| 潘传燕,冯鹏霏,张永德,杨慧赞,张彬,陈福艳,陆专灵,杜雪松,童桂香,罗洪林.2020.尼罗罗非鱼PKCθ蛋白和Spartin蛋白的 原核表达、抗体制备及其组织表达.动物学杂志,55(5):637-646. |
| 尼罗罗非鱼PKCθ蛋白和Spartin蛋白的 原核表达、抗体制备及其组织表达 |
| Prokaryotic Expression, Antibody Preparation and Tissue Expression Analysis of PKCθ and Spartin in Nile Tilapia |
| 投稿时间：2019-10-29 修订日期：2020-09-01 |
| DOI：10.13859/j.cjz.202005012 |
| 中文关键词: 尼罗罗非鱼 PKCθ Spartin 原核表达 多克隆抗体 |
| 英文关键词:Nile Tilapia PKCθ Spartin Prokaryotic expression Polyclonal antibody |
| 基金项目:国家自然科学基金项目（No. 31372553，31760765） |
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| 中文摘要: |
| 为了研究尼罗罗非鱼（Oreochromis niloticus）蛋白激酶C theta（PKCθ）和Spartin的蛋白表达情况，本实验在大肠杆菌（Escherichia coli）中表达和提纯了尼罗罗非鱼PKCθ和Spartin的重组蛋白，并利用日本大耳兔（Oryctolagus cuniculus）制备了相应的多克隆抗体。用间接ELISA技术检测抗体效价，Western Blot鉴定抗体的特异性，并检测其在罗非鱼肝、脾、肠和肌肉组织中的表达情况。结果表明，实验成功构建了原核表达载体pET-B2m-PKCθ和pET-B2m-Spartin，实现了重组蛋白的原核表达和纯化。PKCθ重组蛋白主要存在于包涵体中，分子量约为56 ku；Spartin重组蛋白分子量约为30 ku，以包涵体蛋白的形式存在。获得的多克隆抗体效价均高达1︰512 000，Western Blot检测结果表明，制备的抗体能特异性识别尼罗罗非鱼PKCθ和Spartin多种异构体；PKCθ蛋白在罗非鱼肝、脾、肠与肌肉组织中均有表达；Spartin蛋白在罗非鱼的肝、脾和肠组织中不表达，在肌肉组织中表达。研究表明，尼罗罗非鱼PKCθ和Spartin重组蛋白在大肠杆菌中成功表达，获得了高效价的多克隆抗体，并明确了尼罗罗非鱼PKCθ和Spartin在不同组织中的表达情况，为进一步研究尼罗罗非鱼PKCθ和Spartin的功能及其作用机制奠定了基础。 |
| 英文摘要: |
| In order to study the expression of PKCθ and Spartin in Nile tilapia (Oreochromis niloticus), these two proteins were expressed in Escherichia coli, purified, and then used to immunize Japanese big-ear white rabbits (Oryctolagus cuniculus) according to the conventional method to prepare rabbit anti-PKCθ and rabbit anti-Spartin polyclonal antibody. The titers of rabbit anti-PKCθ and rabbit anti-Spartin antiserum were evaluated by indirect ELISA. The expressions of these two proteins in different issues (liver, spleen, intestine and muscle) of Nile tilapia were detected by Western Blotting. The results showed that the prokaryotic expression vectors of pET-B2m-PKCθ and pET-B2m-Spartin were successfully constructed, the recombinant PKCθ and Spartin proteins were expressed in inclusion body with molecular weight of 56 ku and 30 ku, respectively. The indirect ELISA assay showed that the rabbit anti-PKCθ and rabbit anti-Spartin antisera had a good sensitivity with the titer of 1︰512 000, and Western blotting showed that the polyclonal antibody had good specificity. PKCθ protein was expressed in the liver, spleen, intestine and muscle. Spartin protein was only expressed in the muscle. In this study, the PKCθ and Spartin recombinant proteins of Nile tilapia were expressed and purified successfully, the polyclonal antibodies against PKCθ and Spartin proteins with a high titer were obtained, and the expressions of PKCθ and Spartin in different issues of Nile Tilapia were determined. The current study will shed new light on the functional and mechanism study on PKCθ and Spartin in Nile tilapia. |
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