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

【摘要】：抗菌肽作為機(jī)體防御外來(lái)病原菌的第一道防線,是構(gòu)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(lái)入侵的病原體。近年來(lái),人們從植物、兩棲動(dòng)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(guò)程中會(huì)機(jī)械地傳播到人和動(dòng)物機(jī)體上,自身卻很少感染,說(shuō)明其具有獨(dú)特有效的免疫防御機(jī)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(duì)細(xì)菌、病毒、真菌及癌細(xì)胞有很強(qiáng)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(guān)注的焦點(diǎn)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(dá)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(dǎo)家蠅幼蟲后構(gòu)建的抑制性消減文庫(kù)(SSH)中篩選到三個(gè)未知功能基因片段,即分別命名為MD-UF672、MD-UF21和MLAP,進(jìn)一步采用PCR技術(shù)擴(kuò)增獲得全長(zhǎng)目的基因,而后將基因片段與pMD18-T載體連接進(jìn)行T-A克隆。將BamH?/Xho?雙酶切后的MD-UF672與表達(dá)載體pET-32a(+)連接,EcoR?/Xho?雙酶切后的MLAP基因和MD-UF21分別與表達(dá)載體pET-32a(+)和pGEX-4T連接,構(gòu)建原核重組表達(dá)質(zhì)粒pET-32a(+)-MD-UF672、pET-32a(+)-MLAP和pGEX-4T-MD-UF21。將重組表達(dá)質(zhì)粒分別轉(zhuǎn)入至大腸桿菌BL21感受態(tài)菌株中進(jìn)行誘導(dǎo)表達(dá)。利用Ni-NTA親和層析對(duì)重組蛋白pET-32a(+)-MD-UF672和pET-32a(+)-MLAP進(jìn)行純化,利用GST親和層析對(duì)重組蛋白pGEX-4T-MD-UF21進(jìn)行純化。采用管碟法對(duì)MD-UF672、MD-UF21和MLAP的純化產(chǎn)物進(jìn)行抑菌活性的檢測(cè),隨后檢測(cè)重組蛋白MLAP對(duì)大腸桿菌、沙門氏菌和金黃色葡萄球菌的生長(zhǎng)抑制情況和MIC值,主要實(shí)驗(yàn)結(jié)果如下:1、利用PCR擴(kuò)增得到MD-UF672、MD-UF21和MLAP全長(zhǎng)基因,并對(duì)這三個(gè)基因進(jìn)行了T-A克隆。2、成功構(gòu)建了pET-32a(+)-MD-UF672、pET-32a(+)-MLAP和pGEX-4T-MDUF21的原核重組表達(dá)質(zhì)粒,這三種重組基因均在大腸肝菌表達(dá)系統(tǒng)中獲得表達(dá)。3、純化后的MD-UF672、MD-UF21和MLAP表達(dá)產(chǎn)物經(jīng)過(guò)抑菌活性分析顯示,重組蛋白pET-32a(+)-MLAP對(duì)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(cè)了重組蛋白MLAP對(duì)大腸桿菌、沙門氏菌和金黃色葡萄球菌的生長(zhǎng)抑制情況和MIC值,繪制生長(zhǎng)抑制曲線,MLAP對(duì)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(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 周志江;;生物防腐劑及其在食品防腐中的應(yīng)用[J];保鮮與加工;2015年01期

2 王自蕊;譙仕彥;李波;阮記明;隗黎麗;楊竹青;;飼料中添加天蠶素抗菌肽對(duì)湘云鯽生長(zhǎng)性能、非特異性免疫功能及抗病力的影響[J];動(dòng)物營(yíng)養(yǎng)學(xué)報(bào);2014年07期

3 萬(wàn)玲;王梅梅;孔令聰;裴志花;劉樹明;馬紅霞;;家蠅基因工程抗菌肽的研究進(jìn)展[J];中國(guó)獸藥雜志;2013年10期

4 包義風(fēng);應(yīng)蓮芳;蔣琳;;包涵體蛋白復(fù)性技術(shù)研究進(jìn)展[J];微生物學(xué)免疫學(xué)進(jìn)展;2012年02期

5 金小寶;龔水明;蒲俏虹;朱家勇;褚夫江;梅寒芳;;家蠅抗菌肽Cecropin對(duì)人源性腫瘤細(xì)胞增殖與凋亡的影響[J];中國(guó)熱帶醫(yī)學(xué);2012年03期

6 姜珊;王寶杰;劉梅;蔣克勇;宮魁;王雷;;飼料中添加重組抗菌肽對(duì)吉富羅非魚生長(zhǎng)性能及免疫力的影響[J];中國(guó)水產(chǎn)科學(xué);2011年06期

7 張?chǎng)┑?殷幼平;;昆蟲抗真菌肽的研究進(jìn)展[J];貴州農(nóng)業(yè)科學(xué);2011年11期

8 黨向利;柴一秋;翁宏飚;張文慶;;細(xì)菌誘導(dǎo)家蠅蛹抗菌物質(zhì)的分離純化[J];環(huán)境昆蟲學(xué)報(bào);2011年02期

9 胡榮貴;李改瑞;陸家海;;抗菌肽在抗腫瘤方面的研究進(jìn)展[J];實(shí)用腫瘤雜志;2010年02期

10 王義鵬;賴仞;;昆蟲抗菌肽結(jié)構(gòu)、性質(zhì)和基因調(diào)控[J];動(dòng)物學(xué)研究;2010年01期

相關(guān)會(huì)議論文 前1條

1 李林;陳冰;孫育平;黃燕華;王國(guó)霞;趙紅霞;齊飛;曹俊明;;家蠅抗菌肽的提取純化及對(duì)凡納濱對(duì)蝦抗病力的影響[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)動(dòng)物營(yíng)養(yǎng)學(xué)分會(huì)第七屆中國(guó)飼料營(yíng)養(yǎng)學(xué)術(shù)研討會(huì)論文集[C];2014年

相關(guān)碩士學(xué)位論文 前1條

1 魏詩(shī);抗菌肽對(duì)口腔常見致病菌及biofilm的抑制作用以及抗炎活性的研究[D];遼寧師范大學(xué);2013年

，

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