日本血吸虫信号转导蛋白SjWnt4、SjWnt10a编码基因的克

发布时间：2018-03-18 14:37

本文选题：日本血吸虫　切入点：信号转导蛋白　出处：《中国农业科学院》2007年硕士论文　论文类型：学位论文

【摘要】： 血吸虫病是由血吸虫感染引起的分布广泛、危害严重的人兽共患寄生虫病。血吸虫不同发育阶段虫体呈现不同的基因差异表达模式，导致血吸虫独特的代谢和发育过程及显著的生物学和形态学变化。由Wnt基因家族产物与其它相关基因产物所构成的Wnt信号通路，是细胞发育和生长调节的一个关键途径，对动物的发育起着重要的调节作用。本研究在日本血吸虫性别、期别差异蛋白质组研究的基础上，首次克隆了2个编码信号转导蛋白Wnt的基因，并对这两个基因进行了表达及生物学功能研究。作者首先利用本实验室在双向电泳结合肽质量指纹图谱分析基础上获得的1个Wnt家族蛋白的一段肽序列，以此肽序列为询问序列在日本血吸虫EST库中搜索到2个日本血吸虫的相应EST片段(GenBank登录号AAM89872、AY811118)。根据EST序列，利用RACE技术首次克隆获得日本血吸虫信号转导蛋白Wnt4编码基因Sjwnt4(GenBank登录号DQ643829)和Wnt10a编码基因Sjwnt10a(GenBank登录号DQ643830)。生物信息学分析表明这两个基因编码的蛋白质具有十分典型的Wnt家族蛋白特征：整个蛋白序列中散在着100多个Wnt家族蛋白的保守位点；有23～24个可交连形成二硫键的保守的半胱氨酸残基，其中50％位于蛋白的羧基端：具有三个或四个糖基化位点。序列分析表明Sjwnt4基因的ORF含1311bp，编码436个氨基酸，理论分子量49.6kD，该基因编码的氨基酸序列与日本三角涡虫的Wnt4相似性达43％，与人Wnt4的相似性为37％。实时定量PCR分析显示该基因在14天童虫、19天童虫、31天成虫、44天雌虫及44天雄虫中均有表达，其中19天童虫中的表达量明显高于其它发育阶段，44天雌虫中的表达量明显高于雄虫。成功构建了该基因的重组表达质粒pGEX-4T-2-Sjwnt4，应用大肠杆菌系统进行了表达，，重组蛋白以包涵体形式存在，分子量为76kD，Western blotting显示表达产物能被日本血吸虫成虫抗原免疫兔血清所识别，具有良好的抗原性。Sjwnt10a基因的ORF含1896bp，编码631个氨基酸，理论分子量73.3kD。该基因编码的氨基酸序列与人、鼠的Wnt10a的氨基酸序列相似性都为26％。实时定量PCR分析显示该基因在日本血吸虫19天童虫中表达量最高，在14天童虫和44天雄虫中也有表达，但分别仅为19天童虫表达量的8.8％和5％，而在31天成虫和44天雌虫中没有检测到该基因。本文利用RNAi技术对Sjwnt4基因表达进行了初步地探索，成功筛选出具有较高干扰效果的siRNA小分子S499，抑制率达到86.4％，为后续试验打下基础。应用重组蛋白rSjWnt4免疫BALB／c小鼠，获得了19.90％的减虫率和20.58％的肝组织减卵率。本论文率先开展了血吸虫信号转导蛋白Writ的研究，首次获得两个编码信号转导蛋白Writ的基因，发现这两个基因在童虫期高表达；成功将Sjwnt4在大肠杆菌中进行了表达，在小鼠中初步评估了重组蛋白诱导的免疫保护效果；成功筛选出对Sjwnt4基因具有干扰作用的siRNA。本研究结果，为深入揭示血吸虫生长，特别是童虫发育及性别发育成熟机制提供了新途径，也对研制早期干预血吸虫生长和抗血吸虫生殖产卵的高效疫苗和药物有重要意义。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383

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