日本血吸虫Sj Cyclophilin B基因的克隆、表达及免疫保护效果分析

Cloning and expressing of Cyclophilin B gene from Schistosoma japonnicum and the analysis of immunoprotective effect

摘要:

本研究克隆和表达了日本血吸虫Cyclophilin B (Sj CyPB) 编码基因的cDNA，分析其在日本血吸虫不同发育阶段虫体的表达情况，评估该重组抗原在小鼠体内诱导的抗血吸虫免疫保护效果。本研究以日本血吸虫童虫cDNA为模板，RT-PCR扩增其基因全长cDNA，提交序列到NCBI，登录号为GQ403665。荧光实时定量PCR分析该基因在日本血吸虫不同发育阶段虫体的表达情况，构建重组表达质粒，表达纯化重组蛋白。利用Western blotting检测重组蛋白的抗原性。以重组抗原免疫小鼠，评估其对小鼠诱导的免疫保护效果。结果表明，RT-PCR获得了Sj CyPB编码基因的全长cDNA，其开放阅读框为672 bp。经分析确定其为CyPs家族中的CyPB基因，命名为Sj CyPB。荧光实时定量PCR分析表明，该基因在18 d童虫期表达量最高，32 d次之。构建了重组表达质粒pGEX-6P-1-SjCyPB，并在大肠杆菌中成功表达，表达产物分子量为49.5 kDa。Western blotting试验显示该重组蛋白具有良好的抗原性，在小鼠免疫试验中，与空白对照组比较，免疫组小鼠获得31.5％的减虫率和41.01％的肝脏减卵率。本研究获得了日本血吸虫童虫期高表达的Sj CyPB基因的全长cDNA，成功构建了Sj CyPB原核重组表达质粒，并在大肠杆菌中成功表达，证实该重组抗原在小鼠体内诱导产生了部分免疫保护效果。

Abstract:

The present study was intend to clone and express the cDNA encoding Cyclophilin B(CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.