HLAB2705重链原核表达和活性鉴定.docVIP

HLAB*2705重链的原核表达和活性鉴定 作者：李彦博，孙玉英，郭斯启，孙业平，张秀云，孔繁华，奚永志 【摘要】 本研究探讨HLAB*2705重链的原核表达及活性鉴定。从HLAB*2705全长cDNA中PCR扩增出膜外区片段并克隆进入pGEMT载体，序列测定后构建原核融合表达载体pET32a（+）B*2705，并转化大肠杆菌。Western Blot 和阻断抗体反应鉴定它们在体外是否具有HLAB27抗原活性。结果表明：HLAB*2705重链融合蛋白在大肠杆菌中获得高效融合表达，表达产量占细菌总蛋白的50%以上；通过Western Blot和阻断抗体反应鉴定它们在体外具有HLAB27抗原活性。结论：本研究获得了HLAB*2705重链融合蛋白，为今后的相关研究打下了基础。 【关键词】 原核表达 Prokaryotic Expression of HLAB*2705 Heavy Chain and Identification for Its Activity Abstract In order to investigate the expression of heavy chain of HLAB*2705 in prokaryotic system and identify its activity, the extramembrane gene fragment of HLAB*2705 was amplified from fulllength HLAB*2705 cDNA by PCR and cloned into pGEMT vector. After identification by sequencing, the prokaryotic expressing vector pET32a（+）B*2705 was constructed. The antigenic activity of expressed protein was identified by Western blot and antibody blocking reaction. The results indicated that the fused HLAB*2705 protein expression with high efficiency was obtained. The expressed product was more than 50% of the total bacteria protein. The antigenic activity of expressed protein was confirmed by Western blot and antibody blocking reaction. It is concluded that HLAB*2705 fusion protein are obtained as basis for the further studies. Key words HLAB*2705 heavy chain; prokaryotic expression; antigenic activity 强直性脊柱炎(ankylosing spondylitis，AS )是致残率较高的脊椎关节病变。脊柱炎症病变从骶髂关节逐渐上升颈椎，晚期致关节变形使患者丧失劳动能力，严重危害生活质量［1］。1973年Brewerton和Schlosstein发现HLAB27和AS之间存在着非常强的关联性［2］。以后的许多研究结果都支持 HLAB27与AS之间在遗传学上的联系。首先，HLAB27与AS的关联性在世界上的许多人群中得到重复［3］；其次，B27转基因大鼠和小鼠可产生多系统自发性炎症，与AS非常相似［4，5］。关于HLAB27分子的致病机制有多种假说，例如分子模拟假说［6］和关节源性肽（arthritogenic peptide）假说［7］等，但证据不足。最近的研究表明，HLAB27存在多种异常形式，如缺乏抗原肽的B27β2微球蛋白复合体、游离B27重链单体、B27重链同源二聚体等［8-10］。这些异常B27可作为自身抗原被CD4+T细胞识别［11］或作为配体被巨噬细胞表达的成对免疫球蛋白样受体（paired Iglike receptors, PIR）［12］识别而导致AS发病。在本研究中，我们用大肠杆菌高效表达了HLAB27抗原中的一个亚型－HLAB*2705重链胞外区，并对其活性进行了初步研究。 材料和方法 材料 大肠杆菌DH5α由本室保存， 质粒 pET32a（+）和宿主菌origamiTM（DE3）购自Nova