重组SARS-CoV未知功能小蛋白X5表达、纯化及抗体制备

Expression、 purification and antibody preparation of recombinat SARS-CoV X5 protein

摘要: 将SARS-CoV未知功能小蛋白X5的基因在大肠杆菌中进行诱导表达，获得以包涵体形式存在的重组X5蛋白。通过高浓度尿素变性溶解包涵体，亲和层析法纯化蛋白，然后对其进行尿素梯度透析复性，最终得到的蛋白纯度>95%，终产率93.3 mg·L^{-1。SDS-PAGE电泳和LC-ESI-MS/MS结果表明，该重组蛋白大小与理论相符，序列与GenBank中注册的X5蛋白序列一致，说明该蛋白为目的蛋白。利用纯化的X5蛋白制备得到多克隆抗体，ELISA检测抗体效价为1∶72 900，说明纯化后的蛋白对兔有很强的免疫原性。X5多克隆抗体的成功制备，为将来鉴定X5蛋白的药理学活性奠定了基础。}

Abstract: X5 protein is one of the putative unknown proteins of SARS-CoV. The recombinant protein has been successfully expressed in E.coli in the form of insoluble inclusion body. The inclusion body was dissolved in high concentration of urea. Affinity Chromatography was preformed to purify the denatured protein, and then the product was refolded in a series of gradient solutions of urea. The purified protein was obtained with the purity of >95% and the yield of 93.3 mg·L^{-1. Polyclonal antibody of this protein was obtained, and Western blotting assay indicated that the X5 protein has the strong property of antigen. Sixty-eight percent of the recombinant protein sequence was confirmed by LC-ESI-MS/MS analysis.}