菌丝霉素的串联表达及其菌内抑菌活性分析  

Tandom Expression and Bacteriostatic Activity in vivo of Plectasin

作　　者：管韬[1] 余冰[1,2] 陈代文[1,2] 韩国全[1] 黄志清[1] 毛湘冰[1] 郑萍[1] 毛倩[1] 

机构地区：[1]四川农业大学动物营养研究所,雅安625014 [2]四川农业大学动物抗病营养教育部重点实验室,雅安625014

出　　处：《畜牧兽医学报》2012年第4期620-626,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：生物饲料核心生物技术及产业化关键技术研究与应用(2010GZ0193);教育部"长江学者和创新团队发展计划"项目(IRTO555-5)

摘　　要：构建菌丝霉素(Plectasin,PT)的串联表达菌株,研究各串联体融合表达产物的菌内抑菌活性。根据大肠杆菌表达系统对密码子的偏爱性,人工合成优化后PT基因,构建表达质粒pGEX-PT/pe;并利用同尾酶法分别构建PT基因二聚体表达质粒pGEX-PT/pes2与PT基因三聚体表达质粒pGEX-PT/pes3;然后分别将各表达质粒转化入BL21(DE3)进行IPTG的诱导表达,对各表达蛋白进行SDS-PAGE、Western Blot分析,并测定各融合表达产物的菌內抑菌活性。测序结果表明,串联体扩增到产物大小分别为135、261和387bp的目的基因片段;SDS-PAGE分析各串联表达菌表达的融合蛋白相对分子质量分别约为30、35和40ku,其产量分别占细菌总蛋白的64.5%、26.4%和25.3%。Western Blot分析表明各表达蛋白均具有良好的免疫反应性;菌内抑菌结果显示,各融合表达产物均表现对DH5α的弱抑制作用,但随PT基因串联数的增加,融合表达产物的抑菌效果显著提高,且抑菌时间延长。各串联表达菌株的成功构建与其表达产物的宿主菌抑制作用,为菌丝霉素高效抑菌产品的开发奠定了基础。The study was conducted to construct the tandom expression strains of the plectasin,and to analyze the bacteriostatic activity of the tandom expression products.According to the codon preference in E.coli expression system,the gene of plectasin was synthesized and inserted into plasmid pGEX-4T-1 to construct the recombinant plasmid pGEX-PT/pe.The co-adhesive end restriction and ligation strategy was used to add the repeat unit one by one.The recombinant vectors pGEX-PT/pes2 and pGEX-PT/pes3 with two and three repeat units of plectasin were constructed respectively.The expression vectors of tandom repeat plectasin gene were respectively transformed into E.coli DH5α and induced with IPTG.The expressed protein was detected by SDS-PAGE and Western Blot analyses.Then the bacteriostatic activity was determined.The results showed that the fusion proteins were correctly expressed.DNA sequencing indicated that 135,261 and 387 bp of gene fragments were obtained after amplifying by PCR.SDS-PAGE results showed that the expressed fusion proteins had molecular weight of 30,35 and 40 kDa bands and accounted for 64.5%,26.4% and 25.3% of the total bacterium proteins,respectively.The Western Blot analysis demonstrated that all of the target proteins had immunological activity.The results of bacteriostatic activity in vivo indicated that all of the expressed fusion proteins were definite inhibitors of E.coli DH5α.However,bacteriostatic efficacy of the fusion expression products significantly increased and antibacterial time extended with the increasingly number of plectasin gene.The successful construction of the tandom expression strains and the inhibition of the expression products to host bacteria laid a foundation for product engineering of efficient bacteriostatic effect of plectasin.

关 键 词：菌丝霉素 串联表达 菌内抑菌活性 

分 类 号：Q786[生物学—分子生物学]