家鸡MDH2基因克隆、生物信息学分析及原核表达载体构建  

Cloning,Bioinformatics Analysis,and Prokaryotic Expression Vector Construction of MDH2 in Gallus gallus domesticus

作　　者：袁铭君 李军德[1] 邢建永 高巍 闫士猛 张恬[1] YUAN Ming-jun;LI Jun-de;XING Jian-yong;GAO Wei;YAN Shi-meng;ZHANG Tian(China Academy of Chinese Medical Sciences National Resource Center for Chinese Materia Medica,State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs,Beijing 100700,China;China Resources Sanjiu Medical&Pharmaceutical Co.,Ltd.,Shenzhen 518110,China;China Resources Sanjiu(Lu'an)Traditional Chinese Medicine Industry Development Co.,Ltd.,Lu'an 237300,China)

机构地区：[1]中国中医科学院中药资源中心/道地药材品质保障与资源持续利用全国重点实验室,北京100700 [2]华润三九医药股份有限公司,广东深圳518110 [3]华润三九(六安)中药材产业发展有限公司,安徽六安237300

出　　处：《中国现代中药》2023年第8期1646-1654,共9页Modern Chinese Medicine

基　　金：国家自然科学基金项目(82104346);中国中医科学院科技创新工程项目(CI2021A04012)。

摘　　要：目的:初步探索家鸡苹果酸脱氢酶2编码基因MDH2的序列特征、组织表达及原核表达情况。方法:采用克隆技术与测序技术获取家鸡MDH2基因互补脱氧核糖核酸(cDNA)全长,使用多种在线软件对其进行生物信息学分析,利用实时荧光定量聚合酶链式反应(PCR)技术检测该基因在不同组织中的表达情况,利用同源重组技术构建pET32a(+)-MDH2表达载体,使用异丙基硫代半乳糖苷(IPTG)诱导蛋白表达。结果:从新鲜家鸡砂囊内壁中成功克隆到MDH2基因,序列长度1120 bp,其开放阅读框(ORF)为1014 bp,编码337个氨基酸;MDH2蛋白相对分子质量为35.95 kDa,理论等电点9.17,属于稳定碱性疏水性蛋白。MDH2蛋白含25个磷酸化位点;二级结构主要包括α-螺旋、β-螺旋、无规则卷曲,无信号肽和跨膜结构域。MDH2基因在心、肝、脾、肺、肾、砂囊内壁中都有表达,其中心、肾、砂囊内壁中表达量较高,pET32a(+)-MDH2重组蛋白主要是以包涵体的形式存在。结论:MDH2基因在家鸡体内广泛表达,但其在各组织/器官中表达趋势不同,提示其功能可能具有广泛性与多重性。Objective:To explore the sequence characteristics,tissue expression,and prokaryotic expression of malate dehydrogenase 2 gene(MDH2)in Gallus gallus domesticus.Methods:The full-length cDNA of MDH2 was obtained by cloning and sequencing,and online tools were used to perform the bioinformatics analysis.The expression levels of MDH2 in different tissues were determined by real-time fluorescence quantitative PCR.The pET32a(+)-MDH2 expression vector was constructed by homologous recombination,and the protein expression was induced by isopropyl-β-D-thiogalactoside(IPTG).Results:MDH2 was successfully cloned from the inner wall of a fresh chicken gizzard.MDH2 was 1120 bp in length,with an open reading frame(ORF)of 1014 bp,encoding 337 amino acid residues.MDH2 was a stable alkaline hydrophobic protein with a molecular weight of 35.95 kDa and a theoretical isoelectric point of 9.17.MDH2 protein contained 25 phosphorylation sites,and its secondary structure mainly includedα-helix,β-helix,and random coil.The protein had no signal peptide or transmembrane domain.MDH2 was expressed in the heart,liver,spleen,lung,kidney,and inner wall of the gizzard,with high expression in the heart,kidney,and inner wall of the gizzard.The pET32a(+)-MDH2 recombinant protein mainly existed as inclusion bodies.Conclusion:MDH2 is widely expressed in G.gallus domesticus,and its differential expression in different tissues/organs suggesting the diverse functions.

关 键 词：MDH2 基因克隆 生物信息学 原核表达 

分 类 号：R282.7[医药卫生—中药学]