rhIL-7生物信息学分析及原核系统分泌表达  

Bioinformatics Analysis of rhIL-7 and Expression of Prokaryotic System

作　　者：令瑛 徐雷[1] 李向茸 张海霞 马忠仁 冯若飞[2,3] LING Ying;XU Lei;LI Xiong rong;ZHANG Hai xia;MA Zhong ren;FENG Ruo fei(College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China;Engineering &Technology Research Center for Animal Cells, Lanzhou 730030, China;Key Bioengineering and Technology Laboratory of Nationality Commission, Northwest Minzu University, Lanzhou 730030, China)

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州730030 [2]甘肃省动物细胞工程技术研究中心,甘肃兰州730030 [3]西北民族大学生物工程与技术国家民委重点实验室,甘肃兰州730030

出　　处：《西北民族大学学报（自然科学版）》2017年第2期60-69,共10页Journal of Northwest Minzu University(Natural Science)

基　　金：中央高校基本科研业务费专项资金项目(31920150075);中央专项研究生科研创新项目(Yxm2015205)

摘　　要：通过生物信息学分析并预测hIL7糖基化位点、磷酸化位点、信号肽预测、跨膜蛋白、亲疏水性,构建pGEX-4T-1-hIL-7原核表达载体,转化至大肠杆菌LB21(DE3),优化hIL-7表达条件,利用SDS-PAGE电泳和WesternBlot鉴定重组蛋白.表明hIL-7与猴的同源性一致,是一个含有13个磷酸化位点及3个糖基化位点,具有信号肽的亲水性分泌蛋白.重组载体经DNA测序,显示包含正确的hIL-7编码序列.将pGEX-4T-1-hIL-7重组载体转入大肠杆菌后,经IPTG诱导表达融合蛋白(含GST标签),相对分子量约为43 000 Da,表达形式为包涵体表达.采用单因素方法对诱导时间、诱导温度、IPTG浓度进行考查.在此基础上进行3因素3水平正交实验的统计学优化,得到的最优表达条件为37℃、6 h、IPTG浓度0.5 mmol/L.Construction of recombinant human interleukin7(rhIL7)prokaryotic expression vector,and statistically for rhIL7prokaryotic expression conditions optimization studies to improve yield of rhIL7.Methods:The Bioinformatics analysis and prediction of hIL7glycosylation sites,phosphorylation sites,signal peptide prediction,transmembrane protein,hydrophobicity.Then,the hIL7gene was cloned into pGEX4T1plasmid to construct a prokaryotic expression vector.Th combinant protein was expressed in E.coli BL21(DE3)strain and identified with SDS PAGE and Western-Blot.Results:The hIL7is consistent with the homology of Papio anubis and is a hydrophilic secretory protein with13phosphorylation sites and3glycosylation sites with a signal peptide.The prokaryotic confirmed by sequencing.The expression vector was transformed into E.coli and was expressed in after induction with IPTG.The relative molecular weight of GST hIL7was about43000.The form of expression is an inclusion body expression.And the prokaryotic expression conditions,inoculation amount,IPTG concentration,culture temperature and culture time,which are the key factors for bacteria fermentation,were optimized for improving yield of hIL7.The effects of these four parameters on the hIL7protein yield were studied by single-factor test,respectively.The key parameters of fermentation conditions were studied through the orthogonal experimental at3factors3levels.The expression of the optimal conditions is at37℃,IPTG concentration of0.5mmol/L for6h.Conclusion:hIL7prokaryotic expression vector was constructed successfully,and have determined the optimal expression conditions.

关 键 词：重组人白介素7 原核表达 生物信息学 条件优化 单因素法 正交实验 

分 类 号：S435.131[农业科学—农业昆虫与害虫防治]