日本血吸虫SjIrV1的基因特性及免疫保护效果

Characterization and immunoprotective effect of SjIrV1, a 66 kDa calcium-binding protein from Schistosoma japonicum

摘要:

钙结合蛋白是日本血吸虫生长发育不可缺少的蛋白，具有非常广泛而重要的功能。在课题组日本血吸虫体被表膜蛋白研究基础上，利用PCR技术克隆了中国大陆株日本血吸虫66 kDa钙结合蛋白 (SjIrV1) 编码基因的cDNA序列，BLAST分析与菲律宾株日本血吸虫SjIrV1 cDNA编码序列一致，荧光定量PCR分析表明该基因在童虫和成虫期不同发育阶段均有表达，其中在35 d和42 d成虫中表达量较高，在42 d雌虫中该基因表达水平远高于42 d雄虫。构建重组表达质粒pET28a (+)-SjIrV1，在大肠杆菌中成功诱导表达，重组蛋白主要以可溶性形式存在，通过高效液相色谱法 (RP-HPLC) 以及串联质谱法 (MS/MS) 鉴定所获蛋白为目的蛋白SjIrV1。蛋白质印迹 (Western blotting) 分析结果显示重组蛋白能被感染日本血吸虫鼠血清和免疫鼠血清所识别，SjIrV1蛋白在虫体各发育阶段中均表达。免疫荧光染色实验观察表明SjIrV1主要分布在日本血吸虫成虫的表膜。应用重组蛋白免疫BALB/c小鼠后，免疫鼠血清中检测到较高水平的特异性IgG、IgG1和IgG2a抗体。结果表明SjIrV1可能在日本血吸虫的生长发育过程中起着重要作用。

Abstract:

Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum. Based on our primary study on tegument surface proteins of S. japonicun, a cDNA encoding a 66 kDa calcium-binding protein of S. japonicum (Chinese strain) was cloned, sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S. japonicum. The expression of SjIrV1 were detected by Real-time PCR, using cDNA templates isolated from 7, 14, 21, 28, 35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages, and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms. The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a (+) vector and transformed into competent Escherichia coli BL21 for expression. The recombinant protein was purified using a Ni-NTA purification system, and confirmed by high performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS). Western blotting analysis showed that recombinant SjIrV1 (rSjIrV1) could be recognized by the S. japonicum infected mouse serum and the mouse serum specific to rSjIrV1, respectively. Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms. ELISA test revealed that IgG, IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice. The study suggested that rSjIrV1 might play an important role in the development of S. japonicum.