诱导毕赤酵母分泌表达致倦库蚊防御素Defensin成熟肽

Secretory expression of Culex quinquefasciatus defensin induced in Pichia pastoris

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。

This study aimed to induce secretory expression of Culex quinquefasciatus defensin in Pichia pastoris, which would provided a fundamental basis for further studying its functions in salivary of Aedes albopictu, The cDNA of the mature defensin from Culex quinquefasciatus was cloned by the bioinformation and RT-PCR. Then the Defensin gene was inserted into the downstream of α- factor signal sequence of a Pichia pastoris secreting expression vector pPICZα-A and constructed a recombinant expression vector pPICZα-A-defensin. After being linearized by Sac I restriction enzyme, pPICZα-A-defensin vector was transformed into Pichia pastoris X-33 by eleetroporation. At last, the recombinant strains pPICZα-A-defensin/X-33 was identified by Zeocin resistance screening, PCR, Mut phenotype screening, and Tricine-SDS-PAGE. We found that a recombinant defensin protein of Mr 8 200 was induced and expressed by methanol in supernatants of Pichiapastoris in 24 h after the shake bottle fermentation. In conclusion, defensin of Culex quinquefasciatus in Pichia pastoris may be efficiently expressed and secreted.