日本血吸虫雌雄合抱相关蛋白的分离、鉴定及其DNA疫苗的研究

发布时间：2018-03-14 02:03

本文选题：日本血吸虫　切入点：雌雄合抱　出处：《中南大学》2006年博士论文　论文类型：学位论文

【摘要】： 研究目的血吸虫病是一个严重的全球性公共卫生问题，全球每年超过6亿人口受到血吸虫感染的威胁，约2亿人口被感染。目前，控制血吸虫病的主要策略是用安全有效的药物对感染人群进行化疗，但化疗并不能预防人群的反复再感染，致使疫情易于反弹。当前，国家提出现阶段以控制传染源为主的综合防治新策略，对于湖区血吸虫病疫情的控制将起到重要的作用。日本血吸虫(Schistosoma japonicum，Sj)的一个显著特点是雌雄异体但又终生合抱。雌雄合抱是血吸虫生长发育、成熟、产卵的前提，，而虫卵是造成病理损害和病原体传播的关键因素。阻断传播型抗血吸虫卵胚发育和抗雌虫生殖产卵的研究，一直是本课题组的重要攻关内容。在1999年-2000年我国总理基金血防重点项目结题后进行的全国血吸虫疫苗免疫保护统一检测实验中，由本室构建的天然分子疫苗(1号疫苗)在全国16种候选疫苗中获得了最佳的动物保护效果。本文在此研究基础上，利用双向电泳和质谱技术，鉴定了9个日本血吸虫雌雄合抱相关蛋白，从中选用编码血吸虫雌雄合抱相关蛋白SJCHGC基因为疫苗候选分子，进行克隆、表达及DNA疫苗构建，以考核其作为抗日本血吸虫病疫苗候选分子的潜在价值，为抗日本血吸虫雌雄合抱和抗生殖疫苗的制备及应用提供实验依据。研究方法 1)利用双向凝胶电泳和MALDI-TOF质谱分析等蛋白质组学方法，对比研究了日本血吸虫雄虫在合抱与未合抱状态下蛋白质表达上的差异，并在mRNA水平进行进一步验证。 2)采用生物信息学等技术对获得的日本血吸虫雌雄合抱相关蛋白SJCHGC(Sj22.7)编码基因序列进行开放阅读框(ORF)的寻找，编码氨基酸的推导，蛋白质同源性比较以及二、三级结构的预测。 3)通过用阳离子脂质体Lipofectamine2000将重组质粒pEGFP-C1／SJCHGC转染COS-7细胞，用荧光显微镜直接观察pEGFP-C1／SJCHGC融合蛋白在细胞中的分布和定位，用RT-PCR、SDS-PAGE和Western blot方法验证其mRNA和蛋白的表达。 4)用PCR特异性扩增SJCHGC基因，将其克隆入真核表达载体pcDNA3，构建DNA疫苗pcDNA3／SJCHGC，并通过PCR、双酶切及测序鉴定。将pcDNA3／SJCHGC经脂质体转染Hela细胞，检测SJCHGC蛋白的体外瞬时表达。免疫接种并攻击感染小鼠，检测重组质粒在小鼠肌肉组织中的表达情况，以减虫率、减卵率和每雌肝卵数评价其免疫保护性。 5)用DNA疫苗pcDNA3／SJCHGC免疫接种并攻击感染小鼠，通过ELISA法测定血清中总IgG抗体水平，并以Western blot分析抗SJCHGC特异性抗体。四甲基偶氮唑盐试验(MTT法)检测免疫小鼠T淋巴细胞增殖反应。攻击感染后以脾细胞培养法检测经血吸虫雄虫可溶性抗原(AWAm)刺激后，小鼠脾细胞分泌IFN-γ和IL-4的水平。研究结果 1．采用2-DE和质谱技术分离、鉴定Sj雌雄合抱差异表达蛋白利用双向凝胶电泳和MALDI-TOF质谱分析，获得重复性和分辨率较好的双向凝胶电泳图谱。质谱分析了11个差异表达蛋白质点，获得11个点的肽质量指纹图，通过查询数据库成功鉴定了9个血吸虫雌雄合抱相关蛋白质，大多数差异表达蛋白质功能涉及血吸虫的生长发育、生殖、营养、运动、信号传递等过程。 2．Sj雌雄合抱相关蛋白新基因SJCHGC编码蛋白结构与功能分析通过Internet在线分析和生物信息学软件分析，对新基因SJCHGC的结构和功能有了进一步的认识，序列分析结果提示该cDNA序列含有一个597bp的完整阅读框序列，编码198个氨基酸，其编码蛋白的理论分子量为22.76kDa，等电点为4.84。SJCHGC的编码蛋白含有2个Efh(EF-hand)保守结构功能域，即钙结合基序，属于钙传感器和钙调节基因的超家族成员。功能位点分析显示含有多个磷酸化位点，可能为一重要的胞浆内信号转导分子；基因结构及抗原表位分析显示，该基因具有较好的抗原性。 3．pEGFP-C1／SJCHGC真核表达载体的构建及在COS-7细胞中的表达定位在空载体pEGFP-C1转染组中，COS-7细胞内绿色荧光弥散分布于整个细胞；重组质粒pEGFP-C1／SJCHGC转染组中，绿色荧光分布在细胞胞浆中。RT-PCR结果示，pEGFP-C1／SJCHGC转染的COS-7细胞在622bp处有一条带，而用空质粒pEGFP-C1转染的COS-7细胞未出现条带，表明pEGFP-C1／SJCHGC转染细胞中有SJCHGC基因转录。Western blot的结果也确证了pEGFP-C1／SJCHGC融合蛋白的表达。 4．SJCHGC DNA疫苗的构建、表达及免疫保护性研究经PCR、双酶切及测序鉴定结果证明，成功构建了SJCHGC真核表达重组质粒pcDNA3／SJCHGC。pcDNA3／SJCHGC可在Hela细胞中特异性表达SJCHGC蛋白，并能在小鼠肌肉组织细胞中表达，其表达蛋白能被血吸虫AWAm免疫兔血清识别。免疫保护效果测定显示，DNA疫苗pcDNA3／SJCHGC免疫小鼠后获得了29.70％的减虫率、47.25％肝减卵率、51.77％肠减卵率以及25.90％每雌肝减卵率，具有明显的抗雌虫生殖能力。 5．DNA疫苗pcDNA3／SJCHGC免疫效应机制初探免疫后4周pcDNA3／SJCHGC组小鼠血清中IgG抗体水平开始升高。ELISA法和Western blot示免疫小鼠产生了抗SJCHGC特异性IgG抗体。用AWAm刺激免疫小鼠脾细胞，有明显的T细胞增殖反应。攻击感染后用AWAm刺激，pcDNA3／SJCHGC组脾细胞产生较高水平的Th1型细胞因子IFN-γ，而产生的Th2型细胞因子IL-4水平较其它组低。结论 1)建立了日本血吸虫合抱前后雄虫双向凝胶电泳图谱，并应用质谱技术成功鉴定了9个血吸虫雄虫合抱相关的差异表达蛋白质，其功能涉及血吸虫的生长发育、生殖、营养、运动、信号传递等过程。为血吸虫蛋白质组数据库的建立提供了有意义的数据，为揭示日本血吸虫雌雄合抱的机制提供新的线索和思路。 2)生物信息学分析示：SJCHGC的编码蛋白含有2个Efh(EF-hand)保守结构功能域，即钙结合基序，属于钙传感器和钙调节基因的超家族成员，存在多个潜在抗原表位与特定功能位点，可能为一重要的胞浆内信号转导分子。属首次发现并报导的与日本血吸虫雌雄合抱相关的蛋白质，进一步研究其功能具有重要的生物学意义。 3)重组质粒pEGFP-C1／SJCHGC转染COS-7细胞，可用荧光显微镜直接观察pEGFP-C1／SJCHGC融合蛋白在COS-7细胞中的表达和胞浆中亚细胞定位，细胞所表达的融合蛋白具有血吸虫抗原性，为该基因功能研究提供了线索。 4) DNA疫苗pcDNA3／SJCHGC可诱导小鼠产生较显著的抗血吸虫攻击感染的免疫保护力，减虫率为29.70％，肝减卵率为47.25％，肠减卵率为51.77％，每雌肝减卵率为25.90％，具有明显的抗雌虫生殖能力。提示，该核酸疫苗可作为新的有效抗血吸虫病疫苗候选分子。 5)体液免疫和细胞免疫应答共同参与了DNA疫苗pcDNA3／SJCHGC诱导的保护性免疫作用，其中Th1型优势免疫应答在抗日本血吸虫感染的保护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.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R383;R392

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