本文選題：抗菌肽 + 畢赤酵母　； 參考：《吉林農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：抗菌肽是動物先天免疫系統(tǒng)抵御微生物入侵時產(chǎn)生的陽離子肽類活性物質(zhì),能夠有效防止病菌的侵入,具有廣譜抗菌活性,相對傳統(tǒng)抗生素而言,它不容易引起細(xì)菌耐藥性問題。因此,近年來,抗菌肽的研究受到了國內(nèi)外相關(guān)學(xué)者的高度重視。家蠅(Musca domestica)常生活在多種病原菌孳生的環(huán)境中并能夠攜帶、傳播細(xì)菌,而自身卻很少染病,這緣于其體內(nèi)外抗菌活性物質(zhì)作用的結(jié)果�？咕淖鳛榧蚁夡w內(nèi)強(qiáng)效的抗菌活性物質(zhì)之一,可抑制多種細(xì)菌、真菌、寄生蟲、病毒、腫瘤細(xì)胞,且對正常生物體細(xì)胞無破壞作用。目前,對抗菌肽編碼基因的篩選、表達(dá)及生物學(xué)活性的研究是抗菌肽研究的主要熱點。本研究以致病性雞源大腸桿菌及雞源沙門氏菌誘導(dǎo)家蠅幼蟲抑制性消減文庫(SSH)中篩選并克隆得到的三個全長差異基因(家蠅抗菌肽(MDAP-2)、家蠅未知功能基因(Md-UF4)、家蠅未知功能基因(Md-UF21))為研究基礎(chǔ),采用PCR技術(shù)擴(kuò)增獲得這三個全長差異基因,并進(jìn)一步通過T-A克隆將全長差異基因克隆至pMD18-T載體中,隨后亞克隆至真核表達(dá)載體,構(gòu)建真核重組表達(dá)質(zhì)粒pPIC9K-MDAP-2、pPIC9K-Md-UF4、pPIC9K-Md-UF21,將線性化的重組表達(dá)質(zhì)粒電擊轉(zhuǎn)入畢赤酵母(P.Pastoris)GS115感受態(tài)細(xì)胞內(nèi),將PCR鑒定為陽性的重組酵母菌進(jìn)行甲醇誘導(dǎo)表達(dá),采用SDS-PAGE及Tricine-SDS-PGAE檢測目的基因的表達(dá)情況,并對重組蛋白的誘導(dǎo)表達(dá)溫度及發(fā)酵液pH值進(jìn)行了條件優(yōu)化,經(jīng)鎳柱親和層析純化重組蛋白后,采用管碟法檢測重組蛋白的抑菌活性。主要實驗結(jié)果如下:(1)采用PCR技術(shù)擴(kuò)增獲得MDAP-2、Md-UF4、Md-UF21三個全長差異基因,并成功構(gòu)建三個基因與pMD-18T載體連接的克隆質(zhì)粒。(2)成功構(gòu)建真核重組表達(dá)質(zhì)粒pPIC9K-MDAP-2、pPIC9K-Md-UF4、pPIC9K-Md-UF21。MDAP-2、Md-UF4基因在畢赤酵母表達(dá)系統(tǒng)中獲得表達(dá)。GS115-pPIC9K-MDAP-2最佳誘導(dǎo)表達(dá)條件:誘導(dǎo)時間為96 h,發(fā)酵液pH為7;GS115-pPIC9K-Md-UF4最佳誘導(dǎo)表達(dá)條件:誘導(dǎo)時間為72 h,發(fā)酵液pH為7。(3)純化后MDAP-2、Md-UF4的重組蛋白經(jīng)抑菌活性檢測結(jié)果顯示,MDAP-2重組蛋白對臨床分離的雞源大腸桿菌耐藥株、雞源傷寒沙門氏菌耐藥株均具有抑菌活性;Md-UF4重組蛋白對臨床分離的三株菌均無抑菌活性,仍是家蠅未知功能基因。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S859.79;Q78

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