SPF鸭Mx基因两个可变剪接转录本编码产物抗RNA病毒的差异研究

Mx proteins is one of the IFN-induced antiviral proteins, which made an inbred mouse strain (A2G) that showed an extraordinary high degree of resistance against infection with influenza A viruses, so it were studied extensively. Mx genes were found in most vertebrate and invertebrate species, and have several alleles or isoforms with different antiviral activity which depended on the polymorphism and location in the cell. The duck Mx gene was first cloned by Bazzigher in 1993, and it was no anti- influenza A viruses came from Chicken and Turkey expressed by mouse and chicken cells. But the duck Mx gene was polymorphism and Mx mRNA was increased after infected by some subtype AIV, and the level of mRNA was related with the AIV loading in following studies. .This study aim to compare the different anti-RNA virus activity of Mx gene two alternatively spliced transcripts in SPF duck, based on the deep analysis of the questions and situation of duck Mx gene studies of past. The spatial and temporal difference will be assessed in Mx gene transcription, protein expression and virus loading in vivo and in vitro after the duck or the DEF infected with several RNA virus, and to investigate the relationship of them. Meanwhile the duck Mx gene antiviral activity will be conformed in vitro by siRNA and overexpression. The result of this study will to answer whether the duck Mx protein have the antiviral activity scientifically, systematically and objectively again.

Mx蛋白是ISGs表达产物之一，具有抗多种病毒活性。Mx基因广泛分布于多种脊椎和非脊椎动物体内，存在数量不等的等位基因或亚型，其抗病毒活性受基因多态性、细胞定位等因素的影响。1993年Bazzigher首次克隆鸭Mx基因，并证实鼠源及鸡源细胞表达的鸭Mx蛋白无抗流感病毒的活性，但后续研究表明鸭Mx基因不仅存在多态性，且在某些亚型流感病毒刺激后其转录水平显著增加，与病毒载量呈现正相关。许多科学事实都提示应该重新定义鸭Mx蛋白的抗病毒活性。本项目拟开展SPF鸭Mx基因两个可变剪接转录本编码产物抗RNA病毒的差异研究。研究的主要内容：体内、外感染不同RNA病毒后评价SPF鸭Mx基因两个转录本转录、蛋白表达及病毒载量的时空差异，并探讨三者的相关性，同时利用siRNA和DEF过表达，体外验证鸭Mx蛋白的抗病毒活性，从而科学的、系统的、客观的重新回答鸭Mx蛋白是否具有抗病毒活性这一悬而未解的问题。

Mx基因广泛分布于多种脊椎和非脊椎动物体内，Mx蛋白是ISGs表达产物之一，具有抗多种病毒活性。而鸭Mx蛋白是否具有抗病毒活性尚未定论。本项目克隆了鸭Mx基因，在此基础上表达了其截短蛋白制备相应的多克隆抗体和单克隆抗体，结果表明制备的单克隆抗体可特异性识别真核表达及感染组织中表达的鸭Mx蛋白，利用该单克隆抗体建立检测鸭Mx蛋白的western blot方法，同时建立了检测VSV和DHAV-3病毒载量的q-PCR方法。转染携带鸭Mx基因的慢病毒转移载体，体外研究鸭Mx抗VSV的抗病毒活性，同时分别用DHAV-3感染SPF鸭胚及SPF雏鸭，体内研究鸭Mx抗DHAV-3的抗病毒活性。体外过表达鸭Mx 12h、24h、36h后在感染VSV，在不同检测点VSV病毒载量均低于接毒组，且在接毒后的9h、12h、24h和36h的病毒载量均显著低于接毒组。体内DHAV-3感染研究表明雏鸭的Mx转录水平显著高于鸭胚转录水平，且均以肝脏组织鸭Mx转录水平最高，依次是肠、脑、心、肌肉组织，且肝脏中的病毒载量与鸭Mx的转录水平成反比。总之，无论是体外还是体外研究均表明鸭Mx蛋白起到了抑制VSV及DHAV-3繁殖的作用，其鸭Mx转录水平决定了蛋白表达水平，蛋白表达水平决定了其抗病毒活性，抑制病毒的作用与鸭Mx蛋白的表达量呈正相关，即随着鸭Mx转录及蛋白表达量的增加其抑制VSV及DHAV-3的活性也在加强。