天蚕素A和溶菌酶杂合肽在毕赤酵母中重组表达及其活性分析

Recombinant Expression of Cecropin A and Lysozyme Heteropeptide in Pichia pastoris and Its Activity Analysis

【目的】试验旨在利用毕赤酵母表达新型天蚕素A和溶菌酶杂合肽(CecA-Lyz),并初步探讨重组杂合肽的抑菌活性。【方法】根据毕赤酵母密码子偏好性优化杂合肽CecA-Lyz的编码基因,分析CecA-Lyz蛋白的理化性质,并将优化后的基因插入到质粒pPIC9K-HSA中,构建N-端含有6×His标签和HSA DⅠ&DⅡ区的重组质粒pPIC9K-HSA-CecA-Lzy。利用PmeⅠ线性化重组质粒并电转化毕赤酵母GS115感受态细胞,利用酵母浸出粉胨葡萄糖培养基(YPD)+遗传霉素(G418)筛选重组CecA-Lyz蛋白的高表达菌株,并摸索重组CecA-Lyz蛋白的最适诱导条件,包括甲醇浓度和诱导时间,验证CecA-Lyz的抑菌活性。【结果】经过密码子优化后,CecA-Lyz基因序列的GC含量为48.9%,密码子适应指数(CAI)为0.87,适合在毕赤酵母中分泌表达。重组CecA-Lyz蛋白的预期分子质量为20.8 ku,理论等电点为9.62,理化性质较为稳定。此外,本试验成功地将优化后的目的基因构建到表达载体上并在毕赤酵母中表达重组CecA-Lyz蛋白。重组CecA-Lyz蛋白在摇瓶中的最适诱导甲醇浓度为2.5%,最适诱导时间为72 h。另外,本试验通过TEV酶切的方式获得了N-端不含HSA DⅠ&DⅡ区的重组融合肽。通过平板计数法和牛津杯法初步验证重组CecA-Lyz对大肠杆菌具有抑菌活性,包括抑制养鸭场分离的耐药大肠杆菌的生长,抑菌直径达到1.45 cm,但是对沙门菌和葡萄球菌没有明显的抑制作用。【结论】重组CecA-Lyz融合肽可在毕赤酵母中稳定表达,得到无HSA标签的融合肽,其具有抑制大肠杆菌的作用,为进一步开发抗菌肽提供研究思路和参考。

【Objective】This experiment aimed to produce a novel cecropin A and lysozyme fusion peptide(CecA-Lyz)using Pichia pastoris and to explore its antibacterial activity.【Method】The gene encoding the fusion peptide CecA-Lyz was optimized according to the codon preference of Pichia pastoris and the physicochemical properties of CecA-Lyz was analyzed.The optimized CecA-Lyz gene was inserted into the plasmid pPIC9K-HSA to construct the recombinant plasmid pPIC9K-HSA-CecA-Lyz,which contained a 6×His tag and HSA DⅠ&DⅡregion at N-terminal.The recombinant plasmid was linearized using PmeⅠand electrotransformed into the Pichia pastoris GS115 competent cell.The recombinant strain of high-level expressed CecA-Lyz protein was selected in YPD+G418 plate.Moreover,the optimum induction conditions of recombinant CecA-Lyz protein,including the methanol concentration and induction time were explored,and the antibacterial activity of recombinant CecA-Lyz was verified.【Result】After codon optimization,the GC content of the CecA-Lyz gene was 48.9%,and the codon adaptation index(CAI)was 0.87,which was suitable for secretory expression in Pichia pastoris.The expected molecular weight of CecA-Lyz protein was 20.8 ku,with a theoretical isoelectric point of 9.62 and relatively stable physicochemical properties.Moreover,the optimized CecA-Lyz gene was successfully contructed into expression plamid and produced recombinant CecA-Lyz fusion protein in Pichia pastoris.The optimal methanol concentration for the induction of recombinant CecA-Lyz protein was 2.5%and the optimal induction time was 72 h.In addition,the recombinant fusion peptide without N-terminal HSA DⅠ&DⅡwas obtained by TEV digestion.The recombinant CecA-Lyz was initially shown to have antibacterial activity against Escherichia coli by plate counting and Oxford cup methods,including inhibition of the growth of drug-resistant Escherichia coli isolated from duck farms,with the inhibitory diameter up to 1.45 cm,but no significant inhibition against Salmonella and Staphylococcus.【Conclusion】The recombinant CecA-Lyz fusion peptide could be stably produced in Pichia pastoris to obtain the fusion peptide without HSA tag and with the effect of inhibiting growth of Escherichia coli,which provided research ideas and references for further development of antimicrobial peptides.