摘要
为了明确鸡(Gallus gallus)溴结构域和超末端结构(bromodomain and extra terminal,BET)家族蛋白亚细胞定位及其基因在免疫器官中的表达特性,并为后续研究BET家族蛋白调控鸡相关病毒复制的作用机制提供基础,本研究将前期构建的鸡BRD2、BRD3、BRD4基因重组真核表达载体pEGFP-BRD2、pEGFP-BRD3和pEGFP-BRD4分别转染人(Homo sapiens)胚胎肾细胞(HEK-293T),利用Western blotting和荧光检测鸡BRD2、BRD3、BRD4基因的表达和蛋白的亚细胞定位情况,并利用实时定量PCR(real time quantitative PCR)技术检测1~6月龄鸡免疫器官中BRD2、BRD3和BRD4基因的表达。结果表明,鸡BET家族蛋白融合蛋白EGFP-BRD2、EGFP-BRD3和EGFP-BRD4在细胞中正确表达,且鸡BRD2、BRD3和BRD4蛋白主要定位在细胞核。实时定量PCR检测结果发现,鸡的脾脏和胸腺中BRD2基因的表达量在3月龄时最高,在法氏囊中则在4月龄时最高,表达变化均为“倒V”型;脾脏中BRD3基因的表达量在1月龄时为最高;而鸡的胸腺和法氏囊中,BRD3和BRD4基因的表达量均在6月龄时为最高,其中,BRD3基因在胸腺和法氏囊的表达变化,以及BRD4基因在法氏囊中的表达变化均为逐渐上升的“直线”型;BRD3基因在脾脏中的表达变化,以及BRD4基因在胸腺中的表达变化均为“倒N”型;脾脏中BRD4基因的表达量在3月龄时最高,其表达变化呈先上升后下降的趋势。鸡BET家族基因在不同月龄鸡的脾脏、胸腺和法氏囊中均有表达,但表达量呈现不同的变化趋势,BET家族蛋白主要定位在细胞核。
To clarify the subcellular localization of chicken bromodomain and extra terminal(BET)family proteins and the expression characteristics of the genes in immune organs,and to provide a basis for the follow-up research on the mechanism of BET family proteins regulating chicken related virus replication,The constructed recombinant eukaryotic expression vectors pEGFP-BRD2,pEGFPBRD3 and pEGFP-BRD4 of chicken BRD2,BRD3 and BRD4 gene were transfected into human(Homo sapiens)embryonic kidney cells(HEK-293T)in this study,respectively.Western blotting and fluorescence observation were used to detect the expression of BRD2,BRD3 and BRD4 gene and the subcellular localization of chicken BET family proteins.In addition,real time quantitative PCR was used to detect the expression of BRD2,BRD3 and BRD4 gene in immune organs of 1 to 6 months old chickens.The results showed that the chicken BET family fusion proteins EGFP-BRD2,EGFP-BRD3 and EGFP-BRD4 were correctly expressed in cells,and the chicken BRD2,BRD3 and BRD4 were mainly localized in the nucleus.Real time quantitative PCR detection results showed that the BRD2 gene was the most highly expressed in the spleen and thymus of 3 months old chickens and in the bursa of fabricius of 4 months old chickens,with an“inverted V”expression pattern.The expression of BRD3 gene was the highest in the spleen of 1 month old chickens,and BRD3 and BRD4 genes were the highest in the thymus and bursa of fabricius of 6 months old chickens.Among them,the expression of BRD3 gene in the thymus and bursa of fabricius and BRD4 gene in the bursa of fabricius increased gradually in a“straight line”type,and the expression of BRD3 gene in the spleen of 1 month old chickens and BRD4 gene in the thymus were“inverted N”type.The expression of BRD4 gene was the highest in the spleen of 3 months old chickens,and the expression of BRD4 gene first increased and then decreased.Chicken BET family proteins are mainly located in the nucleus,and their genes were expressed in the spleen,thymus and bursa o
作者
石海英
韩一帆
邢静如
唐宏
段志强
SHI Haiying;HAN Yifan;XING Jingru;TANG Hong;DUAN Zhiqiang(College of Animal Sciences,Guizhou University,Guiyang,550025;Key Laboratory of Ministry of Education for Animal Genetics,Breeding and Reproduction in the Plateau Mountains Region,Guiyang,550025;Key Laboratory of Animal Genetics,Breeding and Reproduction in Guizhou,Guiyang,550025)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2023年第5期491-500,共10页
Genomics and Applied Biology
基金
国家自然科学基金项目(31960698,31760732)
贵州省科学技术基金项目(黔科合基础[2020]1Y134号)共同资助。
关键词
鸡
BET家族蛋白
亚细胞定位
免疫器官
表达特征
Chicken
BET family proteins
Subcellular localization
Immune organ
Expression characteristics
