本文選題：日本血吸蟲(chóng) + 糖酵解�。� 參考：《上海師范大學(xué)》2008年碩士論文

【摘要】： 血吸蟲(chóng)病是由血吸蟲(chóng)感染引起的分布廣泛、危害嚴(yán)重的人獸共患寄生蟲(chóng)病。血吸蟲(chóng)不同發(fā)育階段蟲(chóng)體呈現(xiàn)不同的基因差異表達(dá)模式,導(dǎo)致血吸蟲(chóng)獨(dú)特的代謝和發(fā)育過(guò)程及顯著的生物學(xué)和形態(tài)學(xué)變化。由磷酸甘油酸變位酶(Phosphoglycerate mutas PGAM)基因家族產(chǎn)物與其它相關(guān)基因產(chǎn)物所構(gòu)成的糖代謝途徑,是細(xì)胞能量代謝的一個(gè)關(guān)鍵途徑,對(duì)動(dòng)物的生長(zhǎng)發(fā)育起著重要的作用。本研究在日本血吸蟲(chóng)性別、期別差異蛋白質(zhì)組研究的基礎(chǔ)上,首次克隆了日本血吸蟲(chóng)PGAM基因,并對(duì)這個(gè)基因進(jìn)行了表達(dá)及生物學(xué)功能的初步研究。 作者首先利用本實(shí)驗(yàn)室在雙向電泳結(jié)合肽指紋圖譜分析基礎(chǔ)上獲得的一童蟲(chóng)期差異表達(dá)蛋白的一段肽序列,以此肽序列為詢(xún)問(wèn)序列在日本血吸蟲(chóng)EST庫(kù)中搜索到1個(gè)日本血吸蟲(chóng)的相應(yīng)EST片段(GenBank登錄號(hào)AAL30898.2),根據(jù)該EST序列,設(shè)計(jì)特異引物,利用PCR技術(shù)首次克隆獲得日本血吸蟲(chóng)糖酵解途徑中的相關(guān)蛋白PGAM編碼基因SjPGAM(GenBank登錄號(hào)EU374631)。生物信息學(xué)分析表明這個(gè)基因編碼的蛋白質(zhì)具有十分典型的PGAM家族蛋白特征:在其保守區(qū)域同源性為100%,大部分為催化位點(diǎn)和組氨酸結(jié)合域。在183～190氨基酸處為磷酸甘油酸結(jié)合位點(diǎn)。具有三個(gè)糖基化位點(diǎn)。序列分析表明SjPGAM基因的ORF含1 003bp,編碼250個(gè)氨基酸,理論分子量28.26kD,理論等電點(diǎn)7.01,該基因編碼的氨基酸序列與華支睪吸蟲(chóng)的PGAM相似性達(dá)79%,與人PGAM的相似性為58%。實(shí)時(shí)定量PCR分析顯示該基因在7天童蟲(chóng)、14天童蟲(chóng)、19天童蟲(chóng)、27天成蟲(chóng)、32天成蟲(chóng)、42天雌蟲(chóng)及42天雄蟲(chóng)中均有表達(dá),其中19天和14天童蟲(chóng)中的表達(dá)量明顯高于其它發(fā)育階段。成功構(gòu)建了該基因的重組表達(dá)質(zhì)粒pET28a(+)-SjPGAM,并在大腸桿菌系統(tǒng)中成功地表達(dá),重組蛋白以包涵體形式存在,分子量為31kD,Western blotting顯示表達(dá)產(chǎn)物能被日本血吸蟲(chóng)成蟲(chóng)抗原免疫兔血清所識(shí)別,具有良好的抗原性。應(yīng)用重組蛋白rSjPGAM免疫BALB/c小鼠,獲得了16.48%的減蟲(chóng)率,27.11%的糞便減卵率和28.42%的肝組織減卵率。 本文首次克隆了編碼日本血吸蟲(chóng)PGAM的基因,發(fā)現(xiàn)該基因在童蟲(chóng)期高表達(dá);成功將SjPGAM在大腸桿菌中進(jìn)行了表達(dá),在小鼠中初步評(píng)估了該重組蛋白誘導(dǎo)的免疫保護(hù)效果。本研究為深入探討SjPGAM在血吸蟲(chóng)能量代謝過(guò)程中的作用及童蟲(chóng)發(fā)育生物學(xué)提供了基礎(chǔ),也對(duì)研制早期干預(yù)血吸蟲(chóng)生長(zhǎng)發(fā)育的有效疫苗和藥物提供了新思路。
[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.
【學(xué)位授予單位】：上海師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R383

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