单核细胞增生李斯特菌精氨酸脱亚胺酶基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Arginine Deiminase from Listeria monocytogenes

作　　者：程昌勇[1] 陈健舜[1] 赵寒昕[1] 白帆[1] 方维焕[1] 

机构地区：[1]浙江大学动物预防医学研究所浙江省动物预防医学重点实验室,浙江杭州310029

出　　处：《动物医学进展》2010年第S1期70-73,共4页Progress In Veterinary Medicine

基　　金："十一五"国家科技支撑计划重点项目(2009BADB9B09)

摘　　要：精氨酸脱亚胺酶(ADI)可能介导单核细胞增生李斯特菌抗酸应激,由arcA基因编码。利用分子克隆技术从单核细胞增生李斯特菌10403S扩增得到ADI基因,测序正确后将其插入pET30a表达载体中,构建该基因的原核表达质粒pET30a-ADI,并转化入大肠埃希菌Rosetta中进行诱导表达。核苷酸序列分析显示,ADI基因的完整开放阅读框为1 233 bp,编码410个氨基酸。SDS-PAGE表明:该基因在大肠埃希菌中成功表达,重组蛋白的分子质量约为52.5 ku。为进一步研究精氨酸脱亚胺酶的活性提供了条件,同时也为探索单核细胞增生李斯特菌抗酸应激的机理奠定了重要的基础。Arginine deiminase(ADI),encoded by arcA,is one of the most important enzymes involved in acid tolerance of Listeria monocytogenes.The ADI gene was amplified from L.monocytogenes 10403S and cloned into pMD18-T vector.Sequence analysis showed that the full length ADI gene was 1 233 bp,encoding 410 amino acids.The gene fragment was then subcloned into prokaryotic expression vector pET30a,yielding recombinant expression plasmid pET30a-ADI,and transformed into competent E.coli Rosetta cells.Expression of the target protein was induced with IPTG.SDS-PAGE showed a specific protein band with a mass weight of 55.2 ku.Successful expression of ADI in E.coli provides possibilities for further study of the enzyme activity and its role in acid tolerance mechanism of L.monocytogenes.

关 键 词：单核细胞增生李斯特菌 arcA基因 精氨酸脱亚胺酶 原核表达 

分 类 号：Q789[生物学—分子生物学]