绵羊清道夫受体A启动子活性分析  

Analysis of Promoter Activity of Sheep Scavenger Receptor A

作　　者：曹鑫艳 王钢 张秦川 范文雨 顾兰英 盛金良[1] 贺笋 孙延鸣[1] 张彦兵[1] CAO Xin-yan;WANG Gang;ZHANG Qin-chuan;FAN Wen-yu;GU Lan-ying;SHENG Jin-liang;HE Sun;SUN Yan-ming;ZHANG Yan-bing(College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang,832003,China;TECON Pharmaceutical Co.,Ltd,Urumqi,Xinjiang,830000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]天康制药股份有限公司,新疆乌鲁木齐830000

出　　处：《动物医学进展》2025年第6期13-18,共6页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(32360901,32060133);新疆生产建设兵团指导性科技计划项目(2023ZD042);新疆维吾尔自治区科学基金项目(2022D01B197);新疆维吾尔自治区两区项目(2023LQ03003);石河子大学高层次人才科研启动项目(RCZK202043)。

摘　　要：旨在初步分析绵羊清道夫受体A(SRA)启动子活性调节机理,为进一步探究SRA转录调控机制奠定基础。使用同源重组的方法构建SRA启动子报告质粒,XhoⅠ酶切验证,进一步测序比对;将构建的SRA启动子报告质粒与海肾荧光素酶载体共转染至HEK293T细胞,利用双荧光素酶报告基因试验检测SRA启动子活性。利用化学合成技术合成肿瘤坏死因子α(TNF-α)和白介素6(IL-6)真核表达载体,将SRA报告基因载体分别与干扰素调节因子1(IRF1)、TNF-α和IL-6的表达载体共转染于HEK293T细胞,分析IRF1、TNF-α和IL-6对SRA启动子活性的影响。结果显示,XhoⅠ酶切pGL3-SRA出现预期目的条带和载体片段,测序比对正确,成功构建pGL3-SRA启动子报告基因载体;双荧光素酶报告基因试验证实SRA启动子具有较强的活性,IRF1、TNF-α和IL-6过表达能显著激活SRA的启动子活性(P<0.05,P<0.01)。研究结果为进一步探究IRF1、TNF-α和IL-6对SRA表达调控机制提供了基础资料。This study is to preliminarily analyze the regulatory mechanism of the promoter activity of sheep scavenger receptor A(SRA),laying the foundation for further exploration of the transcriptional regulation mechanism of SRA.The SRA promoter reporter plasmid was construct using homologous recombination method,validated by XhoⅠenzyme digestion,and further sequence alignment;The constructed SRA promoter reporter plasmid was cotransfected with a marine luciferase vector into HEK293T cells,and the SRA promoter activity was detected using a dual luciferase reporter gene assay.The chemical synthesis techniques were used to synthesize eukaryotic expression vectors for tumor necrosis factor alpha(TNF-α)and interleukin-6(IL-6),the SRA reporter gene vector was co-transfected with expression vectors for IRF1,TNF-α,and IL-6 in HEK293T cells,and the effects of IRF1,TNF-αand IL-6 on SRA promoter activity were analyzed.The results showed that XhoⅠenzyme digestion of pGL3 SRA resulted in the expected target band and vector fragment,and sequencing alignment was right.The pGL3 SRA promoter reporter gene vector was successfully constructed;The dual luciferase reporter gene experiment confirmed that the SRA promoter has strong activity,and overexpressions of IRF1,TNF-αand IL-6 can significantly activate the promoter activity of SRA(P<0.01,P<0.01).This provides basic information for further exploring the regulatory mechanisms of IRF1,TNF-αand IL-6 on SRA expression.

关 键 词：绵羊 清道夫受体A 转录调控 启动子活性 双荧光素酶 

分 类 号：S852.21[农业科学—基础兽医学]