家蝇MDAP-2、Md-UF4及Md-UF21基因的真核表达及产物抑菌活性研究

发布时间：2018-05-22 15:49

本文选题：抗菌肽 + 毕赤酵母　；参考：《吉林农业大学》2016年硕士论文

【摘要】：抗菌肽是动物先天免疫系统抵御微生物入侵时产生的阳离子肽类活性物质,能够有效防止病菌的侵入,具有广谱抗菌活性,相对传统抗生素而言,它不容易引起细菌耐药性问题。因此,近年来,抗菌肽的研究受到了国内外相关学者的高度重视。家蝇(Musca domestica)常生活在多种病原菌孳生的环境中并能够携带、传播细菌,而自身却很少染病,这缘于其体内外抗菌活性物质作用的结果。抗菌肽作为家蝇体内强效的抗菌活性物质之一,可抑制多种细菌、真菌、寄生虫、病毒、肿瘤细胞,且对正常生物体细胞无破坏作用。目前,对抗菌肽编码基因的筛选、表达及生物学活性的研究是抗菌肽研究的主要热点。本研究以致病性鸡源大肠杆菌及鸡源沙门氏菌诱导家蝇幼虫抑制性消减文库(SSH)中筛选并克隆得到的三个全长差异基因(家蝇抗菌肽(MDAP-2)、家蝇未知功能基因(Md-UF4)、家蝇未知功能基因(Md-UF21))为研究基础,采用PCR技术扩增获得这三个全长差异基因,并进一步通过T-A克隆将全长差异基因克隆至pMD18-T载体中,随后亚克隆至真核表达载体,构建真核重组表达质粒pPIC9K-MDAP-2、pPIC9K-Md-UF4、pPIC9K-Md-UF21,将线性化的重组表达质粒电击转入毕赤酵母(P.Pastoris)GS115感受态细胞内,将PCR鉴定为阳性的重组酵母菌进行甲醇诱导表达,采用SDS-PAGE及Tricine-SDS-PGAE检测目的基因的表达情况,并对重组蛋白的诱导表达温度及发酵液pH值进行了条件优化,经镍柱亲和层析纯化重组蛋白后,采用管碟法检测重组蛋白的抑菌活性。主要实验结果如下:(1)采用PCR技术扩增获得MDAP-2、Md-UF4、Md-UF21三个全长差异基因,并成功构建三个基因与pMD-18T载体连接的克隆质粒。(2)成功构建真核重组表达质粒pPIC9K-MDAP-2、pPIC9K-Md-UF4、pPIC9K-Md-UF21。MDAP-2、Md-UF4基因在毕赤酵母表达系统中获得表达。GS115-pPIC9K-MDAP-2最佳诱导表达条件:诱导时间为96 h,发酵液pH为7;GS115-pPIC9K-Md-UF4最佳诱导表达条件:诱导时间为72 h,发酵液pH为7。(3)纯化后MDAP-2、Md-UF4的重组蛋白经抑菌活性检测结果显示,MDAP-2重组蛋白对临床分离的鸡源大肠杆菌耐药株、鸡源伤寒沙门氏菌耐药株均具有抑菌活性;Md-UF4重组蛋白对临床分离的三株菌均无抑菌活性,仍是家蝇未知功能基因。
[Abstract]:Antimicrobial peptide is a cationic peptide active substance produced by animal innate immune system to resist microbial invasion. It can effectively prevent bacterial invasion and has broad-spectrum antibacterial activity, compared with traditional antibiotics. It is not easy to cause bacterial resistance problems. Therefore, in recent years, the study of antimicrobial peptides has been highly valued by domestic and foreign scholars. Musca domestica (Musca domestica) often lives in a variety of pathogen breeding environment and can carry and transmit bacteria, but it rarely infects itself, which is due to the effect of antimicrobial active substances in vivo and in vitro. Antimicrobial peptides, as one of the most potent antimicrobial active substances in housefly, can inhibit many bacteria, fungi, parasites, viruses, tumor cells, and have no damage to normal body cells. At present, the screening, expression and biological activity of antimicrobial peptide coding genes are the main focus of antimicrobial peptide research. In this study, three full-length differentially expressed genes (housefly antimicrobial peptide MDAP-2, housefly unknown function gene Md-UF4, Musca domestica) were screened and cloned from pathogenic chicken Escherichia coli and chicken salmonella induced larva suppression subtractive library (SSHs). The known functional gene Md-UF21 is the basis of the study. The three full-length differentially expressed genes were amplified by PCR, and further cloned into pMD18-T vector by T-A cloning, and then subcloned into eukaryotic expression vector. The eukaryotic recombinant expression plasmid pPIC9K-Md-Md-UF21 was constructed. The linearized recombinant expression plasmid was electrocuted into Pichia pastoris P.Pastoris-GS115 receptive cells. The recombinant yeast identified by PCR as positive was induced by methanol. The expression of the target gene was detected by SDS-PAGE and Tricine-SDS-PGAE. The induced expression temperature and pH value of the recombinant protein were optimized. The recombinant protein was purified by nickel column affinity chromatography and the bacteriostatic activity of the recombinant protein was detected by tube-disc method. The main results were as follows: (1) three full-length differentially expressed genes of MDAP-2Md-UF4Md-UF21 were obtained by PCR amplification. The recombinant eukaryotic expression plasmid pPIC9K-Md-Md-UF4 pPIC9K-Md-UF4 pPIC9K-Md-UF2Md-UF4 gene was successfully constructed in Pichia pastoris expression system. GS115-pPIC9K-MDAP-2 was induced in 96 h. The recombinant protein of MDAP-2md-Md-UF4 was purified at 72 h and pH 7.3). The results of bacteriostatic activity test showed that MDAP-2 recombinant protein was resistant to clinical isolates of chicken Escherichia coli. All the resistant strains of Salmonella typhimurium from chicken had antimicrobial activity and Md-UF4 recombinant protein had no antimicrobial activity against the three strains of clinical isolates, which were still unknown functional genes of Musca domestica.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S859.79;Q78

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