细粒棘球绦虫TSP2基因的克隆及生物信息学分析

Cloning and bioinformatic analysis of the TSP2 gene of Echinococcus granulosus

目的克隆细粒棘球绦虫发育相关基因Tetraspain 2-TSP2(TSP2),并对其进行生物学分析。方法通过在线检索WormBas ParaSite网站中Eg细粒棘球绦虫基因组数据库,获得TSP1的cDNA序列并设计特异性引物。提取细粒棘球绦虫原头蚴总RNA,以此模板,RT-PCR扩增TSP2基因,将PCR产物克隆到pMD19-T载体后进行测序,并进行生物信息学分析。通过SYBR Green I qRT-PCR方法分析TSP2基因在原头蚴、包囊壁以及成虫中mRNA的相对表达量。结果克隆的TSP2基因序列与WormBas ParaSite中已登录的细粒棘球绦虫跨膜蛋白EGR05083.1同源性为100%。TSP2基因cDNA全长621个核苷酸,编码206个氨基酸的TSP2蛋白,理论等电点为7.33,不稳定指数为47.50,为不稳定蛋白。脂肪系数为109.71,亲水性疏水性平均值为0.987,为疏水性可溶性蛋白。TSP2编码的蛋白含有4个跨膜区,4个优势B抗原表位,属于跨膜蛋白家族,该蛋白具有3个开放阅读框。qRT-PCR显示,TSP2基因在细粒棘球绦虫原头蚴、包囊壁及成虫中均有转录,转录水平差异无统计学意义(P>0.05)。结论细粒棘球绦虫TSP2基因其编码蛋白含有优势B抗原表位,为包虫病重组免疫诊断抗原和亚单位疫苗的研发奠定了基础。

Objective To clone and biologically analyze the tetraspanin 2(TSP2) gene from Echinococcus granulosus. Methods The cDNA sequence of TSP1 was obtained from an online search of the E. granulosus genome database on the WormBas ParaSite website, and specific primers were designed. The total RNA of E. granulosus protoscoleces was extracted, and the TSP2 gene was amplified with RT-PCR using this template. The PCR product was cloned into a pMD19-T vector for sequencing and bioinformatic analysis. The relative expression of TSP2 mRNA in protoscoleces, cysts walls, and adults was analyzed using SYBR Green I qRT-PCR. Results The cloned TSP2 gene sequence had 100% similarity to the transmembrane protein EGR 05083.1 of E. granulosus according to WormBas ParaSite. The full-length cDNA of TSP2 is 621 nucleotides, encoding 206 amino acids of the TSP2 protein. Its theoretical isoelectric point is 7.33, and its instability index is 47.50. Its aliphatic index was 109.71, and its average hydrophilicity and hydrophobicity was 0.987, indicating that the protein is a hydrophobic soluble protein. The protein encoded by TSP2 contains four transmembrane regions and four dominant B epitopes, and it belongs to the transmembrane protein family. The protein has 3 open reading frames. qRT-PCR indicated that the TSP2 gene was transcribed in protoscoleces, cyst walls, and adult E. granulosus, and differences in the level of transcription were not significant(P>0.05). Conclusion The protein encoded by the TSP2 gene of E. granulosus contains dominant B epitopes. The finding lays the foundation for the development of a recombinant immunodiagnostic antigen and subunit vaccine against hydatid disease.