鸡载脂蛋白A-Ⅰ的多克隆抗体制备及亚细胞定位分析
S855.3; 载脂蛋白A-Ⅰ(apolipoprotein A-Ⅰ,Apo A-Ⅰ)在动脉粥样硬化、病毒感染、脂质代谢等方面发挥重要的调控作用.目前关于鸡载脂蛋白A-Ⅰ(chicken Apo A-Ⅰ,chApo A-Ⅰ)的研究较少,对其生物学功能尚不清楚.本研究在对chApo A-Ⅰ进行生物信息学分析的基础上,通过pET-28a原核表达系统对chApo A-Ⅰ的重组蛋白进行表达和纯化.利用纯化后的重组蛋白免疫小鼠,制备鼠多克隆抗血清(多抗血清),通过酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测其效价,并通过蛋白质印迹法(Wester...
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| Published in | 浙江大学学报（农业与生命科学版） Vol. 50; no. 1; pp. 137 - 146 |
|---|---|
| Main Authors | , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
浙江大学动物医学中心,农业农村部动物病毒学重点实验室,浙江 杭州 310058
01.02.2024
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1008-9209 |
| DOI | 10.3785/j.issn.1008-9209.2023.02.061 |
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| Abstract | S855.3; 载脂蛋白A-Ⅰ(apolipoprotein A-Ⅰ,Apo A-Ⅰ)在动脉粥样硬化、病毒感染、脂质代谢等方面发挥重要的调控作用.目前关于鸡载脂蛋白A-Ⅰ(chicken Apo A-Ⅰ,chApo A-Ⅰ)的研究较少,对其生物学功能尚不清楚.本研究在对chApo A-Ⅰ进行生物信息学分析的基础上,通过pET-28a原核表达系统对chApo A-Ⅰ的重组蛋白进行表达和纯化.利用纯化后的重组蛋白免疫小鼠,制备鼠多克隆抗血清(多抗血清),通过酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测其效价,并通过蛋白质印迹法(Western blot,WB)、间接免疫荧光试验(indirect immunofluorescence assay,IFA)鉴定其反应性,随后利用多抗血清对chApo A-Ⅰ进行亚细胞定位分析.生物信息学分析显示,chApo A-Ⅰ蛋白在第1-18位氨基酸处含有信号肽,由N端的连续α螺旋构成.氨基酸序列同源性分析显示,chApo A-Ⅰ蛋白与火鸡的同源性最高,与鱼类的同源性最低.利用成功表达和纯化的重组蛋白His-chApo A-Ⅰ制备多抗血清,其ELISA效价达1×105以上,与真核表达的chApo A-Ⅰ蛋白有WB和IFA反应性,能识别鸡血清中的Apo A-Ⅰ蛋白,而与鼠、兔、牛、猪血清中的Apo A-Ⅰ蛋白无交叉反应性.利用该多克隆抗体对全长片段chApo A-Ⅰ-FL和不含信号肽的片段chApo A-Ⅰ-NS进行亚细胞定位分析,经共聚焦显微镜观察发现,chApo A-Ⅰ-FL蛋白大多分布在细胞膜附近,而chApo A-Ⅰ-NS蛋白则定位于细胞胞浆中,且多数呈弥散分布.chApo A-Ⅰ蛋白的特异性多克隆抗体和亚细胞定位结果为进一步开展Apo A-Ⅰ的生物学功能研究奠定了基础. |
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| AbstractList | S855.3; 载脂蛋白A-Ⅰ(apolipoprotein A-Ⅰ,Apo A-Ⅰ)在动脉粥样硬化、病毒感染、脂质代谢等方面发挥重要的调控作用.目前关于鸡载脂蛋白A-Ⅰ(chicken Apo A-Ⅰ,chApo A-Ⅰ)的研究较少,对其生物学功能尚不清楚.本研究在对chApo A-Ⅰ进行生物信息学分析的基础上,通过pET-28a原核表达系统对chApo A-Ⅰ的重组蛋白进行表达和纯化.利用纯化后的重组蛋白免疫小鼠,制备鼠多克隆抗血清(多抗血清),通过酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测其效价,并通过蛋白质印迹法(Western blot,WB)、间接免疫荧光试验(indirect immunofluorescence assay,IFA)鉴定其反应性,随后利用多抗血清对chApo A-Ⅰ进行亚细胞定位分析.生物信息学分析显示,chApo A-Ⅰ蛋白在第1-18位氨基酸处含有信号肽,由N端的连续α螺旋构成.氨基酸序列同源性分析显示,chApo A-Ⅰ蛋白与火鸡的同源性最高,与鱼类的同源性最低.利用成功表达和纯化的重组蛋白His-chApo A-Ⅰ制备多抗血清,其ELISA效价达1×105以上,与真核表达的chApo A-Ⅰ蛋白有WB和IFA反应性,能识别鸡血清中的Apo A-Ⅰ蛋白,而与鼠、兔、牛、猪血清中的Apo A-Ⅰ蛋白无交叉反应性.利用该多克隆抗体对全长片段chApo A-Ⅰ-FL和不含信号肽的片段chApo A-Ⅰ-NS进行亚细胞定位分析,经共聚焦显微镜观察发现,chApo A-Ⅰ-FL蛋白大多分布在细胞膜附近,而chApo A-Ⅰ-NS蛋白则定位于细胞胞浆中,且多数呈弥散分布.chApo A-Ⅰ蛋白的特异性多克隆抗体和亚细胞定位结果为进一步开展Apo A-Ⅰ的生物学功能研究奠定了基础. |
| Abstract_FL | Apolipoprotein A-Ⅰ(Apo A-Ⅰ)plays important roles in the regulation of atherosclerosis,viral infections,lipid metabolism and other aspects.However,there are few studies on chicken Apo A-Ⅰ(chApo A-Ⅰ),and its biological function is not well understood.Based on the bioinformatics analysis of chApo A-Ⅰ,this study further performed the expression and purification of the recombinant protein of chApo A-Ⅰ via the pET-28a prokaryotic expression system.Mouse polyclonal antiserum was prepared by immunizing mice with purified recombinant protein.Enzyme-linked immunosorbent assay(ELISA)was used to determine the titer of the polyclonal antiserum,and Western blot(WB)and indirect immunofluorescence assay(IFA)were used to determine its reactivity.Then,the polyclonal antiserum was further applied for subcellular localization analysis of chApo A-Ⅰ.Bioinformatic analysis revealed that the chApo A-Ⅰ protein contains signal peptide at 1-18 amino acids,which is composed of continuous alpha helix at the N-terminal.Homology analysis of amino acid sequences revealed that the chApo A-Ⅰ protein had the highest homology with turkey and the lowest homology with fish.The polyclonal antibody prepared using successfully expressed and purified recombinant protein His-chApo A-Ⅰhad an ELISA titer above 1×105 and specifically reacted with the eukaryotic expressed chApo A-Ⅰ protein in WB and IFA.Particularly,the antibody can recognize the Apo A-Ⅰ protein in chicken serum,but cannot cross-react with Apo A-Ⅰ proteins in the serums of mice,rabbits,cattle or pigs.This polyclonal antibody was further applied for subcellular localization analysis of full-length chApo A-Ⅰ(chApo A-Ⅰ-FL)and chApo A-Ⅰ without signal peptide(chApo A-Ⅰ-NS).Observed by confocal microscope,it was found that chApo A-Ⅰ-FL protein was mainly localized near the cell membrane,but chApo A-Ⅰ-NS protein was localized in the cytoplasm,and most of them were diffusely distributed.The specific polyclonal antibody and the results of subcellular localization of chApo A-Ⅰ provide a basis for further research on the biological function of Apo A-Ⅰ. |
| Author | 张丹 王圣文 周继勇 邬雨倩 郑肖娟 |
| AuthorAffiliation | 浙江大学动物医学中心,农业农村部动物病毒学重点实验室,浙江 杭州 310058 |
| AuthorAffiliation_xml | – name: 浙江大学动物医学中心,农业农村部动物病毒学重点实验室,浙江 杭州 310058 |
| Author_FL | WU Yuqian WANG Shengwen ZHOU Jiyong ZHANG Dan ZHENG Xiaojuan |
| Author_FL_xml | – sequence: 1 fullname: WANG Shengwen – sequence: 2 fullname: ZHANG Dan – sequence: 3 fullname: WU Yuqian – sequence: 4 fullname: ZHOU Jiyong – sequence: 5 fullname: ZHENG Xiaojuan |
| Author_xml | – sequence: 1 fullname: 王圣文 – sequence: 2 fullname: 张丹 – sequence: 3 fullname: 邬雨倩 – sequence: 4 fullname: 周继勇 – sequence: 5 fullname: 郑肖娟 |
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| Keywords | 鸡载脂蛋白A- 原核表达 chicken apolipoprotein A- polyclonal antibody subcellular localization 亚细胞定位 prokaryotic expression 多克隆抗体 |
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| Snippet | S855.3; 载脂蛋白A-Ⅰ(apolipoprotein A-Ⅰ,Apo A-Ⅰ)在动脉粥样硬化、病毒感染、脂质代谢等方面发挥重要的调控作用.目前关于鸡载脂蛋白A-Ⅰ(chicken Apo A-Ⅰ,chApo A-Ⅰ)的研究... |
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| Title | 鸡载脂蛋白A-Ⅰ的多克隆抗体制备及亚细胞定位分析 |
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