旋毛虫5′-nucleotidase基因特征与克隆表达

Sequence characterization and expression of Trichinella spiralis 5′-nucleotidase gene

目的研究旋毛虫5′-nucleotidase基因的序列结构特征及其蛋白克隆表达。方法从旋毛虫获得5′-nucleotidase基因的序列,利用生物信息学方法进行系统性分析,并进行原核表达。结果旋毛虫5′-nucleotidase基因大小为1 653bp,编码550个氨基酸,119个氨基酸残基组成,理论相对分子质量(Mr)为62kD,分子式为C_(2800)H_(4358)N_(742)O_(803)S_(23),等电点为6.13,属于稳定存在的亲水蛋白质。该蛋白含有21个氨基酸组成的信号肽,同时具有跨膜区域,此外还含有3个N-糖基化位点、2个O-糖基化位点、20个磷酸化位点、28个B细胞线性结合位点和13个T细胞结合位点。二级结构中,α-螺旋占43.27%(238个),伸展链占22.73%(125个),β-折叠占7.82%(43个),无规则卷曲占26.18%(144个)。SDS-PAGE显示,5′-nucleotidase基因在原核表达系统中呈可溶性形式表达,重组蛋白大小约为74kD。结论成功克隆了旋毛虫5′-nucleotidase基因,对其进行序列分析和原核表达,为进一步探究5′-nucleotidase蛋白在旋毛虫感染过程中的作用奠定基础。

We aimed to study the sequence structure and cloning and expression of 5′-nucleotidase gene from Trichinella spiralis(T.spiralis).The sequence of 5′-nucleotidase gene was obtained from T.spiralis,analyzed systematically by bioinformatics method,and expressed in prokaryotic expression system and purified.Sequencing analysis showed that the size of 5′-nucleotidase gene was 1 653 bp.The gene encoded a protein of 550 amino acids.The protein was composed of 119 amino acid residues and the molecular formula was C2800H4358N742O803S23.5′-nucleotidase was a stable hydrophilic protein with a predicted molecular mass(Mr)of 62 kD and theoretical pI of 6.13.The protein has signal peptides and a transmembrane region.A total of 3 N-glycosylation sites,2 O-glycosylation sites,20 phosphorylation sites,28 B cells linear binding sites,and 13 T cell binding sites were predicted in 5′-nucleotidase.In the secondary structure,α-helix accounted for 43.27%(238),extension chain accounted for 22.73%(125),β-sheet accounted for 7.82%accounte d for 26.18%(144).SDS-PAGE analysis showed that the 5′-nucleotidase gene could be successfully expressed in prokaryotic expression system.The soluble expression products with about 74 kD were purified by Ni-NTA affinity chromatography.In this study,the 5′-nucleotidase gene from T.spiralis was successfully cloned and analyzed and highly expressed in E.coli.The results laid the foundation for the further study on the role of 5′-nucleotidase of development infection in T.spiralis.