日本血吸虫童虫差异表达蛋白磷酸甘油酸变位酶SjPGAM的研究

发布时间：2018-04-25 22:26

本文选题：日本血吸虫 + 糖酵解　；参考：《上海师范大学》2008年硕士论文

【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。由磷酸甘油酸变位酶(Phosphoglycerate mutas PGAM)基因家族产物与其它相关基因产物所构成的糖代谢途径,是细胞能量代谢的一个关键途径,对动物的生长发育起着重要的作用。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,首次克隆了日本血吸虫PGAM基因,并对这个基因进行了表达及生物学功能的初步研究。作者首先利用本实验室在双向电泳结合肽指纹图谱分析基础上获得的一童虫期差异表达蛋白的一段肽序列,以此肽序列为询问序列在日本血吸虫EST库中搜索到1个日本血吸虫的相应EST片段(GenBank登录号AAL30898.2),根据该EST序列,设计特异引物,利用PCR技术首次克隆获得日本血吸虫糖酵解途径中的相关蛋白PGAM编码基因SjPGAM(GenBank登录号EU374631)。生物信息学分析表明这个基因编码的蛋白质具有十分典型的PGAM家族蛋白特征:在其保守区域同源性为100%,大部分为催化位点和组氨酸结合域。在183～190氨基酸处为磷酸甘油酸结合位点。具有三个糖基化位点。序列分析表明SjPGAM基因的ORF含1 003bp,编码250个氨基酸,理论分子量28.26kD,理论等电点7.01,该基因编码的氨基酸序列与华支睾吸虫的PGAM相似性达79%,与人PGAM的相似性为58%。实时定量PCR分析显示该基因在7天童虫、14天童虫、19天童虫、27天成虫、32天成虫、42天雌虫及42天雄虫中均有表达,其中19天和14天童虫中的表达量明显高于其它发育阶段。成功构建了该基因的重组表达质粒pET28a(+)-SjPGAM,并在大肠杆菌系统中成功地表达,重组蛋白以包涵体形式存在,分子量为31kD,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。应用重组蛋白rSjPGAM免疫BALB/c小鼠,获得了16.48%的减虫率,27.11%的粪便减卵率和28.42%的肝组织减卵率。本文首次克隆了编码日本血吸虫PGAM的基因,发现该基因在童虫期高表达;成功将SjPGAM在大肠杆菌中进行了表达,在小鼠中初步评估了该重组蛋白诱导的免疫保护效果。本研究为深入探讨SjPGAM在血吸虫能量代谢过程中的作用及童虫发育生物学提供了基础,也对研制早期干预血吸虫生长发育的有效疫苗和药物提供了新思路。
[Abstract]:Schistosomiasis is a zoonotic parasitic disease caused by schistosomiasis infection. Different gene expression patterns of Schistosoma japonicum in different developmental stages resulted in distinct metabolic and developmental processes and significant biological and morphological changes. The glycometabolism pathway composed of phosphoglycerate mutas PGAM gene family products and other related gene products is a key pathway of cell energy metabolism and plays an important role in the growth and development of animals. The PGAM gene of Schistosoma japonicum was cloned for the first time on the basis of the study of sex and phase differential proteome of Schistosoma japonicum, and the expression and biological function of this gene were studied. The authors first obtained a sequence of peptides of differentially expressed proteins from a child worm based on two-dimensional electrophoresis and peptide fingerprinting analysis in our laboratory. A corresponding EST fragment of Schistosoma japonicum was found in the EST library of Schistosoma japonicum by using this peptide sequence as a query sequence. According to the EST sequence, a specific primer was designed according to the accession number AAL30898.2 of Schistosoma japonicum. The PGAM encoding gene EU374631 of the glycolysis pathway of Schistosoma japonicum was first cloned by PCR technique. Bioinformatics analysis showed that the protein encoded by this gene had typical PGAM family characteristics: 100 homology in its conserved region, most of which were catalytic site and histidine binding domain. Glyceric acid phosphate binding site was found at the amino acid 183C 190. There are three glycosylation sites. Sequence analysis showed that the ORF of SjPGAM gene contained 1 003bpand encoded 250 amino acids with theoretical molecular weight of 28.26kD.The theoretical isoelectric point was 7.01. The amino acid sequence encoded by this gene was similar to the PGAM of Clonorchis sinensis and human PGAM. Real time quantitative PCR analysis showed that the gene was expressed in both female and male worms on day 7, day 14, day 19, day 27, adult day 32, adult at day 42 and male at day 42, respectively, and the expression levels in 19 and 14 days were significantly higher than those in other developmental stages. The recombinant expression plasmid pET28a-SjPGAM was successfully constructed and successfully expressed in Escherichia coli system. The recombinant protein was expressed as inclusion body, and its molecular weight was 31 kDX Western blotting, which showed that the expressed product could be recognized by the sera of rabbits immunized with Schistosoma japonicum adult antigen. It has good antigenicity. The recombinant protein rSjPGAM was used to immunize BALB/c mice, and the fecal egg reduction rate of 16.48% and liver tissue was 27.11% and 28.42%, respectively. The gene encoding PGAM of Schistosoma japonicum was cloned for the first time, and it was found that the gene was overexpressed in infantile stage, SjPGAM was successfully expressed in Escherichia coli, and the immune protection induced by the recombinant protein was evaluated in mice. This study provides a basis for further study on the role of SjPGAM in the process of energy metabolism of Schistosoma japonicum and the developmental biology of Schistosoma japonicum. It also provides a new idea for the development of effective vaccines and drugs for early intervention of Schistosoma japonicum growth and development.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R383

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