H5N9重组流感病毒株的构建及感染特性分析

发布时间：2018-03-18 05:17

本文选题：反向遗传技术　切入点：甲型流感病毒　出处：《昆明医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：[目的]甲型流感病毒的随机重配和变异对人类可造成极大危害,特别是近年来频发的高致病性禽流感H5型已广泛存在于家禽种群中并具有传染给人类及其他动物的可能性,呈现出不断扩散的趋势。禽流感病毒A型中的H5业型,如H5N1,H5N2,H5N6,H5N8和H5N9在我国及其他亚洲国家和欧洲等地区的鸟类中均相继被检测到,这类病毒除导致禽流感外,其跨物种传播特别是对人类的感染也越来越引起重视。虽然H5N9尚未报道类似于H5N1或H5N8的人类感染病例,但基于1966年的禽类爆发流行和近几年散发病例报道的增多,提示了其作为家禽中主要毒株所带来的重配和变异进而引起跨物种传播的潜在危险性,造成的抗原漂移使得阻止流感流行的药物出现耐药性现象,疫苗接种防控手段也疲于应对。本研究尝试利用反向遗传学技术构建H5N9重组流感病毒并对其感染性特性进行分析。[方法]全基因合成A/Beijing/01/2003(H5N1)禽流感病毒HA基因片段和A/Zhejiang/DTID-ZJU10/2013(H7N9)禽流感病毒NA基因片段,将其分别插入到pHW2000载体中,与携带有A/Puerto Rico/8/34(H1N1)的6个内部基因(M,NS,NP,PA,PB1,PB2)的pHW2000重组质粒一起转染293T和MDCK混合细胞,拯救H5N9重组病毒。以细胞法观察H5N9病毒的致病变效应,通过蛋白免疫印迹实验和免疫荧光验证HA和NA蛋白的表达,血凝实验验证流感病毒HA蛋白的致血凝特性,透射电镜观察重组病毒的形态结构。以滴鼻形式接种BALB/C小鼠进行H5N9重组病毒感染特性研究。[结果]通过细胞病变、核酸测序、HA和NA蛋白免疫印迹实验,免疫荧光等结果的分析,确定利用该反向遗传学系统可以成功获救H5N9病毒。并且经过在细胞上连续传代,测定病毒滴度,血凝效价及在小鼠的致病性实验中,发现重组H5N9病毒在MDCK细胞上复制增殖能力低于相同方法获救的H1N1病毒,也没有引起小鼠有明显的临床症状。H5N9病毒感染后产生血凝抑制抗体的滴度为1:160,低于同种方法重组的H1N1病毒,其滴度为1:320。[结论]本实验利用反向遗传学技术成功构建了 H5N9重组流感病毒,重组病毒HA、NA与亲本病毒相应的核酸序列一致,无基因变异,可以稳定传代。并通过上述实验方法及研究,证明其在体外MDCK细胞内的复制增殖能力和小鼠体内致病力弱于同样方法制备的H1N1。
[Abstract]:[objective] the random reassortment and mutation of influenza A virus can cause great harm to human beings, especially the highly pathogenic avian influenza type H5, which has been widespread in poultry populations in recent years and has the potential to infect humans and other animals. The H5 industry of avian influenza virus A, such as H5N1, H5N1, H5N1, H5, and H5N1 9, has been detected in birds in China, other Asian countries and Europe. In addition to causing avian influenza, Although H5N1 N9 has not yet reported cases of human infection similar to H5N1 or H5N1, it is based on an outbreak of avian epidemics in 1966 and an increase in sporadic cases in recent years, It is suggested that the reassortment and variation of the major strains in poultry may lead to the potential risk of cross-species transmission, resulting in antigen drift leading to resistance to drugs that prevent influenza epidemics. Vaccination and control measures are also struggling. In this study, reverse genetics was used to construct H5N1 N9 recombinant influenza virus and to analyze its infectious properties. [methods] A / Beijing / 01 / 2003 H 5N 1) avian influenza virus HA gene fragment was synthesized and its infectious properties were analyzed. [methods] A / Beijing / 01 / 2003 H 5N 1) avian influenza virus HA gene fragment and. A / Zhejiang / DTID-ZJU 10 / 2013 H7N9) A gene fragment of avian influenza virus, They were inserted into the pHW2000 vector and transfected into 293T and MDCK mixed cells with six pHW2000 recombinant plasmids carrying A / Puerto Rico / 8 / 34 H1 / N1). The pathogenicity of the H5N1 N9 virus was observed by cell method. The expression of HA and na protein was verified by Western blot and immunofluorescence. The hemagglutination characteristics of HA protein of influenza virus were tested by hemagglutination assay. Transmission electron microscope (TEM) was used to observe the morphology and structure of recombinant virus. The infection characteristics of H5N1 N9 virus were studied by nasal drip inoculation of BALB/C mice. [results] by cytopathic acid, nucleic acid sequencing, HA and na Western blot assay, immunofluorescence analysis, etc. Determined that the reverse genetic system could be used to successfully save the H5N1 N9 virus. After successive passage on cells, the titer of the virus, hemagglutination titer and pathogenicity in mice were determined. It was found that the replication and proliferation ability of recombinant H5N1 N9 virus in MDCK cells was lower than that of H1N1 virus rescued by the same method. Nor did it cause significant clinical symptoms in mice. The titer of hemagglutination suppressor antibody after infection was 1: 160, lower than that of H1N1 virus recombined by the same method. [conclusion] the recombinant influenza virus H5N1 N9 was successfully constructed by reverse genetics technique. The nucleic acid sequence of the recombinant virus HAN9 was consistent with that of the parent virus, and there was no genetic variation. It was proved that the replication and proliferation of MDCK cells in vitro and the pathogenicity of mice in vivo were weaker than those prepared by the same method.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

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