H7N9流感假病毒制备条件的优化及其在中和抗体检测中的初步应用
发布时间：2019-06-02 22:25
【摘要】：目的:优化A/Anhui/1/2013(H7N9)流感假病毒的制备条件。方法:采用基于慢病毒载体的假病毒包装系统,通过对质粒组合方式、骨架质粒与包膜质粒配比、质粒复合物与转染试剂配比、换液时间点、维持液血清浓度、假病毒收获时间点等条件的探索,优化H7N9假病毒的包装条件;通过对4种假病毒纯化方案的比较,确定纯化方法;对纯化的假病毒进行Western印迹鉴定和电镜形态观察,了解其生物学特征;通过中和试验了解其在中和抗体检测中的应用情况。结果:假病毒包装条件优化结果表明,以血凝素(HA)与神经氨酸酶(NA)作为膜质粒,采用骨架质粒与膜质粒质量比3:1、质粒复合物与转染试剂质量体积比1:2混合,转染293FT细胞后10 h换含2%胎牛血清的维持液,培养48 h后收获上清,可高效获得流感假病毒AHop-H7N9pp;不同纯化方式获得的假病毒对感染H7N9的雪貂血清的中和抗体检测结果表明,假病毒纯化方法对中和抗体检测灵敏度的影响没有显著性差异;生物学特征分析显示,纯化的假病毒在电镜下具有典型的流感病毒形态,Western印迹能检测到HA和P24蛋白;多种流感疫苗小鼠血清中和试验结果表明该假病毒具有高度特异性,可用于H7N9中和抗体检测。结论:获得了包装有A/Anhui/1/2013(H7N9)HA与NA的假病毒,并确定了高效制备H7N9假病毒的各条件参数,为基于H7N9流感假病毒的中和抗体检测平台的建立奠定了基础。
[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，
本文编号：2491460
[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，
本文编号：2491460
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