本文關(guān)鍵詞： 家蠅幼蟲(chóng) 抗病活性物質(zhì) 未知功能基因 克隆表達(dá) 抑菌活性　出處：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：家蠅幼蟲(chóng)長(zhǎng)期生活在潮濕、富含有機(jī)質(zhì)的環(huán)境中,常攜帶大量的病原體和有害因子,但本身卻很少發(fā)病。據(jù)研究人員預(yù)測(cè),家蠅幼蟲(chóng)獨(dú)特的免疫防御能力源于其體內(nèi)產(chǎn)生的抗病活性物質(zhì)。這些抗病活性物質(zhì)不僅具有廣譜抗菌能力,還有抗真菌、原生動(dòng)物、病毒乃至殺死癌細(xì)胞而不破壞體內(nèi)正常細(xì)胞作用的功能,已成為國(guó)內(nèi)外學(xué)者關(guān)注的焦點(diǎn)。在病原體的誘導(dǎo)刺激下家蠅幼蟲(chóng)可通過(guò)基因?qū)ζ涿庖叻磻?yīng)進(jìn)行調(diào)控,合成多種增量表達(dá)的抗病活性物質(zhì),如抗菌肽、凝集素、溶菌酶、幼蟲(chóng)體外分泌物、幾丁質(zhì)、殼聚糖以及一些未知的活性物質(zhì)等。這些抗病活性物質(zhì)的發(fā)現(xiàn)使研發(fā)新型藥物代替抗生素進(jìn)而緩解病原體耐藥壓力成為可能。本研究分別選取牛多殺性巴氏桿菌、豬肺炎支原體及雞致病性大腸桿菌誘導(dǎo)家蠅幼蟲(chóng)前、后的三個(gè)差異基因?yàn)檠芯繉?duì)象,進(jìn)行其克隆、表達(dá)及表達(dá)產(chǎn)物活性研究。應(yīng)用RACE技術(shù)(Rapid amplification of cDNA ends,RACE)以3'和5'-RACE Ready cDNA為模板克隆牛多殺性巴氏桿菌誘導(dǎo)前后構(gòu)建的抑制性消減文庫(kù)(SSH文庫(kù))中的一個(gè)家蠅幼蟲(chóng)未知功能基因MdU-F501(Musca domestica uncharacterized-F501)全長(zhǎng)序列,測(cè)序分析后應(yīng)用生物信息學(xué)軟件對(duì)MdU-F501的ORF(open reading frame)序列進(jìn)行分析并預(yù)測(cè)其功能,并進(jìn)一步將克隆獲得的MdU-F501全長(zhǎng)序列與原核表達(dá)載體pGEX-4T-1連接,構(gòu)建重組表達(dá)質(zhì)粒MdU-F501-pGEX-4T-1;將豬肺炎支原體誘導(dǎo)家蠅幼蟲(chóng)前后構(gòu)建的SSH文庫(kù)中的一個(gè)家蠅幼蟲(chóng)未知功能基因MdU-132(Musca domestica uncharacterized-132)全長(zhǎng)序列和雞致病性大腸桿菌誘導(dǎo)家蠅幼蟲(chóng)前后構(gòu)建的SSH文庫(kù)中的家蠅幼蟲(chóng)溶菌酶II基因MdL-II(Musca domestica lysozyme-II)全長(zhǎng)序列亞克隆至pET-32a(+)原核表達(dá)載體中,分別構(gòu)建重組表達(dá)質(zhì)粒MdLII-pET-32a和MdU-132-pET-32a。將以上三種重組表達(dá)質(zhì)粒分別轉(zhuǎn)化至E.coli BL21(DE3)感受態(tài)細(xì)胞中,進(jìn)行誘導(dǎo)表達(dá)和SDS-PAGE電泳分析。利用親和層析法對(duì)表達(dá)的融合蛋白進(jìn)行純化,并以牛津杯法檢測(cè)純化蛋白的抑菌活性,主要試驗(yàn)結(jié)果如下:(1)應(yīng)用RACE技術(shù)克隆獲得大小為525bp的MdU-F501全長(zhǎng)ORF序列,生物信息學(xué)分析結(jié)果顯示MdU-F501氨基酸序列無(wú)保守結(jié)構(gòu)域,是一個(gè)未知功能的蛋白,其理論等電點(diǎn)為4.73、疏水性較強(qiáng)、無(wú)明顯跨膜區(qū)域、有信號(hào)肽存在。(2)經(jīng)測(cè)序分析后重組表達(dá)質(zhì)粒MdU-F501-pGEX-4T-1、MdLII-pET-32a和MdU-132-pET-32a構(gòu)建成功,并在E.coli BL21(DE3)感受態(tài)細(xì)胞中獲得可溶性表達(dá)。通過(guò)優(yōu)化IPTG濃度和誘導(dǎo)溫度,確定重組質(zhì)粒MdLII-pET-32a的優(yōu)化表達(dá)條件,即當(dāng)IPTG濃度為0.6 mM,誘導(dǎo)溫度為37℃時(shí)表達(dá)量相對(duì)較高。(3)經(jīng)親和層析法分別純化獲得純度較高且條帶單一的三個(gè)融合蛋白。檢測(cè)三種融合蛋白的抑菌活性,結(jié)果顯示,融合蛋白GST-MdU-F501無(wú)抑菌活性;融合蛋白Trx-MdLII對(duì)豬源鏈球菌耐藥株、雞源大腸桿菌耐藥株和牛多殺性巴氏桿菌耐藥株均有抑制作用,對(duì)豬肺炎支原體無(wú)抑制作用;融合蛋白Trx-MdU-132對(duì)豬源鏈球菌耐藥株和雞源大腸桿菌耐藥株有抑制作用,對(duì)豬肺炎支原體有較弱抑制作用。
[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.

【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】：2015
【分類(lèi)號(hào)】：S852.743

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