[目的]明确鸡肿瘤坏死因子受体相关因子相互作用的具有叉形头相关结构域蛋白(TIFA)和肿瘤坏死因子受体相关因子6(TRAF6)的分子特征及其基因在鸡免疫器官中的表达特性,为后续深入研究TIFA和TRAF6相互作用调控家禽病毒NDV复制的作用机制提供理论依据.[方法]采用生物信息学软件结合实时荧光定量PCR(RT-qPCR)方法,通过分析鸡TIFA和TRAF6蛋白的理化性质、二级结构、三级结构、氨基酸同源性和结构域保守性及其在1~6月龄白来航鸡和新城疫病毒(NDV)感染SPF鸡后第3~5天的免疫器官(脾脏、胸腺和法氏囊),分别检测鸡免疫器官中TIFA和TRAF6基因的表达水平,进而研究鸡TIFA和TRAF6蛋白的分子特征及其基因在免疫器官中的表达特性.[结果]鸡TIFA和TRAF6蛋白分别含有187和545个氨基酸残基,相对分子量约为21.8和61.9 kDa,其二级结构均主要由无规则卷曲和α-螺旋组成.氨基酸同源性分析结果表明,鸡与火鸡TIFA和TRAF6蛋白的氨基酸同源性最高,分别为97.3%和98.2%,而与猪TIFA及小鼠TRAF6蛋白的氨基酸同源性最低,分别为51.1%和74.7%.同时,鸡TIFA蛋白的FHA结构域及TRAF6蛋白的RING、Zinc finger和MATH结构域均在禽类中保守性高.RT-qPCR分析结果表明,鸡TIFA和TRAF6基因在1~6月龄鸡免疫器官中均存在表达,但在脾脏中的相对表达量最高(47.94和10.84),而在法氏囊中最低(8.08和2.01),其中,TIFA基因在1月龄鸡免疫器官中表达量最高(22.15),在6月龄时最低(1.11),呈先减后增再减趋势,整体表达趋势呈倒"N"型模式;TRAF6基因在4月龄鸡免疫器官中表达量最高(7.13),在5月龄时最低(1.05),呈先降后升再降再升趋势,整体表达趋势呈"W"型模式.在NDV感染SPF鸡后的免疫器官中,TIFA和TRAF6基因的相对表达量均显著高于对照,其中,TIFA基因在第3天的鸡脾脏、第4天的鸡胸腺和法氏囊中的表达量最高,而TRAF6基因则在第3天的鸡脾脏、第5天的鸡胸腺和法氏囊中的表达量最高.[结论]鸡TIFA和TRAF6蛋白在禽类中的保守性高,两者在不同月龄鸡和NDV感染鸡的免疫器官中呈不同的表达水平和表达变化趋势,其参与的先天性免疫应答可能在抵抗NDV感染过程中发挥一定作用.
[Objective]The molecular feature of chicken tumor necrosis factor receptor-associated factor(TRAF)-interacting protein with a forked-associated domain(TIFA)and tumor necrosis factor receptor-associated factor 6(TRAF6)and their gene expression characteristics in the chicken immune organs were studied,which provided the theoretical basis for further study on the mechanism of interaction between TIFA and TRAF6 to regulate the replication of poultry virus NDV.[Method]Using bioinformatics software combined with real-time fluores-cence quantitative PCR(RT-qPCR)method,through analyzing the physicochemical properties,secondary structure,tertiary structure,ami-no acid homology and domain conservation of TIFA and TRAF6 proteins in chickens,and their immune organs(spleen,thymus and bursa of fabricius)of 1-6 month-old White Leghorn chickens and SPF chickens infected with Newcastle disease virus(NDV)on the 3rd to 5th day,the expression levels of TIFA and TRAF6 genes in chicken immune organs were detected,and then the molecular characteristics of chicken TIFA and TRAF6 proteins and the expression characteristics of their genes in immune organs were studied.[Result]Chicken TIFA and TRAF6 proteins contained 187 and 545 amino acid residues,respectively,with relative molecular weight about 21.8 and 61.9 kDa.The secondary structure of chicken TIFA and TRAF6 proteins was mainly composed of random coil and α-helix.The TIFA and TRAF6 proteins of chicken had the highest amino acid homology with turkey(97.3%and 98.2%,respectively),but had the lowest amino acid homology with pig TIFA and mouse TRAF6(51.1%and 74.7%,respectively).Meanwhile,the FHA domain in chicken TIFA protein and the RING,Zinc finger and MATH domains in chicken TRAF6 protein had high conservation with other avian species.The results of RT-qPCR showed that both TIFA and TRAF6 genes were expressed in the immune organs of chickens aged 1 to 6 months,but the relative expression levels were the highest in the spleen(47.94 and 10.84),and the lowest in the bursa of fabricius(8.08 and 2.01).The expression level of TIFA gene was the highest in immune organs of 1-month-old chickens(22.15),and the lowest in immune organs of 6-month-old chickens(1.11),showing a trend of first decreasing,increasing and then decreasing,and the overall expression trend showed an inverted'N'type pattern.The expression of TRAF6 gene in the immune organs of chickens at 4 months of age was the highest(7.13),and the lowest(1.05)at 5 months of age,showing a trend of first decreasing,then increasing and then decreasing and increasing again,and the overall expression trend showed a'W'pattern.In the immune organs of SPF chickens infected with NDV,the expression levels of TIFA and TRAF6 genes were significantly increased in comparison to that of the normal group,of which the TIFA gene had the highest expression level in the 3rd-day chicken spleen and the 4th-day chicken thymus and bursa of fabricius,while the TRAF6 gene had the highest expression level in the 3rd-day chicken spleen and the 5th-day chicken thymus and bursa of fabricius,respectively.[Conclusion]Chicken TIFA and TRAF6 proteins were highly conserved in avian species,and their expression levels and expression trends were different in the immune organs of chickens with dif-ferent months of age and NDV-infected chickens.The TIFA and TRAF6 proteins involving in innate immune response might played a certain role in resisting NDV infection.