鸡源沙门氏菌诱导家蝇幼虫所产生部分差异基因的表达与产物活性研究

发布时间：2018-05-02 17:50

本文选题：家蝇 + 乙醇脱氢酶　；参考：《吉林农业大学》2015年硕士论文

【摘要】：抗生素自问世以来,挽救了无数生命,为人类和动物的健康做出了巨大贡献,然而不规范使用抗生素,致使临床致病菌逐渐产生了耐药性。因此,寻找新型、绿色、安全的抗菌剂代替抗生素,已成为当前国内外抗菌药物研究的一项重要内容。抗菌肽(antibacterial peptides)是生物体内产生的一类具有抗菌活性的小分子多肽,是生物先天免疫的重要组成成分,抗菌肽不但具有广谱的抗菌活性,还具有高效的抗真菌、病毒和原虫的活性,倍受国内外学者青睐。而家蝇从幼虫到成虫均表现出很强的环境适应能力,其体内外常携带多种病原菌,却极少出现集体发病的现象,这源于其体内产生的多种抗菌肽及抗菌蛋白。因此,家蝇抗菌肽有可能成为新一代的抗生素替代品。本研究以鸡沙门氏菌诱导家蝇幼虫构建的抑制性消减文库(SSH)中筛选得到的部分差异基因片段为基础,进行全长差异基因的克隆、表达,并对其表达产物的生物活性进行初步研究。以沙门氏菌诱导家蝇幼虫cDNA为模板,采用cDNA末端快速扩增技术(RACE),对乙醇脱氢酶基因MdADH进行PCR扩增和序列分析。将乙醇脱氢酶基因MdADH、家蝇双翅肽基因MdDpt-I、家蝇溶菌酶基因Mdd与表达载体连接,构建重组表达质粒MdADH-pET-28a、MdDpt I-pET-28a和Mdd-pET-32a,转化至大肠杆菌BL21(DE3)中,进行IPTG诱导和SDS-PAGE电泳分析。利用镍离子亲和层析柱对融合蛋白进行纯化,并对融合蛋白进行生物学活性分析,主要实验结果如下:1、家蝇乙醇脱氢酶基因MdADH的ORF序列为807bp,编码268个氨基酸。其氨基酸序列具有短链脱氢酶的保守结构域,属于短链脱氢酶超家族成员(SDR superfamily),具有脱氢酶的活性部位(active site)和辅酶结合位点(NAD(P)binding site)。分子量为29.6ku,理论等电点为7.65,无明显跨膜区,不属于膜蛋白或分泌蛋白,无信号肽区域。2、成功构建了重组表达质粒MdADH-pET-28a、MdDpt I-pET-28a以及Mdd-pET-32a,转化至大肠杆菌BL21(DE3)中,经IPTG诱导表达,对表达产物进行SDS-PAGE电泳分析。借助亲和层析柱纯化方法,成功获得了高纯度的的融合蛋白。3、生物活性鉴定。通过瓦勒霍赫法检测出MdADH融合蛋白具有乙醇脱氢酶活性,MdADH融合蛋白的小鼠的体内试验结果表明,MdADH具有缩短醉酒时间的作用。MdDpt I-pET-28a质粒表达的融合蛋白对临床分离的大肠杆菌与沙门氏菌具有明显的抑制作用,对猪源链球菌无明显的抑菌作用。Mdd-pET-32a质粒表达的融合蛋白对临床分离的大肠杆菌具有明显的抑制作用,对猪源链球菌有一定的抑制作用,对沙门氏菌无抑制作用。
[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.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.7

【参考文献】