【摘要】 目的 :利用分子生物学技术和方法将pRSET -B质粒改建为带有绿色荧光蛋白 (GFP)突变体基因 (GFP -S6 5T)的新型瞬时表达载体pRSET -EGFP ,并在E .coli.BL2 1中得到GFP基因的高效表达。方法 :PCR法从pEGFP质粒克隆GFP -S6 5TcDNA并在 5’末端引入KpnI的位点。将扩增出来的GFP -S6 5T基因和pRSET -B质粒用HindIII和KpnI双酶切后连接构成重组质粒。用化学法把重组质粒转化到E .coli.BL2 1中 ,培养发酵液OD540 =0 .4时加入IPTG诱导GFP -S6 5T基因转录和表达 ,合成绿色荧光蛋白。还对诱导条件进行了优化 ,发酵液OD540 =0 .4加入IPTG可以得到最优表达。结果 :通过Ni2 + 柱亲和层析 ,纯化得到绿色荧光蛋白 ,SDS-PAGE电泳检测 ,分子量为 2 7kDa ,与文献报道值一致。这说明Ni2 + 柱能够有效的纯化表达产物。结论 :成功构建了新型瞬时表达载体pRSET -EGFP ,并且在E .coli.BL2 1中得到高效表达更多还原

【Abstract】 Objective:Using molecular biology method and techniques,the plasmid pRSET-B was constructed to a novelty transient expression vector pRSET-EGFP carrying the mutant gene of green fluorescent protein （GFP-S65T）,and it was expressed in E.coli BL21.Methods:GFP-S65T cDNA was cloned from plasmid pEGFP with Kpn I site attached at the 5’termination of cDNA fragment by PCR.The PCR product of GFP-S65T gene and the plasmid pRSET-B were digested with Hind III and Kpn I,and then ligated as a novelty plasmid.The recombinant plasmid was transformed into E.coli.BL21 by the chemical method.Added IPTG to induce the GFP-S65T transcription and expression,synthesize the green fluorescent protein.The optimum condition of expression was optimized,when the OD₅₄₀ of the culture was 0.4,the expression was the maximum.Results:The protein was purified by the affinity chromatography on a Ni²⁺ column,and the expression products were identified by SDS-PAGE gel electrophoresis.The molecular weight of the protein is about 27kD,was in accord with the referrence,which indicates that the Ni²⁺ column could purify the product efficiently.Conclusion:A novelty transient expression vector pRSET-EGFP was constructed successfully and expressed effectively in E.coli.BL21.更多还原