目的 为研究高致病性H5N8亚型(A/Astrakhan/3212/2020)禽流感病毒的结构,制备相应假病毒,并对其在细胞水平上的感染能力以及血清抗体的中和能力进行初步验证。方法 通过对H5N8亚型病毒的血凝素(HA)和神经氨酸酶(NA)的氨基酸序列分析,将目的HA和NA序列进行优化并提取相应质粒,采用慢病毒包装系统制备H5N8亚型假病毒;使用感染含有α-2,6唾液酸受体的293T-ST6GAL1细胞后的荧光素酶读值、血凝抑制试验、透射电镜以及血清中和试验对制备的假病毒进行验证。结果 成功制备H5N8亚型假病毒,其对293T-ST6GAL1细胞的感染力为103 TCID50/mL,浓缩50倍后血凝活性从16提高到128,透射电镜可观察到典型的流感病毒颗粒,中和试验显示3份H5N8阳性血清中和活性明显高于其他亚型阳性血清以及阴性血清。结论 成功制备高致病性H5N8亚型禽流感假病毒,为未来的疫苗开发和中和抗体筛选提供了重要的实验平台。
Abstract
Objective In order to study the structure of the highly pathogenic H5N8 subtype (A/Astrakhan/3212/2020) avian influenza virus,the corresponding pseudovirus was prepared and its infective ability at the cellular level,as well as the neutralizing ability of serum antibodies,were preliminarily verified. Methods By analyzing the amino acid sequences of the H5N8 subtype (A/Astrakhan/3212/2020) virus hemagglutinin (HA) and neuraminidase (NA),the target HA and NA sequences were optimized and the corresponding plasmid was extracted.Moreover,H5N8 subtype pseudovirus was prepared by a lentivirus packaging system.The fabricated pseudovirus was verified by luciferase readings,hemagglutination inhibition tests,transmission electron microscopy,and serum neutralization tests after infection with 293T-ST6GAL1 cells containing α-2,6 sialic acid receptors. Results H5N8 subtype pseudovirus was successfully prepared,and its infectivity to 293T-ST6GAL1 cells was 103 TCID50/mL.After being 50 times-concentrated,the hemagglutination activity of the pseudovirus increased from 16 to 128.Typical influenza virus particles could be observed by transmission electron microscopy.The neutralization test results showed that the neutralization activity of 3 H5N8 positive sera was significantly higher than that of other positive sera and negative sera. Conclusion This study successfully prepared the highly pathgenic H5N8 subtype avian influenza pseudovirus,which provides an important experimental platform for future vaccine development and screening.
关键词
H5N8亚型禽流感病毒 /
假病毒制备 /
血凝素 /
神经氨酸酶 /
中和试验
Key words
H5N8 subtype avian influenza virus /
Pseudovirus preparation /
Hemagglutinin /
Neuraminidase /
Neutralization test
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基金
安徽省卫生健康委科研项目(AHWJ2021a030);国家重点研发计划项目(2022YFE0110100);传染病诊治国家重点实验室开放基金资助项目(SKLID2021KF04)
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