病原菌诱导家蝇幼虫部分差异基因的原核表达及活性研究

发布时间：2018-05-18 03:30

本文选题：抗菌肽 + 家蝇　；参考：《吉林农业大学》2017年硕士论文

【摘要】：抗菌肽作为机体防御外来病原菌的第一道防线,是构成先天免疫系统的重要组成部分,不需要免疫记忆并能直接有效的消灭外来入侵的病原体。近年来,人们从植物、两栖动物、昆虫甚至人体内分离得到多种具有活性的抗菌肽。家蝇(Musca domestica)生活在病原微生物滋生的环境中,并能携带病原微生物,在接触过程中会机械地传播到人和动物机体上,自身却很少感染,说明其具有独特有效的免疫防御机制。研究显示家蝇体内的抗菌肽/蛋白(AMPs)在家蝇体内免疫中起到了很大的作用,且抗菌物质对细菌、病毒、真菌及癌细胞有很强的抑制作用。因此,家蝇抗菌肽作为一种新型的抗菌类药物逐渐成为人们关注的焦点之一。由于自然界中家蝇的抗菌肽资源有限,因此基因工程法重组表达抗菌肽成为获得AMPs的最有效的方法。本研究分别从牛多杀性巴氏杆菌、致病性鸡源大肠杆菌及鸡源沙门氏菌诱导家蝇幼虫后构建的抑制性消减文库(SSH)中筛选到三个未知功能基因片段,即分别命名为MD-UF672、MD-UF21和MLAP,进一步采用PCR技术扩增获得全长目的基因,而后将基因片段与pMD18-T载体连接进行T-A克隆。将BamH?/Xho?双酶切后的MD-UF672与表达载体pET-32a(+)连接,EcoR?/Xho?双酶切后的MLAP基因和MD-UF21分别与表达载体pET-32a(+)和pGEX-4T连接,构建原核重组表达质粒pET-32a(+)-MD-UF672、pET-32a(+)-MLAP和pGEX-4T-MD-UF21。将重组表达质粒分别转入至大肠杆菌BL21感受态菌株中进行诱导表达。利用Ni-NTA亲和层析对重组蛋白pET-32a(+)-MD-UF672和pET-32a(+)-MLAP进行纯化,利用GST亲和层析对重组蛋白pGEX-4T-MD-UF21进行纯化。采用管碟法对MD-UF672、MD-UF21和MLAP的纯化产物进行抑菌活性的检测,随后检测重组蛋白MLAP对大肠杆菌、沙门氏菌和金黄色葡萄球菌的生长抑制情况和MIC值,主要实验结果如下:1、利用PCR扩增得到MD-UF672、MD-UF21和MLAP全长基因,并对这三个基因进行了T-A克隆。2、成功构建了pET-32a(+)-MD-UF672、pET-32a(+)-MLAP和pGEX-4T-MDUF21的原核重组表达质粒,这三种重组基因均在大肠肝菌表达系统中获得表达。3、纯化后的MD-UF672、MD-UF21和MLAP表达产物经过抑菌活性分析显示,重组蛋白pET-32a(+)-MLAP对临床分离的大肠杆菌、沙门氏菌和金黄色葡萄球菌有抑菌活性。重组蛋白pET-32a(+)-MD-UF672和pGEX-4T-MD-UF21不具有抑菌活性,目前为家蝇未知功能蛋白。4、检测了重组蛋白MLAP对大肠杆菌、沙门氏菌和金黄色葡萄球菌的生长抑制情况和MIC值,绘制生长抑制曲线,MLAP对大肠杆菌、沙门氏菌和金黄色葡萄球菌的MIC值分别为0.012μg/μL、0.003μg/μL和0.025μg/μL。
[Abstract]:As the first line of defense against foreign pathogens, antimicrobial peptides constitute an important part of the innate immune system. They do not require immune memory and can effectively eliminate foreign invading pathogens. In recent years, many active antimicrobial peptides have been isolated from plants, amphibians, insects and even human bodies. Musca domestica (Musca domestica) lives in the breeding environment of pathogenic microorganisms and can carry pathogenic microorganisms. It can spread mechanically to human and animal bodies during contact, but it rarely infects itself, indicating that it has a unique and effective immune defense mechanism. Studies have shown that antimicrobial peptides / proteins (AMPs) play a significant role in housefly immunity, and antimicrobial substances have a strong inhibitory effect on bacteria, viruses, fungi and cancer cells. Therefore, housefly antimicrobial peptide as a new type of antimicrobial drugs has gradually become one of the focus of attention. Because of the limited resources of antimicrobial peptides in nature, recombinant expression of antimicrobial peptides by genetic engineering is the most effective method to obtain AMPs. In this study, three unknown functional gene fragments were screened from bovine Pasteurella multocida, pathogenic Escherichia coli and Salmonella chickens-induced suppression subtractive library (SSH). They were named MD-UF672MD-UF21 and MLAP respectively. The full-length target gene was amplified by PCR and then ligated with pMD18-T vector for T-A cloning. Will BamHU / Xhos? The double enzyme digested MD-UF672 is ligated with the expression vector pET-32a (). The double enzyme digested MLAP gene and MD-UF21 were ligated with the expression vector pET-32a () and pGEX-4T, respectively, and the prokaryotic expression plasmid pET-32a (pET-32a) (pET-32a) and pGEX-4T-MD-UF21 (pGEX-4T-MD-UF21) were constructed. The recombinant expression plasmid was transferred into Escherichia coli BL21 competent strain for induction expression. The recombinant proteins pET-32a (pET-32a) and pET-32a (pET-32a) were purified by Ni-NTA affinity chromatography, and the recombinant protein pGEX-4T-MD-UF21 was purified by GST affinity chromatography. The antimicrobial activity of the purified products of MD-UF672MD-UF21 and MLAP was detected by tube-disc method, and then the growth inhibition and MIC value of recombinant protein MLAP against Escherichia coli, Salmonella and Staphylococcus aureus were detected. The main results are as follows: 1. The full-length MD-UF672MD-UF21 and MLAP genes were amplified by PCR, and the three genes were cloned by T-A clone. The prokaryotic expression plasmids of pET-32a (pET-32a) (MLAP and pGEX-4T-MDUF21) were successfully constructed. These three recombinant genes were all expressed in the E. coli expression system. The purified MD-UF672MD-UF21 and MLAP expression products showed that the recombinant protein pET-32a (pET-32a) was expressed in clinical isolates of Escherichia coli, and the purified MD-UF672 MD-UF21 and MLAP expression products were expressed in E. coli. Salmonella and Staphylococcus aureus have antimicrobial activity. The recombinant protein pET-32a (pET-32a) (pET-32a) and pGEX-4T-MD-UF21 have no bacteriostatic activity. They are currently unknown functional proteins of Musca domestica. The growth inhibition and MIC value of recombinant protein MLAP against Escherichia coli, Salmonella aureus and Staphylococcus aureus were detected. The MIC values of MLAP against Escherichia coli, Salmonella and Staphylococcus aureus were 0.012 渭 g / 渭 L and 0.025 渭 g / 渭 L, respectively.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.6

【参考文献】