本文選題：日本血吸蟲　切入點(diǎn)：雌雄合抱　出處：《中南大學(xué)》2006年博士論文　論文類型：學(xué)位論文

【摘要】： 研究目的 血吸蟲病是一個嚴(yán)重的全球性公共衛(wèi)生問題，全球每年超過6億人口受到血吸蟲感染的威脅，約2億人口被感染。目前，控制血吸蟲病的主要策略是用安全有效的藥物對感染人群進(jìn)行化療，但化療并不能預(yù)防人群的反復(fù)再感染，致使疫情易于反彈。當(dāng)前，國家提出現(xiàn)階段以控制傳染源為主的綜合防治新策略，對于湖區(qū)血吸蟲病疫情的控制將起到重要的作用。日本血吸蟲(Schistosoma japonicum，Sj)的一個顯著特點(diǎn)是雌雄異體但又終生合抱。雌雄合抱是血吸蟲生長發(fā)育、成熟、產(chǎn)卵的前提，，而蟲卵是造成病理損害和病原體傳播的關(guān)鍵因素。阻斷傳播型抗血吸蟲卵胚發(fā)育和抗雌蟲生殖產(chǎn)卵的研究，一直是本課題組的重要攻關(guān)內(nèi)容。在1999年-2000年我國總理基金血防重點(diǎn)項(xiàng)目結(jié)題后進(jìn)行的全國血吸蟲疫苗免疫保護(hù)統(tǒng)一檢測實(shí)驗(yàn)中，由本室構(gòu)建的天然分子疫苗(1號疫苗)在全國16種候選疫苗中獲得了最佳的動物保護(hù)效果。本文在此研究基礎(chǔ)上，利用雙向電泳和質(zhì)譜技術(shù)，鑒定了9個日本血吸蟲雌雄合抱相關(guān)蛋白，從中選用編碼血吸蟲雌雄合抱相關(guān)蛋白SJCHGC基因?yàn)橐呙绾蜻x分子，進(jìn)行克隆、表達(dá)及DNA疫苗構(gòu)建，以考核其作為抗日本血吸蟲病疫苗候選分子的潛在價值，為抗日本血吸蟲雌雄合抱和抗生殖疫苗的制備及應(yīng)用提供實(shí)驗(yàn)依據(jù)。 研究方法 1)利用雙向凝膠電泳和MALDI-TOF質(zhì)譜分析等蛋白質(zhì)組學(xué)方法，對比研究了日本血吸蟲雄蟲在合抱與未合抱狀態(tài)下蛋白質(zhì)表達(dá)上的差異，并在mRNA水平進(jìn)行進(jìn)一步驗(yàn)證。 2)采用生物信息學(xué)等技術(shù)對獲得的日本血吸蟲雌雄合抱相關(guān)蛋白SJCHGC(Sj22.7)編碼基因序列進(jìn)行開放閱讀框(ORF)的尋找，編碼氨基酸的推導(dǎo)，蛋白質(zhì)同源性比較以及二、三級結(jié)構(gòu)的預(yù)測。 3)通過用陽離子脂質(zhì)體Lipofectamine2000將重組質(zhì)粒pEGFP-C1／SJCHGC轉(zhuǎn)染COS-7細(xì)胞，用熒光顯微鏡直接觀察pEGFP-C1／SJCHGC融合蛋白在細(xì)胞中的分布和定位，用RT-PCR、SDS-PAGE和Western blot方法驗(yàn)證其mRNA和蛋白的表達(dá)。 4)用PCR特異性擴(kuò)增SJCHGC基因，將其克隆入真核表達(dá)載體pcDNA3，構(gòu)建DNA疫苗pcDNA3／SJCHGC，并通過PCR、雙酶切及測序鑒定。將pcDNA3／SJCHGC經(jīng)脂質(zhì)體轉(zhuǎn)染Hela細(xì)胞，檢測SJCHGC蛋白的體外瞬時表達(dá)。免疫接種并攻擊感染小鼠，檢測重組質(zhì)粒在小鼠肌肉組織中的表達(dá)情況，以減蟲率、減卵率和每雌肝卵數(shù)評價其免疫保護(hù)性。 5)用DNA疫苗pcDNA3／SJCHGC免疫接種并攻擊感染小鼠，通過ELISA法測定血清中總IgG抗體水平，并以Western blot分析抗SJCHGC特異性抗體。四甲基偶氮唑鹽試驗(yàn)(MTT法)檢測免疫小鼠T淋巴細(xì)胞增殖反應(yīng)。攻擊感染后以脾細(xì)胞培養(yǎng)法檢測經(jīng)血吸蟲雄蟲可溶性抗原(AWAm)刺激后，小鼠脾細(xì)胞分泌IFN-γ和IL-4的水平。 研究結(jié)果 1．采用2-DE和質(zhì)譜技術(shù)分離、鑒定Sj雌雄合抱差異表達(dá)蛋白 利用雙向凝膠電泳和MALDI-TOF質(zhì)譜分析，獲得重復(fù)性和分辨率較好的雙向凝膠電泳圖譜。質(zhì)譜分析了11個差異表達(dá)蛋白質(zhì)點(diǎn)，獲得11個點(diǎn)的肽質(zhì)量指紋圖，通過查詢數(shù)據(jù)庫成功鑒定了9個血吸蟲雌雄合抱相關(guān)蛋白質(zhì)，大多數(shù)差異表達(dá)蛋白質(zhì)功能涉及血吸蟲的生長發(fā)育、生殖、營養(yǎng)、運(yùn)動、信號傳遞等過程。 2．Sj雌雄合抱相關(guān)蛋白新基因SJCHGC編碼蛋白結(jié)構(gòu)與功能分析 通過Internet在線分析和生物信息學(xué)軟件分析，對新基因SJCHGC的結(jié)構(gòu)和功能有了進(jìn)一步的認(rèn)識，序列分析結(jié)果提示該cDNA序列含有一個597bp的完整閱讀框序列，編碼198個氨基酸，其編碼蛋白的理論分子量為22.76kDa，等電點(diǎn)為4.84。SJCHGC的編碼蛋白含有2個Efh(EF-hand)保守結(jié)構(gòu)功能域，即鈣結(jié)合基序，屬于鈣傳感器和鈣調(diào)節(jié)基因的超家族成員。功能位點(diǎn)分析顯示含有多個磷酸化位點(diǎn)，可能為一重要的胞漿內(nèi)信號轉(zhuǎn)導(dǎo)分子；基因結(jié)構(gòu)及抗原表位分析顯示，該基因具有較好的抗原性。 3．pEGFP-C1／SJCHGC真核表達(dá)載體的構(gòu)建及在COS-7細(xì)胞中的表達(dá)定位 在空載體pEGFP-C1轉(zhuǎn)染組中，COS-7細(xì)胞內(nèi)綠色熒光彌散分布于整個細(xì)胞；重組質(zhì)粒pEGFP-C1／SJCHGC轉(zhuǎn)染組中，綠色熒光分布在細(xì)胞胞漿中。RT-PCR結(jié)果示，pEGFP-C1／SJCHGC轉(zhuǎn)染的COS-7細(xì)胞在622bp處有一條帶，而用空質(zhì)粒pEGFP-C1轉(zhuǎn)染的COS-7細(xì)胞未出現(xiàn)條帶，表明pEGFP-C1／SJCHGC轉(zhuǎn)染細(xì)胞中有SJCHGC基因轉(zhuǎn)錄。Western blot的結(jié)果也確證了pEGFP-C1／SJCHGC融合蛋白的表達(dá)。 4．SJCHGC DNA疫苗的構(gòu)建、表達(dá)及免疫保護(hù)性研究 經(jīng)PCR、雙酶切及測序鑒定結(jié)果證明，成功構(gòu)建了SJCHGC真核表達(dá)重組質(zhì)粒pcDNA3／SJCHGC。pcDNA3／SJCHGC可在Hela細(xì)胞中特異性表達(dá)SJCHGC蛋白，并能在小鼠肌肉組織細(xì)胞中表達(dá)，其表達(dá)蛋白能被血吸蟲AWAm免疫兔血清識別。免疫保護(hù)效果測定顯示，DNA疫苗pcDNA3／SJCHGC免疫小鼠后獲得了29.70％的減蟲率、47.25％肝減卵率、51.77％腸減卵率以及25.90％每雌肝減卵率，具有明顯的抗雌蟲生殖能力。 5．DNA疫苗pcDNA3／SJCHGC免疫效應(yīng)機(jī)制初探 免疫后4周pcDNA3／SJCHGC組小鼠血清中IgG抗體水平開始升高。ELISA法和Western blot示免疫小鼠產(chǎn)生了抗SJCHGC特異性IgG抗體。用AWAm刺激免疫小鼠脾細(xì)胞，有明顯的T細(xì)胞增殖反應(yīng)。攻擊感染后用AWAm刺激，pcDNA3／SJCHGC組脾細(xì)胞產(chǎn)生較高水平的Th1型細(xì)胞因子IFN-γ，而產(chǎn)生的Th2型細(xì)胞因子IL-4水平較其它組低。 結(jié)論 1)建立了日本血吸蟲合抱前后雄蟲雙向凝膠電泳圖譜，并應(yīng)用質(zhì)譜技術(shù)成功鑒定了9個血吸蟲雄蟲合抱相關(guān)的差異表達(dá)蛋白質(zhì)，其功能涉及血吸蟲的生長發(fā)育、生殖、營養(yǎng)、運(yùn)動、信號傳遞等過程。為血吸蟲蛋白質(zhì)組數(shù)據(jù)庫的建立提供了有意義的數(shù)據(jù)，為揭示日本血吸蟲雌雄合抱的機(jī)制提供新的線索和思路。 2)生物信息學(xué)分析示：SJCHGC的編碼蛋白含有2個Efh(EF-hand)保守結(jié)構(gòu)功能域，即鈣結(jié)合基序，屬于鈣傳感器和鈣調(diào)節(jié)基因的超家族成員，存在多個潛在抗原表位與特定功能位點(diǎn)，可能為一重要的胞漿內(nèi)信號轉(zhuǎn)導(dǎo)分子。屬首次發(fā)現(xiàn)并報導(dǎo)的與日本血吸蟲雌雄合抱相關(guān)的蛋白質(zhì)，進(jìn)一步研究其功能具有重要的生物學(xué)意義。 3)重組質(zhì)粒pEGFP-C1／SJCHGC轉(zhuǎn)染COS-7細(xì)胞，可用熒光顯微鏡直接觀察pEGFP-C1／SJCHGC融合蛋白在COS-7細(xì)胞中的表達(dá)和胞漿中亞細(xì)胞定位，細(xì)胞所表達(dá)的融合蛋白具有血吸蟲抗原性，為該基因功能研究提供了線索。 4) DNA疫苗pcDNA3／SJCHGC可誘導(dǎo)小鼠產(chǎn)生較顯著的抗血吸蟲攻擊感染的免疫保護(hù)力，減蟲率為29.70％，肝減卵率為47.25％，腸減卵率為51.77％，每雌肝減卵率為25.90％，具有明顯的抗雌蟲生殖能力。提示，該核酸疫苗可作為新的有效抗血吸蟲病疫苗候選分子。 5)體液免疫和細(xì)胞免疫應(yīng)答共同參與了DNA疫苗pcDNA3／SJCHGC誘導(dǎo)的保護(hù)性免疫作用，其中Th1型優(yōu)勢免疫應(yīng)答在抗日本血吸蟲感染的保護(hù)性免疫中起主要作用。
[Abstract]:research objective
Schistosomiasis is a serious global public health problem, with more than 600 million people exposed to the threat of infection, approximately 200 million of the population are infected. At present, the main strategy is to control schistosomiasis chemotherapy for people infected with safe and effective drugs, but the treatment does not prevent schistosome re infection, resulting in easy rebound. The current epidemic situation the new national strategy, comprehensive prevention and control at present to control the infectious source, for the control of schistosomiasis epidemic will play an important role. Schistosoma japonicum (Schistosoma japonicum Sj) is a significant characteristic is gonochorism but lifelong encircle. Male femalewormpairing is schistosome growth, maturation, spawning, and the eggs are the key factors causing pathological damage and pathogen transmission. Research on blocking the spread of anti schistosome and anti fecundity embryos, has been Is an important research content of the project group. In 1999 -2000 China's premier fund key project of schistosomiasis after concluding the national schistosome vaccine unified detection experiment, natural molecular vaccine constructed by our laboratory (No. 1 vaccine) won the best national animal protection in 16 vaccine candidates. Based in this study, using two-dimensional electrophoresis and mass spectrometry, 9 pairing associated proteins of Schistosoma japonicum were identified by encoding Schistosoma japonicum male femalewormpairing related protein SJCHGC gene as a vaccine candidate, was cloned in expression and construction of DNA vaccine and to evaluate their potential as anti schistosomiasis vaccine candidate molecules, for experiment on the basis of preparation and application of anti Schistosoma japonicum male femalewormpairing and anti fecundity vaccine.
research method
1) by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF mass spectrometry proteomics method, a comparative study of the differences in male Schistosoma japonicum and not just encircle state of protein expression, and further verified at the mRNA level.
2) by using bioinformatics for pairing associated proteins of Schistosoma japonicum SJCHGC gene (Sj22.7) encoding sequences of open reading frame (ORF) for derivation of encoding amino acid, protein homology prediction and two, three level structure.
3) the recombinant plasmid pEGFP-C1 / SJCHGC was transfected into COS-7 cells by using cationic liposome Lipofectamine2000 by fluorescence microscopy, direct observation of pEGFP-C1 / SJCHGC fusion protein in the cell distribution and location, with RT-PCR, SDS-PAGE and Western blot method to verify the mRNA and protein expression.
4) gene was amplified by SJCHGC specific PCR, cloned into eukaryotic expression vector pcDNA3 to construct DNA vaccine pcDNA3 / SJCHGC, and by PCR, double enzyme digestion and sequencing. The pcDNA3 / SJCHGC transfected Hela cells, expression of SJCHGC protein was detected in vitro. The instantaneous inoculation and infected mice and to detect the expression of recombinant plasmid in muscle of mice, the worm reduction rate, egg reduction rate and the number of eggs per female liver to evaluate its immune protection.
5 pcDNA3 / SJCHGC) with DNA vaccine immunization and infected mice, determination of total serum IgG antibody level by ELISA method, and the Western blot analysis of anti SJCHGC specific antibody. Four methyl thiazolyl tetrazolium assay (MTT method) were detected T lymphocyte proliferation. After challenge infection in spleen cell culture assay blood fluke male soluble antigen (AWAm) after stimulation of mouse spleen cells secrete IFN- and IL-4.
Research results
1. using 2-DE and mass spectrometry separation, protein identification of Sj pairing differences
Analysis of two-dimensional gel electrophoresis and mass spectrometry using MALDI-TOF, obtain good repeatability and resolution of two-dimensional gel electrophoresis mass spectrometry. 11 differentially expressed protein spots for peptide mass fingerprinting of 11 points, by querying the database successfully identified 9 proteins with unisexual Schistosoma japonicum, protein function relates to growth and development. The majority of differentially expressed reproduction, nutrition, exercise, signal transfer process.
2.Sj analysis of male femalewormpairing related protein SJCHGC gene encoding protein structure and function
Through the online software Internet analysis and bioinformatics analysis, with the further understanding of the structure and function of SJCHGC gene, sequence analysis indicated that the cDNA sequence contains a complete ORF sequence of 597bp, encoding 198 amino acids, its encoding protein theoretical molecular weight is 22.76kDa and isoelectric point of 4.84.SJCHGC encoding protein contains 2 Efh (EF-hand) conserved functional domains, namely calcium binding motif, which belongs to the calcium sensor and the calcium regulated superfamily of genes. Analysis showed that functional sites containing multiple phosphorylation sites, may be an important intracellular signaling molecule; gene structure and epitope analysis this gene, with good antigenicity.
Construction of 3.pEGFP-C1 / SJCHGC eukaryotic expression vector and expression and localization in COS-7 cells
In plasmid pEGFP-C1 transfection group, COS-7 cells, green fluorescence dispersed throughout the cell; recombinant plasmid pEGFP-C1 / SJCHGC transfection group, the distribution of green fluorescence in the cytoplasm of.RT-PCR showed that the pEGFP-C1 / SJCHGC transfected COS-7 cells have a band at 622bp, and transfected with the empty plasmid pEGFP-C1 in COS-7 cells no band showed that transcription of SJCHGC gene.Western blot pEGFP-C1 / SJCHGC in the transfected cells confirmed the expression of pEGFP-C1 / SJCHGC fusion protein.
Construction, expression and immunological protection of 4.SJCHGC DNA vaccine
By PCR, double enzyme digestion and sequencing results showed that SJCHGC was successfully constructed a recombinant eukaryotic expression plasmid pcDNA3 / SJCHGC.pcDNA3 / SJCHGC specific expression of SJCHGC protein in Hela cells, and can be expressed in skeletal muscle cells, the expression of AWAm protein can be schistosome immune rabbit sera. The immune protective effect determination showed that the mice immunized with DNA vaccine pcDNA3 / SJCHGC were obtained after 29.70% of worm reduction rate and 47.25% liver egg reduction rates of 51.77% and 25.90% intestinal reduction rate of eggs per female liver egg reduction rate, has obvious anti fecundity.
A preliminary study on the immune effect mechanism of 5.DNA vaccine pcDNA3 / SJCHGC
The IgG level pcDNA3 / SJCHGC group 4 weeks after immunization in mice serum began to increase.ELISA and Western blot in mice immunized by anti SJCHGC specific IgG antibody by AWAm. Stimulation of immune spleen cells, T cell proliferation. After being infected by AWAm stimulation, Th1 type cytokines IFN- high level pcDNA3 / SJCHGC group of spleen cells, the Th2 cytokine IL-4 levels than that of other groups.
conclusion
1) before and after a male Schistosoma japonicum 2-DE pattern was established, and the application of mass spectrometry successfully identified 9 male worm pairing associated differentially expressed proteins, whose functions are involved in schistosome growth and development, reproduction, nutrition, exercise, signal transfer process. To provide meaningful data for the establishment of the proteome of Schistosoma and provide new clues and ideas for revealing the mechanisms of Schistosoma japonicum male femalewormpairing.
2) bioinformatic analysis showed that SJCHGC encoding protein contains 2 Efh (EF-hand) conserved functional domains, namely calcium binding motif, which belongs to the calcium sensor and calcium regulated superfamily genes, there are a number of potential epitopes with specific functional sites, may be important in intracellular signal transduction for the first time. The molecular found associated with Schistosoma japonicum male femalewormpairing protein and reported, has important biological significance to further study its function.
3) the recombinant plasmid pEGFP-C1 / SJCHGC was transfected into COS-7 cells, and the expression of direct observation of cytoplasmic pEGFP-C1 / SJCHGC fusion protein in COS-7 cells. The subcellular localization by fluorescence microscope, cells express the fusion protein with antigenicity of Schistosoma, to study the gene function and provided clues.
4 pcDNA3 / SJCHGC) DNA vaccine against Schistosoma can produce protective immunity was induced in mice, the worm reduction rate was 29.70%, liver egg reduction rate was 47.25%, intestinal egg reduction rate of 51.77% per female liver egg reduction rate was 25.90%, with obvious anti fecundity ability. The results indicated that the nucleic acid the vaccine can be used as a new effective anti schistosomiasis vaccine candidate molecules.
5) humoral immunity and cellular immune response participate in the protective immunity induced by DNA vaccine pcDNA3 / SJCHGC. Th1 dominant immune response plays a major role in the protective immunity against Schistosoma japonicum infection.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R383;R392

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 ;血吸蟲感染與實(shí)驗(yàn)性大腸癌發(fā)病關(guān)系的探討(摘要)[J];腫瘤學(xué)雜志;1980年04期

2 曾慶仁,張悟澄,周金春;釘螺組織冷浸液免疫小鼠抗日本血吸蟲感染的實(shí)驗(yàn)報告[J];中國寄生蟲學(xué)與寄生蟲病雜志;1987年04期

3 葉愛民,張悟澄,吳志良,陳碧珍;日本血吸蟲感染與HBV感染關(guān)系的初探[J];中國血吸蟲病防治雜志;1993年05期

4 劉述先;;日本血吸蟲疫苗候選抗原GST的研究及重組GST抗原的應(yīng)用[J];醫(yī)學(xué)研究雜志;1996年07期

5 張超威;;哺乳動物感染曼氏血吸蟲、日本血吸蟲和埃及血吸蟲后的病理變化比較[J];國際醫(yī)學(xué)寄生蟲病雜志;1990年02期

6 朱國正;汪英華;雷觀愚;劉瑞三;;東方田鼠的實(shí)驗(yàn)室飼養(yǎng)及其抗血吸蟲感染特性[J];實(shí)驗(yàn)動物與比較醫(yī)學(xué);1991年04期

7 余炳楨,黃遠(yuǎn)程,容t

本文編號：1609112