曼氏裂头蚴糖原磷酸化酶的生物学特性分析及功能域克隆、表达

Analysis of the biological characteristics,gene cloning,and protein expression of glycogen phosphorylase in functional domains from Sparganum mansoni

目的通过生物信息学软件分析曼氏裂头蚴(Sparganum mansoni,Sm)糖原磷酸化酶(glycogen phosphorylase,GP)的生物学特性,并对SmGP功能域(fSmGP)进行基因克隆及蛋白原核表达。方法利用生物信息学相关软件,分析Sm GP基因的生物信息学特征。通过PCR扩增得到功能域序列片段,克隆至原核表达载体pET-28α,用异丙基-β-D-硫代半乳糖苷诱导表达fSmGP,并对表达蛋白进行western blot鉴定。结果Sm GP蛋白由519个氨基酸残基组成,相对分子质量为59.80 kDa,等电点为6.95,由C、H、N、O、S 5种原子组成。SmGP的序列与其他物种的氨基酸序列一致性在70%以上。其氨基酸序列中具有32个磷酸化位点、10个潜在的B细胞表位和15个潜在的T细胞表位。在二级结构中,以α螺旋为主,三维空间结构分析显示以二聚体形式存在,且分子中的功能位点空间分布集中。PCR、双酶切鉴定、测序结果均表明pET-28α-fSmGP的功能区重组质粒构建成功;western blot结果显示成功诱导fSmGP蛋白表达。结论SmGP属于Glycosyltransferase-GTB-type超基因家族,是一个高度保守的蛋白分子,但在蛋白分子的C端是功能区域较为集中和表位集中的区域,表达该功能域为后续对fSmGP免疫学分析等实验提供了指导,同时也为研究曼氏裂头蚴的糖代谢规律奠定了一定基础。

This study aimed to analyze the biological characteristics of glycogen phosphorylase(Sm GP)from Sparganum mansoni by bioinformatics analysis,clone the gene and express the functional region of Sm GP(f Sm GP).Used bioinformatics software,the bioinformatics characteristics of Sm GP were analyzed.f Sm GP was amplified by PCR and cloned into the prokaryotic expression vector pET-28α.Expression of the functional region of Sm GP was induced by isopropyl-β-D-thiolactoside,and the recombinant protein was analyzed by western blot.The GP was composed of 519 amino acids.The relative molecular weight and isoelectric point were 59.8 kDa and 6.95,respectively.The molecular components of Sm GP comprised C,H,N,O and S atoms.Compared with GP from other species,the amino acid sequence similarity was greater than 70%.The amino acid sequence contained 32 phosphorylation sites,10 potential B cell epitopes and 15 potential T cell epitopes.In the secondary structure,theαhelix was the main structure.Three-dimensional structural analysis showed that the protein exists in dimeric form,and the functional sites are located in proximity in the molecule.A recombinant plasmid for f Sm GP expression was successfully constructed by PCR,double enzyme digestion and sequencing analysis.Western blotting showed that f Sm GP was expressed in E.coli.Sm GP belongs to the glycosyltransferase-GTB-type supergene family and is a relatively conserved protein.The functional region and epitopes are found in the C-terminus of the molecule.The expression of this functional region should provide guidance for further immunological analysis and also lay a foundation for exploring glucose metabolism in Sparganum mansoni throughout its life cycle stages.