摘要
胎球蛋白B(FETUB)是一种新型的脂肪因子/肝因子,是由肝脏产生并释放入血液的半胱氨酸蛋白酶抑制剂,其在糖脂代谢中起重要作用.本研究目的是构建鸡的FETUB基因真核表达载体,在LMH细胞过表达FETUB后研究其对其他脂类相关基因表达的影响.采用荧光定量PCR检测FETUB基因在鸡各种不同组织中的表达量,以肝脏组织总RNA为模板,RT-PCR扩增鸡FETUB基因编码区,并构建真核表达重组载体,转染LMH细胞后分别提取总蛋白和总RNA,Western Blot检测重组蛋白表达,荧光定量PCR检测脂类代谢相关基因的表达.结果显示,FETUB基因在鸡11种不同组织中有表达,其中在肝脏中表达量极高,小肠和肾脏中表达较高,而在肌胃、脾脏、脂肪中表达量较低.通过RT-PCR获得鸡FETUB基因CDS,构建真核表达载体pCMV-3Tag-9-ggaFETUB,在LMH中成功融合表达FETUB-MYC.FETUB基因过表达使LMH细胞LPL和ITIH2基因表达均显著下调,而对APOA4、DGAT2、FASN、ACACA四个基因的表达没有显著影响.研究结果表明FETUB基因通过抑制LPL和ITIH2基因表达来影响脂类代谢,为进一步分析鸡的FETUB基因的功能奠定基础.
Fetuin B(FETUB),a novel adipokine/hepatokine,is a cysteine protease inhibitor produced by the liver and released into the blood.Fetuin B plays an important role in glycolipid metabolism.The purpose of this study was to construct an eukaryotic expression vector of FETUB gene in chickens,and to study the effects of over-expression of FETUB in LMH on the expression of other lipid-related genes.Fluorescence quantitative PCR was used to detect the expression levels of FETUB gene in various tissues of chicken.The total RNA of liver tissue was used as the template to amplify the coding region of FETUB gene in chicken by RT-PCR,and the eukaryotic expression recombinant vector was constructed.Total protein and total RNA were extracted from LMH cells after transfection.The recombinant protein was detected by Western Blot,and the expression of lipid metabolism-related genes was detected by fluorescence quantitative PCR.The results show that FETUB gene was expressed in 11 kinds of chicken tissues,with high expression in liver,low expression in muscle stomach,spleen and fat,and no expression in glandular stomach.Chicken FETUB cDNA was obtained by RT-PCR,the eukaryotic expression vector pCMV-3Tag-9-ggaFETUB was constructed,and FETUB-MYC was successfully fusion expressed in LMH.Over-expression of FETUB gene significantly down-regulated the expressions of LPL and ITIH2 genes in LMH cells,but had no significant effect on the expressions of APOA4,DGAT2,FASN and ACACA genes.The indication that FETUB gene affected lipid metabolism by inhibiting LPL and ITIH2 gene expression after LMH over-expression lays a foundation for further analysis of the function of FETUB gene in chickens.
作者
刘晨
徐庶勋
魏志恒
彭鑫
刘世昊
徐璐
郁建锋
顾志良
LIU Chen;XU Shuxun;WEI Zhiheng;PENG Xin;LIU Shihao;XU Lu;YU Jianfeng;GU Zhiiang(School of Biology and Food Engineering,Changshu Institute of Technology,Changshu 215500,China)
出处
《常熟理工学院学报》
2024年第2期39-45,共7页
Journal of Changshu Institute of Technology
基金
江苏省自然科学基金项目“RNA去甲基酶FTO对鸡肝脏细胞脂类代谢和脂肪沉积作用机制研究”(BK20191476)。
