【摘要】 目的克隆鉴定猪带绦虫胰岛素受体TsIR-4810基因,并表达其LBD区。方法设计猪带绦虫胰岛素受体TsIR-4810特异性引物,并分段进行RT-PCR扩增。TsIR-4810的完整ORF序列经BLAST同源比对,SMART预测其结构域,SWISS-MODEL同源建模,并用PhyML构建系统发育树。将TsIR-4810的LBD区克隆至pET-30a中进行诱导表达,表达产物经SDS-PAGE和Western-blot鉴定。结果获得的目的基因完整的ORF为4 920bp,其编码蛋白的氨基酸序列与其他生物的胰岛素受体一样,具有相对保守的分泌信号肽、受体L1和L2结构域、FnⅢ结构域、跨膜区和受体酪氨酸激酶域。构建的LBD重组菌株诱导表达了63ku的目的蛋白,与预期大小相符,且主要以包涵体形式存在。结论 TsIR-4810为猪带绦虫胰岛素受体基因,其LBD区的成功表达,将为开展基于胰岛素受体LBD区的新型疫苗和药物奠定基础。更多还原

【Abstract】 Taenia solium insulin receptor 4810(TsIR-4810)was identified,and its ligand binding domain(LBD)was cloned and expressed in the present study.Two pairs of specific primers were designed and used to amplify overlapping segments of the complete TsIR-4810 receptor gene.The complete ORF was confirmed by sequencing.Homology analysis of TsIR-4810 gene was conducted using BLAST and the domain of its encoding protein was predicted by SMART software.Tertiary structure for TsIR-4810 was predicted by SWISS-MODEL.Phylogenetic analysis was performed using PhyML.LBD fragment of TsIR-4810 gene was cloned into the PET-30 avector and expressed after induction with IPTG.The expressed product was identified by SDS-PAGE and Western-blot analysis.The full length cDNA of TsIR-4810 was 4 920 bp.The comprehensive analyses of its amino acid sequence revealed the peptide showed the common features shared by other members of the insulin family,including an insulin-like motif,signal sequence and conserved domain of tyrosine kinase family.The recombinant protein was successfully expressed in E.coli BL21(DE3)and the molecular mass of the protein was estimated to be 63 kDa.Western blotting with anti-His antibody revealed a specific reactivity with recombinant protein and showed the successful expression in the prokaryotic system.The results showed that TsIR-4810 identified was insulin receptor from T.solium,providing the foundation for further studies on its physiological function and development of new vaccines and biological drugs.更多还原