H5N1禽流感病毒HA假病毒的构建及特性研究

摘要

目的 构建包膜蛋白为H5N1禽流感病毒HA蛋白的假病毒,对其生物学特性进行研究,并将其初步应用于H5N1禽流感病毒的血清检测.方法 将我国分离的高致病性H5N1禽流感病毒的HA基因插入真核表达质粒,得到pLP-HA,与假病毒构建体系的三种质粒pLP1,pLF2和pEmGFP,瞬时共转染人胚肾细胞293T,48 h收集假病毒上清,对其感染性,血凝活性进行测定,并应用于微量中和实验.同时,构建了优化HA基因的假病毒以及一株含有越南禽流感病毒HA基因的假病毒,进行比较.结果 电镜下观察到假病毒颗粒的存在;Western-Blot表明HA蛋白存在于假病毒颗粒中;HA假病毒与野生型活病毒的微量中和实验相比,两者结果具有很好的相关性.结论 成功构建了不同高致病性H5N1禽流感病毒HA蛋白的假病毒,所构建的假病毒可以应用于微量中和实验.研究发现不同禽流感病毒株HA蛋白假病毒的包装效率不同,并且真核表达优化基因并不能显著提高假病毒颗粒包装效率.%Objective To construct pseudovirus beating H5N1 HA based on a lentivirus vector system.Then we study the biological feature of the pseudovirus.With the newly established viral particles,we performed the serological tests.Methods H5N1 avian influenza vires that isolated from human case was cloned to construct pLP-HA,then pLP-HA co-transfected with lentivirus vector plasmids pLP1,pLP2 and pEmGFP into 293T cells.The supematant was harvested 48h post-transfection.Concentrated by super centrifuge,the pseudotyped viruses were analyzed by infection test,HA test and micro-neutralization test.At the same time,optimized HA gene and a Vietnam H5N1 HA gene were used to construct pseudotyped virus for comparison.Results Pseudotyped virus particles can be observed with electronic microscope.Western-blot revealed that HA glycoprotein can be expressed in the virions. Our neutralization assay by using the pseudovimses was comparable with the conventional microneutralization assay with wild-type viruses.A hish degree of correlation was detected.Conclusion Pseudotyped Viruses coated with HA of H5N1 High Pathogenic Avian lnfluenza were successfully constructed;it can be used to for the microneutralization assay.The HA gene from different sources affect the efficiency of the packaging of the pseudovirus.But the optimized HA gene can not obviously improve packaging efficiency of the pseudovims.