西尼罗病毒特异性抗原片段筛选及ELISA方法的研究

目的筛选西尼罗病毒特异抗原片段,并建立其ELISA快检方法。方法参考亲水性、抗原性、可塑性、表面可及性及二级结构信息,对西尼罗病毒抗原表位进行系统分析,预测得分值较高的抗原区域经原核表达、Western blot筛选及亲和层析纯化后,包被ELISA微孔板,对ELISA反应条件进行系统优化,建立其ELISA快检方法。结果预测获病毒特异性抗原表位29个,对其中11段进行了表达,经筛选,获得西尼罗病毒特异抗原片段及西尼罗与乙型脑炎病毒共同抗原片段各一段。利用西尼罗病毒特异抗原片段WnAg16建立了检测西尼罗病毒抗体的ELISA方法,与所试相近病毒无交叉反应,S/N比值均在23以上,对鼠抗西尼罗病毒多抗的检出下限达1∶204 800。结论利用特异性抗原初步建立了检测西尼罗病毒抗体的ELISA方法。

Primary screening of West Nile virus specific antigens for ELISA

Objective To screen and identify the West Nile virus specific antigens and to establish a fast ELISA detection method for West Nile virus antibody. Methods Bioinformatic software DNAstar and ANTHEP- ROT were used to analyze the hydrophilicity, flexihility, surface prohahility, antigenicity and secondary structure of West Nile virus antigen epitopes. Based on the epitopes location and amino acid sequence similari- ty, specific antigen segments of West Nile virus were predicted and expressed using prokaryotic expression system. The antigenicity was tested by western blot. After the purification, these antigen segments were used for ELISA. Results 29 specific epitopes of West Nile virus were forecasted, and 11 antigenic fragments were expressed successfully in E. coli. After screening, one specific antigen WnAg16 was achieved and used for the detection of antibody by ELISA. There was no cross reaction when tested with antibodies of other similar viruses. The S/N ratio was found to be above 23. The sensitivity was as high as 1 ： 204800 for antibodies of mouse West Nile virus. Conclusion One West Nile virus specific antigen is obtained and the sensitivity of this antigen for ELISA is very high.