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目的在毕赤酵母中分泌表达具有抗菌活性的杂合抗菌肽Cecropin P1-Dermaseptin S4(CP1-DS4)。方法根据毕赤酵母密码子的偏爱性优化Cecropin P1和Dermaseptin S4两抗菌肽基因序列,通过SOE-PCR拼接,构建克隆载体pMD18T-cp1-ds4并测序;pMD18T-cp1-ds4经EcoRⅠ、XbaⅠ双酶切后构建重组表达载体pPICZαA-cp1-ds4,测序验证开放阅读框(ORF)正确后经SacⅠ线性化处理,然后电转化毕赤酵母X-33(800V,1mm电转化杯,15s电击1次)并利用Zeocin筛选阳性重组子。重组子接种BMGY、BMMY培养基,采用5%的甲醇诱导表达,采用Tricine-SDS-PAGE分析表达上清,表达上清浓缩后进行抑菌试验。结果在本研究中拼接引物经SOE-PCR拼接后得到优化的长度为120bp的Dermaseptin S4基因基因片段,该片段与Cecropin P1基因连接形成约197bp杂合抗菌肽片段,成功构建pPICZαA-cp1-ds4表达载体,电转化获得重组酵母菌pPICZαA-cp1-ds4/X-33,经诱导后可产生分子质量单位约为9.7ku的目的多肽,该多肽对大肠埃希菌和金黄葡萄球菌均有抑制作用。结论杂合肽CP1-DS4可以在毕赤酵母X-33中融合分泌表达,且具有抗菌活性。
Aim To express Cecropin P1-Dermaseptin S4 (CP1-DS4) with antibacterial activity in Pichia pastoris. Methods The sequences of Cecropin P1 and Dermaseptin S4 were optimized by the codon usage of Pichia pastoris. The cloned vector pMD18T-cp1-ds4 was cloned by SOE-PCR. The pMD18T-cp1-ds4 was digested by EcoRⅠ, XbaⅠ The recombinant expression vector pPICZαA-cp1-ds4 was constructed and sequenced. The ORF of the recombinant plasmid was verified by sequencing and then linearized by SacⅠ. The recombinant plasmid was transformed into Pichia pastoris X-33 (800V, Zeocin was used to screen positive recombinants. The recombinant cells were inoculated with BMGY and BMMY medium and induced with 5% methanol. The supernatant was analyzed by Tricine-SDS-PAGE. The supernatant was concentrated and then tested for antibacterial activity. Results In the present study, splicing primers were spliced by SOE-PCR to obtain a 120 bp fragment of Dermaseptin S4 gene. The fragment was linked with Cecropin P1 gene to form an approximately 197 bp hybrid antimicrobial peptide fragment. The pPICZαA-cp1-ds4 expression was successfully constructed The recombinant plasmid pPICZαA-cp1-ds4 / X-33 was obtained by electrotransformation into E.coli and E.coli. The recombinant plasmid pPICZαA-cp1-ds4 / X-33 was induced by electroporation to produce a target polypeptide with a mass unit of about 9.7ku, which could inhibit both Escherichia coli and Staphylococcus aureus. Conclusion The hybrid peptide CP1-DS4 can be fused and secreted in Pichia pastoris X-33 and has antibacterial activity.