hHGF-αcDNA的克隆及表达载体的构建  

Cloning of hHGF-α cDNA and construction of its expre ssion vector

作　　者：党素英[1] 程牛亮[1] 牛勃[1] 王惠珍[1] 赵建滨[1] 张祖珣[2] 

机构地区：[1]山西医科大学生物化学与分子生物学教研室,太原030001 [2]首都医科大学生物化学教研室

出　　处：《山西医科大学学报》2001年第2期100-103,共4页Journal of Shanxi Medical University

基　　金：山西省归国留学人员科研基金资助项目

摘　　要：目的　定点突变法扩增制备完整hHGF αcDNA ,克隆并构建其原核表达载体。方法与结果　以含有人HGFcDNA全序列的质粒 pRC/CMV hHGF为模板 ,设计合成一对特异引物进行聚合酶链反应 (PCR) ,扩增hHGF αcDNA基因 ,琼脂糖凝胶电泳检测 ,结果显示扩增得到了 1 34kb长的目的基因产物。将扩增产物以限制性内切酶XhoⅠ酶将其切为 386bp、95 4bp两个片段 ,分别以EcoRⅠ /XhoⅠ、XhoⅠ /BamHⅠ与 pBSKS载体连接重组 ,对目的基因分段克隆后进行序列测定 ,结果表明除通过定点突变引入的起始密码ATG、终止密码子TAA、TGA及EcoRⅠ、BamHⅠ识别位点外 ,扩增得到的PCR产物序列与Nakamura等报道的HGFcDNA中不包括前体序列的α链部分完全相同。将以EcoRⅠ /XhoⅠ、XhoⅠ /BamHⅠ从pBSKS HGF α 386、pBSKS HGF α 95 4中切出的 386bp、95 4bp两个片段以EcoRⅠ /BamHⅠ与pBV2 2 0连接构建重组表达质粒 pBV2 2 0 HGF α ,限制性内切酶图谱分析显示HGF αcDNA插入到载体pBV2 2 0的EcoRⅠ /BamHⅠ位点 ,插入方向正确。温度诱导表达 ,SDS PAGE显示一分子量与单体hHGF α链吻合的蛋白带 ,证明表达质粒构建成功。结论　设计制备了hHGF α链cDNA ,克隆建立了hHGF α链原核表达质粒。Objective To obtain and clone hHGF α cDNA and construct its expression vector.Methods and Results The templat e pl asmid pRC/CMV hHGF and a pair of specific primers were used in polymerase chain reaction(PCR)to amplify the goal gene,the hHGF α cDNA.A 1 34 kb fragment was observed on the agarose electrophoresis map.The purified product of PCR was dige st ed with XhoⅠ and two fragments of 386 bp and 954 bp were obtained.They were in serted into pBSKS at EcoRⅠ/XhoⅠ sites and XhoⅠ/BamHⅠ sites respectively. Thus, two subcloned plasmids pBSKS HGF 386 and pBSKS HGF 954 were constructed an d used for sequencing.The results showed that the sequence of the product of PCR was in consistent with the hHGF α Nakamura reported except the added start co don ATG,tandem stop codon T and TGA and restriction endonuclease EcoRⅠ and BamH Ⅰ recognition sites.The two fragments of EcoRⅠ XhoⅠ(386 bp) and XhoⅠ B amHⅠ(954 bp)were recovered from pBSKS HGF386 and pBSKS HGF954 and inserted to ge ther into the EcoRⅠ/BamHⅠ sites of pBV220,a prokaryotic expression vector, t o construct the expression plasmid.The analysis of multiple RE mapping indicated that the recombinant expression plasmid vector has correct cloning orientation an d perfect structure in accord with authors' designation.After inducing the E.c oli tran sformed with expression plasmid to express the goal protein by changing temperat ure, HGF α chain expression was identified by SDS PAGE.Conclusion The hH GF α cDNA is perfectly designed and cloned.A procaryotic expression vector of hHGF α is perfectly designed and constructed.

关 键 词：肝细胞生长因子 克隆 CDNA 基因表达 载体构建 

分 类 号：Q785[生物学—分子生物学]