共表达MEL和Ec-cLYZ重组表达质粒的构建及其重组蛋白的抑菌效果评价

Construction of recombinant plasmid co-expressing MEL and Ec-cLYZ and evaluation of its recombinant protein’s antibacterial activity

为了表达一种高效低毒的广谱抑菌蛋白,试验通过T2A连接肽连接蜂毒肽(MEL)与斜带石斑鱼c型溶菌酶(Ec-cLYZ)基因,并克隆至pPICZαA质粒上构建重组表达质粒;将鉴定正确的重组表达质粒用限制性内切酶SacⅠ线性化后电转化至毕赤酵母表达菌株GS115感受态细胞中,构建毕赤酵母表达菌株;利用0.5%甲醇诱导表达,收集上清液进行冻干浓缩并纯化,采用Western-blot法检测重组蛋白的表达情况,BCA蛋白浓度测定试剂盒测定重组蛋白浓度,试管二倍稀释法分别检测不同浓度重组蛋白对兔红细胞的溶血活性,牛津杯法评价重组蛋白的抑菌效果。结果表明:成功构建出重组表达质粒pPICZαA-Ec-cLYZ-MEL、pPICZαA-Ec-cLYZ和pPICZαA-MEL及毕赤酵母表达菌株GS115/pPICZαA-Ec-cLYZ-MEL、GS115/pPICZαA-Ec-cLYZ和GS115/pPICZαA-MEL;经甲醇诱导,GS115/pPICZαA-Ec-cLYZ-MEL表达出分子质量为16.5,3.4 ku的重组蛋白,GS115/pPICZαA-Ec-cLYZ表达出分子质量为16.5 ku的重组蛋白,GS115/pPICZαA-MEL表达出分子质量为3.4 ku的重组蛋白;上清液中重组蛋白Ec-cLYZ-MEL、Ec-cLYZ、MEL的浓度分别为35.49,28.73,27.31 mg/L;重组蛋白Ec-cLYZ-MEL、Ec-cLYZ、MEL在浓度为150 mg/L时对兔红细胞的溶血率分别为2.3%、2.9%、79.0%;停乳链球菌对重组蛋白Ec-cLYZ-MEL高度敏感,表皮葡萄球菌对重组蛋白Ec-cLYZ-MEL中度敏感,金黄色葡萄球菌和大肠杆菌对重组蛋白Ec-cLYZ-MEL低度敏感,且重组蛋白Ec-cLYZ-MEL对测试菌株的抑菌效果优于重组蛋白Ec-cLYZ和MEL。说明重组蛋白Ec-cLYZ-MEL对红细胞无溶血活性且具有良好的抑菌活性。

In order to express a broad-spectrum bacteriostatic protein with high efficiency and low toxicity, epinephelus coioides melittin(MEL) gene of grouper and c type lysozyme(Ec-cLYZ) gene were linked via T2A and cloned into the vector pPICZαA to construct the recombinant plamids pPICZαA-Ec-cLYZ-MEL. The correctly identified recombinant expression plasmid was linearized by restriction endonuclease SacⅠ and was electrotransformed into receptive cells of Pichia pastoris GS115 to construct Pichia pastoris expression strains. The recombinant strains were induced by 0.5% methanol. The supernatant was collected for freeze-drying, concentration and purification. The expression of the recombinant protein was verified by Western-blot. The protein concentration was determined by BCA method. Hemolytic activity of different concentrations of recombinant protein on rabbit erythrocytes was detected by test tube double dilution method. The antibacterial activity of the recombinant protein was evaluated by Oxford cup method. The results showed that the recombinant plasmids pPICZαA-Ec-cLYZ-MEL, pPICZαA-Ec-cLYZ and pPICZαA-MEL were successfully constructed;the expression strains GS115/pPICZαA-Ec-cLYZ-MEL, GS115/pPICZαA-Ec-cLYZ and GS115/pPICZαA-MEL. After induction, GS115/pPICZαA-Ec-cLYZ-MEL expressed protein with molecular masses of 16.5, 3.4 ku, GS115/pPICZαA-Ec-cLYZ expressed protein with molecular masses of 16.5 ku, and GS115/pPICZαA-MEL expressed protein with molecular masses of 3.4 ku. The concentrations of recombinant protein Ec-cLYZ-MEL, Ec-cLYZ, and MEL in the supernatant were 35.49, 28.73, 27.31 mg/L, respectively. The hemolysis of rabbit erythrocytes by recombinant protein Ec-cLYZ-MEL, Ec-cLYZ, and MEL treated at the concentration of 150 mg/L was 2.3%, 2.9%, and 79.0%, respectively.Streptococcus alactiae was highly sensitive to recombinant protein Ec-cLYZ-MEL,Staphylococcus epidermidis moderately sensitive to recombinant protein Ec-cLYZ-MEL,Staphylococcus aureus and Escherichia coli were moderately sensitive to recombinant protein Ec-cLYZ-MEL, and recombinant protein Ec-cLYZ-MEL had better antibacterial zone effects than recombinant protein Ec-cLYZ-MEL. The results indicated that the recombinant protein Ec-cLYZ-MEL had no hemolytic activity on erythrocytes and had good bacteriostatic activity.