自身核抗原U1SnRNP70kD多肽分子中U1RNA结合功能区的cDNA克隆及其在大肠杆菌中的表达

U1RNA Binding Domain of Autoantigen U1SnRNP 70kD:cDNA Cloning and its Expression in E.coli

本研究采用逆转录－ＰＣＲ技术克隆自身核抗原Ｕ１ＳｎＲＮＰ７０ｋＤ多肽分子中Ｕ１ＲＮＡ结合功能区（Ｕ１ＲＮＡｂｉｎｄｉｎｇｄｏｍａｉｎ，Ｕ１ＢＤ）的ｃＤＮＡ，经ＤＮＡ测序证实以后定向插入原核表达载体ＰＧＥＸ－２Ｔ，进而导入大肠杆菌中表达重组蛋白。免疫印迹法研究表明：９６％（４８／５０）的抗Ｕ１ＳｎＲＮＰ７０ｋＤ抗体阳性血清能够识别该重组蛋白，证实Ｕ１ＢＤ重组蛋白具有Ｕ１ＳｎＲＮＰ７０ｋＤ抗原性，而且Ｕ１ＢＤ是Ｕ１ＳｎＲＮＰ７０ｋＤ多肽上主要抗原表位区域，能够被大多数抗Ｕ１ＳｎＲＮＰ７０ｋＤ抗体阳性血清识别。这为今后对Ｕ１ＢＤ抗原表位精确定位，分析特定抗原表位与疾病的相关性以及制备重组抗原用于临床检测等研究奠定基础。

By adapting reverse transcription-PCR technology,we obtained the cDNA cloneencoding U1RNA binding domain(U1BD)of the autoantigen U1SnRNP 70kD polypeptlde,using HeLa cell total RNA as template.Confirmed by DNA sequencing,the target U1BD cDNA was subcloned unindirectionally into the bacterial expression plasmid PGEX-2T and then transformed into E.coli to express the recombinant protein.Further analysis by immunoblotting demonstrated that 96%(48/50)of antiU1SnRNP 70kD positive sera can recognize the recombinant protein.Thus,it was shown that the recombinant protein of U1BD exhibited the antigenicity of U1SnRNP70kD, and it may be a major epitope region that can be recognized by a majority of anti-U1SnRNP 70kD antibodies.