奶牛SREBP1蛋白在乳腺上皮细胞的表达定位及对SCD1基因启动子的转录调控

【目的】固醇调节元件结合蛋白1(SREBP1)作为核转录因子,对于细胞脂肪合成酶基因的表达发挥着重要的调控作用。论文旨在奶牛乳腺上皮细胞中研究SREBP1对于SCD1基因启动子的转录调控作用,为进一步明确SREBP1对于靶基因的转录调控机制提供理论基础。【方法】以荷斯坦奶牛乳腺组织的c DNA为模板,采用分段克隆的方法获得SREBP1基因的编码序列,通过重组酶与pc DNA3.1载体进行重组环化构建pc DNA3.1-SREBP1表达载体,将构建的载体测序验证后提取质粒,转染奶牛乳腺上皮细胞。以EIF3K基因为内参基因,采用荧光定量PCR检测SREBP1基因m RNA的表达差异;采用免疫荧光的方法对SREBP1进行标记,以DAPI复染细胞核,激光共聚焦观察SREBP1蛋白的亚细胞定位;转染含有不同调控元件的SCD基因启动子,同时转染1.0μg pc DNA3.1-SREBP1作为处理,荧光素酶报告基因系统分析启动子活性;分别转染0.25、0.5和1μg的pc DNA3.1-SREBP1载体,分析p GL3-SCD 2和p GL3-SCD3启动子活性与SREBP1之间的量效关系。【结果】分段克隆得到的PCR产物分别为1 170、1 116、363和900 bp的片段,经过与pc DNA3.1载体重组后获得pc DNA3.1-SREBP1表达载体,经酶切和测序验证,发现除1个无义突变外,与标准序列完全相同,整个序列长度达到3 510bp;将pc DNA3.1-SREBP1载体转染乳腺上皮细胞后,Real-time PCR检测发现与转染空载体的对照组相比,SREBP1基因的m RNA表达倍数增强130.4倍(P0.001);激光共聚焦观察发现,DAPI染色的细胞核呈蓝色,免疫荧光标记的SREBP1呈绿色,二者融合后呈现青色,共定位在乳腺上皮细胞核中;启动子活性检测发现,与p GL3-SCD1、p GL3SCD 2相比,SREBP1处理能够极显著增加p GL3-SCD3、p GL3-CD4启动子的活性(P0.001),分别比对照组提高了1.0倍和0.7倍,进一步分析发现,在用0.25—1μg的pc DNA3.1-SREBP1处理后,与p GL3-SCD2的启动子活性持续下降相比,p GL3-SCD3的启动子活性从59.81上升到108.43(P0.001),二者存在剂量效应关系,结合SCD2和SCD 3启动子上主要的结构差异SRE元件(5′-AGCAGATTGCG-3′),推测此序列可能是SREBP1调控SCD基因启动子转录的结合序列。【结论】克隆构建奶牛SREBP1基因表达载体,亚细胞定位SREBP1蛋白主要在乳腺上皮细胞核中,SREBP1可以与SRE调控元件结合促进SCD1基因启动子的转录。     

Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection

To better understand the basis for human immunodeficiency virus type 1 (HIV-1) persistence and latency, the form in which viral DNA exists in the peripheral T lymphocyte reservoir of infected individuals was investigated. In asymptomatic individuals, HIV-1 was harbored predominantly as full-length, unintegrated complementary DNA. These extrachromosomal DNA forms retained the ability to integrate upon T cell activation in vitro. In patients with acquired immunodeficiency syndrome (AIDS), there was an increase in integrated relative to extrachromosomal DNA forms. By analysis of DNA from patient lymphocyte subpopulations depleted of human lymphocyte antigen-Dr receptor-positive cells, quiescent T cells were identified as the source of extrachromosomal HIV-1 DNA. Thus quiescent T lymphocytes may be a major and inducible HIV-1 reservoir in infected individuals.     

牛FoxO1基因启动子转录调控分析

为鉴定牛叉头框转录因子1(Forkhead box,FoxO1)的核心启动子区及其关键转录因子,利用PCR扩增牛FoxO1基因的启动子序列,同时设计7个逐段缺失片段引物扩增其启动子逐段缺失片段,进一步将其构建双荧光素酶报告载体并分别转染小鼠C2C12和3T3-L1细胞系,通过检测不同缺失片段荧光素酶报告载体的活性,以确定牛FoxO1启动子的核心区域;使用Genomatix和JASPAR在线软件预测牛FoxO1基因启动子核心区域的关键转录因子,采用定点突变试验在小鼠C2C12细胞系中初步鉴定预测的关键转录因子对FoxO1的转录调控作用。结果表明：1）成功扩增了牛FoxO1的启动子区1 920bp序列,获得了FoxO1基因启动子序列的7个逐段缺失片段序列;2）经双荧光素酶报告试验进一步证实,牛FoxO1基因核心启动子位于-285/-27区域;3）预测并通过定点突变试验鉴定出MEF2A、KLF4、HOXA5和KLF5转录因子对FoxO1基因的转录活性具有关键调控作用。综上,本研究初步鉴定出牛FoxO1基因启动子核心区（-285/-27）内转录因子MEF2A、KLF4、HOXA5和KLF5对F...     

cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily

Interleukin-1 alpha and -1 beta (IL-1 alpha and IL-1 beta) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1 alpha and IL-1 beta in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.     

Studies on Immunoregulation of Cytokine of Chickens after Inoculation with Marek''''s Disease Vaccines

The spleenic and thymus T lymphocyte proliferaitivc reactions (TLPR) were enhanced, the intcrlcukin 2(IL-2) inductive activity was raised, and the IL-2 receptor (IL-2R) inductive expression was increased significantly in chickens after inoculation with trivalcnt Marck's disease vaccine in comparison with control chickens and those after vaccination with herpcsvirus of turkey (HVT) vaccine. In chickens after inoculation with HVT vaccine, in contrast with control chickens, the splcenic TLPR was strengthened and IL-2R inductive expression was hoisted significantly, the thymus TLPR, splccnic and thymus T lymphocyte IL-2 inductive activity, the thymus T lymphocyte IL-2R inductive expression were not significantly increased.     

内皮PAS1蛋白(EPAS1)基因的研究进展

内皮PAS1蛋白(EPAS1/HIF-2)是细胞应对缺氧时关键的转录因子,在生物体生理及病理过程中有重要作用。文中对EPAS1的结构、分布、表达调控及在病理过程中的作用、高原低氧适应性方面进行了综述,为今后进一步研究EPAS1提供了参考和借鉴。     

不同Se源对奶牛繁殖性能和免疫功能的影响

30头健康、经直肠检查确定已妊娠的黑白花母牛 ,分成 5组 ,其中 4组为处理组 (T1 、T2 、T3、T4 ) ,1组作为对照组(CK) .各处理组分别添加 4种 Se添加剂 ,即 Se酵母 (Sel-Plex5 0 ,Alltech产品 )、混合 Se化合物 (70 % Se酵母 +3 0 %亚硒酸钠 )、Se氨基酸络合物 (主要是 Se蛋氨酸 )和亚硒酸钠 .试验持续了 1 68d,在此期间 ,对血液 GSH-Px活性、血清 Se水平、淋巴细胞转化率和 T细胞数做了测定 ,同时观察了某些繁殖性能的变化 .结果表明 :(1 ) GSH-Px活性和血清 Se水平 ,T1 、T2 和 T3显著高于 CK(P<0 .0 1 ) ,而 T4 亦高于 CK(P<0 .0 5 ) ;T1 显著高于 T2 、T3和 T4 (P<0 .0 5 ) .(2 )补 Se后第 70天测得的淋巴细胞转化率和 T细胞数 ,T1 和 T2 显著高于 CK(P<0 .0 1 ) ,而 T3、T4 高于 CK(P<0 .0 5 ) ;同时 T1 、T2 和 T3的淋巴细胞转化率和 T细胞数亦高于 T4 (P<0 .0 5 ) .(3 )补 Se明显改善了某些繁殖性能 ,如减少了每次受孕的配种次数并降低了诸如胎衣不下和子宫内膜炎等产科疾病的发病率 .试验也证明 ,补充有机 Se可明显提高母牛抗感染 ,促进淋巴细胞增殖 ,增进嗜中性白细胞的吞噬作用