中国农业科学 ›› 2010, Vol. 43 ›› Issue (7): 1531-1538 .doi: 10.3864/j.issn.0578-1752.2010.07.027

• 研究简报 • 上一篇    

日本血吸虫Sj CyclophilinA基因的克隆、表达及其生物学功能研究

彭金彪,韩宏晓,洪 炀,王欣之,石耀军,傅志强,刘金明,林矫矫

  

  1. (中国农业科学院上海兽医研究所/农业部动物寄生虫学重点开放实验室)

  • 收稿日期:2009-08-25 修回日期:2010-01-21 出版日期:2010-04-01 发布日期:2010-04-01
  • 通讯作者: 林矫矫

Cloning, Expression and Biological Function Analysis of the Novel Gene Sj Cyclophilin A of Schistosoma Japonicum

PENG Jin-biao, HAN Hong-xiao, HONG Yang, WANG Xin-zhi, SHI Yao-jun, FU Zhi-qiang,LIU Jin-ming, LIN Jiao-jiao
  

  1. (中国农业科学院上海兽医研究所/农业部动物寄生虫学重点开放实验室)

  • Received:2009-08-25 Revised:2010-01-21 Online:2010-04-01 Published:2010-04-01
  • Contact: LIN Jiao-jiao

摘要:

【目的】克隆和表达了日本血吸虫CyclophilinA(Sj CyPA)编码基因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。【方法】从实验室构建的7天童虫消减cDNA文库中,PCR扩增一EST序列的基因全长cDNA,提交到NCBI,登录号为GQ403666。应用荧光实时定量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,以pET28a(+)为载体构建重组表达质粒,并在大肠杆菌中表达。诱导、表达、纯化和复性重组蛋白,测定其PPIase活性。利用Western blot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】PCR获得了Sj CyPA编码基因的全长cDNA,其开放阅读框为519 bp。荧光实时定量PCR分析表明,该基因在13 d童虫表达量最高,为童虫期高表达基因。构建了重组表达质粒pET28a(+)-Sj CyPA,并在大肠杆菌中成功表达。复性重组蛋白具有PPIase活性。Western blot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18.72%的减虫率和44.6%的肝脏减卵率。【结论】获得了日本血吸虫童虫期高表达的Sj CyPA基因的全长cDNA,成功构建了Sj CyPA原核重组表达质粒,在大肠杆菌中成功表达,纯化复性得到有PPIase活性的Sj CyPA重组蛋白,并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。

关键词: 日本血吸虫, Cyclophilin A(CyPA), 克隆和表达, PPIase活性, 免疫保护效果

Abstract:

【Objective】 The present study was intended to clone a cDNA encoding cyclophilin A in Schistosoma japonicum and subsequently investigate its molecular functions as well as immunoprotective potential as a vaccine candidate for schistosomasis . 【Method】 Polymerase chain reaction (PCR) technique was employed to amplify the full-length cDNA encoding cyclophilin A of schistosomula (Sj CyPA) by employing a schistosomula specific enrichment cDNA library as the template. The expression profiles of Sj CyPA were determined at several different development stages by using real-time RT-PCR. The cDNA containing the open reading frame (ORF) of CyPA was subcloned into a pET28a(+) vector and the recombinant plasmid was transformed into competent E.coil/BL21 for producing recombinant protein. The PPIase activity of recombinant protein was determined by chymotrypsin-coupled chromogenic assay, and its antigenicity was confirmed by Western blot. The immunoprotective potential immunized by recombinant Sj CyPA in mice was also evaluated in the present study. 【Result】 The length of cDNA containing Sj CyPA ORF is 519 base pairs, encoding 172 amino acids. As determined by real-time PCR, the highest expression of Sj CyPA was observed at the transcript level at 13-day schistosomula stage, indicating that Sj CyPA was a abundant expression gene at schistosomula stage. The expressions of Sj CyPA both at transprict level and at protein level were not significantly alternated upon the treatment by cyclosporin A as determined by real time PCR and Western blot. Expectly, chymotrypsin- coupled chromogenic assay confirmed that Sj CyPA had PPIase activity and Western blot indicated that Sj CyPA was able to induce specific antibodies. Additionally, animal experiment showed that 18.72% worm reduction and 44.6% egg reduction were achieved in mice vaccinated with recombinant CyPA protein, respectively. 【Conclusion】 A full-length cDNA encoding Sj CyPA was obtained and its molecular characterizations were preliminarily investigated. Moreover, it was observed that the vaccination of recombinant protein of Sj CyPA could induce certain potential against schistosome schistosomasis.

Key words: Schistosoma japonnicum, CyclophilinA (Sj CyPA), gene clone and expression, PPIase activity, immunoprotective effect