鸭Toll样受体3基因克隆及功能研究

发布时间：2018-04-16 02:06

本文选题：北京鸭 + TLR3　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：目的:先天性免疫是机体感知并体抵抗病原的第一道防线,Toll样受体(Toll-like receptor,TLR)是模式识别受体的重要组成部分,能够感知病原体相关模式分子,启动天然免疫应答。目前关于鸭Toll样受体3(Toll-like receptor 3,TLR3)的结构及功能了解不多,本研究从克隆北京鸭TLR3基因出发,利用生物信息学技术预测其结构及功能,制备鸭TLR3多克隆抗体和建立TLR3荧光定量PCR方法,利用上述技术评估鸭内源性TLR3抗病毒功能。方法:(1)采用RT-PCR技术从北京鸭外周血单个核细胞中克隆出北京鸭TLR3,命名为duTLR3。利用生物信息学技术预测其结构及功能;(2)采用PCR方法扩增鸭TLR3基因胞外区,将其克隆至原核表达载体pET-32a(+)中,转化大肠杆菌BL21(DE3)菌株,IPTG诱导,表达重组TLR3蛋白,并采用从包涵体中纯化重组蛋白。PCR扩增得到972 bp的TLR3基因胞外区,用纯化的重组TLR3蛋白免疫试验兔,制备TLR3的多克隆抗体,用Western blot和ELISA检测其特异性及效价;(3)根据鸭TLR3的基因序列保守区设计特异性引物,以鸭β肌动蛋白mRNA为内参,建立一种检测鸭Toll样受体3 mRNA表达水平的荧光定量PCR方法;(4)采用上述已建立的方法在细胞和组织水平检测TLR3的抗病毒功能。结果:(1)克隆了北京鸭TLR3基因,预测分析了duTLR3具有典型的TLR家族结构特征;(2)成功表达了TLR3基因胞外区,并制备了特异性良好的多克隆抗体;(3)定量检测方法的标准曲线在1×102~1×108 copies/μL范围内,具有良好的线性关系,相关系数和扩增效率均在0.9以上。熔解曲线显示,PCR产物为单峰,具有高度扩增特异性。重复性结果表明,该方法组内、组间变异系数均小于3%,最低检测浓度达100copies/μL。TLR3在鸭组织器官中的表达和分布情况检测结果表明,TLR3在鸭子的各种组织或器官中均有分布,其中以气管的表达水平最高,而在脾脏的表达水平最低;(4)DEF和组织水平检测发现,TLR3及其下游细胞因子表达与DRV(Duck reovirus)滴度呈正相关。结论:初步证实鸭TLR3与DRV存在相互作用,通过下游信号分子和抗病毒蛋白发挥其抗病毒功能。
[Abstract]:Aim: innate immunity is the first line of defense against pathogens, Toll-like receptor (TLRR) is an important part of pattern recognition receptor, which can sense pathogen-related model molecules and initiate innate immune response.At present, little is known about the structure and function of duck Toll like receptor 3(Toll-like receptor 3 (TLR3). Based on the cloning of TLR3 gene of Peking duck, this study used bioinformatics technology to predict its structure and function, to prepare duck TLR3 polyclonal antibody and to establish the method of TLR3 fluorescence quantitative PCR.To evaluate the antiviral function of duck endogenous TLR3 using the above technique.Methods TLR3 was cloned from the peripheral blood mononuclear cells of Beijing duck by RT-PCR technique and named duTLR3.The extracellular region of duck TLR3 gene was amplified by PCR and cloned into the prokaryotic expression vector pET-32a (). The recombinant TLR3 protein was induced by the transformation of Escherichia coli BL21DE3 strain.The extracellular region of TLR3 gene was amplified by PCR and purified from inclusion body. The purified recombinant TLR3 protein was used to immunize rabbits to prepare the polyclonal antibody to TLR3.Western blot and ELISA were used to detect its specificity and titer. According to the conserved region of duck TLR3 gene sequence, specific primers were designed, and duck 尾 -actin mRNA was used as internal control.A fluorescent quantitative PCR method for detecting the expression of duck Toll like receptor 3 mRNA was established. The established method was used to detect the antiviral function of TLR3 at cell and tissue level.Results the TLR3 gene of Peking duck was cloned, and the characteristic of TLR family structure of duTLR3 was predicted. (2) the extracellular domain of TLR3 gene was successfully expressed.The standard curve of the method was in the range of 1 脳 10 ~ 2 and 1 脳 10 ~ 8 copies/ 渭 L, and the correlation coefficient and amplification efficiency were above 0.9.The melting curve showed that the PCR product was a single peak with high amplification specificity.The results of repeatability showed that the coefficient of variation between groups was less than 3, and the lowest detection concentration was 100copies/ 渭 L.TLR3 in duck tissues and organs. The results showed that TLR3 was distributed in all tissues and organs of ducks.The expression of TLR3 and its downstream cytokines were positively correlated with the titer of DRV(Duck reovirus.Conclusion: the interaction between duck TLR3 and DRV was preliminarily confirmed, and its antiviral function was played by downstream signal molecules and antiviral proteins.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S834.81

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