本文選題：家蠅　切入點(diǎn)：白假絲酵母菌　出處：《貴州醫(yī)科大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:利用家蠅與人類(lèi)機(jī)會(huì)性致病真菌白假絲酵母菌(Candida albicans)的互作探討宿主與病原體的相互作用,篩選家蠅(Musca domestica)幼蟲(chóng)中腸應(yīng)對(duì)白假絲酵母菌侵染的應(yīng)答基因,研究家蠅3日齡幼蟲(chóng)中腸圍食膜蛋白質(zhì)組成成分以及在應(yīng)對(duì)白假絲酵母菌侵染后的變化,進(jìn)一步對(duì)其中篩選的圍食膜蛋白基因(Md Pt1)進(jìn)行研究,為深入研究家蠅幼蟲(chóng)腸道抵御真菌侵染的分子機(jī)制提供理論基礎(chǔ)。方法:1.采用新一代高通量測(cè)序技術(shù)對(duì)經(jīng)口感染白假絲酵母菌和空白對(duì)照的家蠅幼蟲(chóng)中腸進(jìn)行測(cè)序分析,并篩選差異表達(dá)基因;利用生物信息學(xué)工具對(duì)轉(zhuǎn)錄組測(cè)序得到的基因進(jìn)行了功能注釋、分類(lèi)以及參與的信號(hào)通路展示。應(yīng)用熒光定量PCR技術(shù)驗(yàn)證選定的20個(gè)差異表達(dá)基因。2.解剖健康家蠅3日齡幼蟲(chóng)中腸圍食膜,并解剖白假絲酵母菌侵染24h后(MD-T)和PBS對(duì)照組(MD-C)的圍食膜,通過(guò)聚丙烯酰胺凝膠電泳檢測(cè)分離提取的圍食膜蛋白,將整條泳道上的蛋白從頂?shù)降浊懈?膠在酶解消化后做液相色譜-串聯(lián)質(zhì)譜分析鑒定。3.用RT-PCR方法從家蠅cDNA中獲取Md Pt1基因,利用生物信息學(xué)分析工具預(yù)測(cè)、分析基因編碼的蛋白質(zhì)的結(jié)構(gòu)與生物學(xué)功能。將Md Pt1基因克隆到原核表達(dá)質(zhì)粒pET-28a(+)中,在大腸桿菌BL-21/DE3中誘導(dǎo)表達(dá),表達(dá)產(chǎn)物經(jīng)純化后用以免疫SD大鼠制備免疫血清。用蛋白印跡(Western Blotting)方法檢測(cè)重組蛋白的幾丁質(zhì)結(jié)合特性。結(jié)果:1.在白假絲酵母菌侵染24h后,中腸上皮組織通過(guò)轉(zhuǎn)錄組測(cè)序和生物信息學(xué)分析,共獲得3121個(gè)差異表達(dá)基因,其中表達(dá)上調(diào)基因1501個(gè),下調(diào)基因1620個(gè);通過(guò)對(duì)差異表達(dá)基因的GO分析,有2531個(gè)差異表達(dá)基因能夠GO富集,在生物過(guò)程本體顯著富集的是代謝過(guò)程、蛋白質(zhì)水解和氨基聚糖的代謝過(guò)程;在細(xì)胞組分本體顯著富集的是蛋白酶體核心復(fù)合體、蛋白酶體復(fù)合體以及細(xì)胞外區(qū)域;在分子功能本體中顯著富集的是催化活性、溶菌酶活性以及水解酶活性。KEGG Pathway分析發(fā)現(xiàn),共有1809個(gè)DEGs能夠富集到115個(gè)代謝通路,差異基因主要參與到營(yíng)養(yǎng)物質(zhì)代謝、氧化磷酸化以及免疫調(diào)節(jié)等通路。通過(guò)對(duì)選取的20個(gè)DEGs進(jìn)行qRT-PCR分析驗(yàn)證結(jié)果表明,所選基因表達(dá)模式與高通量測(cè)序結(jié)果完全一致。2.從健康的3日齡幼蟲(chóng)圍食膜共鑒定到374個(gè)蛋白質(zhì),分子量分布在8.225 kDa至996.065 kDa之間,等電點(diǎn)為3.83至11.24之間。家蠅幼蟲(chóng)飼喂白假絲酵母菌24h后解剖PM并提取蛋白質(zhì)進(jìn)行質(zhì)譜鑒定,在MD-T與MD-C組中分別有335、302個(gè)蛋白被成功鑒定,共有409個(gè)不重復(fù)蛋白質(zhì),其中MD-T組單獨(dú)鑒定蛋白質(zhì)221個(gè),MD-C組單獨(dú)鑒定蛋白質(zhì)188個(gè),兩組同時(shí)表達(dá)114個(gè),與對(duì)照相比,兩組中分子量小于30kDa的條帶差異變化明顯,顯示這些蛋白在家蠅幼蟲(chóng)對(duì)白假絲酵母菌的免疫過(guò)程中發(fā)揮了重要作用。鑒定到的大多數(shù)差異蛋白蛋白質(zhì)主要涉及免疫、消化與營(yíng)養(yǎng)代謝以及PM結(jié)構(gòu)有關(guān)。根據(jù)GO注釋分析顯示,這些蛋白質(zhì)主要參與模式識(shí)別結(jié)合、多聚糖的結(jié)合,圍食膜的結(jié)構(gòu)組成和幾丁質(zhì)結(jié)合等相關(guān)功能。3.成功獲得Md Pt1基因,cDNA全長(zhǎng)1063bp,其開(kāi)放閱讀框ORF長(zhǎng)711bp,編碼236個(gè)氨基酸,N端有20個(gè)氨基酸的信號(hào)肽序列;其蛋白質(zhì)理論分子量為26503.7 Da,等電點(diǎn)為4.65;結(jié)構(gòu)預(yù)測(cè)分析顯示其包含3個(gè)ChtBD2結(jié)構(gòu)域。RT-PCR檢測(cè)顯示Md Pt1只在家蠅幼蟲(chóng)表皮、中腸和氣管中有表達(dá)。Md Pt1蛋白具有幾丁質(zhì)結(jié)合活性,并只能夠被強(qiáng)變性劑6M尿素和2%SDS+5%β-巰基乙醇洗脫,屬于PM第三類(lèi)蛋白。結(jié)論:1.成功建立白假絲酵母菌侵染家蠅幼蟲(chóng)的腸道感染,比較轉(zhuǎn)錄組學(xué)分析獲得參與免疫應(yīng)答相關(guān)基因;2.374個(gè)PM蛋白在健康的家蠅3日齡幼蟲(chóng)被鑒定,白假絲酵母菌侵染24h后,在MD-T和MD-C分別鑒定到335、302個(gè)PM蛋白,白假絲酵母菌侵染后圍食膜的變化比較大;3.成功克隆Md Pt1基因,驗(yàn)證其具有幾丁質(zhì)結(jié)合活性�？傊�,本研究結(jié)果從宿主與白假絲酵母菌之間的相互作用,揭示了家蠅幼蟲(chóng)中腸上皮組織和圍食膜在應(yīng)對(duì)白假絲酵母菌侵染過(guò)程中的作用,為深入理解家蠅幼蟲(chóng)腸道免疫提供理論基礎(chǔ)。
[Abstract]:Objective: using the housefly and human opportunistic pathogenic fungus Candida albicans (Candida albicans) the interaction of interaction between host and pathogen, screening of housefly (Musca domestica) larvae midgut response gene of Candida albicans infection, changes of 3 day old larvae of Musca domestica peritrophic membrane protein composition and in response to the white Candida infection, further screening of the peritrophic membrane protein gene (Md Pt1) were studied to provide theoretical basis for further research on the molecular mechanism of intestinal fungal infection against housefly larvae. Methods: 1. using a new generation of high-throughput sequencing were sequenced by oral infection of housefly larvae midgut and blank yeast the control of Candida, and screening of differentially expressed genes; using bioinformatics tools for transcriptome sequencing of gene functional annotation, classification and participation The signal pathway of.2. gene display. Anatomical health 3 day old larvae of Musca domestica peritrophic membrane of 20 differential expression by fluorescence quantitative PCR technology to verify the selected, and anatomy of Candida albicans infection after 24h (MD-T) and PBS control group (MD-C) of the peritrophic membrane, extraction of peritrophic membrane proteins by polyacrylamide gel the electrophoresis separation, the lanes on the protein from top to bottom cut, glue in enzymatic digestion after tandem mass spectrometry analysis and identification of.3. using RT-PCR method to obtain the Md Pt1 gene from housefly cDNA in liquid chromatography, using bioinformatics analysis tools to predict, structure and biological function of protein analysis of gene encoding. The Md Pt1 gene was cloned into prokaryotic expression plasmid pET-28a (+), induced expression in Escherichia coli BL-21/DE3. The expression products were purified by preparation of immune serum to immune SD rats. By Western blotting (Western Blotting) detection method Chitin binding properties of the recombinant protein. Results: 1. in Candida albicans infection after 24h, midgut epithelium by transcriptome sequencing and bioinformatics analysis, a total of 3121 differentially expressed genes, including 1501 up-regulated genes and 1620 down regulated genes; gene expression analysis by GO of difference gene can GO enrichment 2531 differentially expressed, in the biological process ontology is significantly enriched the metabolic process, metabolic process of protein hydrolysis and glycosaminoglycan; proteasome core complex is in the cellular component ontology significantly enriched the proteasome complex and extracellular region; significantly enriched in molecular function ontology is the catalytic activity, lysozyme activity.KEGG Pathway and hydrolase activity analysis showed that a total of 1809 DEGs can be enriched to 115 metabolic pathways, these genes mainly involved in metabolism, oxidative phosphorylation And the immune regulatory pathway. Through the 20 selected DEGs qRT-PCR analysis results showed that the selected gene expression patterns and high-throughput sequencing results in.2. from 3 instar larvae of healthy peritrophic membrane of 374 proteins were identified, the molecular weight distribution between 8.225 kDa and 996.065 kDa, etc for 3.83 to 11.24. Housefly larvae feeding of Candida albicans 24h dissection after extraction of protein PM and identified in MD-T and MD-C group respectively, 335302 proteins were identified successfully, a total of 409 not repeat protein, MD-T protein identification alone group 221, MD-C group identified 188 separate protein at the same time, two groups of 114, compared with the control group, two groups in the change of molecular weight less than 30kDa with significant differences, showed that these proteins in the immune process of housefly larvae on Candida albicans has played an important role. Most of these proteins identified proteins mainly related to immune, digestive and nutritional metabolism and structure of PM. According to GO annotation analysis showed that these proteins were mainly involved in pattern recognition with combination of polysaccharide, peritrophic membrane structure and chitin binding and other related functions.3. Md succeeded Pt1 cDNA gene, full-length 1063bp, its open ORF long reading frame 711bp, encoding 236 amino acids N terminal signal peptide sequence of 20 amino acids; the theory of protein molecular weight of 26503.7 Da, isoelectric point was 4.65; structure prediction analysis shows that it contains 3 ChtBD2 domain Md Pt1.RT-PCR detected only in the epidermis of housefly larvae, the expression of.Md protein with Pt1 chitin binding activity in the midgut and trachea, and can only be strong denaturant urea 6M and 2%SDS+5% beta mercaptoethanol elution, belonging to the PM third protein. Conclusion: 1. the successful establishment of Candida albicans The infection of housefly larvae intestinal infection, comparative transcriptome analysis to get involved in the immune response related genes; 2.374 PM protein in healthy 3 day old larvae of housefly were identified, Candida albicans infection after 24h in MD-T and MD-C were identified to 335302 PM proteins, changes of Candida albicans infection after peritrophic the film is relatively large; 3. successful cloning of Md Pt1 gene, verifies the chitin binding activity. In conclusion, the results of this study from the host with Candida albicans interaction, reveals the housefly larvae midgut epithelium and peritrophic membrane in response to Candida albicans infection in effect, provide a theoretical basis for further understanding of housefly larvae gut immunity.

【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R379
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本文編號(hào)：1603548