| 小鼠髓过氧化物酶(MPO)基因克隆分析及真核表达载体的构建 |
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| 引用本文： | 甘泽,王琪,王媛媛,赵彩英,李怡娜,张全伟,马友记,张勇,赵兴绪.小鼠髓过氧化物酶(MPO)基因克隆分析及真核表达载体的构建[J].中国兽医学报,2021(1):129-136,142. |
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| 作者姓名： | 甘泽 王琪 王媛媛 赵彩英 李怡娜 张全伟 马友记 张勇 赵兴绪 |
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| 作者单位： | ;1.甘肃农业大学动物医学学院;2.甘肃农业大学生命科学与技术学院;3.甘肃农业大学动物科学技术学院 |
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| 基金项目： | 甘肃省教育厅科研基金资助项目;甘肃省引导科技创新专项基金资助项目(GSCXZX-2019)。 |
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| 摘 要： | 髓过氧化物酶(myeloperoxidase,MPO)是一种血红素辅基蛋白酶,是血红素过氧化物酶超家族成员之一,参与多种疾病的发生和发展过程。本研究旨在克隆小鼠MPO基因CDS全长序列,检测该基因在小鼠神经细胞的表达谱。首先利用RT-PCR法与克隆载体克隆小鼠MPO基因CDS全长序列,对其进行生物信息学分析;然后构建真核表达载体pEGFP-C1-MPO,瞬时转染小鼠神经元细胞,通过qRT-PCR、Western blot和免疫荧光检测MPO在神经细胞的表达及分布规律。结果显示,小鼠MPO基因CDS全长2171 bp,编码718个氨基酸,与章鱼、美洲灰熊、马、人、非洲爪蟾、扬子鳄等物种的氨基酸序列同源性均高于79.25%。转染细胞后,试验组MPO的mRNA和蛋白表达水平极显著高于阴性对照组(P<0.01),表明小鼠MPO基因的真核表达载体pEGFP-C1-MPO构建及转染成功;免疫荧光结果显示MPO蛋白主要分布于神经元细胞质中。过表达MPO后,nNOS的mRNA水平显著高于对照组(P<0.05),而NADPH表达水平显著低于对照组(P<0.05)。总之,在体外过表达MPO可促进nNOS表达,抑制NADPH的表达,这为今后深入研究MPO的功能奠定理论基础。
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| 关 键 词： | 小鼠 MPO基因 NNOS NADPH |
Cloning,analysis and construction of eukaryotic expression vector of mouse myeloperoxidase (MPO) gene |
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| GAN Ze,WANG Qi,WANG Yuanyuan,ZHAO Caiying,LI Yina,ZHANG Quanwei,MA Youji,ZHANG Yong,ZHAO Xingxu.Cloning,analysis and construction of eukaryotic expression vector of mouse myeloperoxidase (MPO) gene[J].Chinese Journal of Veterinary Science,2021(1):129-136,142. |
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| Authors: | GAN Ze WANG Qi WANG Yuanyuan ZHAO Caiying LI Yina ZHANG Quanwei MA Youji ZHANG Yong ZHAO Xingxu |
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| Institution: | (College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,China;College of Animal Science and Technology,Gansu Agricultural University,Lanzhou 730070,China) |
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| Abstract: | Myeloperoxidase(MPO)is a heme co-protease and a member of the heme peroxidase superfamily.It is regulated by many growth factors,exists in a variety of cells,and is involved in the development of various diseases.In this study,the aim was to clone the CDS sequence of mouse MPO gene,and then to detect the gene expression profile in mouse nerve cells.The mouse full-length CDS sequence of MPO gene was obtained by RT-PCR and analyzed by bioinformatics.The eukaryotic expression vector pEGFP-C1-MPO was constructed and transfected into mouse neuronal cells.qRT-PCR,Western blot and immunofluorescence were used to detect the expression and distribution of MPO in nerve cells.The results showed that the CDS of mouse MPO gene was 2171 bp in length and encoded 718 amino acids.Its amino acid sequence homology to Octodon degus,Ursus arctos horribilis,Equus caballus,human,Alligator sinensis and Xenopus tropicalis was no less than 79.25%.After transfection,the mRNA and protein expression levels of MPO in the eukaryotic expression vector group were significantly higher than those in the negative control group(P<0.01),indicating that the eukaryotic expression vector pEGFP-C1-MPO was successfully constructed and transfected.The results of immunofluorescence assay showed that MPO protein was mainly distributed in the cytoplasm.In the MPO eukaryotic expression vector group,the mRNA level of nNOS was significantly higher than that of the negative control group(P<0.05),while the expression level of NADPH was significantly lower than that of the negative control group(P<0.05).In summary,the overexpression of MPO gene in vitro promotes the expression of nNOS and inhibits the expression level of NADPH.Our results lay a theoretical foundation for further research on the function of MPO in the future. |
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| Keywords: | 小鼠 MPO基因 nNOS NADPH |
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