病原体诱导前后家蝇幼虫差异基因的表达及表达产物活性研究

发布时间：2018-01-21 13:43

本文关键词： 家蝇幼虫抗病活性物质未知功能基因克隆表达抑菌活性　出处：《吉林农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：家蝇幼虫长期生活在潮湿、富含有机质的环境中,常携带大量的病原体和有害因子,但本身却很少发病。据研究人员预测,家蝇幼虫独特的免疫防御能力源于其体内产生的抗病活性物质。这些抗病活性物质不仅具有广谱抗菌能力,还有抗真菌、原生动物、病毒乃至杀死癌细胞而不破坏体内正常细胞作用的功能,已成为国内外学者关注的焦点。在病原体的诱导刺激下家蝇幼虫可通过基因对其免疫反应进行调控,合成多种增量表达的抗病活性物质,如抗菌肽、凝集素、溶菌酶、幼虫体外分泌物、几丁质、壳聚糖以及一些未知的活性物质等。这些抗病活性物质的发现使研发新型药物代替抗生素进而缓解病原体耐药压力成为可能。本研究分别选取牛多杀性巴氏杆菌、猪肺炎支原体及鸡致病性大肠杆菌诱导家蝇幼虫前、后的三个差异基因为研究对象,进行其克隆、表达及表达产物活性研究。应用RACE技术(Rapid amplification of cDNA ends,RACE)以3'和5'-RACE Ready cDNA为模板克隆牛多杀性巴氏杆菌诱导前后构建的抑制性消减文库(SSH文库)中的一个家蝇幼虫未知功能基因MdU-F501(Musca domestica uncharacterized-F501)全长序列,测序分析后应用生物信息学软件对MdU-F501的ORF(open reading frame)序列进行分析并预测其功能,并进一步将克隆获得的MdU-F501全长序列与原核表达载体pGEX-4T-1连接,构建重组表达质粒MdU-F501-pGEX-4T-1;将猪肺炎支原体诱导家蝇幼虫前后构建的SSH文库中的一个家蝇幼虫未知功能基因MdU-132(Musca domestica uncharacterized-132)全长序列和鸡致病性大肠杆菌诱导家蝇幼虫前后构建的SSH文库中的家蝇幼虫溶菌酶II基因MdL-II(Musca domestica lysozyme-II)全长序列亚克隆至pET-32a(+)原核表达载体中,分别构建重组表达质粒MdLII-pET-32a和MdU-132-pET-32a。将以上三种重组表达质粒分别转化至E.coli BL21(DE3)感受态细胞中,进行诱导表达和SDS-PAGE电泳分析。利用亲和层析法对表达的融合蛋白进行纯化,并以牛津杯法检测纯化蛋白的抑菌活性,主要试验结果如下:(1)应用RACE技术克隆获得大小为525bp的MdU-F501全长ORF序列,生物信息学分析结果显示MdU-F501氨基酸序列无保守结构域,是一个未知功能的蛋白,其理论等电点为4.73、疏水性较强、无明显跨膜区域、有信号肽存在。(2)经测序分析后重组表达质粒MdU-F501-pGEX-4T-1、MdLII-pET-32a和MdU-132-pET-32a构建成功,并在E.coli BL21(DE3)感受态细胞中获得可溶性表达。通过优化IPTG浓度和诱导温度,确定重组质粒MdLII-pET-32a的优化表达条件,即当IPTG浓度为0.6 mM,诱导温度为37℃时表达量相对较高。(3)经亲和层析法分别纯化获得纯度较高且条带单一的三个融合蛋白。检测三种融合蛋白的抑菌活性,结果显示,融合蛋白GST-MdU-F501无抑菌活性;融合蛋白Trx-MdLII对猪源链球菌耐药株、鸡源大肠杆菌耐药株和牛多杀性巴氏杆菌耐药株均有抑制作用,对猪肺炎支原体无抑制作用;融合蛋白Trx-MdU-132对猪源链球菌耐药株和鸡源大肠杆菌耐药株有抑制作用,对猪肺炎支原体有较弱抑制作用。
[Abstract]:Housefly larvae long live in moist, rich in organic matter in the environment, often carrying a large number of pathogens and harmful factors, but rarely incidence. Researchers predict that the disease resistance substances immunity to its unique source of housefly larvae produced in vivo. These disease resistance substances not only has broad-spectrum antibacterial and antifungal ability. Protozoa, virus, and even kill cancer cells without damaging normal cells in vivo function, has become the focus of attention of scholars at home and abroad. The pathogen induced by housefly larvae under gene on the immune response regulation, the synthesis of a variety of incremental expression of disease resistance substances, such as antibacterial peptide, lysozyme, lectin, larvae in vitro secretions, chitin, chitosan and some unknown substances. These disease resistance substances the discovery of new drugs instead of antibiotics To relieve pressure resistant pathogens as possible. This study selected bovine Pasteurella multocida, Musca domestica larvae induced by Mycoplasma hyopneumoniae and Chicken Pathogenic Escherichia coli, the three genes as research object and its clone, expression and activity of the product. The application of RACE Technology (Rapid amplification of cDNA ends. RACE 3'and 5'-RACE Ready) with cDNA as the template clone suppression subtractive library of bovine Pasteurella multocida induced and constructed (SSH Library) a housefly larvae of unknown function gene in the MdU-F501 (Musca domestica uncharacterized-F501) sequence, after sequencing analysis using bioinformatics software MdU-F501 ORF (open reading frame) sequence analysis and prediction of its function, and further cloned the full-length sequence of MdU-F501 was obtained with the prokaryotic expression vector pGEX-4T-1 to construct recombinant plasmid Md connection. U-F501-pGEX-4T-1; pig mycoplasma pneumonia induced by an unknown function gene MdU-132 of housefly larvae SSH Library of Musca domestica larvae before and after the construction of (Musca domestica uncharacterized-132) II gene MdL-II lysozyme of housefly larvae SSH Library of Musca domestica larvae before and after construction of induction and full-length sequence of Chicken Pathogenic Escherichia coli (Musca domestica lysozyme-II) sequence was subcloned into pET-32a (+) prokaryotic expression vector respectively, the recombinant expression plasmid MdLII-pET-32a and MdU-132-pET-32a. will be more than three kinds of recombinant plasmids were transformed into E.coli BL21 (DE3) competentcells, analyzed the induced expression and SDS-PAGE electrophoresis. Affinity chromatography on the expression of the fusion protein was purified using, and to detect the Oxford cup method and purification of antibacterial the activity of protein. The main results are as follows: (1) application of RACE clone size for 525bp MdU-F5 01 full-length ORF sequences, bioinformatics analysis showed that MdU-F501 amino acid sequence of non conserved domain, is a protein of unknown function. Its isoelectric point was 4.73, without obvious hydrophobicity, transmembrane domain, signal peptide. (2) by sequencing after recombinant expression plasmid MdU-F501-pGEX-4T-1, MdLII-pET-32a and MdU-132-pET-32a was constructed successfully, and in E.coli BL21 (DE3) competentcells obtained soluble expression. By optimizing the IPTG concentration and induction temperature, to determine the optimal expression conditions of the recombinant plasmid MdLII-pET-32a, when IPTG concentration was 0.6 mM, the expression induced by relatively high temperature is 37 degrees centigrade. (3) were purified by affinity chromatography to obtain high purity and a single band of three fusion protein. The fusion protein detection of three kinds of antibacterial activity, results showed that the antibacterial activity of GST-MdU-F501 fusion protein; fusion protein Trx-MdLII of porcine chain Aureus resistant strains, Escherichia coli resistant strains and bovine Pasteurella multocida strains were inhibited, had no inhibitory effect on Mycoplasma hyopneumoniae; fusion protein Trx-MdU-132 has inhibitory effect on Streptococcus suis resistant strains and resistant strains of Escherichia coli, a weak inhibition on mycoplasma pneumonia of swine.

【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】：S852.743

【参考文献】