| 任雅宁,周冬梅,袁存银,廖媛芬,强士豪,罗金丹,崔玉宝.粉尘螨线粒体样苹果酸脱氢酶基因cDNA克隆和分子特征分析[J].,2023,(20):3815-3819 |
| 粉尘螨线粒体样苹果酸脱氢酶基因cDNA克隆和分子特征分析 |
| cDNA Cloning and Molecular Characterization of Mitochondrial-like Malate Dehydrogenase Protein Gene of Dermatophagoides Farinae |
| 投稿时间：2023-03-31 修订日期：2023-04-28 |
| DOI：10.13241/j.cnki.pmb.2023.20.003 |
| 中文关键词: 粉尘螨 苹果酸脱氢酶 基因克隆 |
| 英文关键词: Dermatophagoides farina Mitochondrial-like malate dehydrogenase protein Gene clone |
| 基金项目:国家自然科学基金项目（81971511）；无锡市太湖人才计划高端人才（2020THRC-GD-7）；无锡市科技局"太湖之光"科技攻关（Y20212006）；无锡市卫生计生科研重大项目（Z201701） |
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| 中文摘要: |
| 摘要 目的：获取粉尘螨线粒体样苹果酸脱氢酶蛋白（mitochondrial-like malate dehydrogenase，mMDH）的编码基因并了解其分子特征。方法：以粉尘螨Total RNA为模板，RT-PCR扩增获得mMDH编码基因后、构建原核表达质粒，转化E.coil BL21(DE3)感受态细胞中，IPTG诱导表达，SDS-PAGE和Western Blot鉴定目的蛋白表达情况。采用NCBI、EXPASY在线生物信息学软件分析该基因编码蛋白质的生物学特征。结果：获得粉尘螨线粒体样苹果酸脱氢酶蛋白的编码基因，全长1032bp。构建的原核表达质粒pET28a(+)-mMDH，经转化和诱导表达后，SDS-PAGE和Western Blot可见目的蛋白条带。该基因编码的蛋白质由343个氨基酸组成，相对分子质量（Mr）36014.54Da，亚细胞定位主要在细胞质和细胞核，含有34个磷酸化位点（19个丝氨酸，12个苏氨酸和3个酪氨酸）。糖基化预测结果显示其含有2个N-糖基化位点（123位存在糖基化位点NASI和151位存在糖基化位点NSTV）和1个0-糖基化位点。二级结构主要为?琢-螺旋和无规则卷曲。三维建模可观察到该蛋白为二聚体结构，CD-Search保守区域分析后显示其属于NADB-Rossmann家族，具有MDH-glyoxysomal-mitochondrial结构域。PyMol可视化后可在三维结构中观察到保守区域位点。将该基因推导出的氨基酸序列进行Blast获得同源基因，粉尘螨与屋尘螨、梅氏嗜霉螨进化关系较近，独成一簇。结论：获得粉尘螨线粒体样苹果酸脱氢酶蛋白的编码基因全长及其原核表达质粒，并对其生物学特征进行分析，为进一步探讨该基因的生理功能、开发尘螨控制措施奠定基础。 |
| 英文摘要: |
| ABSTRACT Objective: To obtain the cDNA coding for the mitochondrial-like malate dehydrogenase protein (mMDH) of Dermatophagoides farinae and to characterize that protein. Methods: By using the primers designed according to the sequence for mMDH 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-thiogalactopyranoside (IPTG) and identified by SDS-PAGE and Western Blot. The structure analyses were conducted by NCBI and EXPASY. Results: The product of amplification with RT-PCR showed a clear band on agarose gel electrophoresis, and nucleotide sequencing of the pET28a(+)-mMDH plasmid yielded a coding gene 1032 bp. Once the plasmid was transformed into E. coil and its expression was induced with IPTG, a specific band was produced on SDS-PAGE and Western Blot. Bioinformatic analysis of the protein encoded by this gene consists of 343 amino acids with a relative molecular mass of 36014.54 Da, mainly localized to the nucleus and cytoplasm. The protein contain 34 potential phosphorylation sites, including nineteen Threonine, twelve Serine and three Tyrosine. The glycosylation prediction results showed that it contained two N-glycosylation sites (NASI at position 123 and NSTV at position 151) and one 0-glycosylation site. And it's advanced structure consisted of random coil(35.57%), α-helix(39.94%), extended chain(18.37%)，and β-turn(6.12%). The protein was observed as a dimeric structure by 3D modeling, and CD-Search conserved region analysis revealed that it belongs to the NADB-Rossmann family with MDH-glyoxysomal-mitochondrial structural domain conserved region sites were observed in the 3D structure after PyMol visualization. The deduced amino acid sequence of the gene was Blast to obtain homologous genes, which clustered with D. pteronyssinus and Euroglyphus maynei in the phylogenetic tree. Conclusion: The full-length cDNA of the mitochondrial-like malate dehydrogenase protein in D. farinae and its prokaryotic expression plasmid were obtained for the first time, and the molecular features of the gene are demonstrated using bioinformatics analyses, which provide insights into further studies on the gene. |
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