大片吸虫钙结合蛋白2基因的克隆表达及其表达产物的免疫活性研究

Cloning and expression of calcium-binding protein 2 gene of Fasciola gigantica and study on immuno-reactivity of the expressed protein

通过克隆、表达大片吸虫钙结合蛋白2(FgCaBP2)基因,旨在探索其作为诊断抗原的可行性。在分析大片吸虫蛋白组数据的基础上,根据肝片吸虫CaBP2基因组序列和转录组数据,设计以NdeⅠ和XhoⅠ作为酶切位点的引物。以大片吸虫总RNA为模板,通过RT-PCR获得FgCaBP2基因,对其进行测序、生物信息学分析。构建pET-30a-FgCaBP2重组表达载体,在大肠杆菌Transetta(DE3)感受态细胞中完成诱导、表达;对纯化的目的蛋白进行SDS-PAGE和Western-blot分析,并用目的蛋白免疫家兔,制备多克隆抗体。结果表明,FgCaBP2基因的开放阅读框长570 bp,编码189个氨基酸残基。生物信息学预测显示,FgCaBP2主要以α螺旋为主,β折叠较少,有7个β转角、4个较长的无规则卷曲、含有4个亲水区、具有较多的B细胞抗原表位。SDS-PAGE分析表明,FgCaBP2呈现出可溶性表达。Western-blot分析表明,25 ku大小的特异性目的蛋白条带能够很好地被大片吸虫感染的阳性水牛血清识别,具有良好的免疫反应性。本研究成功克隆了FgCaBP2基因,并获得了可溶性重组蛋白,该蛋白具有良好的免疫原性和免疫反应性,是潜在的大片吸虫病诊断抗原。

The objectives of the present study were to clone and express the gene of Fasciola gigantica calcium binding protein 2(FgCaBP2),and to explore the protein’s potential as a diagnostic antigen.Based on the F.gigantica transcriptome data and the genomic sequence of Fasciola hepatica CaBP2,primers containing restriction sites for NdeⅠand XhoⅠwere designed.The FgCaBP2 gene was obtained by RT-PCR amplification using total RNA from F.gigantica as a template,and its identification and bioinformatics analysis were performed.The recombinant expression vector pET-30 a-FgCaBP2 was constructed,then induced and expressed in Escherichia coli Transetta(DE3)competent cells.The purified recombinant FgCaBP2 was purified and analyzed by SDS-PAGE and Western-blot.After the rabbits were immunized with the recombinant FgCaBP2,polyclonal antibody was prepared.The results showed that the ORF of FgCaBP2 was 570 bp in length,encoding 189 amino acid residues.FgCaBP2 had manyα-helix,but only a fewβ-sheets,7β-turns,and 4 longer irregular curls,4 hydrophilic regions and many B-cell epitopes.SDS-PAGE analyses indicated FgCaBP2 exhibited soluble expression.Western-blot results showed that the target protein had a specific band at 25 ku and had a good recognition effect with the positive sera from buffaloes infected with F.gigantica.In conclusion,the FgCaBP2 gene was cloned,and its expressed recombinant protein had good immuno-reactivity,indicating that FgCaBP 2 could be a potential diagnostic antigen.