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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。
Calcium-binding protein is an indispensable protein for the growth and development of Schistosoma japonicum, and has a very wide and important function. Based on the study of the surface membrane proteins of Schistosoma japonicum, the cDNA sequence of the 66 kDa calcium-binding protein (SjIrV1) gene of Chinese mainland Schistosoma japonicum was cloned by PCR. The BLAST analysis was consistent with that of SjIrV1 cDNA of S. japonicum The results of real-time quantitative PCR showed that the gene was expressed at different developmental stages of the schistosomiasis and adult stages, of which the expression was higher on the 35th day and the 42th day adults than that on the 42th day Male Recombinant plasmid pET28a (+) - SjIrV1 was constructed and successfully expressed in E. coli. The recombinant protein mainly existed in soluble form and identified by high performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS / MS) Protein is the target protein SjIrV1. Western blotting analysis showed that the recombinant protein was recognized by the sera of infected Schistosoma japonicum and the serum of immunized mice, and SjIrV1 protein was expressed in all developmental stages of the parasite. Immunofluorescence staining experiments showed that SjIrV1 mainly distributed in the adult Schistosoma japonicum surface membrane. After immunization of BALB / c mice with recombinant protein, higher levels of specific IgG, IgG1 and IgG2a antibodies were detected in immunized rat serum. The results showed that SjIrV1 may play an important role in the growth and development of Schistosoma japonicum.