發(fā)布時間：2018-05-14 12:28

  本文選題：草魚 + 草魚呼腸孤病毒　； 參考：《上海海洋大學(xué)》2016年博士論文

【摘要】：草魚(Ctenopharyngodon idellus)是中國傳統(tǒng)的養(yǎng)殖品種,養(yǎng)殖規(guī)模較大。草魚出血病制約了我國草魚產(chǎn)量的提升,該病是由草魚呼腸孤病毒(Grass Carp Reovirus,GCRV)感染引起的病毒性疾病。GCRV病毒為呼腸孤病毒科(Reoviridae)、水生呼腸孤病毒屬(Aquareovirus),無囊膜,具有雙鏈RNA結(jié)構(gòu),最外層衣殼蛋白由VP5和VP7組合形成。本研究通過鑒定GCRV病毒外衣殼蛋白與宿主受體的相互作用關(guān)系,闡釋了GCRV吸附和進(jìn)入宿主的機(jī)制,主要研究結(jié)果如下:1.GCRV利用Laminin receptor(Lam R)介導(dǎo)其識別吸附宿主細(xì)胞我們通過酵母雙雜交實(shí)驗(yàn),篩選草魚cDNA文庫中與GCRV外衣殼蛋白VP5或VP7相互作用的蛋白,發(fā)現(xiàn)GCRV-VP5能夠與層黏連蛋白受體蛋白(Laminin receptor,LamR)在酵母雜交系統(tǒng)中發(fā)生相互作用,而VP7沒有篩選到類似的受體蛋白。利用Race擴(kuò)增技術(shù)我們獲得了草魚LamR的基因全長;激光共聚焦顯微鏡觀察顯示,GCRV病毒粒子吸附CIK細(xì)胞后,能夠與細(xì)胞膜上的LamR發(fā)生共定位;原核表達(dá)系統(tǒng)純化的LamR-gst與GCRV病毒粒子進(jìn)行Dot-blot overlay表明,LamR-gst能夠與GCRV發(fā)生特異性的吸附。利用真核轉(zhuǎn)染系統(tǒng)表達(dá)GCRVVP5和GCRV-VP7 GFP融合蛋白與LamR-gst,進(jìn)行GST Pull-Down和Dot-blot overlay等實(shí)驗(yàn)表明,LamR能夠與GCRV-VP5發(fā)生相互作用,而與GCRV-VP7不能發(fā)生相互作用;RNAi敲降CIK細(xì)胞中的LamR后,吸附感染GCRV顯示,LamR敲降后能夠顯著性的降低GCRV的吸附感染效率;制備LamR的多抗,封閉CIK細(xì)胞后發(fā)現(xiàn),能夠顯著性的降低GCRV的感染效率;推測GCRV利用外層衣殼蛋白VP5來識別宿主細(xì)胞膜上的受體LamR,從而介導(dǎo)其吸附宿主細(xì)胞;2.EGCG((-)-epigallocatechin-3-gallate)能夠拮抗GCRV感染宿主細(xì)胞基于現(xiàn)有研究顯示,EGCG能夠結(jié)合細(xì)胞膜上的LamR,體外等離子共振實(shí)驗(yàn)表明,EGCG和LamR具有顯著的結(jié)合活性。我們嘗試?yán)�用EGCG封閉宿主細(xì)胞膜上的LamR干擾GCRV的吸附途徑,從而拮抗GCRV感染。實(shí)驗(yàn)結(jié)果表明,隨著封閉CIK細(xì)胞的EGCG或GTE濃度的升高,GCRV吸附和感染的效率顯著2性的降低。通過不同濃度EGCG和EGCG粗提物(Green tea,GTE)處理CIK細(xì)胞后,檢測CIK細(xì)胞的生理活性,評價了EGCG和GTE的安全濃度。綜上,EGCG和GTE能夠有效的拮抗GCRV吸附和感染宿主細(xì)胞。3.GCRV依賴于Clathrin-dependent內(nèi)吞途徑進(jìn)入CIK細(xì)胞本研究應(yīng)用多種內(nèi)吞途徑抑制劑處理CIK細(xì)胞后,感染GCRV探討其進(jìn)入水平,研究了GCRV進(jìn)入宿主細(xì)胞的機(jī)制。實(shí)驗(yàn)結(jié)果顯示,GCRV感染CIK細(xì)胞能夠被氯丙嗪(Chlorpromazine)顯著性抑制,而Filipin III、Nystatin和LY294002對其沒有干擾效果,推測其可能利用了Clathrin途徑。氯喹(Chloroquine)和氯化銨(Ammonium chloride)預(yù)處理CIK細(xì)胞能夠顯著性的降低GCRV的感染進(jìn)入效率,表明GCRV進(jìn)入宿主細(xì)胞依賴于酸性的內(nèi)吞途徑。Dynasore抑制劑干擾CIK細(xì)胞中dynamin,實(shí)驗(yàn)表明GCRV需要依賴于Dynamin參與的內(nèi)吞途徑。共聚焦顯微鏡觀察GCRV病毒粒子和Clathrin定位情況表明,GCRV病毒粒子能夠與Clathrin蛋白共定位。綜上,我們證實(shí)GCRV病毒粒子進(jìn)入宿主細(xì)胞需要在低pH環(huán)境下,通過Dynamin蛋白的輔助依賴于clathrin介導(dǎo)的內(nèi)吞途徑完成。
[Abstract]:Grass carp (Ctenopharyngodon idellus) is a traditional Chinese breed and has a large scale of culture. Grass carp hemorrhage restricts the improvement of Chinese grass carp production. The disease is a viral disease caused by the Grass Carp Reovirus (GCRV) infection, the.GCRV virus is called the reovirus family (Reoviridae) and the aquatic reovirus (Aquare). Ovirus), without the capsule, with double stranded RNA structure, the outer shell protein is formed by the combination of VP5 and VP7. This study explains the mechanism of GCRV adsorption and entry to host by identifying the interaction relationship between the GCRV virus outer shell protein and the host receptor. The main results are as follows: 1.GCRV uses Laminin receptor (Lam R) to mediate the identification of the adsorption lodging. We screened the interaction between the grass carp cDNA library and the GCRV coat protein VP5 or VP7 by the yeast two hybrid experiment. It was found that GCRV-VP5 could interact with the laminin receptor protein (Laminin receptor, LamR) in the yeast hybrid system, and VP7 did not screen a similar receptor protein. We obtained the full length of the gene of the grass carp LamR; the laser confocal microscopy showed that the GCRV virus particles adsorbed the CIK cells and were able to co localize with the LamR on the cell membrane. The Dot-blot overlay of the LamR-gst and GCRV virus particles purified by the prokaryotic expression system showed that LamR-gst could be adsorbed specifically with GCRV. The nuclear transfection system expresses GCRVVP5 and GCRV-VP7 GFP fusion protein and LamR-gst, and the experiments of GST Pull-Down and Dot-blot overlay show that LamR can interact with GCRV-VP5 and can not interact with GCRV-VP7. The efficiency of adsorption of infection, LamR polyclonal antibody, closed CIK cells, can significantly reduce the infection efficiency of GCRV; it is speculated that GCRV uses the outer capsid protein VP5 to identify the receptor LamR on the host cell membrane and mediate its adsorption of host cells; 2.EGCG ((-) -epigallocatechin-3-gallate) can antagonize the host cell base of GCRV infection. The existing research shows that EGCG can combine with LamR on the cell membrane. In vitro plasma resonance experiments show that EGCG and LamR have significant binding activity. We try to block GCRV infection by EGCG blocking the LamR interference on the cell membrane of the host cell, thus antagonizing GCRV infection. The experimental results show that the EGCG or GTE concentration of the closed CIK cells increases with the increase of the concentration of EGCG or GTE. The efficiency of GCRV adsorption and infection was significantly reduced. The physiological activity of CIK cells was detected by the treatment of CIK cells with different concentrations of EGCG and EGCG (Green tea, GTE), and the safe concentration of EGCG and GTE was evaluated. The approach entered CIK cell based on the application of a variety of endocytic pathway inhibitors to treat CIK cells. Infection GCRV explored its entry level and studied the mechanism of GCRV entering the host cell. The experimental results showed that GCRV infected CIK cells could be significantly inhibited by chlorpromazine (Chlorpromazine), and Filipin III, Nystatin, and LY294002 had no interference effect. It is presumed that it may use the Clathrin pathway. The pretreatment of CIK cells with chloroquine (Chloroquine) and ammonium chloride (Ammonium chloride) can significantly reduce the entry efficiency of GCRV infection, indicating that GCRV entering the host cell depends on the acidic endocytosis pathway that.Dynasore inhibitors interfere with dynamin in CIK cells, and the experiment indicates that GCRV needs to be dependent on Dynam. In participates in endocytosis pathway. Confocal microscope observation of GCRV virus particles and Clathrin localization shows that GCRV virus particles can co localize with Clathrin protein. To sum up, we confirm that GCRV virus particles enter into host cells in low pH environment and rely on clathrin mediated endocytosis through the aid of Dynamin protein.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S943

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