具备中和中东呼吸综合征冠状病毒活性的单克隆抗体的制备

PREPARATION OF MONOCLONAL ANTIBODY NEUTRALIZING MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS

目的制备并鉴定对中东呼吸综合征冠状病毒(MERS-CoV)具有中和活性的单克隆抗体,为进一步开展MERS-CoV感染与免疫保护机制研究,以及发展诊断与治疗手段奠定基础。方法 MERS-CoV S蛋白受体结合区与人IgG Fc片段融合表达的重组蛋白免疫BALB/c小鼠,将免疫后小鼠脾细胞与SP2/0骨髓瘤细胞融合,通过ELISA筛选出阳性克隆,随后通过多次亚克隆筛选出稳定分泌特异性抗体的杂交瘤细胞株,制备单抗腹水并进行抗体效价测定及亚类鉴定;通过MERS-CoV假病毒及活病毒中和试验筛选出中和单抗。结果获得了10株能够稳定分泌抗原特异性单抗的杂交瘤细胞株。制备的腹水单抗亚类均为IgG1;其中7株单抗抗体ELISA效价达到10 000以上,并有1株单抗对MERS-CoV具有良好的中和活性。结论本研究获得了1株对MERSCoV具有良好中和活性的单克隆抗体。

Objective To produce and evaluate the monoclonal antibody neutralizing Middle East Respiratory Syndrome Coronavirus（MERS-CoV）,in order to lay a foundation for further research of the mechanism and prevention of MERS-CoV infection. Methods The BALB /c mice were immunized with recombinant fusion protein linking receptor binding domain of MERS-CoV S protein to Fc of human IgG,and the fusion of lymphocyte and myeloma SP2 /0 cells was performed by conventional method. Positive clones were screened by ELISA,and hybridoma cell lines with stable secretion of specific antibody were obtained through subclonal method. Ascites antibodies were produced and purified,followed by characterization and identification. Pseudovirus- and live-virus-based neutralization assay were carried out to screen monoclonal antibodies with neutralizing activiy against MERS-CoV. Results Ten hybridoma cell lines with stable secretion of specific antibody were obtained. All prepared ascitic monoclonal antibodies belonged to the IgG1 subtype,while 7 of those ascites have high antibody titers reaching above 10 000.There was one strain of monoclonal antibody further proved to have neutralizing activity. Conclusion One strain of monoclonal antibody with good neutralizing activity against MERS- CoV was obtained.