酿酒酵母表达中华绒鳌蟹甲壳肽及其抗菌功能研究  

Eriocheir Sinensis Crustin Expressed in Saccharomyces Cerevisiae and Study on Its Antibacterial Function

作　　者：杜婕 张容彬 徐浩鹏 周广帅 Du Jie;Zhang Rongbin;Xu Haopeng;Zhou Guangshuai(Jiangsu Vocational College of Agriculture and Forestry,Jiangsu,212400)

机构地区：[1]江苏农林职业技术学院,江苏212400

出　　处：《当代化工研究》2021年第22期1-6,共6页Modern Chemical Research

基　　金：江苏省高等学校大学生创新创业训练计划项目(NO.202013103018Y);江苏农林职业技术学院大学生创新创业训练计划项目(NO.202013103018Y);江苏农林职业技术学院青年扶持项目(NO.2020kj011)资助。

摘　　要：抗菌肽是宿主抵御外来病原入侵时在先天免疫中的重要效应因子。作为一种抗菌蛋白,甲壳动物中的甲壳肽是其先天免疫系统的组成成分。在本研究中,中华绒鳌蟹(Eriocheir sinensis)甲壳肽Crustin(以下简称EsCrustin)在酵母真核系统中表达。先将EsCrustin的ORF克隆到质粒载体pHAC181中,然后通过同源重组技术将重组质粒整合到酿酒酵母菌株GAL1-ScRCH1的GAL1启动子下游以启动表达。重组蛋白在其C末端含有3个HA标签,经Westernblot分析表明重组蛋白MrCrustin的分子量约为20kDa。重组蛋白的最小抑菌浓度(MIC)和最小杀菌浓度(MBC)值分别为0.467-0.921μM和0.887-1.135μM。重组蛋白几乎可以凝集除铜绿假单胞菌外的多种革兰氏阴性及阳性细菌。此外,重组蛋白EsCrustin对嗜水气单胞菌有明显的抑制作用,在琼脂平板上出现较大抑菌圈。以上研究结果表明,本研究中酵母表达的重组蛋白对部分革兰氏阳性菌和革兰氏阴性菌均表现出抗菌活性。因此,重组蛋白EsCrustin是一种具有杀菌功能的抗菌肽,它在参与甲壳类先天免疫,抵抗病原细菌和病毒的入侵过程中发挥重要作用。Antimicrobial peptides are important effectors in the host innate immune response against microbial invasion.As an antimicrobial protein,crustin had been described in crustaceans as a component of the innate immune system.In the present study,the Eriocheir sinensis Crustin was expressed in the yeast eukaryotic system.The ORF of EsCrustin was cloned into the plasmid vector pHAC181,then the recombinant plasmids was integrate into the GAL1 promoter downstream of Saccharomyces cerevisiae strain GAL1-ScRCH1 by homologous recombination technique.The resulting recombinant EsCrustin consist of a 3×HA-tag at its C-terminal while Western Blot analysis indicated the molecular weights of the recombinant EsCrustin was estimated to be about 20 kDa.The MIC and MBC values of the recombinant protein were examined in ranging from 0.467-0.921μM and 0.887-1.135μM,respectively.The recombinant EsCrustin protein agglutinated almost all bacteria except Pseudomonas aeruginosa.Furthermore,the agar plate diffusion method also showed recombinant EsCrustin can inhibition the Aeromonas hydrophila for the inhibition zone appeared obviously.The results demonstrated the recombinant protein exhibited antimicrobial activity against both some Gram-positive bacteria and Gram-negative bacteria.All of these results suggest that EsCrustin is a potential antimicrobial peptide,which is involved in crustacean innate immune system to resist the invasion of pathogenic bacteria and virus.

关 键 词：酿酒酵母 真核表达 中华绒鳌蟹 甲壳肽 抗菌功能 

分 类 号：S[农业科学]