抗SARS冠状病毒中和活性单克隆抗体的筛选及其结合位点分析

Screening and epitope mapping of monoclonal antibodies to severe acute respiratory syndrome-associated coronavirus

目的建立能分泌具有中和活性的抗SARSCoV单克隆抗体(McAb)细胞株,制备有中和活性的抗SARSCoVMcAb,用于SARSCoV感染的早期特异诊断和进行SARSCoV结构蛋白的功能研究。方法用灭活纯化SARSCoV(BJ01株)免疫BALBc小鼠,通过细胞融合技术,建立能稳定分泌SARS病毒McAb的杂交瘤细胞系,然后用中和试验进一步筛选分泌具有中和活性的抗SARSCoVMcAb细胞株。利用SARSCoV的S和N蛋白的不同长度肽段通过ELISA鉴定McAb的特异结合区域,分析S和N蛋白不同区域诱导产生抗体的能力和特性。结果建立了12株能稳定分泌抗SARSCoVMcAb的杂交瘤细胞株,间接免疫荧光法检测抗体效价在1∶320～1∶20480之间,其中6株具有较好的中和SARSCoV的能力。具有中和活性的McAb中,3株是针对S蛋白的,2株是针对N蛋白的,其中抗S蛋白的中和抗体活性比抗N蛋白的中和抗体活性高,另外1株可能是针对SARSCoV的其他结构蛋白的。进一步对6株抗S蛋白的McAb的特异抗原结合表位分析,发现其中3株结合位点在S蛋白第12～311氨基酸之间,2株在第310～535氨基酸之间,后者中和效价高于前者,另1株是针对S2蛋白的,SARS病毒的受体结合区正位于第310～535氨基酸这一区段。具有中和活性的McAb通过间接免疫荧光、ELISA、酶标免疫组化法和胶体金方法对SARS病毒进行检测,都显示出高度的特异性和良好亲和力。结论成功制备了具中和活性的抗SARSCoV单克隆抗体,并分别测定了McAb对S蛋白和N蛋白的特异抗原结合活性,初步确定了S蛋白的中和表位。为今后SARS病毒的特异诊断、结构蛋白的功能分析和重组疫苗的研制奠定了较好的基础。

Objective To establish a hybridoma cell line which can secrete monoclonal antilbodies against SARS-CoV and to screen the antibodies with neutrolizing bio-function. Methods BALB/c mice were immunized with inactivated BJ01 strain of SARS-CoV, cell lines secreting anti-SAILS McAbe were established with hybridoma technique. McAb specialized binding epitope to SARS-CoV were identified by ELISA with different segment of S and N proteins of SARS-CoV. Results A panel of 12 McAbs was obtained by hybridoma cell lines and 6 of them showed high neutralizing activities. These McAbe could recognize SARS-CoV-infected ceils by immunofluorescence analysis（WA）. The titers were between 1：320- 1：20 480. Among the McAbs with neutralizing activities, three were targeted to S protein and 2 targeted N protein, one was targeted to other protein of SARS-CoV. As for the 6 McAbs specialized to S protein, 3 were to Sl.la subunit（AA 12- 311） and 2 to S1.1bsubunit（AA 310-535）, another 1 was to S2. McAbs with neutralizing activity were used to identify SARS-CoV antigen by IFA, ELISA, ISH and ICG, all of them showed high specificity and high affinity. Conclusion We established 12 McAbs by immunizing BALB/c mice with inactivated BJ01 strain of SARS-CoV, 6 of these McAbs had neutralizing activities. The neutralizing epitope on S protein of SARS-CoV were also demonstrated with these McAbs. These resuits would be useful for specific diagnosis, and vaccine designing of SARS-CoV.