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-3 gene 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℃ and induction time 5 h,higher protein expression could be obtained.Conclusion The 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.
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