融合域点突变的埃博拉病毒GP蛋白重组新城疫病毒的构建与免疫评价

Construction and immune evaluation of a recombinant Newcastle disease virus expressing Ebola virus GP protein mutated at fusion domain

扎伊尔型埃博拉病毒(EBOV)的感染会引起人类以严重出血和发热为主要症状的传染病,死亡率高达90%,对公共卫生构成严重威胁。EBOV囊膜蛋白(GP)是病毒主要的免疫原性蛋白。本团队前期的研究表明,以新城疫病毒(NDV)为载体构建表达EBOV GP蛋白的重组病毒rLa-EBOVGP改变了NDV的侵入方式。为了研制出安全性更高的埃博拉候选疫苗株,本研究将EBOV GP蛋白的融合域I532突变为R后插入NDV LaSota疫苗株(rLa)基因组中,通过反向遗传学操作拯救表达EBOV GP(I532R)蛋白的重组NDV,并将经PCR鉴定正确的重组病毒在鸡胚中连续传代检测了其的体外遗传稳定性。RT-PCR鉴定结果显示,EBOV突变的GP(I532R)蛋白基因正确插入了NDV基因组中,表明获得了重组病毒rLa-EBOVGP(I532R);在鸡胚中连续传20代后,经RT-PCR扩增EBOV GP(I532R)基因后经测序鉴定,结果显示rLa-EBOVGP(I532R)能够保持良好的遗传稳定性。利用蔗糖梯度超速离心法纯化rLa、rLa-EBOVGP和rLa-EBOVGP(I532R)病毒,分别进行western blot检测。结果显示,EBOV GP和GP(I532R)蛋白均能够正确表达;将rLa、rLa-EBOVGP和rLa-EBOVGP(I532R)分别感染BHK-21细胞后,利用激光共聚焦试验分析EBOV GP(I532R)的表达和定位,结果显示GP(I532R)能够表达且位于BHK-21细胞的细胞膜上;对纯化病毒利用免疫电镜观察病毒颗粒,结果可见rLa-EBOVGP(I532R)与亲本病毒rLa结构特征相似,且GP(I532R)蛋白能够嵌合在重组病毒粒子表面;生长动力学试验结果表明,rLa-EBOVGP(I532R)与亲本株rLa在鸡胚中的生长特性基本一致,且能够稳定增殖;分别将重组病毒rLa-EBOVGP和rLa-EBOVGP(I532R)经肌肉注射和滴鼻两种途径免疫小鼠,经稀释血清固定病毒的方法分别检测各组小鼠针对NDV和EBOV GP蛋白的中和抗体。结果显示rLa-EBOVGP(I532R)能够诱导小鼠产生较高的针对NDV和EBOV GP蛋白的中和抗体。以上结果表明rLa-EBOVGP(I532R)具有作为防控EBOV感染的储备性候选疫苗株的潜力。本研究为进一步研究GP(I532R)的生物学功能奠定了基础。

Zaire Ebola virus(EBOV)can cause severe hemorrhagic fever in humans with a case fatality rate of up to 90%and poses a significant threat to public health.Envelope protein(GP)is the main immunogenic protein of EBOV.Our previous study showed that the incorporation of EBOV GP protein into NDV particles significantly altered the entry of NDV into cells.In order to develop a safer Ebola vaccine,the GP of EBOV was mutated into GP(I532R),and a recombinant NDV expressing the mutant Zaire Ebola virus glycoprotein rLa-EBOVGP(I532R)was rescued via reverse genetics.The recombinant virus rLa-EBOVGP(I532R)was continuously passaged in embryonated chicken eggs and its genetic stability during in vitro propagation was evaluated.RT-PCR results showed that EBOV GP(I532R)was correctly inserted into the NDV,which indicated that rLa-EBOVGP(I532R)was rescued successfully.The recombinant virus was passaged 20 times in embryonated chicken eggs,and the EBOV GP(I532R)in the virus from each passage was examined by RT-PCR and sequencing.The result showed that the GP(I532R)gene could maintain its genetic stability throughout the propagation in embryonated chicken eggs.rLa,rLa-EBOVGP,and rLa-EBOVGP(I532R)were purified by sucrose gradient centrifugation followed by western blot detection.The results showed that GP(532R)could be expressed correctly.The localization of the GP(I532R)at the cell membrane of BHK-21 cells infected with the recombinant virus was confirmed by confocal assay.Transmission electron microscopy revealed that the rLa-EBOVGP(I532R)had the same morphological characteristics as those of rLa and observed the expression of GP(I532R)on the surface of the chimeric virus.The growth kinetic curve of rLa-EBOVGP(I532R)in embryonated chicken eggs was similar to that of parent strain.Mice immunization assay demonstrated that intranasal and intramuscular immunization with rLa-EBOVGP(I532R)could induce NVD specific neutralizing antibody and EBOV GP specific neutralizing antibody significantly in mice.The above results indicate that rLa-EBOVGP(I532R)has the potential to be a vaccine candidate against EBOV infection.This study also lays a foundation for further study of the biological function of GP(I532R).