HLA-B* 2705分子结构对其在原核细胞可溶性表达影响的研究  

HLA-B* 2705 molecular structure affects its soluble expression in prokaryotic cells

作　　者：代东发[1,2] 袁方[2] 于坤[1,2] 梁飞[2] 刘楠[2] 龙娟[2] 赵晓[2] 奚永志[2] 孙玉英[2] 

机构地区：[1]安徽医科大学解放军307临床学院,北京100071 [2]军事医学科学院附属医院免疫学实验室暨国家生物医学分析中心免疫分析实验室,北京100071

出　　处：《军事医学》2013年第8期578-581,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(81071448)

摘　　要：目的研究人类白细胞抗原(human leukocyte antigen,HLA)-B* 2705分子结构对其在原核细胞可溶性表达的影响。方法采用RT-PCR法将HLA-B* 2705基因的全长、胞外区和α1、α2功能区分别进行克隆并连接到pET-32a(+)表达载体。经IPTG诱导表达,并经超声破碎、层析和透析纯化蛋白,再经体外微量细胞淋巴毒反应检测蛋白质活性。结果成功构建3种pET-32a(+)表达载体,经IPTG诱导后,HLA-B* 2705基因全长和胞外区主要以包涵体的形式表达,可溶性蛋白的量较低;而α1和α2功能区则以高效可溶性形式表达,通过体外微量细胞淋巴毒实验证实,HLA-B* 2705的α1和α2功能区可溶性蛋白可与HLA-B27抗体特异性结合,并成功阻断补体介导的细胞毒作用。结论α1和α2功能区能够在原核细胞中高效表达可溶性蛋白,并具有蛋白功能,而HLA-B* 2705基因全长和胞外区主要以包涵体的形式表达,表明蛋白质的大小和空间结构对于其可溶性表达具有重要影响。Objective To investigate the effect of molecular structure of HLA-B＊ 2705 on its soluble expression in prokaryotic cells.Methods HLA-B＊ 2705 full-length,extracellular domains and α 1 and α 2 domains encoded cDNA were amplified by RT-PCR method and cloned into the pET-32a（＋） expression vector.After being induced by IPTG,the expression product was disrupted by the ultrasonic cell disruptor and purified with chromatography and dialysis before its biological reactivity was detected by microlymphocytotoxicity reaction in vitro.Results Three pET-32a（＋） expression vectors were constructed successfully and induced by IPTG.HLA-B＊ 2705 full-length and extracellular domains were mainly expressed in the form of inclusion bodies with a small amount of soluble protein;and the α 1 and α 2 domains were mainly expressed in a soluble form.In microlymphocytotoxicity reaction,the α 1 and α 2 domains could specifically bind to HLA-B27 antibody and successfully block complement-dependent cytotoxicity.Conclusion The α 1 and α 2 domains can be efficiently expressed in a soluble form in E.coli,while HLA-B＊ 2705 gene full-length and extracellular domains are mainly expressed in the form of inclusion bodies,suggesting that the size and structure of a protein have important influence on its soluble expression.

关 键 词：HLA-B＊ 2705 分子结构 可溶性表达 包涵体 

分 类 号：R392.1[医药卫生—免疫学]