粉尘螨谷氧还蛋白-1样蛋白基因原核表达质粒的构建及结构分析

Construction plasmid and Structural analysis of the glutaredoxin-1-like protein gene of Dermatophagoides farinae

目的获得粉尘螨谷氧还蛋白-1样蛋白(Glutaredoxin-1,Grx1)的编码基因并构建表达质粒,对该基因及其编码蛋白质进行结构预测。方法以粉尘螨总RNA为模板,RT-PCR扩增获得编码基因后插入原核表达载体pET28a(+),构建的重组质粒转化入E.coil BL21(DE3)感受态细胞中,用异丙基-b-D-硫代半乳糖苷(isopropyl-beta-Dthiogalactoside,IPTG)诱导表达,SDS-PAGE分析表达产物。采用生物信息学软件分析该基因及其编码蛋白的结构等生物学特征。结果获得的粉尘螨Grx1编码基因全长315bp,构建的原核表达质粒pET28a(+)-Grx1转化入大肠埃希菌感受态细胞后经IPTG诱导,表达相对分子质量为25.9×10^(3)的重组蛋白。生物信息学分析该基因编码的蛋白质由104个氨基酸组成,主要分布于细胞核(占87.0%)。该蛋白含有11个潜在磷酸化位点(4个苏氨酸,6个丝氨酸和1个酪氨酸),二级结构为α-螺旋(45.19%)、无规则卷曲(31.73%)、延伸链(14.42%)和β-转角(8.65%)。将该基因推导出的编码氨基酸序列进行Blast获得同源基因与屋尘螨谷胱甘肽样C8样蛋白(Dermatophagoides pteronyssinus glutaredoxin-C8-like)同源性最高(83.52%)。结论获得了粉尘螨Grx1的编码基因及其原核表达质粒,生物信息学分析该蛋白含有潜在的磷酸化位点且主要分布于细胞核,可为该基因的生理功能研究及粉尘螨的防制提供参考。

Objective To obtain the cDNA coding for the glutaredoxin-1-like protein(Grx1)of Dermatophagoides farinae and to characterize that protein.Method By using the primers designed according to the sequence for Glrx1 inferred from annotation of D.farinae transcriptome data,the cDNA was amplified by RT-PCR from total RNA of D.farinae and inserted into pET28a(+),transformed into E.coil BL21(DE3),expressed with the induction of isopropyl-beta-D-thiogalactoside(IPTG)and identified by SDS-PAGE.The structure analyses were conducted by using software and tools online.Results The product of amplification with RT-PCR showed a clear band on agarose gel electrophoresis,and nucleotide sequencing of the pET28a(+)-Grx1 plasmid yielded a coding gene 315 bp.Once the plasmid was transformed into E.coil and its expression was induced with IPTG,a specific band was produced on SDS-PAGE with a relative molecular mass of 25.9×10^(3).Bioinformatic analysis of the protein encoded by this gene consists of 104 amino acids,subcellularly localized to the nucleus(87.0%).The protein contains 11 potential phosphorylation sites,including four Threonine,six Serine and one Tyrosine.And it's advanced structure consisted of random coil(31.73%),α-helix(45.19%),extended chain(14.42%),andβ-turn(8.65%).Blast of the deduced amino acid sequence of this gene yielded homologous genes with the highest homology to D.pteronyssinus glutaredoxin-C8-like up to 83.52%.Conclusion We obtained the coding gene and prokaryotic expression plasmid for Grx1 of D.farinae.Bioinformatics analysis showed that this protein contains potential phosphorylation sites that distributed in the nucleus,providing references for the physiological function research about Grx1 and control measures for D.farinae.