重组SARS-CoV核衣壳蛋白的表达纯化及其单克隆抗体的制备鉴定

Expression and purification of recombinant nucleocapsid protein of SARS Coronavirus and preparation of its monoclonal antibodies

目的目的表达纯化SARS冠状病毒(SARS-CoV)的核衣壳蛋白(nucleocapsid N蛋白),并制备出高安全性、高特异性的针对该蛋白的单克隆抗体(McAb),为SARS的早期快速诊断提供有力的抗体工具。方法在不接触病原体的前提下,采用全基因合成方式分别将SARS-CoV N蛋白编码基因第1-549位碱基(N端)和第496-1269位碱基(C端)直接合成至原核表达载体pET32a(+),表达、纯化重组蛋白(分别记为N1蛋白、N2蛋白),并以纯化蛋白免疫BALB/c小鼠,制备特异性针对N蛋白的单克隆抗体,以间接ELISA法对所需的杂交瘤细胞株进行配对筛选,分析其亚类,以Western blot和间接免疫荧光法鉴定单克隆抗体特异性。结果成功表达并纯化SARS-CoV N1、N2蛋白,筛选出7株抗SARS-CoV N1蛋白及2株抗SARS-CoVN2蛋白的单克隆抗体杂交瘤细胞株,IgG亚类鉴定6株为IgG1,2株为IgG2b,1株为IgG3,Western blot及间接免疫荧光证实所获的单克隆抗体可与SARS-CoV N蛋白发生特异性反应。结论重组SARS-CoV N蛋白成功表达及纯化,并由此获得了SARS-CoV特异性单克隆抗体,为SARS预防检测和发病机制研究奠定基础。

To express and purify the nucleocapsid protein （N protein） of SARS Coronavirus （SARS-CoV） and to generate monoclonal antibodies with high security and specificity against N protein, genes encoding N protein were separated into two parts according to the prediction of antigenicty, the first part designed from 1 to 549 base pairs（N-terminus）, the second from 496 to 1269 base pairs（C-terminus）. They were obtained by nucleotide synthesis and were cloned into expression vector pET32a（＋） respectively. Proteins then were induced to expression by IPTG and purified through Ni-NTA. Balb/c mice were immunized with purified recombinant protein to prepare McAb by hybridoma technique and the McAb was selected by indirect ELISA. The specificity of McAb was identified by Western blot and indirect fluorescence assay. Results showed that target proteins were successfully expressed and purified and seven hybridoma cell lines secreting monoclonal antibodies against SARS-CoV N1 and two hybridoma cell lines secreting monoclonal antibodies against SARS-CoV N2 were obtained. About the immunoglobulin subclass of McAb, six were IgG1, two were IgG2b and one was IgG3. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with SARS-CoV N protein. It is concluded that the recombinant SARS-CoV N protein were expressed and purified and nine specific McAb against SARS-CoV N protein were obtained successfully in the present study.