本文選題：抗菌肽 + 家蠅��； 參考：《吉林農(nóng)業(yè)大學》2017年碩士論文

【摘要】：抗菌肽作為機體防御外來病原菌的第一道防線,是構成先天免疫系統(tǒng)的重要組成部分,不需要免疫記憶并能直接有效的消滅外來入侵的病原體。近年來,人們從植物、兩棲動物、昆蟲甚至人體內(nèi)分離得到多種具有活性的抗菌肽。家蠅(Musca domestica)生活在病原微生物滋生的環(huán)境中,并能攜帶病原微生物,在接觸過程中會機械地傳播到人和動物機體上,自身卻很少感染,說明其具有獨特有效的免疫防御機制。研究顯示家蠅體內(nèi)的抗菌肽/蛋白(AMPs)在家蠅體內(nèi)免疫中起到了很大的作用,且抗菌物質(zhì)對細菌、病毒、真菌及癌細胞有很強的抑制作用。因此,家蠅抗菌肽作為一種新型的抗菌類藥物逐漸成為人們關注的焦點之一。由于自然界中家蠅的抗菌肽資源有限,因此基因工程法重組表達抗菌肽成為獲得AMPs的最有效的方法。本研究分別從牛多殺性巴氏桿菌、致病性雞源大腸桿菌及雞源沙門氏菌誘導家蠅幼蟲后構建的抑制性消減文庫(SSH)中篩選到三個未知功能基因片段,即分別命名為MD-UF672、MD-UF21和MLAP,進一步采用PCR技術擴增獲得全長目的基因,而后將基因片段與pMD18-T載體連接進行T-A克隆。將BamH?/Xho?雙酶切后的MD-UF672與表達載體pET-32a(+)連接,EcoR?/Xho?雙酶切后的MLAP基因和MD-UF21分別與表達載體pET-32a(+)和pGEX-4T連接,構建原核重組表達質(zhì)粒pET-32a(+)-MD-UF672、pET-32a(+)-MLAP和pGEX-4T-MD-UF21。將重組表達質(zhì)粒分別轉(zhuǎn)入至大腸桿菌BL21感受態(tài)菌株中進行誘導表達。利用Ni-NTA親和層析對重組蛋白pET-32a(+)-MD-UF672和pET-32a(+)-MLAP進行純化,利用GST親和層析對重組蛋白pGEX-4T-MD-UF21進行純化。采用管碟法對MD-UF672、MD-UF21和MLAP的純化產(chǎn)物進行抑菌活性的檢測,隨后檢測重組蛋白MLAP對大腸桿菌、沙門氏菌和金黃色葡萄球菌的生長抑制情況和MIC值,主要實驗結果如下:1、利用PCR擴增得到MD-UF672、MD-UF21和MLAP全長基因,并對這三個基因進行了T-A克隆。2、成功構建了pET-32a(+)-MD-UF672、pET-32a(+)-MLAP和pGEX-4T-MDUF21的原核重組表達質(zhì)粒,這三種重組基因均在大腸肝菌表達系統(tǒng)中獲得表達。3、純化后的MD-UF672、MD-UF21和MLAP表達產(chǎn)物經(jīng)過抑菌活性分析顯示,重組蛋白pET-32a(+)-MLAP對臨床分離的大腸桿菌、沙門氏菌和金黃色葡萄球菌有抑菌活性。重組蛋白pET-32a(+)-MD-UF672和pGEX-4T-MD-UF21不具有抑菌活性,目前為家蠅未知功能蛋白。4、檢測了重組蛋白MLAP對大腸桿菌、沙門氏菌和金黃色葡萄球菌的生長抑制情況和MIC值,繪制生長抑制曲線,MLAP對大腸桿菌、沙門氏菌和金黃色葡萄球菌的MIC值分別為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.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.6

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