摘要
利用ＰＣＲ技术和ＤＮＡ体外重组方法，将内皮素Ａ型受体（ＥｎｄｏｔｈｅｌｉｎｔｙｐｅＡｒｅｃｅｐｔｏｒ，ＥＴＡＲ）胞内区ｃＤＮＡ克隆到ｐＵＣ１８载体中进行序列分析。结果表明，ＤＮＡ序列与预期结果完全相符。然后用低熔点琼脂糖回收插入到ｐＵＣ１８载体中的ＥＴＡＲ胞内区ＤＮＡ片段，连接到ｐＧＥＸ２Ｔ融合蛋白表达载体的凝血酶位点下游，转化大肠杆菌ＪＭ１０３，得到的重组质粒命名为２Ｔ／ＥＴＡＲ。ＪＭ１０３（２Ｔ／ＥＴＡＲ）经３０℃培养、ＩＰＴＧ诱导明显表达出ＥＴＡＲ胞内区融合蛋白，表达产物主要以包涵体形式存在。包涵体经变性和复性后，用亲和层析一步纯化法获得了较纯的ＥＴＡＲ胞内区融合蛋白。
With PCR technology and methods of in vitro DNA recombination,the cDNA for the intracellular region of Endothelin type A receptor (ET AR) was cloned into the pUC18 vector.The result of its DNA sequencing analysis was the same as expected.Then the DNA fragment containing the cellular region of ET AR was recovered from low-melting agarose gel and ligated into the downstream of the thrombin site of the pGEX-2T fusion protein expression vector.The above construce was transformed into E.coli JM103.The resulting recombinant plasmid was designated 2T/ET AR.JM103(2T/ET AR),when cultured at 30℃ and induced by IPTG,expressed an apparent fusion protein of the cellular region of ET AR,mainly in the form of inclusion bodies.After the denaturation and renaturation of the inclusion bodies,the expression product was purified to higher purity by one-step affinity chromatography,laying a good foundation for the functional study of the cellular region of ET AR.
出处
《微生物学免疫学进展》
1998年第4期1-4,共4页
Progress In Microbiology and Immunology
