牛PAI-1基因真核表达载体构建、生物信息学分析及功能初探  

Construction of Eukaryotic Expression Vector, Bioinformatics Analysis of Cattle PAI-1 Gene and Preliminary Study of its Function

作　　者：李锰 赵静[1,2] 刘红羽[1,2] 安雯 王军[1,2] 吕文发[1,2] LI Meng;ZHAO Jing;LIU Hongyu;AN Wen;WANG Jun;LYU Wenfa(Joint Laboratory of Modern Agricultural Technology International Cooperation,Ministry of Education,Jilin Agricultural University,Jilin Changchun 130118,China;Key Lab of Animal Production,Product Quality and Security,Ministry of Education,Jilin Agricultural University,Jilin Changchun 130118,China)

机构地区：[1]吉林农业大学现代农业技术教育部国际合作联合重点实验室,吉林长春130118 [2]吉林农业大学动物生产与产品质量安全教育部重点实验室,吉林长春130118

出　　处：《中国畜牧杂志》2020年第8期184-189,共6页Chinese Journal of Animal Science

基　　金：国家自然科学基金项目(31772600)。

摘　　要：本研究旨在构建牛纤溶酶原激活物抑制剂1(Plasminogen activator inhibitor-1,PAI-1)基因真核表达载体、生物信息学分析并对其功能进行初探。实验参照GenBank数据库公布的牛PAI-1基因的CDS序列信息设计引物,通过RT-PCR获得PAI-1基因片段,将基因片段与pMD-19-T载体连接转化DH5α感受态细胞,构建pMD-19-T-PAI-1克隆载体,双酶切回收基因片段,构建pcDNA3.1-PAI-1真核表达载体,经双酶切、测序鉴定构建成功。将表达载体转染至293T细胞,48 h后进行荧光定量PCR(RT-qPCR)和Western Blot检测PAI-1以及凋亡相关基因Bcl2、Bax和Caspase-3的表达。结果显示:pcDNA3.1-PAI-1真核表达载体测序结果与GenBank数据库公布的牛PAI-1基因CDS序列完全匹配,生物信息学分析发现PAI-1基因编码区全长1 209 bp,编码402个氨基酸,存在35个潜在磷酸化位点,主要分布在细胞外。过表达PAI-1可显著提高293T细胞中PAI-1 mRNA和蛋白表达,抗凋亡基因Bcl2 mRNA和蛋白水平显著升高,促凋亡基因Bax和Caspase-3 mRNA和蛋白水平显著降低。The aim of this experiment is to construct cattle PAI-1 gene eukaryotic expression vector, bioinformatics analysis and preliminary study of its function. The primers were designed according to the CDS sequence information of the cattle PAI-1 gene published in the GenBank database. The PAI-1 gene was obtained by reverse transcription PCR(RT-PCR). It was cloned into pMD-19-T vector, and further transformed into DH5α, then restriction enzyme digestion and recovery of PAI-1 gene and construction of pcDNA3.1-PAI-1 eukaryotic expression vector. The pcDNA3.1-PAI-1 identified by restriction enzyme digestion and sequencing. The pcDNA3.1-PAI-1 was transfected into 293 T cells. The expression of apoptosis-related genes Bcl2, Bax, and Caspase-3 was detected by fluorescence quantitative PCR(RT-qPCR) and western blot 48 hours later. The results showed that the sequencing results of the pcDNA3.1-PAI-1 eukaryotic expression vector exactly matched the CDS sequence of the cattle PAI-1 gene published in the GenBank database. The sequence of PAI-1 gene CDS was 1 209 bp, encoding 402 amino acids, containing 35 potential phosphorylation sites. The subcellular localization was mainly distributed in the extracellular. Overexpression of PAI-1 could significantly increase the expression of PAI-1 mRNA and protein in 293 T cells. The mRNA and protein levels of Bcl2 were significantly increased, and the mRNA and protein levels of Bax and Caspase-3 were significantly reduced. Conclusion: The cattle pc DNA3.1-PAI-1 eukaryotic expression vector was successfully constructed and showed anti-apoptotic function in 239 T cells.

关 键 词：颗粒细胞 PAI-1基因 真核表达载体 细胞凋亡 

分 类 号：S823.2[农业科学—畜牧学]