细粒棘球绦虫TSP 3基因的生物信息学分析及原核表达

Bioinformatics analysis and prokaryotic expression of TSP 3 gene of Echinococcus granulosus

目的为分析细粒棘球绦虫(Echinococcus granulosus)TSP3基因的生物信息学特性并表达其非跨膜区蛋白。方法本研究利用PCR扩增Eg-TSP3全长基因及非跨膜区序列,采用生物信息学软件对Eg-TSP3蛋白的理化性质、结构特点及亲缘进化关系进行分析;利用RT-QPCR分析TSP3在细粒棘球绦虫不同发育阶段中的相对转录水平;并构建重组质粒,经双酶切鉴定,将其转化至大肠埃希菌E.coli BL21(DE3)后,采用SDS-PAGE检测TSP3蛋白的表达,通过western blot对蛋白进行抗原性分析。结果生物信息学分析结果显示,Eg-TSP3基因全长681 bp,编码226个氨基酸,蛋白分子式为C_(1113)H_(1772)N_(270)O_(297)S_(20),无信号肽,有4个跨膜区,理论相对分子质量约24×10^(3),含有5个B细胞抗原表位,为稳定的胞内蛋白。Eg-TSP3的氨基酸序列与绦虫属的氨基酸序列一致性为90%以上,进化关系中两者亲缘性极近,与人的氨基酸序列一致性为42.2%,进化关系中两者亲缘性较远。TSP3基因在细粒棘球绦虫的原头蚴、包囊壁以及成虫阶段均有转录,且转录水平无显著差异。双酶切鉴定及测序分析,证明成功构建重组质粒pET32a-TSP3,重组质粒转化BL21后成功诱导表达蛋白,Western blot结果显示,该蛋白具有良好的反应原性。结论本研究初步表明Eg-TSP3蛋白是一个具有潜在研究价值的疫苗免疫靶点,为进一步研究囊型包虫病的疫苗候选分子奠定了基础。

Objective To analyze the bioinformatics characteristics of Echinococcus granulosus TSP3 gene and express its non-transmembrane domain protein.Methods In this study,the full-length gene and non-transmembrane region sequences of Eg-TSP3 were amplified by PCR,and the physicochemical properties,structural characteristics and phylogenetic relationship of Eg-TSP3 protein were analyzed by bioinformatics software.The relative transcription level of TSP3 in different developmental stages of E.granulosus was analyzed by RT-QPCR.The recombinant plasmid was constructed and identified by double enzyme digestion,and transformed into E.coli BL21(DE3).The expression of TSP3 protein was detected by SDS-PAGE,and the antigenicity of the protein was analyzed by western blot.Results Bioinformatics analysis showed that the full length of Eg-TSP3 gene was 681 bp,encoding 226 amino acids.The protein molecular formula was C_(1113)H_(1772)N_(270)O_(297)S_(20),with no signal peptide and four transmembrane regions.The theoretical molecular weight was about 24 ku,and there were five B cell antigen epitopes,which were stable intracellular proteins.The amino acid sequence of Eg-TSP3 was more than 90%consistent with the amino acid sequence of Taenia.In the evolutionary relationship,the two were closely related,and the consistency with human amino acid sequence was 42.2%.In the evolutionary relationship,the two were far related.TSP3 gene was transcribed in protoscoleces,cyst walls and adult stages of E.granulosus,and there was no significant difference in the transcription level.Double enzyme digestion and sequencing analysis showed that the recombinant plasmid pET32 a-TSP3 was successfully constructed.The recombinant plasmid was transformed into BL21 and the expression protein was successfully induced.Western blot results showed that the protein had good reactivity.Conclusion This study preliminarily showed that Eg-TSP3 protein was a vaccine immune target with potential research value,which laid the foundation for further study on vaccine candidate molecules of cystic echinococcosis.