本文選題：反向遺傳技術(shù)　切入點：甲型流感病毒　出處：《昆明醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]甲型流感病毒的隨機(jī)重配和變異對人類可造成極大危害,特別是近年來頻發(fā)的高致病性禽流感H5型已廣泛存在于家禽種群中并具有傳染給人類及其他動物的可能性,呈現(xiàn)出不斷擴(kuò)散的趨勢。禽流感病毒A型中的H5業(yè)型,如H5N1,H5N2,H5N6,H5N8和H5N9在我國及其他亞洲國家和歐洲等地區(qū)的鳥類中均相繼被檢測到,這類病毒除導(dǎo)致禽流感外,其跨物種傳播特別是對人類的感染也越來越引起重視。雖然H5N9尚未報道類似于H5N1或H5N8的人類感染病例,但基于1966年的禽類爆發(fā)流行和近幾年散發(fā)病例報道的增多,提示了其作為家禽中主要毒株所帶來的重配和變異進(jìn)而引起跨物種傳播的潛在危險性,造成的抗原漂移使得阻止流感流行的藥物出現(xiàn)耐藥性現(xiàn)象,疫苗接種防控手段也疲于應(yīng)對。本研究嘗試?yán)梅聪蜻z傳學(xué)技術(shù)構(gòu)建H5N9重組流感病毒并對其感染性特性進(jìn)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,將其分別插入到pHW2000載體中,與攜帶有A/Puerto Rico/8/34(H1N1)的6個內(nèi)部基因(M,NS,NP,PA,PB1,PB2)的pHW2000重組質(zhì)粒一起轉(zhuǎn)染293T和MDCK混合細(xì)胞,拯救H5N9重組病毒。以細(xì)胞法觀察H5N9病毒的致病變效應(yīng),通過蛋白免疫印跡實驗和免疫熒光驗證HA和NA蛋白的表達(dá),血凝實驗驗證流感病毒HA蛋白的致血凝特性,透射電鏡觀察重組病毒的形態(tài)結(jié)構(gòu)。以滴鼻形式接種BALB/C小鼠進(jìn)行H5N9重組病毒感染特性研究。[結(jié)果]通過細(xì)胞病變、核酸測序、HA和NA蛋白免疫印跡實驗,免疫熒光等結(jié)果的分析,確定利用該反向遺傳學(xué)系統(tǒng)可以成功獲救H5N9病毒。并且經(jīng)過在細(xì)胞上連續(xù)傳代,測定病毒滴度,血凝效價及在小鼠的致病性實驗中,發(fā)現(xiàn)重組H5N9病毒在MDCK細(xì)胞上復(fù)制增殖能力低于相同方法獲救的H1N1病毒,也沒有引起小鼠有明顯的臨床癥狀。H5N9病毒感染后產(chǎn)生血凝抑制抗體的滴度為1:160,低于同種方法重組的H1N1病毒,其滴度為1:320。[結(jié)論]本實驗利用反向遺傳學(xué)技術(shù)成功構(gòu)建了 H5N9重組流感病毒,重組病毒HA、NA與親本病毒相應(yīng)的核酸序列一致,無基因變異,可以穩(wěn)定傳代。并通過上述實驗方法及研究,證明其在體外MDCK細(xì)胞內(nèi)的復(fù)制增殖能力和小鼠體內(nèi)致病力弱于同樣方法制備的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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R373

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