本文選題：雞傳染性支氣管炎病毒 + 雞胚腎細胞　； 參考：《東北農業(yè)大學》2017年碩士論文

【摘要】：雞傳染性支氣管炎(Infectious bronchitis,IB)是由雞傳染性支氣管炎病毒(Infectious bronchitis virus,IBV)引起的一種急性、高度接觸性傳染病,被認為是危害全球養(yǎng)禽業(yè)的主要病毒性傳染病之一。雞胚腎(Chicken embryo kidney,CEK)細胞源于IBV易感宿主,相較于其他細胞系更貼近于IBV的感染環(huán)境,且對IBV具有極高的敏感性,接種后能觀察到明顯的細胞病變,CEK已成為研究IBV感染細胞致病過程及其機制的適宜宿主細胞模型。脂筏是質膜中富含膽固醇和鞘脂的微結構域,具有介導病毒感染、信號轉導、蛋白分選等生物學功能。膽固醇作為脂筏的重要組分,可維持脂筏的穩(wěn)定結構并參與脂筏功能的發(fā)揮。研究表明,膽固醇在某些病毒感染細胞時扮演著非常重要的作用,包括病毒的進入、吸附和釋放過程,但膽固醇在IBV感染過程中的作用還并未有報道。本實驗分別研究了細胞膜和病毒囊膜膽固醇在IBV M41株感染CEK細胞過程中的作用。首先檢測了膽固醇萃取劑甲基-β-環(huán)糊精(MβCD)對CEK細胞活性的影響,結果發(fā)現(xiàn),當MβCD濃度小于10 m M時,不會對CEK細胞活性造成明顯影響,所以本實驗中所用的MβCD最大濃度為10 m M。同時進行膽固醇含量測定,當MβCD濃度為10 m M時,細胞膜膽固醇含量下降至37%,補充外源膽固醇后,膽固醇濃度會得到相應恢復,當充補膽固醇濃度為200μM時,膽固醇含量恢復至接近MβCD處理前。MβCD還能有效去除IBV囊膜膽固醇,用10m M MβCD處理病毒后,病毒囊膜膽固醇含量下降至約66%。本實驗利用Real Time PCR、病毒滴度試驗和Western Blot方法檢測了細胞膜膽固醇在IBV M41株感染CEK細胞不同過程中的作用。結果表明,加入不同濃度MβCD去除細胞膜膽固醇,會以劑量依賴性的方式抑制IBV的進入,病毒感染力和IBV N蛋白的表達量也會梯度下降。為進一步確定該抑制作用是由于去除膽固醇引起的,用10 mM MβCD去除細胞膜膽固醇后再補充外源膽固醇,結果顯示,隨著補充膽固醇濃度的增加,IBV感染力和蛋白表達水平均得到相應恢復,說明去除膽固醇可抑制IBV進入CEK細胞,且該抑制作用是可逆的。本實驗還研究了膽固醇在IBV吸附到CEK細胞和從CEK細胞釋放過程中的作用,結果顯示,隨著MβCD濃度增大,IBV的吸附和釋放過程均受到劑量依賴性的抑制。同時,將CEK細胞轉染GPI-pEGFP后感染IBV,結果顯示IBV與GPI錨定蛋白可共定位在同一個CEK細胞膜上,說明IBV可定位到細胞膜脂筏。病毒囊膜膽固醇在很多病毒進入細胞的過程中也扮演重要的作用。本實驗將不同濃度MβCD處理后的IBV M41株感染CEK細胞,結果發(fā)現(xiàn)IBV感染力無明顯趨勢性變化,IBV N蛋白表達量也無顯著性變化,證明病毒囊膜膽固醇并不是IBV進入CEK細胞所必須的。綜上所述,IBV感染CEK細胞過程中需要細胞膜膽固醇參與,而不需要病毒囊膜膽固醇。
[Abstract]:Infectious bronchitis (IBV) is an acute and highly contagious disease caused by infectious bronchitis virus (IBV). It is considered to be one of the major viral infectious diseases in poultry industry worldwide. Chicken embryo kidney1 cells originate from susceptible hosts of IBV, and are more close to IBV infection environment than other cell lines, and highly sensitive to IBV. After inoculation, it was observed that the obvious cytopathic changes had become a suitable host cell model for studying the pathogenetic process and mechanism of IBV infection. Lipid rafts are microdomains rich in cholesterol and sphingolipid in plasma membrane, which can mediate virus infection, signal transduction, protein sorting and so on. Cholesterol, as an important component of lipid raft, can maintain the stable structure of lipid raft and play an important role in the function of lipid raft. Studies have shown that cholesterol plays a very important role in the process of virus infection, including virus entry, adsorption and release, but the role of cholesterol in the process of IBV infection has not been reported. The effects of membrane and viral envelope cholesterol on the infection of IBV M41 strain CEK cells were studied in this experiment. The effect of cholesterol extractant methyl 尾 -cyclodextrin M 尾 CD on the activity of CEK cells was investigated. The results showed that when the concentration of M 尾 CD was less than 10 mm, the activity of CEK cells would not be significantly affected. Therefore, the maximum concentration of M 尾 CD in this experiment was 10 mm. At the same time, cholesterol content was measured. When the concentration of M 尾 CD was 10 mm, the cholesterol content of cell membrane decreased to 37. After supplementation of exogenous cholesterol, the cholesterol concentration recovered accordingly, and when the filling cholesterol concentration was 200 渭 M, the cholesterol content of the cell membrane decreased to 37 渭 M. Cholesterol content returned to close to that before treatment with M 尾 CD, and the cholesterol of IBV capsule membrane was removed effectively. After treated with 10 mm M 尾 CD, the cholesterol content of virus capsule membrane decreased to about 66. In this study, Real Time PCR, virus titer test and Western Blot were used to detect the effect of cell membrane cholesterol on the different processes of IBV M41 cell infection in CEK cells. The results showed that adding different concentrations of M 尾 CD to remove membrane cholesterol inhibited the entry of IBV in a dose-dependent manner, and the viral infectivity and the expression of IBV N protein decreased in a gradient. In order to further confirm that the inhibition was caused by cholesterol removal, 10 mm M 尾 CD was used to remove membrane cholesterol and then replenish exogenous cholesterol. With the increase of cholesterol supplementation, the infectivity and protein expression level of IBV recovered accordingly, which indicated that removal of cholesterol could inhibit the entry of IBV into CEK cells, and the inhibition effect was reversible. The effect of cholesterol on the adsorption of IBV to CEK cells and its release from CEK cells was also studied. The results showed that the adsorption and release of IBV were inhibited in a dose-dependent manner with the increase of M 尾 CD concentration. At the same time, CEK cells were transfected with GPI-pEGFP. The results showed that IBV and GPI anchoring proteins could be co-located on the same CEK cell membrane, indicating that IBV could be located on the membrane lipid raft. Viral envelope cholesterol also plays an important role in the entry of many viruses into cells. In this study, CEK cells were infected with IBV M41 strain treated with different concentrations of M 尾 CD. The results showed that there was no obvious trend change in IBV infectivity and no significant change in the expression of IBV N protein, which proved that viral envelope cholesterol was not necessary for the entry of IBV into CEK cells. In conclusion, membrane cholesterol is required in the process of CEK cell infection, but not the viral envelope cholesterol.
【學位授予單位】：東北農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.31

【參考文獻】

相關期刊論文 前10條

1 崔麗萍;;雞傳染性支氣管炎的防制[J];養(yǎng)殖與飼料;2017年02期

2 張梅;;雞傳染性支氣管炎的診斷及治療[J];中國畜牧獸醫(yī)文摘;2016年11期

3 肖福生;施小生;吳傳東;王海寶;鄒小金;施從福;;雞傳染性支氣管炎的防制[J];養(yǎng)殖與飼料;2016年09期

4 周生;姜逸;唐夢君;俞燕;程旭;高明燕;沈欣悅;李建梅;;雞傳染性支氣管炎病毒毒力變異的分子機制研究進展[J];中國家禽;2016年07期

5 孫曉麗;朱毅;;細胞膜脂筏結構蛋白質組與血管內皮細胞功能調控[J];天津醫(yī)藥;2015年08期

6 馬坤;王玉娟;王俊峰;;流感病毒在脂筏上組裝與出芽[J];生物化學與生物物理進展;2015年06期

7 尤永君;張國中;劉月煥;王友;梁武;劉興彩;沈元;;2010—2012年中國部分地區(qū)雞傳染性支氣管炎流行病學調查[J];畜牧獸醫(yī)學報;2015年02期

8 賀會利;魏小兵;歐長波;徐敏;閆藝婷;劉明成;勾肖晶;劉興友;;雞傳染性支氣管炎病毒研究進展[J];動物醫(yī)學進展;2014年07期

9 李廣興;王新;潘龍;杜威威;黃小丹;孫剛;楊貴君;任曉峰;;IBV HH06株N基因原核表達及間接ELISA方法的建立[J];東北農業(yè)大學學報;2014年05期

10 鄧玄威;趙越;;雞傳染性支氣管炎的防制[J];養(yǎng)殖技術顧問;2014年04期

相關博士學位論文 前2條

1 朱勇U，

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