为研究猪CD81基因特征及对其进行原核表达,本研究从猪PBMC的总RNA中扩增获得CD81全基因,测序后经比对分析表明,与已报道的序列同源性为99.7％～100％,与牛羊的同源性约为92％,其次为灵长类(约90％),大鼠和小鼠最低(约87％).氨基酸序列分析其SEL和LEL区为主要变异区,尤其LEL区在不同物种间变异较大.进一步扩增其LEL区基因并以pGEX-4T-1为载体构建重组表达质粒,经IPTG诱导表达重组蛋白,其主要以包涵体形式存在.Western blot结果显示,重组蛋白以GST融合蛋白形式表达.%CD81 is a widely expressed cell-surface protein involved in a variety of biologic responses. To study the genetic characteristics of porcine CD81 (pCD81) and the expression of pCD81 large extracellular loop (LEL) region, the pCD81 gene was amplified by RT-PCR from ConA stimulated porcine peripheral blood mononuclear cells, and cloned into the pMD18-T vector for sequencing. Sequence alignment and phylogenetic analysis showed that the identity of pCD81 with those of pig, cattle, sheep, human, monkey, rat and mouse in GenBank were 99.7 to 100%, 92.1%, 92.3%, 90.3%, 89.3%, 86.5% and 87.2%, respectively. Deduced ammo acid sequences analysis indicated that SEL and LEL regions were mainly divergent regions, especially in the LEL region, whereas other regions were largely conserved. Furthermore, the LEL gene was amplified and cloned into pGEX-4T-l for expression in E.coli. SDS-PAGE and western blot assays demonstrated that the recombinant protein was expressed mainly in form of inclusion body. The expression of CD81 LEL region would facilitate further studies on the role of CD81 in swine pathogen infection.
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