特异性SARS-CoV N蛋白单克隆抗体的制备及抗原表位初步分析

目的：用分段表达纯化的SARS冠状病毒（SARS-CoV）的核衣壳蛋白（nucleocapsid N蛋白）制备针对该蛋白不同区域抗原表位的高特异性单克隆抗体（McAb），初步定位单克隆抗体识别表位所在区域。方法：用分段表达纯化的SARS～CoV的N蛋白（分别记为N1蛋白、N2蛋白）分别免疫Balb／c小鼠制备McAb，通过检测其亚类、效价及相对亲和力鉴定McAb的生物学特性，以Western blot鉴定单克隆抗体特异性，并对McAb结合表位进行初步分析。结果：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，腹水效价10^5以上；亲和常数达10^8以上。Western blot证实所获的单克隆抗体可与SARS-CoVN蛋白发生特异性反应。ELISA相加实验结果显示2株N1 McAb识别相同的抗原表位，其余均识别不同的抗原表位。结论：通过分段表达纯化的SARS-CoVN蛋白而制备的特异性针对SARS-CoV N蛋白不同区域抗原表位的单克隆抗体中，N1单抗识别N蛋白N端（1-549bp），N2单抗识别N蛋白C端（496～1269bp），可初步定位单克隆抗体识别表位所在区域。

Preparation of monoclonal antibodies against SARS coronavirus nucleocapsid protein and preliminary analysis of antigenic epitopes

Objective:To prepare monoclonal antibodies with high specificity against different regions of N protein and to analyze antigenic epitopes of SARS-CoV N protein.Methods: Balb/ c mice were immunized with purified different region N protein to prepare McAb by hybridoma technique and select by indirect ELISA.Both of these McAbs were characterized for their antibody titre in the culture supernatant as well as ascites,class,subclass and affinity analysis.The antigen specificity for McAb were evaluated and confirmed by Western blot.The binding site and capability of McAb were observed by ELISA additive assay.Results: 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 showed that the McAbs reacted specifically with SARS-CoV N protein.ELISA additive assay indicated that two antibodies against SARS-CoV N1 recognized same epitopes,while others recognized distinct epitopes.Conclusion: We successfully obtained nine specific McAbs against the different region SARS-CoV N protein after expression and purification of the different part SARS-CoV N protein.It shows that the N1 McAb recognizs the N-terminus of the N protein whereas the N2 McAb recognizs the C-terminus.