摘要
目的研究细粒棘球绦虫(Echinococcus granulosus,Eg)TSP基因家族TSP11基因的分子特性及其在虫体不同发育期的差异表达,为细粒棘球绦虫疫苗的研发奠定基础。方法从NCBI GenBank数据库中获得TSP11基因序列并设计特异性引物,以Eg原头节RNA为模板进行RT-PCR。将PCR产物克隆到pMD19-T载体后测序,通过生物信息学软件分析预测TSP11基因编码蛋白的结构与功能;通过SYBR GreenⅠqRT-PCR检测TSP11基因在虫体原头蚴以及成虫mRNA相对转录情况。结果生物信息学分析TSP11基因全长765个核苷酸,编码蛋白含254个氨基酸,理论等电点为8.91,为稳定蛋白分子。编码TSP11基因的氨基酸序列共含有3个跨膜区域,推测含有7个优势B抗原表位,与已登录的细粒棘球绦虫TSP11序列(XP024352489.1)同源性为99.61%。qRT-PCR显示TSP11基因在原头蚴及成虫阶段均有表达,且表达水平差异无统计学义(P>0.05)。结论成功克隆了细粒棘球绦虫TSP11基因,该基因在Eg原头蚴及成虫期均有表达,其编码蛋白为稳点蛋白,含有B细胞抗原表位,为进一步揭示其分子生物学功能奠定了基础。
Objective To explore the molecular characteristics of the TSP11 gene and its differential expression at different developmental stages of Echinococcus granulosus in order to provide a foundation for the development of an E. granulosus vaccine. Methods The sequence of the TSP11 gene and the amino acid sequence of the TSP11 protein were obtained from the NCBI database, and the sequence was cloned from hydatid protoscoleces using RT-PCR. The product was then cloned into a pMD19-T vector and sequenced. The software Prot-Param, SignaIP 4.1, and TMHMM were used to predict the physicochemical properties, the signal peptides, and the transmembrane domains of that protein. The secondary structure of the protein was analyzed using the program SOMPA, and the tertiary structure of the protein that TSP11 codes for was modeled using Expasy tools available online. The relative level of mRNA transcription in E. granulosus in different stages was analyzed using SYBR Green I qRT-RCR. Results Results indicated that the TSP11 gene contains 792 nucleotides and encodes 254 amino acids. It has a theoretical isoelectric point of 8.91 and is a stable protein. The TSP11 gene contained three transmembrane regions and had seven dominant B cell epitopes. The gene sequence was 99.61% similar to a reported TSP11 gene(XP024352489.1). qRT-PCR indicated that the TSP11 gene was expressed in both the protoscolex and adult stages, and differences in the level of gene expression were not significant(P>0.05). Conclusion The TSP11 gene of E. granulosus was successfully cloned, and the gene was expressed in both the protoscolex and adult stages. The TSP11 gene was a stable protein and contained B cell epitopes. These findings have provided a foundation to further elucidate its molecular biological function.
作者
王炜烨
王云菲
陆宝燕
马勋
张艳艳
孟季蒙
王正荣
薄新文
WANG Wei-Ye;WANG Yun-Fei;LU Bao-yan;MA Xun;ZHANG Yan-yan;MENG Ji-meng;WANG Zheng-rong;BO Xin-wen(College of Animal Science and Technology,Shihezij China 832000;State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding,Animal Husbandry and Veterinary Institute,Xinjiang Academy of Agricultural and Reclamation Sciences,Shiheziy China 832000)
出处
《中国病原生物学杂志》
CSCD
北大核心
2020年第1期25-31,共7页
Journal of Pathogen Biology
基金
国家重点基础研究发展计划项目(973计划)(No.2015CB150300)
国家自然科学基金项目(No.31860701,31360608)
新疆生产建设兵团国际科技合作项目(No.2017BC003)。
