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目的诱导毕赤酵母(Pichia pastoris)分泌表达表达致倦库蚊(Culex quinquefasciatus)防御素(defensin)成熟肽,为进一步研究致倦库蚊defensin的抗病原作用、机理及其应用奠定基础。方法利用生物信息和RT-PCR技术克隆了编码致倦库蚊防御素的成熟肽全基因序列,并克隆到毕赤酵母甲醇诱导型分泌表达载体pPICZα-A的α-factor信号肽序列的下游,构建成pPICZα-A-defensin重组表达载体,表达载体经SacⅠ线性化处理后电击转化毕赤酵母X-33感受态细胞,转化子经Zeocin抗性筛选,菌落PCR,Mut表型鉴定和Tricine-SDS-PAGE分析,成功获得了能够有效表达重组defensin的pPICZα-A-Defensin/X-33甲醇利用表型工程菌。结果在甲醇诱导24 h后pPICZα-A-Defensin/X-33的工程菌分泌表达了预期Mr8 200大小的致倦库蚊防御素成熟重组肽。结论致倦库蚊防御素可以在毕赤酵母中诱导分泌表达。
Objective To induce Pichia pastoris to express and express mature peptide of Culex quinquefasciatus defensin, which will lay the foundation for the further study on the antipathogenic effect, mechanism and application of Defensin from Culex quinquefasciatus. Methods The full-length cDNA sequence of the mature peptide encoding Culex pipiens quinquefasciatus was cloned by bioinformatics and RT-PCR techniques and cloned into the downstream of α-factor signal peptide sequence of pichia pastoris methanol-inducible secretion expression vector pPICZα-A. Recombinant pPICZα-A-defensin expression vector was constructed. The expression vector was transformed into Pichia pastoris X-33 by electroporation. The transformants were screened by Zeocin resistance, colony PCR, Mut phenotype and Tricine-SDS -PAGE analysis, successfully obtained pPICZα-A-Defensin / X-33 methanol utilization engineering bacteria capable of effectively expressing recombinant defensin. RESULTS: The pPICZα-A-Defensin / X-33 engineered bacteria secreted and expressed Mr8 200-sized mature Z. marcescens defensin at 24 h after methanol induction. Conclusion Culex pipiens quinquefasciatus can induce secretion in Pichia pastoris.