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目的通过系统的生物信息学分析和实验验证,筛选黄热病毒的特异抗原片段,为免疫诊断试剂的研制奠定基础。方法分别利用DNAStar及ANTHEPROT软件对黄热病毒蛋白进行分析,参考亲水性、抗原性、可塑性、表面可及性及二级结构信息,对黄热病毒可能的共有及特异性抗原表位进行系统的预测分析,分析其在不同毒株中的保守性,并对预测得分值较高的抗原区域进行RT-PCR扩增,利用pMal-c2x原核表达系统进行原核表达,Western blot验证其免疫学反应原性,检测阳性抗原片段经亲和纯化后,包被ELISA微孔板,进一步验证其免疫学反应特异性及检测敏感性。结果其中27段抗原片段获高效表达,经Western blot筛选及ELISA进一步验证,原核表达抗原片段YFV22表现良好特异抗,可检测低至1∶12 800倍稀释的黄热病毒多克隆抗体,与所试其他参考病毒多克隆抗体无交叉反应。结论经系统筛选、验证,获黄热病毒特异性抗原片段1段,为其免疫学诊断试剂的研制奠定了基础。
Objective To screen specific fragments of yellow fever virus by bioinformatics analysis and experimental verification, and lay a foundation for the development of immunodiagnostic reagents. Methods The yellow fever virus protein was analyzed by using DNAStar and ANTHEPROT software respectively. The possible common and specific epitopes of yellow fever virus were systematically analyzed with reference to hydrophilicity, antigenicity, plasticity, surface accessibility and secondary structure information , And analyzed their conservativeness in different strains. RT-PCR was carried out on the region of the antigen with higher predicted score, prokaryotic expression was carried out by using the prokaryotic expression system pMal-c2x, and the immunology Reactivity. The positive antigen fragments were purified by affinity chromatography and then coated on an ELISA microplate to further verify their immunological specificity and sensitivity. Results The 27 antigen fragments were highly expressed. Western blot and ELISA confirmed that the prokaryotic expression fragment YFV22 showed good specificity and specificity, and could detect polyclonal antibodies of yellow fever virus down to 800 times Other reference polyclonal antibodies did not cross-react. Conclusion The system was screened and validated to obtain a fragment of yellow fever virus-specific antigen, which laid the foundation for the development of immunological diagnostic reagents.