诱生型NO合酶基因真核表达载体的构建  

Construction of an Enkaryotic Expression Vector Carrying an Inducible Nitric Oxide Synthase Gene

作　　者：臧梦维[1,2] 胡萍 刘枝俏[1,2] 刘景生 彭学贤 

机构地区：[1]中国医学科学院基础医学研究所,中国协和医科大学基础医学院 [2]中国科学院微生物研究所

出　　处：《基础医学与临床》1997年第5期42-50,共9页Basic and Clinical Medicine

基　　金：国家自然科学基金;高等学校博士点基金

摘　　要：采用分子克隆技术，设计并构建来源ＲＡＷ２６４．７巨噬细胞ｉＮＯＳ基因ｃＤＮＡ的真核表达载体。以ＨｉｎｃⅡ＋ＮｏｔⅠ双酶解ｐＫＳｉＮＯＳ，电泳回收３９７３ｂｐ编码ｉＮＯＳ的ｃＤＮＡ序列，克隆入真核表达载体ｐＲｃ／ＣＭＶ的ＨｉｎｄⅢ和ＮｏｔⅠ位点。用ＥｃｏＲⅠ和ＳａｌⅠ酶切鉴定，初步筛选２＃和８＃克隆为重组子ｐＣＭＶｉＮＯＳ。经ＮｏｔⅠ和ＫｐｎＩ＋Ｓａ１Ⅰ酶切结果表明，重组克隆外源ｉＮＯＳ基因插入位点和片段大小正确。以ＥｃｏＲＶ＋ＸｂａⅠ、ＥｃｏＲＶ＋ＳｃａⅠ、ＢａｍＨⅠ＋ＸｂａⅠ或ＢａｍＨⅠ＋ＢｇｌⅡ双酶切图谱分析，均证明ｉＮＯＳ基因正向插入真核表达载体。用ｉＮＯＳ基因特异性引物，ＰＣＲ分析质粒ｐＣＭＶｉＮＯＳ，扩增出４５１ｂｐ片段，提示重组质粒含有ｉＮＯＳ基因序列。ｐＣＭＶｉＮＯＳ特点是携有ｉＮＯＳ的ｃＤＮＡ序列，包括编码ｉＮＯＳ开放阅读框架，但删除５＇端部分非编码区，有利于高效表达。还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。因此，重组表达质粒ｐＣＭＶｉＮＯＳ的构建可望为研究ｉＮＯＳ基因真核表达调控，阐明ＮＯ／ｉＮＯＳ调节神经、心血管和免疫系统的分子机理?In this report,an enkaryotic cell expression vector which contains the cDNA encoding an inducible nitric oxide synthase(iNOS) from RAW264.7 macrophages was designed and constructed using the molecular biologic procedures.The plasmid pBluscript Ⅱ KS(+)iNOS was digested with double enzymes,Hinc Ⅱ and Not Ⅰ. A 3973bp DNAfragment carrying the iNOS gene was purified from agarose electrophoresis gels and ligated to the polylinker sites betweem Hind Ⅲ and Not Ⅰ in the pRc/CMV,an enkaryotic expression vector in directional cloning.2 out of 14 transformants,clone2 # and clone8 #,were identified with the cleavage of EcoR Ⅰ and Sal Ⅰ to be a recombinant plasmid pCMV iNOS.The restriction analysis of pCMV iNOS with Not Ⅰ or Kpn Ⅰ+Sal Ⅰ revealed that the position and size of cDNA insertion were consistent with the sequence of prediction.Furthermore,the plasmid pCMV iNOS was analyzed by restriction endonuleases such as EcoR Ⅴ+Xba Ⅰ,EcoRⅤ+Sca Ⅰ,BamH Ⅰ+Xba Ⅰor BamH Ⅰ+Bgl Ⅱ.It was verified that iNOS gene was inserted into pRc/CMV in sense orientation.PCR analysis was carried on using the pCMV iNOS plasmid DNA as a template and the specific primers corresponding to cDNA sequence of iNOS,The result demonstrated that amplification product was a 451bp fragment,thus indicating the presence of iNOS coding sequence in the recombinant vector.The characterization of the expression plasmid displays that the plasmid pCMV iNOS carries the cDNA seqence that may encode the overall 3432 nucleotide open reading frame of iNOS,but delete the partial 5' un translated regions,which should be useful for the functional gene expression at high levels.lts structure also 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.Construction of plasmid pCMV iNOS will be extremely valuable,and provide a novel thought and strategy to s

关 键 词：诱生型 一氧化氮合酶 分子克隆 基因表达 

分 类 号：Q782[生物学—分子生物学]