猪带绦虫胰岛素受体TsIR-1316的鉴定及其配体结合结构域的表达

Identification of Taenia solium Insulin Receptor TsIR-1316 and Expression of Its Ligand Binding Domain

目的分析鉴定猪带绦虫(Taenia solium)胰岛素受体TsIR-1316的结构特征,并对其配体结合结构域(LBD)进行表达。方法参考GeneDB数据库猪带绦虫基因组注释信息,设计特异性引物,克隆获得TsIR-1316基因,用BLASTN和BLASTP分别对其核苷酸和氨基酸序列进行同源性比对,并用在线软件对该蛋白的信号肽和结构域进行预测分析。将其LBD克隆至原核表达载体pET-30a(+),表达、纯化后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,用猪囊尾蚴阳性血清和TsIR-LBD免疫兔血清进行蛋白质印迹(Western blotting)鉴定。结果 TsIR-1316的开放阅读框为5 196 bp,编码1 732个氨基酸,与多房棘球绦虫(Echinococcus multilocularis)的同源性为84%,具有典型的酪氨酸激酶家族的结构特征和"V"型三级结构。SDS-PAGE结果显示,所表达的重组蛋白相对分子质量(M_r)为59 000,与预期一致。Western blotting检测结果显示,重组蛋白可与猪囊尾蚴阳性血清及TsIR-LBD免疫兔血清发生特异性反应,在M_r59 000处出现特异条带。结论克隆并鉴定了猪带绦虫TsIR-1316基因,其LBD的表达产物可被猪囊尾蚴阳性血清所识别,具有良好的反应原性。

Objective To characterize the structure of insulin receptor of Taenia solium（TsIR-1316） and express its ligand binding domain（LBD）. Methods Primers for TsIR-1316 were designed according to the genomic data of T. solium, and the TsIR-1316 gene was amplified by PCR. The nucleotide and amino acid sequences of TsIR-1316 were aligned using BLASTN and BLASTP, and the putative signal peptide and structure domains were predicted. The LBD fragment of TsIR-1316 was cloned into the pET-30a（＋） vector and expressed. The expressed proteins were purified, separated by SDS-PAGE and analyzed with Western blotting using cysticercus cellulosae-positive serum and TsIR-LBD-immunized rabbit serum. Results The open reading frame of TsIR-1316 was 5 196 bp, encoded a protein of 1 732 amino acids which had a typical conserved domain of tyrosine kinase family, was 84% homologous with Echinococcus multilocularis, and had a“V”-shaped tertiary structure. As expected, SDS-PAGE showed that the expressed protein had a band at Mr 59 000. Western blotting showed that the recombinant protein had specific reactions with cysticercus cellulosae positive serum and TsIR-LBD immunized rabbit serum, resulting in a specific band at Mr 59 000. Conclusion The TsIR-1316 gene was successfully cloned and identified. The expressed protein of TsIR-1316 LBD can be recognized by cysticercus cellulosae positive serum, which suggests a good antigenicity of this protein.