本文選題：日本血吸蟲　切入點：信號轉(zhuǎn)導(dǎo)蛋白　出處：《中國農(nóng)業(yè)科學(xué)院》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 血吸蟲病是由血吸蟲感染引起的分布廣泛、危害嚴(yán)重的人獸共患寄生蟲病。血吸蟲不同發(fā)育階段蟲體呈現(xiàn)不同的基因差異表達模式，導(dǎo)致血吸蟲獨特的代謝和發(fā)育過程及顯著的生物學(xué)和形態(tài)學(xué)變化。由Wnt基因家族產(chǎn)物與其它相關(guān)基因產(chǎn)物所構(gòu)成的Wnt信號通路，是細胞發(fā)育和生長調(diào)節(jié)的一個關(guān)鍵途徑，對動物的發(fā)育起著重要的調(diào)節(jié)作用。本研究在日本血吸蟲性別、期別差異蛋白質(zhì)組研究的基礎(chǔ)上，首次克隆了2個編碼信號轉(zhuǎn)導(dǎo)蛋白Wnt的基因，并對這兩個基因進行了表達及生物學(xué)功能研究。 作者首先利用本實驗室在雙向電泳結(jié)合肽質(zhì)量指紋圖譜分析基礎(chǔ)上獲得的1個Wnt家族蛋白的一段肽序列，以此肽序列為詢問序列在日本血吸蟲EST庫中搜索到2個日本血吸蟲的相應(yīng)EST片段(GenBank登錄號AAM89872、AY811118)。根據(jù)EST序列，利用RACE技術(shù)首次克隆獲得日本血吸蟲信號轉(zhuǎn)導(dǎo)蛋白Wnt4編碼基因Sjwnt4(GenBank登錄號DQ643829)和Wnt10a編碼基因Sjwnt10a(GenBank登錄號DQ643830)。 生物信息學(xué)分析表明這兩個基因編碼的蛋白質(zhì)具有十分典型的Wnt家族蛋白特征：整個蛋白序列中散在著100多個Wnt家族蛋白的保守位點；有23～24個可交連形成二硫鍵的保守的半胱氨酸殘基，其中50％位于蛋白的羧基端：具有三個或四個糖基化位點。序列分析表明Sjwnt4基因的ORF含1311bp，編碼436個氨基酸，理論分子量49.6kD，該基因編碼的氨基酸序列與日本三角渦蟲的Wnt4相似性達43％，與人Wnt4的相似性為37％。實時定量PCR分析顯示該基因在14天童蟲、19天童蟲、31天成蟲、44天雌蟲及44天雄蟲中均有表達，其中19天童蟲中的表達量明顯高于其它發(fā)育階段，44天雌蟲中的表達量明顯高于雄蟲。成功構(gòu)建了該基因的重組表達質(zhì)粒pGEX-4T-2-Sjwnt4，應(yīng)用大腸桿菌系統(tǒng)進行了表達，，重組蛋白以包涵體形式存在，分子量為76kD，Western blotting顯示表達產(chǎn)物能被日本血吸蟲成蟲抗原免疫兔血清所識別，具有良好的抗原性。Sjwnt10a基因的ORF含1896bp，編碼631個氨基酸，理論分子量73.3kD。該基因編碼的氨基酸序列與人、鼠的Wnt10a的氨基酸序列相似性都為26％。實時定量PCR分析顯示該基因在日本血吸蟲19天童蟲中表達量最高，在14天童蟲和44天雄蟲中也有表達，但分別僅為19天童蟲表達量的8.8％和5％，而在31天成蟲和44天雌蟲中沒有檢測到該基因。 本文利用RNAi技術(shù)對Sjwnt4基因表達進行了初步地探索，成功篩選出具有較高干擾效果的siRNA小分子S499，抑制率達到86.4％，為后續(xù)試驗打下基礎(chǔ)。應(yīng)用重組蛋白rSjWnt4免疫BALB／c小鼠，獲得了19.90％的減蟲率和20.58％的肝組織減卵率。 本論文率先開展了血吸蟲信號轉(zhuǎn)導(dǎo)蛋白Writ的研究，首次獲得兩個編碼信號轉(zhuǎn)導(dǎo)蛋白Writ的基因，發(fā)現(xiàn)這兩個基因在童蟲期高表達；成功將Sjwnt4在大腸桿菌中進行了表達，在小鼠中初步評估了重組蛋白誘導(dǎo)的免疫保護效果；成功篩選出對Sjwnt4基因具有干擾作用的siRNA。本研究結(jié)果，為深入揭示血吸蟲生長，特別是童蟲發(fā)育及性別發(fā)育成熟機制提供了新途徑，也對研制早期干預(yù)血吸蟲生長和抗血吸蟲生殖產(chǎn)卵的高效疫苗和藥物有重要意義。
[Abstract]:Schistosomiasis is caused by schistosome infection is widespread, serious zoonotic parasitic disease. Gene expression pattern of different presentation of Schistosoma worms from different development stage, resulting in Schistosoma unique metabolic and developmental processes and significant biological and morphological changes. Wnt signaling pathway composed of Wnt gene family related gene products and other products that is a key way to regulate cell growth and development, plays an important role in regulating the development of the animal. In this study, Schistosoma japonicum gender based stage difference proteome research, first cloned 2 genes encoding signal transduction protein Wnt, and the two genes were studied the expression and biological function.
A peptide sequence in the laboratory by the author in the two-dimensional gel electrophoresis and peptide mass fingerprinting analysis of 1 Wnt proteins were obtained on the basis of this, to ask sequence in the peptide sequence of Schistosoma japonicum EST library to search 2 Schistosoma japonicum corresponding EST fragment (GenBank accession No. AAM89872, AY811118). According to the sequence of EST. By using the technology of RACE was cloned from Schistosoma japonicum signal transduction protein Wnt4 encoding gene Sjwnt4 (GenBank accession No. DQ643829) and Wnt10a gene encoding Sjwnt10a (GenBank accession No. DQ643830).
Bioinformatics analysis showed that the two genes encoding protein with Wnt protein family very typical features: the entire protein sequences interspersed with more than 100 conserved sites of Wnt protein family; 23~24 can even form two disulfide conserved cysteine residues, of which 50% is located in the carboxyl terminal protein: three one or four N-glycosylation sites. The sequence analysis showed that Sjwnt4 gene containing ORF 1311bp, encoding 436 amino acids. The theoretical molecular weight of 49.6kD, the amino acid sequence of the gene encoding the planarian Wnt4 similarity was 43%, and the similarity of Wnt4 37%. real-time quantitative PCR analysis showed that 14 genes in Tiantong insects, 19 schistosomula, 31 days from 44 days, were expressed and 44 female worms in which the expression of the 19 days, the amount of schistosomula was significantly higher than that in other developmental stages, the expression of 44 days in females was significantly higher than that of males. Work constructed the recombinant expression plasmid pGEX-4T-2-Sjwnt4, application of Escherichia coli system for the expression of recombinant protein existed in inclusion body. The molecular weight of 76kD, Western and blotting showed that the expression product can be Schistosoma japonicum immune rabbit serum antigen recognition, has good antigenicity of.Sjwnt10a gene containing ORF 1896bp, encoding 631 amino acids the amino acid sequence, and the theory of the molecular weight of 73.3kD. gene encoding the amino acid sequences in mouse Wnt10a of 26%. real-time quantitative PCR analysis showed that the gene of Schistosoma japonicum schistosomula in 19 days, the highest expression level in 14, and 44 days schistosomula were also expressed in insects, but were only 19 schistosomula the expression of 8.8% and 5%, and in 31 days and 44 days of adult females did not detect the gene.
This paper uses the RNAi technology on Sjwnt4 gene expression in a preliminary exploration, we screened siRNA small molecule S499 has high interference effect, the inhibition rate reached 86.4%, which lay the foundation for the follow-up test. The application of recombinant protein rSjWnt4 immune BALB / c mice, the liver tissue of 19.90% worm reduction rate and egg reduction rate of 20.58%.
This paper first carried out research on Schistosoma japonicum signal transduction protein Writ, encoding a two signal transduction protein Writ gene for the first time, found in the schistosomulum high expression of these two genes; Sjwnt4 successfully expressed in Escherichia coli in mice, preliminary assessment of the protective immunity induced by recombinant protein; screening with the interference of Sjwnt4 gene siRNA. and the results of this study, in order to further reveal the growth of Schistosoma japonicum schistosomula, especially the development and sexual maturation mechanism provides a new way, also has important significance for the development of early intervention of schistosome growth and fecundity, anti schistosomiasis vaccines and drugs.

【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R383

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