鸡载脂蛋白A-Ⅰ的多克隆抗体制备及亚细胞定位分析

doi:10.3785/j.issn.1008-9209.2023.02.061

浙江大学学报（农业与生命科学版）

2024, Vol. 50

Issue (1): 137-146 DOI: 10.3785/j.issn.1008-9209.2023.02.061

动物科学与动物医学

鸡载脂蛋白A-Ⅰ的多克隆抗体制备及亚细胞定位分析

王圣文(

),张丹,邬雨倩,周继勇,郑肖娟(

)

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

Preparation of polyclonal antibodies and subcellular localization analysis of chicken apolipoprotein A-Ⅰ

Shengwen WANG(

),Dan ZHANG,Yuqian WU,Jiyong ZHOU,Xiaojuan ZHENG(

)

Key Laboratory of Animal Virology, Ministry of Agriculture and Rural Affairs, Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China

全文: PDF(4557 KB) HTML

摘要：

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。

关键词： 鸡载脂蛋白A-Ⅰ; 原核表达; 多克隆抗体; 亚细胞定位

Abstract:

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-Ⅰ had 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.

Key words: chicken apolipoprotein A-Ⅰ prokaryotic expression polyclonal antibody subcellular localization

收稿日期: 2023-02-06 出版日期: 2024-03-01

CLC:

S855.3

基金资助: 国家自然科学基金项目(31672555)

通讯作者: 郑肖娟 E-mail: wangshengwen@zju.edu.cn;zhengxiaojuan@zju.edu.cn

作者简介: 王圣文（https://orcid.org/0000-0001-9324-6795），E-mail：wangshengwen@zju.edu.cn

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引用本文:

王圣文,张丹,邬雨倩,周继勇,郑肖娟. 鸡载脂蛋白A-Ⅰ的多克隆抗体制备及亚细胞定位分析[J]. 浙江大学学报（农业与生命科学版）, 2024, 50(1): 137-146.

Shengwen WANG,Dan ZHANG,Yuqian WU,Jiyong ZHOU,Xiaojuan ZHENG. Preparation of polyclonal antibodies and subcellular localization analysis of chicken apolipoprotein A-Ⅰ. Journal of Zhejiang University (Agriculture and Life Sciences), 2024, 50(1): 137-146.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2023.02.061 或 https://www.zjujournals.com/agr/CN/Y2024/V50/I1/137

载体名称 Vector name	引物名称 Primer name	引物序列（5 $′$ →3 $′$ ） Primer sequence (5 $′$ →3 $′$ )
pET-28a	pET-chApo A-Ⅰ-NS-BamHⅠ-F	GCGGATCCCGCTCCTTCTGGCAGCACGAT
pET-28a	pET-chApo A-Ⅰ-NS-SalⅠ-R	GCGTCGACTCAGGCCACGGACTTCTGGAGTT
pCI-neo	pCI-chApo A-Ⅰ-FL-XhoⅠ-F	GCCTCGAGATGAGAGGCGTGCTGGTGA
	pCI-chApo A-Ⅰ-NS-XhoⅠ-F	GCCTCGAGATGCGCTCCTTCTGGCAGCA
	pCI-chApo A-Ⅰ-XbaⅠ-R	GCTCTAGATCAGGCCACGGACTTCTGGAGTT

表1 用于chApo A-Ⅰ蛋白表达载体构建的PCR引物

图1 chApo A-Ⅰ的生物信息学分析A. chApo A-Ⅰ蛋白的信号肽预测；B. chApo A-Ⅰ蛋白的跨膜区预测；C. chApo A-Ⅰ蛋白的二级结构预测；D. chApo A-Ⅰ蛋白的三级结构预测；E.不同物种间Apo A-Ⅰ蛋白氨基酸序列的系统发育分析。

图2 用于原核和真核表达载体构建的chApo A-Ⅰ基因扩增及重组质粒双酶切鉴定A. PCR扩增结果；B.双酶切鉴定结果；C.表达载体构建示意图。M：DNA分子量标志物。泳道1～3依次代表chApo A-Ⅰ-FL（pCI-neo载体）、chApo A-Ⅰ-NS（pCI-neo载体）、chApo A-Ⅰ-NS（pET-28a载体）基因的PCR扩增产物，泳道4～6依次代表pCI-chApo A-Ⅰ-FL、pCI-chApo A-Ⅰ-NS、pET-28a-chApo A-Ⅰ。

图3 His-chApo A-Ⅰ重组蛋白的表达和纯化鉴定A～B.重组蛋白诱导表达后的SDS-PAGE（A）、WB（B）鉴定；C～D.纯化后重组蛋白的SDS-PAGE（C）、WB（D）鉴定。M：蛋白质分子质量标志物。泳道1代表未经IPTG诱导的质粒，泳道2代表经IPTG诱导16 h的质粒，泳道3代表重组蛋白过柱前样品，泳道4～7依次代表经10、20、40、60 mmol/L咪唑溶液洗脱后的重组蛋白样品。

图4 利用His-chApo A-Ⅰ重组蛋白免疫小鼠制备的多抗血清的ELISA效价

图5 chApo A-Ⅰ多克隆抗体的WB和IFA反应性鉴定及与不同物种血清中Apo A-Ⅰ蛋白的交叉反应性鉴定A.多克隆抗体的WB反应性；B.多克隆抗体与不同物种血清中Apo A-Ⅰ蛋白的交叉反应性；C.多克隆抗体的IFA反应性。M：蛋白质分子质量标志物。泳道1代表未转染的DF-1细胞，泳道2～3依次代表转染pCI-neo、pCI-chApo A-Ⅰ-FL的DF-1细胞，泳道4代表His-chApo A-Ⅰ重组蛋白，泳道5～9依次代表鼠、兔、牛、猪、鸡血清。

图6 chApo A-Ⅰ蛋白在DF-1细胞中的亚细胞定位

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