SARS冠状病毒S蛋白基因片段的表达及其单克隆抗体的制备-东南大学学报医学版

SARS冠状病毒S蛋白基因片段的表达及其单克隆抗体的制备

单位：东南大学发育与疾病相关基因教育部重点实验室, 遗传与发育生物学系, 江苏南京 210009

分类号：R392.11

出版年·卷·期（页码）：2009·28·第一期（18-21）

摘要：

目的: 表达SARS冠状病毒S蛋白片段,并制备特异性单克隆抗体(mAb)。方法: 原核表达SARS冠状病毒S蛋白片段,从SARS病毒cDNA中扩增S蛋白(N端205至419aa)基因片段,测序结果正确。将该基因片段插入pQE-30中,构建重组质粒pQE-30-S1m,以重组质粒转化 E.coli M15诱导表达His-S1m蛋白。纯化后免疫BALB/c小鼠,经融合、筛选制备特异性mAb。结果: 表达并纯化了重组蛋白His-S1m,获得1株抗His-S蛋白的mAb1H2。Ig亚类鉴定为IgG1a,轻链为κ型。经Western blotting检测,mAb1H2与S蛋白发生特异性反应,而与His蛋白无交叉反应。结论: 成功表达了重组S蛋白并制备了特异性mAb,为SARS-CoV的早期诊断及进一步研究S蛋白的功能奠定了基础。

Objective To generate the recombinant fragment at N-terminal residues 205 to 419aa of SARS coronavirus (SARS-CoV) Spike (S) protein,and the monoclonal antibody specific for S protein.Methods The S gene fragment of SARS-CoV was constructed by PCR techniques and expressed in E.coli.Furthermore,purified recombinant protein was used to immune BALB/c mice.The resulting monoclonal antibody was screened from hybridomas,followed by identification of specificity in ELISA and Western blotting.Results Recombinant S1m protein of SARS-CoV was constructed,expressed and purified.One hybridomas producing mAb specific to S1m protein was generated successfully.It was IgG1a(κ).Conclusion One specific mAb against S1m protein of SARS-CoV has been established,which can be used in S protein research and the early diagnosis of SARS-CoV infection.

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