三疣梭子蟹PtCrustin3成熟肽在毕赤酵母中的重组DNA表达

Recombinant DNA Expression of PtCrustin3 Mature Peptide from Portunus trituberculatus in Pichia pastoris

Crustin抗菌肽是一类由甲壳动物表达的具有抗菌作用的小分子肽,它们在动物抵御病原微生物侵染的免疫系统中发挥了重要作用,被认为是替代抗生素的良好候选者。Crustin的N端含有一个信号序列,与之相连接的C端为成熟肽序列,内含一个WAP(Whey acidic protein)结构域,为Crustin的活性区域;根据信号肽与WAP结构域之间的结构差异,可以将Crustin分为三种类型。本文以三疣梭子蟹(Portunus trituberculatus)I型Crustin的一个同工型"Pt Crustin3"的成熟肽(mPtCrustin3)为研究对象,通过RT-PCR从鳃中克隆到编码mPtCrustin3的cDNA,并通过巢式PCR对其添加EcoRⅠ和XbaⅠ酶切位点以及6×His标签。以pPICZaA为真核表达载体,成功构建了重组表达载体pPICZaA-mPtCrustin3,电转入毕赤酵母X-33,在29℃、250 r/m的条件下,经1.0%甲醇诱导表达96 h后,获得的表达产物经Tricine-SDS-PAGE分析,证明分子量约为11.6 k D的重组体mPtCrustin3在毕赤酵母X-33中被成功表达。

Crustins are series of small molecular antimicrobial peptides expressed mainly in crustaceans.They play important roles in the animal immune system against pathogenic microorganism infection,thus,they are considered to be good candidates to replace antibiotics.The N-terminal of Crustin contains a signal sequence and its C-terminal contains a mature peptide sequence including the WAP(whey acidic protein)domain.The mature peptide is responsible for bioactivity of Crustin.According to structural differences of the region between signal peptide and WAP domain,Crustin can be divided into three types.The present study focused on the mPtCrustin3,the mature peptide of an isoform for Crustin typeI from Portunustri tuberculatus.The cDNA encoding mPtCrustin3was cloned from gill of P.tuberculatus by RT-PCR.EcoR I site,and XbaⅠsite and6×His tag were added to5’and3’of this cDNA,respectively,by nested PCR.This target fragment was ligated to pPICZ?A vector to construct a recombinant expression vector pPICZ?A-mPtCrustin3.Then thepPICZ?A-mPtCrustin3was transformed into competent Pichia pastoris X-33cells.Recombinant mPtCrustin3was inducedwith1.0%(V/V)methanol at29℃,250r/m for96h.Tricine-SDS-PAGE analysis demonstrated that the expressed product was the recombinant mPtCrustin3with molecular weight of about11.6kD.