鸡Visfatin基因真核表达载体的构建、组织表达和功能研究  

Construction of Eukaryotic Expression Vector,Tissue Expression and Function of Visfatin Gene in Chicken

作　　者：刘佳玲 魏志恒 彭鑫 刘世昊 邵芳[2] 徐璐 郁建锋[1] 顾志良[1] LIU Jiaing;WEI Zhiheng;PENG Xin;LIU Shihao;SHAO Fang;XU Lu;YU Jianfeng;GU Zhiiang(College of Biology and Food Engineering,Changshu University of Technology,Suzhou,Jiangsu 215500;The Second People's Hospital of Changzhou,Nanjing Medical University,Changzhou,Jiangsu 213003)

机构地区：[1]常熟理工学院生物与食品工程学院,江苏苏州215500 [2]南京医科大学附属常州市第二人民医院,江苏常州213003

出　　处：《中国家禽》2024年第7期18-25,共8页China Poultry

基　　金：江苏省自然科学基金项目(BK20191476)。

摘　　要：研究旨在构建鸡Visfatin基因的真核表达载体,并在LMH细胞中过表达,揭示LMH中Visfatin对脂类代谢相关基因的影响。试验采用RT-PCR扩增鸡Visfatin的编码区片段,构建鸡Visfatin基因的真核表达载体,转染LMH细胞后经Western blot对表达产物鉴定,用荧光定量PCR检测LMH细胞中过表达Visfatin基因对脂类代谢相关基因影响,检测不同组织Visfatin基因的表达情况。结果显示:扩增到的鸡Visfatin基因CDS全长1 479 bp,其编码493个氨基酸,鸡Visfatin蛋白的化学式为C2513H3912N656O751S8,分子量为55 587.13 u,其理论等电点(p I)为6.66,根据稳定性的相关参数分析,该蛋白的不稳定系数小于40,表明Visfatin蛋白是一种相对稳定的蛋白质,鸡Visfatin蛋白的脂肪指数为84.58,总平均亲水性为-0.455,提示其具有一定的亲水性;构建的p CMV-3Tag-9-gga-Visfatin真核表达载体在LMH细胞成功表达出Visfatin-Myc融合蛋白,Visfatin基因过表达后极显著下调LPL和NMRK1基因表达;鸡12种组织中的Visfatin基因在胸肌组织中表达量较高。研究结果表明,Visfatin基因通过下调LPL和NMRK1基因表达对脂类代谢进行调控。The aim of this study was to construct eukaryotic expression vector of chicken Visfatin gene and overexpress it in LMH cells to reveal the effects of Visfatin on lipid metabolism related genes in LMH.The coding region fragment of Visfatin gene was amplified by RT-PCR and the eukaryotic expression vector of chicken Visfatin gene was constructed.After transfection into LMH cells,the expression products were identified by Western blot.The effect of overexpression of Visfatin gene on expression of lipid metabolism related genes in LMH cells was detected,and the expression of Visfatin gene in various tissues was detected by fluorescent quantitative PCR.The results showed that the full length of the amplified chicken Visfatin gene CDS was 1479 bp,encoding 493 amino acids.The chemical formula of chicken Visfatin protein was C2513H3912N656O751S8,its molecular weight was 55587.13 u,and its theoretical isoelectric point(pI)was 6.66.The instability coefficient of the protein was less than 40,indicating that it was a relatively stable protein.The fat index of chicken Visfatin protein was 84.58,and the total average hydrophilicity was-0.455,suggesting that it had a certain hydrophilicity.The constructed pCMV-3Tag-9-gga-Visfatin eukaryotic expression vector successfully expressed Visfatin-MYC fusion protein in LMH cells,and the expression of LPL and NMRK1 genes was signif-icantly down-regulated after Visfatin gene overexpression.The expression of Visfatin gene was higher in pectoral muscle of all the 12 chicken tissues.These results indicated that Visfatin gene regulates lipid metabolism by down-regulating the expression of LPL and NMRK1 genes.

关 键 词：鸡 Visfatin基因 LMH细胞 LPL基因 NMRK1基因 

分 类 号：S831.2[农业科学—畜牧学]