【摘要】 目的构建重组多价人精子表位肽原核表达质粒,优化其在大肠杆菌中的表达条件。方法用化学合成法合成多价人精子表位肽基因。该基因经PCR扩增,BamHI和XhoI双酶切后,克隆至GST融合表达载体PGEX-4T-1获得重组质粒。将该重组质粒转化E.coli BL21(DE3),经IPTG诱导表达。利用SDS-PAGE电泳和AlphaEase凝胶电泳图像分析系统,观察在摇瓶发酵条件下改变培养基组成、诱导温度、诱导时机、诱导剂浓度和诱导时间等条件对GST-多价人精子表位肽融合蛋白表达量的影响。结果构建了重组多价人精子表位肽原核表达载体。在摇瓶实验中,优化的表达条件为:TB培养基,诱导温度37℃,在菌体对数生长的中后期进行诱导,IPTG诱导终浓度0.05 mmol/L,诱导时间5 h。优化后GST-多价人精子表位肽融合蛋白的表达量占菌体总蛋白的30.2%,且主要以可溶形式表达。结论成功构建重组多价人精子表位肽原核表达质粒,重组表达载体在E.coli BL21(DE3)内主要表达可溶形式的GST-多价人精子表位肽融合蛋白,在优化条件下可获得较高的表达量。更多还原

【Abstract】 OBJECTIVE To construct the prokaryotic expression recombinant plasmid PGEX-4T-1/AG10-IR9-YLP12 and optimize the expression conditions of GST-AG10-IR9-YLP12 fusion protein in E.coli.METHODS The AG10-IR9-YLP12 gene was synthesized chemically.The target gene was amplified by polymerase chain reaction(PCR).After being digested with BamHI and XhoI,the target gene was cloned into GST fusion expression vector PGEX-4T-1.E.coli BL21(DE3)was transfected with the recombinant plasmid and GST-AG10-IR9-YLP12 fusion protein was induced by IPTG.Under the shake flask fermentation conditions,the factors that may be involved in the recombinant gene expression such as culture medium,induction temperature,induction period,inducer concentration and induction time on the expression level of target protein were investigated with SDS-PAGE electrophoresis and AlphaEase gel electrophoresis image analysis system.RESULTS The prokaryotic expression recombinant plasmid PGEX-4T-1/AG10-IR9-YLP12 was constructed.In shake flask experiments,the optimizing conditions for the expression of GST-AG10-IR9-YLP12 fusion protein were as follows:using TB as the medium,induction temperature 37℃,induction in the middle and late logarithmic growth of bacteria,the final induction concentration of IPTG 0.05mmol/L,induction time 5h.The expressed GST-AG10-IR9-YLP12 fusion protein accounted for 30.2 percent of the bacterial total protein,mainly in a soluble form.CONCLUSION The prokaryotic expression recombinant plasmid PGEX-4T-1/AG10-IR9-YLP12 has successfully been constructed.The recombinant plasmid PGEX-4T-1/AG10-IR9-YLP12 expresses GST-AG10-IR9-YLP12 fusion protein that is mainly in a soluble form.Under optimizing conditions,high expression level of the fusion protein is obtained.更多还原