【作者】 童光志； 华荣虹； 周艳君； 王云峰； 刘金霞； 杨金雨； 华玉卓； 安同庆；

【Author】 TONG Guang-zhi, HUA Rong-hong, ZHOU Yan-jun, WANG Yun-feng, HUA Yu-zhuo, AN Tong-qin, LlU Jin-xia, YANG Jin-yu(National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China)

【机构】 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室；

【摘要】 2002年11月,第一例严重急性呼吸综合征(severe acute respiratory syndrome,SARS)病例在中国广东省佛山市出现。之后,SARS迅速传播至其它国家和地区,导致数千人死亡。基于世界卫生组织(WHO)的协调,在世界范围内由十三个实验室成立了SARS研究网络,在它们的通力合作下,WHO于2003年4月16日宣布SARS的病源是一种新的冠状病毒,为SARS冠状病毒(SARS-CoV)。SARS-CoV是一种单链正义RNA病毒,有囊膜结构。病毒基因组约29.7Kb,基因组的组织形式与其它冠状病毒相似,有11个开放阅读框,主要结构蛋白有纤突蛋白(S)、囊膜蛋白(M)、小囊膜糖蛋白(E)和核蛋白(N),无HE蛋白。SARS-CoV的基因序列和蛋白的氨基酸序列与其它已知的冠状病毒的同源性很低。SARS-CoV的纤突蛋白(spike protein)是病毒粒子的一种主要结构蛋白。它在介导病毒粒子与宿主细胞受体的结合以及诱导中和抗体中起重要作用。在本实验中我们通过生物信息学分析并预测了6个纤突蛋白B细胞表位(S1 S2 S3 S4 S5 S6)。利用6个预测的表位我们首先构建了一个多表位嵌合基因,多表位嵌合基因合成后插入原核表达质粒pGEX-6p-1的多克隆位点BamHI与XhoI之间,构建成GST融合表达质粒。重组质粒转化宿主菌BL21后经IPTG诱导后得到了高效表达。Western blot分析结果表明重组融合蛋白可与SARS病人康复血清反应。用重组融合蛋白免疫BALB/c小鼠,经两次加强免疫后,取小鼠脾脏淋巴细胞与SP2/0骨髓瘤细胞融合制备杂交瘤细胞。用ELISA方法筛选出了两株稳定分泌单克隆抗体的杂交瘤细胞株D3C5株和D3D1。并且分析了两株单克隆抗体的特性。亚型分析表明其中D3C5是IgG2b,D3D1是IgG1。轻链均为kappa链。ELISA分析表明D3C5与SARS-CoV全抗原反应。Western blot分析结果表明单抗D3D1可以特异地识别SARS-CoV中的大小约为150kD的条带。又将6个预测表位分别与GST融合表达。在这6个融合蛋白中GST-S5可以和单克隆抗体D3C5反应,GST-S2可以和单抗D3D1反应。Western blot分析表明两个单抗所识别的表位均为线性表位。两个表位分别位于SARS-CoV纤突蛋白的第447至458氨基酸残基和789至799氨基酸残基。两个表位的其它特性仍虽进一步分析。近来有研究表明SARS-CoV的功能性受体是血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)。并且有报道显示纤突蛋白中的一个193氨基酸残基的片段(318-510AA)可以比纤突蛋白的S1结构域(12-672AA)更能有效地结合ACE2。并且在这个片段中第454氨基酸残基在与受体结合中是不可改变的。我们所鉴定的表位D3D1(447-458)刚好中位于这一结构域中。所以我们的结果可以为进一步分析SARS-CoV纤突蛋白功能以及防治SARS的研究提供基础。更多还原

【Abstract】 A novel coronavirus has been identified as the pathogen of severe acute respiratory syndrome (SARS). Based on the work of world SARS research net, WHO declared a novel coronavirus is the pathogen of SARS in 16th April 2003, and referred it to severe acute respiratory syndrome-coronavirus (SARS-CoV). SARS-CoV is enveloped, positive-sense, ssRNA virus. The genome of SARS-CoV is about 29.7Kb in length, has 11 open reading frame, and the genome organization is similar to that of other coronaviruses. The gene sequence and amino acids sequence have very low homology with any other known animal coronavirus. The spike (S) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. It plays an important role in interaction with receptor and inducing neutralizing antibodies. In the study, six epitopes (S1 S2 S3 S4 S5 S6) of the spike protein of SARS-CoV were predicted by bioinformatics analysis. Firstly, a multi-epitopes chimeric antigen gene was constructed and synthesized. The chimeric antigen gene then fused to downstream GST gene in pGEX-6p-1. The western blot assay demonstrated that SARS patient convalescent serum could recognize the recombinant fusion protein. BALB/c mice were immunized with the purified recombinant fusion protein. After two booster immunizations, splenocytes were fused with SP20 myeloma cells to generation hybridomas. Two positive monoclonal antibody secreting hybridomas cell lines were selected by ELISA screening. And the two antibodies were further characterized. Among these antibodies, D3C5 is IgG2b, D3D1 is IgG1. The light chains both were kappa. D3C5 reactive with SARS-CoV sonicates with ELISAanalysis and have cross-reaction with PEDV, TGEV and IBV. In western blot analysis, D3D1specifically recognized a band of about 150 kD. And six predicted epitopes gene fused to GST and expressed in E.coli BL21 respectively. Among six fusion proteins, GST-S5 reacted with monoclonal antibody D3C5 and GST-S2 reacted with monoclonal antibody D3D1 against spike protein of SARS-CoV. The results indicated that the epitopes recognized by monoclonal antibody D3D1 and D3C5 are all lineal epitopes. The two epitopes correspond the sequence of 447 to 458 and 789-799 amino acid of spike protein of SARS-CoV respectively. Though MAb D3C5 have cross reaction with other coronavirus in this study, epitope D3C5 (789aa-799aa) shared no significant homology with other coronavirus. The characters of MAb D3C5 and epitope D3C5 need further investigation. The functional receptor for the SARS coronavirus is angiotensin-converting enzyme 2 (ACE2). The recently report demonstrated that a 193-amino-acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). And the residue 454 (aspartic acid) is unmutationable in the receptor binding reactivity. The epitope D3D1 (447-458) we identified here locate spang in receptor binding domain. So our results could provide useful information for further analysis the function of spike protein and prophylaxis of SARS. Identification of B-cell epitope of spike protein of SARS-CoV could provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for the control of severe acute respiratory syndrome.更多还原