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目的鉴定、表达猪带绦虫(Taenia solium)丝氨酸蛋白酶抑制剂(Tsserpin B6),探索其作为诊断抗原的可行性。方法利用猪带绦虫基因组和转录组数据设计Tsserpin B6特异引物,RT-PCR扩增获得Tsserpin B6基因,对其DNA、氨基酸序列进行生物信息学分析,利用Clustal X1.83进行序列比对,并用MEGA6.0进行系统进化分析。构建pET-30a-Tsserpin B6重组表达载体,在大肠埃希菌(Escherichia coli)BL21(DE3)中诱导表达。纯化表达的蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),用猪囊尾蚴阳性血清进行蛋白质印迹(Western blotting)鉴定。结果Tsserpin B6开放阅读框为1 131 bp,编码376个氨基酸。其编码的氨基酸序列具有serpin较为保守的反应中心环和特征性结构域(NEEGAE和FTVDHPFLF),存在9个潜在的线性B淋巴细胞抗原表位。Tsserpin B6的表达产物相对分子质量(M_r)为53 000,主要以包涵体形式存在。Western blotting检测结果显示,所表达的蛋白可与猪囊尾蚴阳性血清发生特异性反应,在M_r 53 000处产生特异条带。结论克隆了Tsserpin B6基因,其表达产物能被猪囊尾蚴阳性血清所识别。
Objective To identify and express the Taenia solium serine protease inhibitor (Tsserpin B6) and explore its feasibility as a diagnostic antigen. Methods Tsserpin B6 specific primers were designed based on the genome and transcriptome of Taenia monocytogenes. Tsserpin B6 gene was amplified by RT-PCR. The DNA sequence and amino acid sequence were analyzed by bioinformatics. The sequences were aligned by Clustal X1.83 and sequenced with MEGA6 .0 Perform systematic evolutionary analysis. The pET-30a-Tsserpin B6 recombinant expression vector was constructed and induced in Escherichia coli BL21 (DE3). The purified protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using Cysticercus cellulosae positive sera. Results The Tsserpin B6 open reading frame was 1 131 bp, encoding 376 amino acids. Its encoded amino acid sequence has serpin center and characteristic domains (NEEGAE and FTVDHPFLF) which are more conservative. There are nine potential linear B lymphocyte epitopes. The relative molecular mass (M_r) of Tsserpin B6 was 53 000, mainly in the form of inclusion bodies. The result of Western blotting showed that the expressed protein could specifically react with the positive serum of Cysticercus cellulosae, producing a specific band at M_r 53 000. Conclusion The Tsserpin B6 gene was cloned and its expression product was recognized by the positive serum of Cysticercus cellulosae.