發(fā)布時間：2018-05-02 17:50

  本文選題：家蠅 + 乙醇脫氫酶　； 參考：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：抗生素自問世以來,挽救了無數(shù)生命,為人類和動物的健康做出了巨大貢獻(xiàn),然而不規(guī)范使用抗生素,致使臨床致病菌逐漸產(chǎn)生了耐藥性。因此,尋找新型、綠色、安全的抗菌劑代替抗生素,已成為當(dāng)前國內(nèi)外抗菌藥物研究的一項重要內(nèi)容�？咕�(antibacterial peptides)是生物體內(nèi)產(chǎn)生的一類具有抗菌活性的小分子多肽,是生物先天免疫的重要組成成分,抗菌肽不但具有廣譜的抗菌活性,還具有高效的抗真菌、病毒和原蟲的活性,倍受國內(nèi)外學(xué)者青睞。而家蠅從幼蟲到成蟲均表現(xiàn)出很強的環(huán)境適應(yīng)能力,其體內(nèi)外常攜帶多種病原菌,卻極少出現(xiàn)集體發(fā)病的現(xiàn)象,這源于其體內(nèi)產(chǎn)生的多種抗菌肽及抗菌蛋白。因此,家蠅抗菌肽有可能成為新一代的抗生素替代品。本研究以雞沙門氏菌誘導(dǎo)家蠅幼蟲構(gòu)建的抑制性消減文庫(SSH)中篩選得到的部分差異基因片段為基礎(chǔ),進行全長差異基因的克隆、表達(dá),并對其表達(dá)產(chǎn)物的生物活性進行初步研究。以沙門氏菌誘導(dǎo)家蠅幼蟲cDNA為模板,采用cDNA末端快速擴增技術(shù)(RACE),對乙醇脫氫酶基因MdADH進行PCR擴增和序列分析。將乙醇脫氫酶基因MdADH、家蠅雙翅肽基因MdDpt-I、家蠅溶菌酶基因Mdd與表達(dá)載體連接,構(gòu)建重組表達(dá)質(zhì)粒MdADH-pET-28a、MdDpt I-pET-28a和Mdd-pET-32a,轉(zhuǎn)化至大腸桿菌BL21(DE3)中,進行IPTG誘導(dǎo)和SDS-PAGE電泳分析。利用鎳離子親和層析柱對融合蛋白進行純化,并對融合蛋白進行生物學(xué)活性分析,主要實驗結(jié)果如下:1、家蠅乙醇脫氫酶基因MdADH的ORF序列為807bp,編碼268個氨基酸。其氨基酸序列具有短鏈脫氫酶的保守結(jié)構(gòu)域,屬于短鏈脫氫酶超家族成員(SDR superfamily),具有脫氫酶的活性部位(active site)和輔酶結(jié)合位點(NAD(P)binding site)。分子量為29.6ku,理論等電點為7.65,無明顯跨膜區(qū),不屬于膜蛋白或分泌蛋白,無信號肽區(qū)域。2、成功構(gòu)建了重組表達(dá)質(zhì)粒MdADH-pET-28a、MdDpt I-pET-28a以及Mdd-pET-32a,轉(zhuǎn)化至大腸桿菌BL21(DE3)中,經(jīng)IPTG誘導(dǎo)表達(dá),對表達(dá)產(chǎn)物進行SDS-PAGE電泳分析。借助親和層析柱純化方法,成功獲得了高純度的的融合蛋白。3、生物活性鑒定。通過瓦勒霍赫法檢測出MdADH融合蛋白具有乙醇脫氫酶活性,MdADH融合蛋白的小鼠的體內(nèi)試驗結(jié)果表明,MdADH具有縮短醉酒時間的作用。MdDpt I-pET-28a質(zhì)粒表達(dá)的融合蛋白對臨床分離的大腸桿菌與沙門氏菌具有明顯的抑制作用,對豬源鏈球菌無明顯的抑菌作用。Mdd-pET-32a質(zhì)粒表達(dá)的融合蛋白對臨床分離的大腸桿菌具有明顯的抑制作用,對豬源鏈球菌有一定的抑制作用,對沙門氏菌無抑制作用。
[Abstract]:Since the introduction of antibiotics, it has saved countless lives and made great contributions to the health of human beings and animals. However, antibiotics are not used regularly, resulting in the gradual emergence of drug resistance in clinical pathogens. Therefore, to find new, green and safe antibiotics instead of antibiotics has become an important content of the current research on antibacterial drugs both at home and abroad. Antibacterial peptide (antibacterial peptides) is a kind of small molecular polypeptide with antibacterial activity in organism. It is an important component of biological innate immunity. Antibacterial peptide not only has broad-spectrum antibacterial activity, but also has high antifungal activity, highly effective antifungal, virus and protozoa activity. It has a strong ability to adapt to the environment, and it often carries a variety of pathogenic bacteria in vivo and in the body, but rarely occurs collectively. This is derived from a variety of antimicrobial peptides and antibacterial proteins produced in its body. Therefore, the antibacterial peptide of housefly may become a substitute for the new generation of antibiotics. This study inhibits the construction of housefly larvae induced by Salmonella chicken. On the basis of the partial differential gene fragment selected in the sexual subtractive library (SSH), the whole length differentially gene was cloned and expressed, and the biological activity of the expression product was preliminarily studied. The cDNA of the housefly larvae was induced by Salmonella, and the cDNA terminal rapid amplification technique (RACE) was used to expand the PCR dehydrogenase gene MdADH. The ethanol dehydrogenase gene MdADH, the housefly Diptera gene MdDpt-I and the housefly lysozyme gene Mdd were connected with the expression vector, and the recombinant expression plasmid MdADH-pET-28a, MdDpt I-pET-28a and Mdd-pET-32a were transformed into the Escherichia coli BL21 (DE3), and the IPTG inducement and SDS-PAGE electrophoresis analysis were carried out. The nickel ion affinity chromatography column was used. The fusion protein was purified and the biological activity of the fusion protein was analyzed. The main experimental results were as follows: 1, the ORF sequence of the MdADH of the housefly ethanol dehydrogenase gene was 807bp and encoded 268 amino acids. The amino acid sequence had the conservative domain of the short chain dehydrogenase, which belonged to the member of the short chain dehydrogenase superfamily (SDR superfamily) and had dehydrogenation. The active site of the enzyme (active site) and coenzyme binding site (NAD (P) binding site). The molecular weight of the enzyme is 29.6ku, the theoretical isoelectric point is 7.65, and there is no obvious transmembrane region. It does not belong to the membrane protein or secretory protein, without the signal peptide region.2. The recombinant expression plasmid MdADH-pET-28a, MdDpt I-pET-28a, and Mdd-pET-32a are successfully constructed and converted to Escherichia coli. In the process of IPTG induced expression, the expression products were analyzed by SDS-PAGE electrophoresis. With the help of affinity chromatography column purification, high purity fusion protein.3 and bioactivity identification were successfully obtained. The activity of MdADH fusion protein with ethanol dehydrogenase was detected by valerhhh method. The results of mice in vivo of MdADH fusion protein showed that MdADH The fusion protein expressed by the.MdDpt I-pET-28a plasmid, which has the effect of shortening the time of drunkenness, has an obvious inhibitory effect on the clinical isolates of Escherichia coli and Salmonella. The fusion protein expressed by.Mdd-pET-32a plasmid, which has no obvious bacteriostasis on Streptococcus suis, has obvious inhibitory effect on the clinical isolates of Enterobacter. Streptococcus has a certain inhibitory effect and has no inhibitory effect on Salmonella.

【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】：2015
【分類號】：S859.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 梁永利;;天然抗菌肽的來源及分類[J];安徽農(nóng)業(yè)科學(xué);2006年18期

2 萬玲;傅小蒙;唐艷;孫小寧;裴志花;劉樹明;馬紅霞;;家蠅幼蟲防御素基因MddⅠ的克隆與原核表達(dá)[J];中國獸醫(yī)科學(xué);2013年12期

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本文編號：1834889