated into the Hind III /Xbl I cloning site of the pcDNA 3 expression plasmid. Results The restriction analysis of pcDNA 3 iNOS with Hind III and Xbl I revealed that the position and size of cDNA iNOS insertion were consistent with the sequence of prediction. PCR analysis was carried on using the pcDNA 3 iNOS plasmid DNA as a template and the specific primers corresponding to cDNA sequence of iNOS, the result demonstrated that amplification product was 462bp fragment, indicating the presence of iNOS coding sequence in the recombinant vecter. Conclusions Recombinant plasmid carries human iNOS cDNA sequence, which includes a cytomegalovirus promoter, a simian virus 40 polyadenylation site and a neomycin phosphotransferase (neo) selectable marker so that it may be generally available for the use to transfect into a variety of mammaliam cells, it can be used to study the gene regulation of karyocyte expression of iNOS.