摘要
利用基因重组技术，成功地构建了人ＭＤＲＩ基因片断的表达载体ｐＧＥ－Ｈｍｄｒ－１，用氯化钙方法转化Ｅ．ｃｏｌｉＨＢ１０１，挑选２０个克隆扩增培养，经提质粒，酶切，Ａｇａｒｏｓｅ电泳检测证实：其中４个克隆含有重组质粒，选用含重组质粒载体的菌株扩增培养，经ＩＰＴＧ诱导，超声波破碎，抽提蛋白，ＧＳＨ－ａｇａｒｏｓｅ亲和层析纯化，ＳＤＳ－ｐＡＧＥ电泳检测证实：此重组载体已表达所需的目的蛋白（ＧＳＴ－ｍｄｒ融合蛋白）．该表达载体的构建，为基因工程制备人ＭＤＲ基因产物Ｐ－糖蛋白奠定了基础，纯化的Ｐ－糖蛋白将用作抗原制备特异的抗Ｐ－糖蛋白的单克隆抗体，也可用于筛选逆转ＭＤＲ药物的底物。
y means of gene reconbinant techniques , we have successfally constructed an expressionvector pGE-Hmdr- l which expresses a fusion protein of Glutathione-S-Transferase(GST)andhuman MDR1 gene fragment.The plasmid pGE-Hmdr-1 was transformated to E. coli HB10lby standard techniques(CaCl2 method ).2 0 cfones were picked from Master LB plate(containampicillin 50 mg/L)for amplification incubation in liquor LB medium(contain ampicillin 50 mg/L).Plasmids were prepared by alkaline lysis method. It was demonstrated that there were 4 clones containing recomb inant expression vector in 2 0 clones byrestrict ion endon uclease digestion(BamH l and EcoR l )and agarose electrphoresis detection. We chose strain E. coli HB101-6 which contained recombinant expression vector for amplification incubation and induced fusion protein expression by adding IPTG. The protein was extracted from the harve sted bacteria. Then the fusion protein was purified by the GST-agarose affinity absorption. It was demonstrat-ed that the interesting fusion protein had been expressed by SDS-pAGE and coomassie brilliant blue staining detection. Construction of this expression vector may be one of the methods to pre-pare human MDR gene product p-glycoprotein with gene engineering techniques. Purified p-gly-coprotein will be an antigen for preparing specifical monoclonal antibody against the p-glycopro-tein in human multidrug-resistance(MDR).
出处
《昆明医学院学报》
1995年第3期1-5,共5页
Journal of Kunming Medical College
基金
国家八五重点攻关项目
关键词
基因重组
基因
P糖蛋白
多药耐药
Gene recombinant Multidrug-resistant gene Expression ector P-glycoprotein
