重组人可溶性Tim-3原核表达载体的构建及其重组蛋白的优化表达  

Construction and Optimized Expression of Recombinant Human sTim-3-EGFP Fusion Protein

作　　者：白少云[1] 卿吉琳[2] 刘振[1] 谭源福[1] 陈治中[3] 

机构地区：[1]广西医科大学第一附属医院神经外科,南宁530021 [2]广西壮族自治区人民医院妇产科,南宁530021 [3]广西壮族自治区人民医院检验科,南宁530021

出　　处：《华中科技大学学报（医学版）》2017年第3期276-280,290,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81260007);广西卫生厅课题资助项目(No.Z2012316)

摘　　要：目的构建人可溶性Tim-3(sTim-3)的重组质粒表达载体pET28a-sTim-3-EGFP,在大肠埃希菌中进行诱导表达并对其表达条件进行优化。方法取健康人外周血提取总RNA,RT-PCR扩增Tim-3基因胞外段序列,克隆入已构建的表达载体pET28a-EGFP中,鉴定阳性克隆。重组质粒转化BL21(DE3),通过调整IPTG浓度、诱导时机、诱导温度与诱导时间优化蛋白表达条件。结果成功构建原核表达载体pET28a-sTim-3-EGFP,并转化BL21(DE3)进行诱导表达。温度和IPTG浓度对蛋白表达量影响不大;在培养至A600nm值约为0.6~0.8时于25℃诱导5h可获得较高的蛋白表达。结论成功表达可溶性重组蛋白sTim-3-EGFP,为该蛋白的进一步纯化生产及应用奠定了良好基础。Objective To construct and optimize the condition for inducing expression of recombinant human sTim-3-EGFP in E.coli.Methods Total RNA was extracted from peripheral blood of healthy people.The extracellular fragment of human Tim-3gene was amplified by RT-PCR and cloned into the constructed prokaryotic expression vector pET28a-EGFP,and identified by PCR,double digestion and sequencing.The recombinant plasmid was transformed into BL21（DE3）to optimize protein expression by adjusting IPTG concentration,induction starting time,induction temperature and induction expression time.Results The prokaryotic expression vector pET28a-sTim-3-EGFP was successfully constructed and transformed into BL21（DE3）for expression.The temperature and IPTG concentration had little effect on the protein expression.When the A600 nm value was about 0.6-0.8,temperature 25°C and induction time 5h,higher protein expression could be obtained.ConclusionThe human sTim-3-EGFP is successfully constructed and expressed in vitro.This study provides a good experimental basis for further purification,production and application of Tim-3.

关 键 词：TIM-3 原核表达 增强绿色荧光蛋白 融合蛋白 

分 类 号：R394[医药卫生—医学遗传学]