寨卡病毒NS1单克隆抗体制备及其在临床诊断中的应用

目的用寨卡病毒非结构蛋白NS1免疫小鼠并制备鼠源单克隆抗体,建立捕获ELISA方法检测寨卡病毒NS1蛋白。方法合成NS1基因序列构建真核表达载体pcDNA3.1-NS1,转染HEK293细胞收集上清,经Ni+柱亲和层析法纯化NS1蛋白。以纯化的NS1重组蛋白为抗原免疫BALB/c小鼠,取脾脏进行细胞融合,筛选阳性细胞株,经酶联免疫吸附检测抗体活性和特异性。结果成功表达并纯化NS1重组蛋白,获得7株单克隆抗体,筛选非竞争单抗组合建立捕获ELISA法,该方法可特异性识别NS1重组蛋白,最低检出限为1 ng/ml,且未与其他黄病毒属病毒发生交叉反应。结论成功制备特异性抗寨卡病毒NS1蛋白的鼠源单克隆抗体,建立捕获ELISA法,为寨卡疫情的进一步防控奠定了基础。

Preparation of monoclonal antibodies against Zika virus NS1 protein and application in early diagnosis of Zika virus infection

Objective To prepare monoclonal antibodies against the NS1 protein of Zika virus and develop a capture ELISA method for detection of ZIKV NS1 protein.Methods The recombinant eukaryotic expression plasmid pcDNA3.1-NS1 containing NS1 gene of Zika virus was constructed,then the NS1 protein was expressed based on transfected HEK293 cells and purified by Ni+affinity chromatography.BALB/c mice were immunized subcutaneously with purified NS1 protein.Then their spleen cells were isolated and fused with SP2/0 cells.The hybridoma cells secreted monoclonal antibodies against NS1 protein were screened.The reactivity and specificity of the antibodies were preliminarily analyzed with ELISA.Results The NS1 protein of Zika virus was successfully expressed,and 7 mouse monoclonal antibodies which had good activity with NS1 protein were obtained.Two paired antibodies having no competitiveness were screened to develop a capture ELISA method for detecting ZIKV,and the sensitivity for recombined NS1 protein was 1 ng/ml,showing no cross reaction with other flavivirus.Conclusion Monoclonal antibodies specific for ZIKV NS1 protein were successfully prepared,and a capture ELISA method for detecting ZIKV was developed,laying the foundation for further detecting methods and epidemic control of Zika virus.