曼氏裂头蚴6-磷酸葡萄糖酸脱氢酶的基因克隆、蛋白表达及转录水平分析

Biological characteristics,protein expression,and transcriptional analysis of 6-phosphogluconate dehydrogenase from sparganum

目的分析曼氏迭宫绦虫中6-磷酸葡萄糖酸脱氢酶(Sm 6PGDH)的潜在的生物学特征和获得纯化蛋白。方法通过NCBI和ExPASy网站中在线软件,预测Sm 6PGDH基因序列的生物信息学特征。通过PCR技术扩增得到Sm 6PGDH的全长序列和构建pET-28a(+)-Sm 6PGDH重组质粒,利用双酶切和测序手段鉴定重组质粒。利用western blot鉴定重组蛋白和定量PCR分析在不同浓度葡萄糖培养的裂头蚴体内的转录水平。结果Sm 6PGDH蛋白由491个氨基酸组成,理论相对分子质量约为53.84 kDa。该蛋白分子中无信号肽。Sm 6PGDH序列与绦虫、人的同源基因一致性分别为75%和65%以上。Sm 6PGDH氨基酸序列中具有39个磷酸化位点、1个O-糖基化位点和9个B细胞线性表位。通过原核蛋白表达,成功得到可溶性的融合蛋白,并且在不同浓度葡萄糖培养的裂头蚴体内Sm 6PGDH的转录水平升高。结论构建了Sm 6PGDH重组质粒并获得纯化蛋白,为在裂头蚴的糖代谢的规律研究中提供了物质保障。

This study was aimed at obtaining purified 6-phosphogluconate dehydrogenaseprotein from sparganum and analyzingits potential biological characteristics.The bioinformatics characteristics of Sm 6PGDH gene sequences were predicted with NCBI and ExPASy online software.The full-length sequence of Sm 6PGDH was amplified by PCR,and the recombinant plasmid pET-28a(+)-Sm 6PGDH was constructed.In addition,the recombinant plasmid was analyzed through double restriction enzyme digestion and sequencing.Western blotting was used to identify the recombinant protein,and real time PCR was performed to analyze the transcriptional level of Sm 6PGDH in sparganum treated with different concentrations of glucose.Sm 6PGDH protein was composed of 491 amino acids,and its theoretical molecular weight was approximately 53.84 kDa.The protein has no signal peptide.Sm 6PGDH shared 75%and 65%sequence identity with the homologous tapeworm and humangenes,respectively.The amino acid sequence of Sm 6PGDH has 39 phosphorylation sites,1 o-glycosylation site,and 9 B cell linear epitopes.We successfully constructed the recombinant plasmid,induced Sm 6PGDH protein expression,and obtained the purified protein.The soluble protein was successfully obtained through prokaryotic expression,and the transcriptional level of Sm 6PGDH increased through culture of sparganum in different concentrations of glucose.The recombinant plasmid was successfully constructed,and the purified protein was obtained,thus supporting future study of the glycometabolism regulation of sparganum.