摘要
Crustin主要分布于甲壳动物中,是一种富含半胱氨酸的小分子抗菌肽,在甲壳动物的先天免疫系统中发挥重要作用。根据crustin的一级结构特征可以将其分为不同的类型,本文以三疣梭子蟹(Portunus trituberculatus)的PtCrustin2成熟肽为研究对象,通过构建毕赤酵母表达系统,以期实现PtCrustin2成熟肽在毕赤酵母中的重组DNA表达。首先,从其鳃中提取总RNA,通过RT-PCR得到编码PtCrustin2成熟肽的cDNA(m Pt Crustin2),并在其5'和3'端分别引入EcoRⅠ和NotⅠ限制性内切酶位点;然后将此片段与表达载体p PIC9K连接,构建重组表达载体pPIC9K-m Pt Crustin2;电转入毕赤酵母GS115细胞后,以不同浓度的G418筛选到高拷贝酵母转化子,经0.5%甲醇诱导表达和固化金属离子亲和层析(IMAC)分离,获得了纯化的重组体mPt Crustin2,Tricine-SDS-PAGE分析显示其分子量约10.5 k Da;抑菌实验证明重组体mPtCrustin2对金黄色葡萄球菌和铜绿假单胞菌具有一定的抑菌效果。本研究首次实现了三疣梭子蟹Pt Crustin2成熟肽在毕赤酵母中的重组DNA表达,为进一步研究提供参考。
Mainly distributing in crustaceans＇ cells,crustins are small cysteine-rich antimicrobial peptides playing important roles in innate immune system of crustaceans. Crustins can be divided into several types according to their first structures. This paper takes Pt Crustin2 mature peptide from swimming crab（ Portunus trituberculatus） as researching target in order to realize recombinant DNA expression of the Pt Crustin2 mature peptide in Pichia pastoris,through the construction of a P. pastoris expression system. First,total RNA was extracted from the gill of swimming crab. The c DNA encoding m Pt Crustin2 mature peptide was amplified by RT-PCR,and induced EcoR Ⅰ and Not Ⅰrestriction endonuclease sites onto its 5＇ and 3＇ ends respectively. Then linked this target fragment onto the expression vector p PIC9 K to construct the recombinant expression vector p PIC9K-m Pt Crustin2,after it was e-transformed into P. pastoris GS115 cell,and after screened and cultured with YPD medium containing different concentration of G418,high-copying yeast transformant was obtained and induced to express recombinant m Pt Crustin2 through 0. 5% methanol and immobilized metal affinity chromatography（ IMAC） to isolate and obtained purified recombinant m Pt Crustin2.Tricine-SDS-PAGE analysis indicated its molecular mass was 10. 5 k Da. The inhibition experiment had proved that the recombinant m Pt Crustin2 had a certain inhibition effect against Staphylococcus aureus and Pseudomonas aeruginosa.The present study for the first time realized the expression of recombinant DNA of mature peptide from swimming crab（ P. trituberculatus） in P. pastoris that lays foundation for further after-study.
出处
《微生物学杂志》
CAS
CSCD
2016年第5期26-31,共6页
Journal of Microbiology
基金
上海市教育委员会产学研项目(15CXY30)
农业部都市农业(南方)重点实验室开放基金项目(UA201307)
