GCRV宿主表面受体的鉴定及其介导病毒侵入途径的研究

发布时间：2018-05-14 12:28

本文选题：草鱼 + 草鱼呼肠孤病毒　；参考：《上海海洋大学》2016年博士论文

【摘要】：草鱼(Ctenopharyngodon idellus)是中国传统的养殖品种,养殖规模较大。草鱼出血病制约了我国草鱼产量的提升,该病是由草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV)感染引起的病毒性疾病。GCRV病毒为呼肠孤病毒科(Reoviridae)、水生呼肠孤病毒属(Aquareovirus),无囊膜,具有双链RNA结构,最外层衣壳蛋白由VP5和VP7组合形成。本研究通过鉴定GCRV病毒外衣壳蛋白与宿主受体的相互作用关系,阐释了GCRV吸附和进入宿主的机制,主要研究结果如下:1.GCRV利用Laminin receptor(Lam R)介导其识别吸附宿主细胞我们通过酵母双杂交实验,筛选草鱼cDNA文库中与GCRV外衣壳蛋白VP5或VP7相互作用的蛋白,发现GCRV-VP5能够与层黏连蛋白受体蛋白(Laminin receptor,LamR)在酵母杂交系统中发生相互作用,而VP7没有筛选到类似的受体蛋白。利用Race扩增技术我们获得了草鱼LamR的基因全长;激光共聚焦显微镜观察显示,GCRV病毒粒子吸附CIK细胞后,能够与细胞膜上的LamR发生共定位;原核表达系统纯化的LamR-gst与GCRV病毒粒子进行Dot-blot overlay表明,LamR-gst能够与GCRV发生特异性的吸附。利用真核转染系统表达GCRVVP5和GCRV-VP7 GFP融合蛋白与LamR-gst,进行GST Pull-Down和Dot-blot overlay等实验表明,LamR能够与GCRV-VP5发生相互作用,而与GCRV-VP7不能发生相互作用;RNAi敲降CIK细胞中的LamR后,吸附感染GCRV显示,LamR敲降后能够显著性的降低GCRV的吸附感染效率;制备LamR的多抗,封闭CIK细胞后发现,能够显著性的降低GCRV的感染效率;推测GCRV利用外层衣壳蛋白VP5来识别宿主细胞膜上的受体LamR,从而介导其吸附宿主细胞;2.EGCG((-)-epigallocatechin-3-gallate)能够拮抗GCRV感染宿主细胞基于现有研究显示,EGCG能够结合细胞膜上的LamR,体外等离子共振实验表明,EGCG和LamR具有显著的结合活性。我们尝试利用EGCG封闭宿主细胞膜上的LamR干扰GCRV的吸附途径,从而拮抗GCRV感染。实验结果表明,随着封闭CIK细胞的EGCG或GTE浓度的升高,GCRV吸附和感染的效率显著2性的降低。通过不同浓度EGCG和EGCG粗提物(Green tea,GTE)处理CIK细胞后,检测CIK细胞的生理活性,评价了EGCG和GTE的安全浓度。综上,EGCG和GTE能够有效的拮抗GCRV吸附和感染宿主细胞。3.GCRV依赖于Clathrin-dependent内吞途径进入CIK细胞本研究应用多种内吞途径抑制剂处理CIK细胞后,感染GCRV探讨其进入水平,研究了GCRV进入宿主细胞的机制。实验结果显示,GCRV感染CIK细胞能够被氯丙嗪(Chlorpromazine)显著性抑制,而Filipin III、Nystatin和LY294002对其没有干扰效果,推测其可能利用了Clathrin途径。氯喹(Chloroquine)和氯化铵(Ammonium chloride)预处理CIK细胞能够显著性的降低GCRV的感染进入效率,表明GCRV进入宿主细胞依赖于酸性的内吞途径。Dynasore抑制剂干扰CIK细胞中dynamin,实验表明GCRV需要依赖于Dynamin参与的内吞途径。共聚焦显微镜观察GCRV病毒粒子和Clathrin定位情况表明,GCRV病毒粒子能够与Clathrin蛋白共定位。综上,我们证实GCRV病毒粒子进入宿主细胞需要在低pH环境下,通过Dynamin蛋白的辅助依赖于clathrin介导的内吞途径完成。
[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.

【学位授予单位】：上海海洋大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S943

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