HLA-B*2704重链胞外功能区基因片段的克隆表达及其生物学功能的初步鉴定

Cloning, Expression and Identification of HLA-B*2704 Extracellular Heavy Chain

目的:克隆及可溶性表达HLA-B*2704重链胞外功能区,并对其生物学功能进行初步鉴定。方法:以HLA-B位点全长cDNA为模板,用PCR-SSP方法扩增HLA-B*2704重链胞外区cDNA,经测序鉴定后与pET32a可溶性原核表达载体构建其重组原核表达系统,并在大肠杆菌BL21中表达,采用Western印迹及微量淋巴细胞毒阻断实验初步鉴定该蛋白的特异性及其生物学特性。结果:扩增出HLA-B*2704重链胞外功能区cDNA片段,构建的HLA-B*2704 cDNA-pET32a可溶性原核表达载体可在大肠杆菌BL21表达系统中得到较好表达,表达量约占菌体总蛋白的40%;通过Western印迹鉴定了表达蛋白的特异性;通过微量淋巴细胞毒阻断实验发现该重组蛋白具有生物活性并可特异性阻断HLA-B27阳性细胞的微量淋巴细胞毒反应。结论:在大肠杆菌中表达了具有生物学活性的HLA-B*2704重链胞外功能区,为强直性脊柱炎的机理研究及特异性阻断药物的筛选提供了实验基础和新的靶标。

Objective： To investigate the feasibility of cloning and expression of soluble HLA-B＊2704 extracellu- lar heavy chain and its preliminary biological functions. Methods： Full length cDNA of HLA-B locus was used as template to clone and amplify HLA-B＊2704 extracellular heavy chain by PCR-SSP, and the correct sequence of HLA-B＊2704 cDNA confirmed by DNA sequencing was inserted into pET32a soluble prokaryotic expression vec- tor. The expression of HLA-B＊2704 extracellular heavy chain was tested by Western blot, and its biological func- tion was detected through micro-lymphocyte toxicity blockage experiment. Results： We successfully amplified the cDNA fragments of HLA-B＊2704 extracellular heavy chain and constructed the fragment into soluble prokaryotic expression vector of pET32a. By using E.coli BL21 expression system, the total protein expression was around 40%. Western blot identified the HLA-B＊2704 specificity of the expressed protein, and micro-lymphocytes block- ing experiment revealed that the protein possessed the biological activity to specifically block the B27 specific cyto- toxicity reaction. Conclusion： HLA-B＊2704 extracellular heavy chain with biological activity was successfully ob- tained in E.coli, which will be of some help to the investigation of the mechanism of ankylosing spondylitis as well as for the selection of specific blockage drugs.