鼠疫耶尔森菌YopJ蛋白的基因密码子优化及异源表达纯化  

Codon optimization and heterologous expression and purification of the YopJ protein from Yersinia pestis

作　　者：郭一星 杨银龙 马玥 岳盈盈[3] 宋楠楠[3] 王玮玮 贾海红 李翠玲[3] 李冰清[3] GUO Yi-xing;YANG Yin-long;MA Yue;YUE Ying-ying;SONG Nan-nan;WANG Wei-wei;JIA Hai-hong;LI Cui-ling;LI Bing-qing(School of Medicine and Life Sciences,University of Jinan,Shandong Academy of Medical Sciences,Jinan,China 250062;Women and Childrens Hospital of the City of Linyi;Institute of Basic Medicine,Shandong Academy of Medical Sciences)

机构地区：[1]济南大学,山东省医学科学院医学与生命科学学院,山东济南250062 [2]临沂市妇女儿童医院 [3]山东省医学科学院基础医学研究所

出　　处：《中国病原生物学杂志》2019年第9期1058-1061,1080,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31500050,31800054)

摘　　要：目的优化鼠疫耶尔森菌YopJ蛋白基因密码子并在大肠埃希菌中进行异源表达纯化。方法依据大肠埃希菌密码子偏好性对鼠疫耶尔森菌YopJ基因进行全基因序列优化,并设计引物。分别以密码子优化前后的鼠疫耶尔森菌YopJ蛋白的基因为模板,将包含YopJ(22aa-288aa)和YopJ(22aa-250aa)的基因片段连接到载体pGI01中。重组质粒转化到大肠埃希菌BL21菌株中,扩大培养后,IPTG诱导目的蛋白表达。提取大肠埃希菌表达蛋白并使用镍离子柱进行纯化。结果鼠疫耶尔森菌YopJ密码子优化前YopJ(22aa-288aa)及YopJ(22aa-250aa)在大肠埃希菌内表达水平较低,密码子优化后YopJ(22aa-288aa)及YopJ(22aa-250aa)在大肠埃希菌内大量表达可溶性YopJ蛋白。结论对鼠疫耶尔森菌YopJ蛋白进行基因密码子优化能够提高其在大肠埃希菌内的表达量。获得的鼠疫耶尔森菌YopJ蛋白具有可溶性,为进一步研究其结构和功能奠定了基础。Objectives YopJ is the key effector protein of Yersinia pestis.The study of YopJ is conducive to further revealing the pathogenic mechanism of Y.pestis.This study aims to optimize the codons of the YopJ gene of Yersinia pestis,to express them heterogeneously in Escherichia coli,to purify them,and to obtain a highly pure YopJ protein in order to lay the foundation for analysis of the protein’s structure and study of its function.Methods 1)The full-length sequence of the YopJ gene of Y.pestis was optimized based on E.coli codon preference and synthesized.2)Primers were designed.Using the gene of Y.pestis YopJ protein before and after codon optimization as a template,gene fragments containing YopJ(amino acids 22-288)and YopJ(amino acids 22-250)were ligated to the vector pGL01 3)The recombinant plasmid was transformed into the E.coli BL21 strain,and expression of the target protein was induced with IPTG at low temperatures after extended culture.4)The protein expressed by E.coli was extracted and preliminarily purified on a nickel ion column.Results Before codon optimization,the level of expression of YopJ(amino acids 22-288)and YopJ(amino acids 22-250)in E.coli was very low.After codon optimization,the level of expression was much higher,and the YopJ protein remained in soluble form in E.coli.Conclusion Expression of the YopJ protein in E.coli can be greatly increased using codon optimization.Obtaining the YopJ protein from Y.pestis in soluble form has laid the foundation for further study of the structure and function of this protein.

关 键 词：鼠疫耶尔森菌 YopJ蛋白 基因密码子优化 异源表达 

分 类 号：R378.61[医药卫生—病原生物学]