DHAV-1和DHAV-3感染SPF绍鸭胚Mx基因表达差异的研究

发布时间：2018-07-29 10:23

【摘要】：干扰素是脊椎动物机体抵抗病毒感染的第一道防线,作为I-IFN的指示标志,抗黏液病毒基因(myxovirus-resistant,Mx)具有较高的研究价值。目前关于鸭Mx的研究较少,本研究初步揭示了DHAV-1/-3感染鸭胚后,鸭胚部分组织器官中Mx与鸭IFN-α及相应病毒载量之间的关系,为深入研究鸭的抗病毒免疫机制提供必要的理论基础。本研究以感染DHAV-1的绍鸭肾组织的cDNA为模板,设计特异性引物,通过PCR扩增获得绍鸭Mx基因的CDs,并将其1435-2148bp亚克隆至pET-30a(+)原核表达载体中,在大肠杆菌表达系统中进行表达,获得重组鸭Mx蛋白(r Dup ET-30a-Mx),蛋白大小约为35k Da,利用Ni-NTA亲和层析获得纯化的目的蛋白。以纯化的rDupET-30a-Mx作为免疫原,免疫新西兰大白兔,制备了兔抗鸭Mx蛋白多克隆抗体,经I-ELISA检测,抗体效价在1:102400以上。以重组蛋白为检测抗原的Western blot结果显示,制备的多抗能识别成年鸭和鸭胚中的Mx蛋白。分别以10~3ELD_(50)的DHAV-1和DHAV-3接于15日龄SPF鸭胚上,在接毒后3h、6h、12h、24h、48h取其心、肝、胃、肠、肌肉,q PCR检测各组织中Mx、IFN-α及DHAV-1和DHAV-3的m RNA的相对表达量,Western blot检测Mx蛋白的表达水平。结果显示,在心、肝、胃、肠、肌肉中,Mx与IFN-α的mRNA在3-12h变化趋势基本一致;在心、肝、胃中,随着Mx m RNA的上升,相应的病毒载量有所下降。
[Abstract]:Interferon is the first line of defense against virus infection in vertebrates. As an indicator of I-IFN, anti-myxovirus-resistantMx gene is of great value. At present, there are few studies on duck MX. This study preliminarily revealed the relationship between MX and duck IFN- 伪 and the corresponding viral load in some tissues and organs of duck embryo after DHAV-1/-3 infection, which provides a necessary theoretical basis for further study of duck anti-virus immune mechanism. In this study, using the cDNA of Shao duck kidney tissue infected with DHAV-1 as a template, specific primers were designed to amplify the Mx gene of Shao duck by PCR amplification, and its 1435-2148bp was subcloned into the prokaryotic expression vector pET-30a () and expressed in Escherichia coli expression system. The recombinant duck Mx protein (r Dup ET-30a-Mx) was obtained. The protein size was about 35 kDa. The purified protein was purified by Ni-NTA affinity chromatography. The polyclonal antibody against duck Mx protein was prepared by immunizing New Zealand white rabbits with purified rDupET-30a-Mx. The antibody titer was over 1: 102400 by I-ELISA. The Western blot results using recombinant protein as antigen showed that the polyclonal antibody could recognize MX protein in adult duck and duck embryo. DHAV-1 and DHAV-3 of 10x3ELD50 were inoculated on 15-day-old SPF duck embryos respectively. The expression of MxtIFN- 伪 and DHAV-1 and DHAV-3 mRNA in tissues were detected by Western blot after inoculation for 48 h, liver, stomach, intestine, muscle, liver, stomach and intestine were collected at 3 h, 6 h, 12 h and 24 h after inoculation, and the expression level of MX protein was detected by Western blot. The results showed that the expression of MxtIFN- 伪 and DHAV-1 and DHAV-3 were detected by Western blot. The results showed that the mRNA of MX and IFN- 伪 in heart, liver, stomach, intestine and muscle were basically the same in 3-12 h, while in heart, liver and stomach, the corresponding viral load decreased with the increase of Mx m RNA.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.32

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