H5N1亞型禽流感病毒以巨胞飲作用侵入細(xì)胞的研究
發(fā)布時(shí)間：2018-10-30 10:07
【摘要】：禽流感(Avian Influenza)是由A型流感病毒(Influenza A Virus)引起的禽類傳染病。根據(jù)HA和NA的抗原性,禽流感病毒分為16個(gè)HA亞型和9個(gè)NA亞型。根據(jù)毒力差異可分為高致病性禽流感病毒、低致病性禽流感病毒。自1997年高致病性禽流感(HPAI)H5N1亞型爆發(fā)以來,該病毒造成禽類的大量死亡,并且能夠感染人類。如何有效地防治H5N1高致病性禽流感病毒成為研究的重點(diǎn)和熱點(diǎn)。為啟動(dòng)復(fù)制,病毒須將自身基因組運(yùn)輸?shù)剿拗骷?xì)胞內(nèi)。少數(shù)病毒與細(xì)胞膜直接融合將自身基因組釋放至細(xì)胞(例如HIV),而大部分病毒則通過內(nèi)吞作用進(jìn)入細(xì)胞。由于細(xì)胞存在多種內(nèi)吞途徑,每種病毒采用的具體的內(nèi)吞途徑也不盡相同。研究表明,流感病毒侵入細(xì)胞是通過HA蛋白與細(xì)胞表面的唾液酸受體結(jié)合,經(jīng)過網(wǎng)格蛋白和小窩蛋白介導(dǎo)的侵入途徑進(jìn)入細(xì)胞,而Dynamin蛋白是網(wǎng)格蛋白和小窩蛋白介導(dǎo)內(nèi)吞的關(guān)鍵蛋白。本研究為了研究病毒內(nèi)吞作用的侵入途徑,采用AIV A/duck/Guangxi/35/2001(DK/35)(H5N1)、rDK/35(Q226L/G228S)(H5N1)、rDK35/HA-A160T(H5N1)作為主要研究用毒株,加入Dynamin蛋白的抑制劑Dynasore,通過間接免疫熒光觀察病毒侵入情況。結(jié)果表明,在胎牛血清存在的情況下,流感病毒能夠采用不依賴于Dynamin的侵入方式,即流感病毒能夠采用不依賴于網(wǎng)格蛋白和小窩蛋白介導(dǎo)的侵入方式侵入細(xì)胞。本研究進(jìn)一步采用巨胞飲作用抑制劑5-(N-乙基-N-異丙基)阿米洛利EIPA,觀察病毒侵入情況,結(jié)果表明,巨胞飲作用在不依賴于網(wǎng)格蛋白和小窩蛋白介導(dǎo)的侵入體外培養(yǎng)的細(xì)胞中起到一定作用。此外,去除細(xì)胞表面的唾液酸受體,觀察病毒侵入情況。結(jié)果表明,唾液酸受體在病毒不依賴于Dynamin的侵入方式中起著一定作用。同樣,采用不同受體結(jié)合性病毒感染A549時(shí),巨胞飲作用同樣發(fā)揮一定作用。在Dynasore存在的情況下,采用DK/35感染DF1、MDCK,結(jié)果表明不同種源細(xì)胞中,巨胞飲在病毒侵入時(shí)發(fā)揮一定作用。本實(shí)驗(yàn)所驗(yàn)證的巨胞飲在流感病毒侵入細(xì)胞時(shí)可能發(fā)揮的作用,為流感病毒的感染機(jī)理的研究提供了實(shí)驗(yàn)依據(jù),對(duì)于后續(xù)的流感病毒的防控提供了一定的思路。
[Abstract]:Avian influenza (Avian Influenza) is a avian infectious disease caused by influenza A virus (Influenza A Virus). According to the antigenicity of HA and NA, avian influenza virus is divided into 16 HA subtypes and 9 NA subtypes. According to virulence difference can be divided into highly pathogenic avian influenza virus, low-pathogenic avian influenza virus. Since the outbreak of the highly pathogenic avian influenza (HPAI) H5N1 subtype in 1997, the virus has caused a large number of deaths in poultry and can infect humans. How to effectively prevent and cure H5N1 highly pathogenic avian influenza virus has become the focus and hotspot of research. To initiate replication, the virus must transport its own genome into the host cell. A few viruses are directly fused with cell membranes to release their genomes into cells (such as HIV), while most viruses enter cells through endocytosis. Due to the existence of multiple endocytosis pathways, the specific endocytosis pathways used by each virus are different. Studies have shown that influenza virus invades cells through a sialic acid receptor binding to the HA protein, and enters the cell via a griddle protein and nest protein mediated invasion pathway. Dynamin protein is the key protein of endocytosis mediated by grid protein and fossa protein. In this study, AIV A/duck/Guangxi/35/2001 (DK/35) (H5N1), rDK/35 (Q226L/G228S) (H5N1), rDK35/HA-A160T (H5N1) were used to study the invasion pathway of virus endocytosis. Dynasore, an inhibitor of Dynamin protein, was used to observe the invasion of the virus by indirect immunofluorescence. The results showed that in the presence of fetal bovine serum, influenza virus could invade the cells without Dynamin, that is, the influenza virus could invade the cells in a manner independent of grid protein and nest protein. In this study, 5- (N-ethyl-N-isopropyl) amiloride EIPA, was further used to observe the invasion of the virus. Megacyte plays a role in the invasion of cells independent of grid protein and fossa protein in vitro. In addition, the sialic acid receptor on the cell surface was removed and virus invasion was observed. The results showed that sialic acid receptors play a role in the viral invasion independent of Dynamin. Similarly, when A549 was infected with different receptor-binding viruses, giant cell drink also played a role. In the presence of Dynasore, the results of DF1,MDCK, infection with DK/35 showed that megakyne played a role in virus invasion in different provenance cells. The possible role of giant cell drink in the invasion of influenza virus provides experimental basis for the study of the infection mechanism of influenza virus and provides some ideas for the prevention and control of subsequent influenza virus.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65
，
本文編號(hào)：2299697
[Abstract]:Avian influenza (Avian Influenza) is a avian infectious disease caused by influenza A virus (Influenza A Virus). According to the antigenicity of HA and NA, avian influenza virus is divided into 16 HA subtypes and 9 NA subtypes. According to virulence difference can be divided into highly pathogenic avian influenza virus, low-pathogenic avian influenza virus. Since the outbreak of the highly pathogenic avian influenza (HPAI) H5N1 subtype in 1997, the virus has caused a large number of deaths in poultry and can infect humans. How to effectively prevent and cure H5N1 highly pathogenic avian influenza virus has become the focus and hotspot of research. To initiate replication, the virus must transport its own genome into the host cell. A few viruses are directly fused with cell membranes to release their genomes into cells (such as HIV), while most viruses enter cells through endocytosis. Due to the existence of multiple endocytosis pathways, the specific endocytosis pathways used by each virus are different. Studies have shown that influenza virus invades cells through a sialic acid receptor binding to the HA protein, and enters the cell via a griddle protein and nest protein mediated invasion pathway. Dynamin protein is the key protein of endocytosis mediated by grid protein and fossa protein. In this study, AIV A/duck/Guangxi/35/2001 (DK/35) (H5N1), rDK/35 (Q226L/G228S) (H5N1), rDK35/HA-A160T (H5N1) were used to study the invasion pathway of virus endocytosis. Dynasore, an inhibitor of Dynamin protein, was used to observe the invasion of the virus by indirect immunofluorescence. The results showed that in the presence of fetal bovine serum, influenza virus could invade the cells without Dynamin, that is, the influenza virus could invade the cells in a manner independent of grid protein and nest protein. In this study, 5- (N-ethyl-N-isopropyl) amiloride EIPA, was further used to observe the invasion of the virus. Megacyte plays a role in the invasion of cells independent of grid protein and fossa protein in vitro. In addition, the sialic acid receptor on the cell surface was removed and virus invasion was observed. The results showed that sialic acid receptors play a role in the viral invasion independent of Dynamin. Similarly, when A549 was infected with different receptor-binding viruses, giant cell drink also played a role. In the presence of Dynasore, the results of DF1,MDCK, infection with DK/35 showed that megakyne played a role in virus invasion in different provenance cells. The possible role of giant cell drink in the invasion of influenza virus provides experimental basis for the study of the infection mechanism of influenza virus and provides some ideas for the prevention and control of subsequent influenza virus.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65
，
本文編號(hào)：2299697
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