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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。
Objective To construct prokaryotic expression plasmid of recombinant multivalent human sperm epitope peptide and optimize its expression in E. coli. Methods Synthesis of multivalent human sperm epitope peptide gene by chemical synthesis. The gene was amplified by PCR, double digested with BamHI and XhoI, and cloned into GST fusion expression vector PGEX-4T-1 to obtain a recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG. SDS-PAGE electrophoresis and AlphaEase gel electrophoresis image analysis system were observed under shaking flask fermentation conditions change the composition of the medium, the induction temperature, induction time, concentration of induction and induction time and other conditions on GST-polyvalent human sperm epitope peptide Effect of fusion protein expression level. Results Recombinant multivalent human sperm epitope peptide prokaryotic expression vector was constructed. In shaking flask experiments, the optimum conditions were as follows: TB medium, induction temperature 37 ℃, in the middle and late logarithmic growth phase induction, IPTG induction final concentration 0.05 mmol / L, induction time 5 h. The optimized GST-polyvalent human sperm-epitope peptide fusion protein expressed 30.2% of total bacterial proteins and expressed mainly in soluble form. Conclusion The prokaryotic expression plasmid of recombinant multivalent human sperm epitope peptide was successfully constructed. The recombinant expression vector mainly expressed soluble GST-polyvalent human sperm epitope fusion protein in E. coli BL21 (DE3) under optimized conditions Get a higher expression level.