【摘要】：目的:優(yōu)化A/Anhui/1/2013(H7N9)流感假病毒的制備條件。方法:采用基于慢病毒載體的假病毒包裝系統(tǒng),通過對質(zhì)粒組合方式、骨架質(zhì)粒與包膜質(zhì)粒配比、質(zhì)粒復(fù)合物與轉(zhuǎn)染試劑配比、換液時(shí)間點(diǎn)、維持液血清濃度、假病毒收獲時(shí)間點(diǎn)等條件的探索,優(yōu)化H7N9假病毒的包裝條件;通過對4種假病毒純化方案的比較,確定純化方法;對純化的假病毒進(jìn)行Western印跡鑒定和電鏡形態(tài)觀察,了解其生物學(xué)特征;通過中和試驗(yàn)了解其在中和抗體檢測中的應(yīng)用情況。結(jié)果:假病毒包裝條件優(yōu)化結(jié)果表明,以血凝素(HA)與神經(jīng)氨酸酶(NA)作為膜質(zhì)粒,采用骨架質(zhì)粒與膜質(zhì)粒質(zhì)量比3:1、質(zhì)粒復(fù)合物與轉(zhuǎn)染試劑質(zhì)量體積比1:2混合,轉(zhuǎn)染293FT細(xì)胞后10 h換含2%胎牛血清的維持液,培養(yǎng)48 h后收獲上清,可高效獲得流感假病毒AHop-H7N9pp;不同純化方式獲得的假病毒對感染H7N9的雪貂血清的中和抗體檢測結(jié)果表明,假病毒純化方法對中和抗體檢測靈敏度的影響沒有顯著性差異;生物學(xué)特征分析顯示,純化的假病毒在電鏡下具有典型的流感病毒形態(tài),Western印跡能檢測到HA和P24蛋白;多種流感疫苗小鼠血清中和試驗(yàn)結(jié)果表明該假病毒具有高度特異性,可用于H7N9中和抗體檢測。結(jié)論:獲得了包裝有A/Anhui/1/2013(H7N9)HA與NA的假病毒,并確定了高效制備H7N9假病毒的各條件參數(shù),為基于H7N9流感假病毒的中和抗體檢測平臺的建立奠定了基礎(chǔ)。
[Abstract]:Objective: to optimize the preparation conditions of A/Anhui/1/2013 (H7N9) influenza pseudovirus. Methods: the pseudovirus packaging system based on lentivirus vector was used to maintain the concentration of serum through plasmid combination, the ratio of skeleton plasmid to envelope plasmid, the ratio of plasmid complex to transfection reagent, the time point of liquid change, and the maintenance of liquid serum concentration. The conditions such as harvest time of false virus were explored, and the packaging conditions of H7N9 pseudovirus were optimized. Through the comparison of four purification schemes of pseudovirus, the purification methods were determined, the purified pseudovirus was identified by Western imprinting and observed by electron microscope, and its biological characteristics were understood, and its application in neutralizing antibody detection was understood by neutralization test. Results: the results showed that hemagglutinin (HA) and neuraminidase (NA) were used as membrane plasmid, the mass ratio of skeleton plasmid to membrane plasmid was 3 鈮，

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