【摘要】 目的 比较抗菌肽（antimicrobial peptide,AMP）CM25-6在毕赤酵母与酿酒酵母2种表达体系中的表达效果及抑菌活性，旨在为其高效异源表达体系的筛选提供依据。方法 将CM25-6基因分别亚克隆至毕赤酵母表达载体pGAPZαA和酿酒酵母表达载体pYES2/CT/α-factor中，构建重组质粒，并分别转化至毕赤酵母SMD1168和酿酒酵母INVSc1中，Western blot法鉴定重组蛋白的特异性；在YPD培养基中进行扩大培养，经SDS-PAGE及BCA法动态检测重组蛋白的表达情况，并采用牛津杯法测定发酵上清液的抑菌活性。重组蛋白经Ni²⁺螯合亲和层析纯化后，通过微量肉汤稀释法测定纯化产物的最小抑菌浓度（minimum inhibitory concentration,MIC）。结果 经PCR法和测序鉴定，2种重组酵母构建成功。重组毕赤酵母及重组酿酒酵母分泌的重组蛋白均可与小鼠抗His-tag单克隆抗体发生特异性结合，在YPD培养基中扩大表达后的相对分子质量均约8 480，且表达量于发酵60 h时达最高，分别达2.13和1.02 g/L。重组蛋白纯化产物对大肠埃希菌、铜绿假单胞菌和无乳链球菌均具有显著抑菌活性，且来源于毕赤酵母的纯化产物抑菌活性更高，其MIC（7.85～15.7μmol/L）低于来源于酿酒酵母的纯化产物（15.05～30.2μmol/L）。结论 AMP CM25-6在毕赤酵母系统中的表达量及产物抗菌活性均优于酿酒酵母系统，表明毕赤酵母更适合用于该AMP的异源表达与生产开发。更多还原

【Abstract】 ObjectiveTo compare the expression of antimicrobial peptide（AMP） CM25-6 in two expression systems of Pichia pastoris and Saccharomyces cerevisiae and its antimicrobial activity, so as to provide a basis for the screening of its efficient heterologous expression system.MethodsThe CM25-6 gene was subcloned into the Pichia pastoris expression vector pGAPZαA and the Saccharomyces cerevisiae expression vector pYES2/CT/α-factor, and the recombinant plasmids were constructed and transformed into Pichia pastoris SMD1168 and Saccharomyces cerevisiae INVSc1, respectively. The recombinant proteins were identified for specificity by Western blot. The culture was expanded in YPD medium, the expression of the recombinant proteins was dynamically detected by SDS-PAGE and BCA method, and the antibacterial activity of the fermentation supernatant was determined by Oxford cup method. After the recombinant proteins were purified by Ni²⁺chelating affinity chromatography, the minimum inhibitory concentration（MIC） of the purified products was determined by micro broth dilution method.ResultsThe two recombinant yeasts were successfully constructed as identified by PCR and sequencing. The recombinant proteins secreted by recombinant Pichia pastoris and recombinant Saccharomyces cerevisiae exhibited specific binding to mouse anti-His-tag monoclonal antibody. The relative molecular mass was about 8 480 for both recombinant proteins after expanded expression in YPD medium, and the expression reached the highest levels of 2. 13 and1. 02 g/L at 60 h of fermentation, respectively. The purified products of the recombinant proteins showed significant antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Streptococcus agalactiae, and the purified product derived from Pichia pastoris had higher antibacterial activity, with the MIC value（7. 85-15. 7 μmol/L） lower than that of the purified product derived from Saccharomyces cerevisiae（15. 05-30. 2 μmol/L）.ConclusionThe expression level and antibacterial activity of AMP CM25-6 in Pichia pastoris system are better than those in Saccharomyces cerevisiae system, indicating that Pichia pastoris is more suitable for heterologous expression and production development of AMP CM25-6.更多还原