本文選題：血吸蟲雌蟲　切入點：抑制性消減雜交技術(shù)　出處：《安徽醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：血吸蟲病(schistosomiasis)是由血吸蟲(Schistosoma)感染引起的一種分布廣泛、危害嚴(yán)重的人畜共患寄生蟲病。血吸蟲為吸蟲中罕見的雌雄異體形態(tài)特征,雌雄蟲體之間的相互作用對血吸蟲性器官的發(fā)育和產(chǎn)卵起著至關(guān)重要的作用,血吸蟲雌蟲性器官的發(fā)育成熟和產(chǎn)卵必須通過雌雄蟲合抱這一生物學(xué)表象方可完成。成熟雌蟲所產(chǎn)生的大量蟲卵而引起的臟器病變是造成宿主主要病理損害和疾病傳播的前提。本研究目的為篩選鑒定日本血吸蟲雌蟲在合抱前期(感染后16天)、合抱初期(感染后18天)和合抱后期(感染后24天)差異表達(dá)基因,確定其為雌蟲生殖發(fā)育相關(guān)的關(guān)鍵分子,加深對合抱的血吸蟲性發(fā)育成熟和產(chǎn)生活性蟲卵的理解,為控制血吸蟲病的流行和危害尋找新的靶標(biāo)。 方法：應(yīng)用日本血吸蟲感染模型,給以每只昆明鼠感染70條左右尾蚴,分別于14、15、16、17和18天剖殺小鼠,灌注法收集蟲體,觀察計數(shù)該時間段雌雄合抱的對數(shù),確定血吸蟲雌雄合抱前和雌雄合抱初期的具體時間。再應(yīng)用抑制性消減雜交技術(shù)(suppression subtractive hybridization,SSH))篩選日本血吸蟲雌蟲在雌雄合抱前(感染后16天)、合抱初期(感染后18天)和合抱后期(感染后24天)的差異表達(dá)基因。先用合抱初期的雌蟲cDNA作為檢測子(tester),合抱前期的雌蟲cDNA作為驅(qū)趕子(driver),檢測子消減驅(qū)趕子富集合抱初期的高表達(dá)基因,驅(qū)趕子消減檢測子富集合抱前期的高表達(dá)基因；再用合抱后期的雌蟲cDNA作為檢測子,合抱初期的雌蟲cDNA作為驅(qū)趕子,檢測子消減驅(qū)趕子富集合抱后期的高表達(dá)基因的,驅(qū)趕子消減檢測子富集在合抱初期高表達(dá)基因,從而找出血吸蟲雌蟲合抱前、合抱初期以及合抱后期的差異表達(dá)基因。 利用生物信息學(xué)分析工具對上述所有篩選出的差異基因所編碼的蛋白進(jìn)行GO功能分類和KEGG代謝通路分析,依據(jù)分析結(jié)果推測其中28個可能是參與雌蟲生殖發(fā)育密切相關(guān)的基因,再用RT-qPCR (Real time quantity polymerase chain reaction)以不同發(fā)育期的雌蟲cDNA為模板對該28個基因進(jìn)行進(jìn)一步的驗證。 從血吸蟲雌蟲中分離卵巢,提取卵巢總RNA,再分別卵巢cDNA和成蟲從cDNA為模板,使用RT-qPCR觀察這些與生殖發(fā)育相關(guān)基因在卵巢的表達(dá)量。 結(jié)果：根據(jù)小鼠感染后不同時間血吸蟲合抱發(fā)生狀態(tài)的試驗,確定血吸蟲感染小鼠16天時為合抱前期,18天時為合抱初期,24天為合抱后期(性器官基本發(fā)育成熟)。用SSH技術(shù)成功構(gòu)建了日本血吸蟲雌蟲合抱初期和合抱前期的正、反向消減文庫,即相對于合抱前期的合抱初期高表達(dá)基因cDNA文庫和相對于合抱初期的合抱前期高表達(dá)基因cDNA文庫。同法還構(gòu)建了血吸蟲感染雌蟲合抱后期與合抱初期的正、反向消減文庫,即相對于合抱初期的合抱后期高表達(dá)基因cDNA文庫和相對于合抱后期的合抱初期高表達(dá)cDNA文庫。 對相對于合抱前期的合抱初期高表達(dá)基因、相對于合抱初期的合抱前期高表達(dá)基因、相對于合抱初期的合抱后期高表達(dá)基因以及相對于合抱后期的合抱初期高表達(dá)基因進(jìn)行測序,去除重復(fù)序列、序列拼接之后,分別獲得112個、96個、195和172個高表達(dá)基因序列標(biāo)簽(Expressed Sequence Tag, EST),通過與數(shù)據(jù)庫進(jìn)行比對,得到有明確注釋信息的分別有92、80、103和90個EST序列。 對這些EST所編碼蛋白的分子功能、細(xì)胞定位、參與的生物學(xué)過程進(jìn)行基因本體論(Gene Ontology,GO)注釋。 相對于合抱前期的合抱初期高表達(dá)基因主要參與的生物學(xué)過程為環(huán)狀化合物代謝過程、含氮化合物的代謝過程、細(xì)胞大分子生物合成過程、氧化-還原過程等。 相對于合抱初期的合抱前期高表達(dá)基因主要參與的生物學(xué)過程包括含氮化合物的代謝過程、磷代謝過程、環(huán)狀化合物代謝過程、小分子代謝、轉(zhuǎn)運過程等。 相對于合抱初期合抱后期高表達(dá)基因主要參與的生物學(xué)過程如轉(zhuǎn)運、碳水化合物的代謝過程、核苷酸代謝、小分子代謝過程等。 相對于合抱后期的合抱初期高表達(dá)基因主要參與的生物學(xué)過程包括轉(zhuǎn)運,核苷酸代謝、磷代謝、跨膜轉(zhuǎn)運等。 使用京都基因與基因組百科全書(Kyoto Encyclopedia of Genes and Genomes, KEGG)對相對于合抱前期的合抱初期高表達(dá)基因、相對于合抱初期的合抱前期高表達(dá)基因、相對于合抱初期的合抱后期高表達(dá)基、以及相對于合抱后期的合抱初期高表達(dá)基因編碼蛋白所參與的信號通路進(jìn)行分析。KEGG分析發(fā)現(xiàn)相對于合抱前期的合抱初期高表達(dá)基因和相對于合抱初期的合抱前期高表達(dá)基因所編碼蛋白參與76條信號通路；從合抱后期高表達(dá)基因而相對于合抱初期的合抱后期高表達(dá)基因和相對于合抱后期的合抱初期高表達(dá)基因這所編碼蛋白參與79條信號通路。多個高表達(dá)基因集中參與上述信號通路主要包括代謝通路,氧化磷酸化過程,內(nèi)質(zhì)網(wǎng)蛋白質(zhì)加工過程,特別是發(fā)現(xiàn)了與生殖發(fā)育相關(guān)的信號通路高表達(dá)基因。 在這些信號通路中篩選出28個可能與生殖發(fā)育信號通路相關(guān)的基因進(jìn)行熒光定量PCR的進(jìn)一步驗證,結(jié)果18個基因經(jīng)驗正與消減雜交結(jié)果一致。其中有與生殖發(fā)育直接相關(guān)的參與泌乳素和雌激素信號通路的酪氨酸激酶(tyrosine kinases, Tks)、熱休克蛋白70(Heat shock protein70,HSP70)和熱休克蛋白90(Heat shock proteins90, HSP90)、TGF-β信號通路相關(guān)的Type V collagen蛋白、參與Wnt信號通路的早老素蛋白、參與酵母細(xì)胞的發(fā)育周期的NIPBL蛋白(Nipped-β-like protein)和參與Hippo信號通路中的mob蛋白。其中HSP70、HSP90和早老素蛋白(presenilin)在合抱后期的雌蟲的基因水平明顯上調(diào),而酪氨酸激酶、Type V collagen蛋白、NIPBL蛋白和mob蛋白在合抱初期雌蟲的基因水平明顯上調(diào)。 RT-qPCR技術(shù)對雌蟲生殖器官卵巢cDNA和雌蟲成蟲cDNA的Tyk、HSP90、 Type V collagen蛋白、早老素蛋白、NIPBL蛋白(Nipped-β-like protein)和mob蛋白的分析,結(jié)果顯示NIPBL蛋白和早老素蛋白在卵巢高表達(dá)。 結(jié)論：本實驗通過抑制性消減雜交技術(shù)成功構(gòu)建了日本血吸蟲雌蟲合抱前期、合抱初期、合抱后期的消減cDNA文庫。篩選出7個可能直接與血吸蟲雌雄合抱和產(chǎn)卵相關(guān)的分子包括Tks、HSP70和HSP90、Type V collagen蛋白、早老素蛋白、NIPBL蛋白、mob蛋白等。其中有的Tks、HSP70參與血吸蟲生殖發(fā)育的功能已經(jīng)有文獻(xiàn)報道,Type V collagen蛋白、早老素蛋白、NIPBL蛋白、mob蛋白參與血吸蟲生殖發(fā)育的功能尚有待進(jìn)一步明確。
[Abstract]:Objective: schistosomiasis (schistosomiasis) by schistosome infection (Schistosoma) is a kind of distribution caused by widespread, serious zoonotic parasitic diseases. Schistosomiasis characterized dioecious fluke in rare form, the interaction between the male and female worms of Schistosoma mansoni organ development and oviposition plays a vital role in female Schistosoma japonicum organ maturation and spawning by male and female worms of the biological image to complete. Organ lesions in a large number of eggs produced by mature females is caused by the main pathological damage caused by the premise of host and the spread of the disease. The purpose of this study is to screening and identification of Schistosoma japonicum in a previous (16 days after infection), a early (18 days after infection) and a late (24 days after infection) of differentially expressed genes identified as female reproductive development related key points, deepen the The development of Schistosoma japonicum and the understanding of the production of active insect eggs will find new targets for the control of the epidemic and harm of schistosomiasis.
Methods: application of Schistosoma japonicum infection model, give each about 70 Kunming mice infected with cercariae, respectively at 14,15,16,17 and 18 days mice were killed, perfusion worms collected, observation log counting the time of pairing, determine the specific time of Schistosoma japonicum male femalewormpairing before and at the beginning of the pairing. Then using suppression subtractive hybrid Technology (suppression subtractive hybridization), SSH) screening of Schistosoma japonicum in male femalewormpairing before (16 days after infection), from early (18 days after infection) and a late (24 days after infection) of the gene difference. By early female cDNA as detection from sub (tester), a pre the female cDNA as driver (driver), sub sub subtractive gene expression detection them enrichment of early gene expression from them, cut detection early and encircle enrichment; encircle late cDNA females For the early detection, a female cDNA as driver, high gene expression detection sub cut them later from enrichment, them are enriched in the early detection of a subtractive gene expression, so as to find the bleeding of female just before and after a period of initial pairing of differentially expressed genes.
Analysis tools for gene encoding all selected proteins were analyzed by GO and KEGG functional classification of metabolic pathways using biological information, according to the analysis results that 28 may be involved in female reproductive development related gene, then RT-qPCR (Real time quantity polymerase chain reaction) in different developmental stages of the female cDNA the template for further validation of the 28 genes.
Separation of ovaries from female schistosome, ovarian total RNA extraction, respectively cDNA and cDNA from adult ovary as template, RT-qPCR was used to observe these reproductive development related genes in ovarian expression.
Results: according to the test of mice infected with different time after the occurrence of a schistosome, determine the mice infected with Schistosoma japonicum 16 days for 18 days to encircle early, just 24 days earlier, a late (basically mature organ). Using SSH technology successfully constructed Japanese blood sucking females from early and early is a the reverse SSH Library, that is, relative to the cDNA gene library of high expression and early pre pairing pairing with respect to the initial stage of high head from gene expression cDNA library. The same method was also constructed from late early and a female schistosome infection, reverse subtractive library, which is relative to the initial stage of high head from gene expression cDNA library and to from late early from high cDNA expression library.
The relative to the early early high expression of a folded gene, relative to the initial stage of high expression from a gene, relative to the high expression of early and late genes from a high relative to the expression of late genes from early pairing sequencing, removing repetitive sequences, sequence, respectively 112, 96, 195 and 172 high gene expression sequence tags (Expressed Sequence Tag, EST), by comparison with the database, get clear annotations were 92,80103 and 90 EST sequences.
Gene Ontology (GO) annotations for the molecular functions of these EST proteins, cell location, and the biological processes involved in these proteins.
Compared with the previous stage to encircle a cyclic compound metabolic process of high expression genes mainly involved in biological processes, metabolism of nitrogenous compounds in the process of cell, macromolecular biosynthesis, redox process.
Compared with the high expression of the early stage of biological processes from pairing genes mainly involved in metabolic processes including nitrogen, phosphorus metabolism, metabolism of cyclic compounds, small molecule metabolism, transport and so on.
From early stage of high relative to encircle expressed genes mainly involved in biological process such as transport, metabolism, carbohydrate metabolism of nucleotides, small molecule metabolism process.
From late early compared to the high expression of biological processes from genes mainly involved in nucleotide metabolism, including transport, phosphorus metabolism, transmembrane transport and so on.
Using the Kyoto Encyclopedia of genes and genomes (Kyoto Encyclopedia of Genes and Genomes, KEGG) on the early high relative to a pairing gene expression relative to the initial stage of high expression of a folded gene, relative to the initial stage of high expression from a base, as well as to the signal pathway of genes encoding proteins involved in a late early expression from high analysis.KEGG analysis found that compared to the previous pairing high expressed genes and early pairing with respect to the high expression of early encircle genes encoding proteins involved in pre pairing 76 signal pathways; from late gene expression relative to a great early stage of high gene expression and relative to a high expression of a late gene encoding this protein from early involvement 79 signal pathways. A number of highly expressed genes involved in the signaling pathway of the main focus To include the metabolic pathway, oxidative phosphorylation, endoplasmic reticulum protein processing, especially the discovery and reproductive and developmental signaling pathways associated gene expression.
Selected to further verify the 28 possible fluorescence quantitative PCR and reproductive development gene related signaling pathways in these signaling pathways, the 18 genes are consistent with the experience of subtractive hybridization. Among the reproduction and development is directly related to the participation of prolactin and estrogen receptor tyrosine kinase signal pathway (tyrosine kinases, Tks), heat shock protein 70 (HSP70 Heat shock protein70) and heat shock protein 90 (Heat shock, proteins90, HSP90), Type V collagen protein TGF- beta signaling pathway, Wnt signaling pathway involved in presenilin proteins, involved in the developmental cycle of yeast cells NIPBL protein (Nipped- beta -like protein) and involved in Hippo signaling pathway the mob protein. The HSP70, HSP90 and presenilin (presenilin) at the gene level from late females was significantly increased, while the Type V tyrosine kinase, collagen protein, NIPBL protein And mob protein in Schistosoma japonicum early gene level females were significantly increased.
RT-qPCR technology on female reproductive organs of female adult ovarian cDNA and cDNA Tyk, HSP90 Type, V collagen protein, presenilin protein, NIPBL protein (Nipped- beta -like protein) and mob protein analysis, the results showed that NIPBL protein and presenilin protein highly expressed in ovary.
Conclusion: This study successfully constructed by suppression of Schistosoma japonicum from early subtractive hybridization from early, late a subtractive cDNA library. Select 7 may directly with the molecular male femalewormpairing of schistosome and oviposition related including Tks, HSP70 and HSP90, Type V, collagen protein, presenilin protein, NIPBL protein, mob protein. Some Tks, HSP70 is involved in schistosome reproductive development has been reported in the literature, Type V, collagen protein, presenilin protein, NIPBL protein, mob protein is involved in schistosome reproductive development function remain to be further clarified.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R532.21

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