分子营养 Molecular Nutrition

猪磷酸酪氨酸互作结构域1基因的融合表达及其多克隆抗体的制备和鉴定

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  • 四川农业大学动物营养研究所, 动物抗病营养教育部重点实验室, 成都 611130
陈小玲(1977- ),女,江西永新人,副教授,主要从事动物分子营养和生物饲料开发研究。E-mail:xlchen@sicau.edu.cn

收稿日期: 2014-05-07

  网络出版日期: 2014-11-17

基金资助

国家自然科学基金青年科学基金项目(31201811);四川农业大学双支团队项目

Fusion Expression of Porcine Phosphotyrosine Interaction Domain Containing 1 Gene and Preparation, Characterization of Its Polyclonal Antibody

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  • Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu 611130, China

Received date: 2014-05-07

  Online published: 2014-11-17

摘要

本文旨在通过原核表达获得猪磷酸酪氨酸互作结构域1(pPID1)重组蛋白,并制备pPID1多克隆抗体。将pPID1基因插入pET28a(+),构建重组pET28a(+)-pPID1大肠杆菌表达质粒,然后将重组质粒pET28a(+)-pPID1转化到大肠杆菌BL21感受态细胞中,获得的重组子以不同异丙基硫代-β-D-半乳糖苷(IPTG)浓度、温度和时间诱导,确定pPID1融合蛋白表达的最适条件。将表达产物经镍离子-亚氨基二乙酸(Ni2+-IDA)亲和层析纯化后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MSMS)鉴定。同时,将纯化获得的pPID1融合蛋白免疫SD大鼠,制备pPID1多克隆抗体,并检测抗体效价。结果表明:pPID1融合蛋白表达的最佳条件为30 ℃以0.1 mmol/L IPTG诱导4 h;纯化的融合蛋白经MALDI-TOF-MSMS鉴定为pPID1;特异性的pPID1多克隆抗体成功制备,抗体效价为1:20 480。本试验成功制备了高纯度的重组pPID1及其多克隆抗体。

本文引用格式

陈小玲, 王欢, 黄志清, 周波, 贾刚, 刘光芒, 赵华 . 猪磷酸酪氨酸互作结构域1基因的融合表达及其多克隆抗体的制备和鉴定[J]. 动物营养学报, 2014 , 26(11) : 3349 -3355 . DOI: 10.3969/j.issn.1006-267x.2014.11.022

Abstract

The aim of this experiment was to construct recombinant protein of porcine phosphotyrosine interaction domain containing 1 (pPID1) by prokaryotic expression system and prepare polyclonal antibody against the recombinant pPID1. pPID1 gene was cloned into the prokaryotic expression vector pET28a(+). The recombinant expression plasmid pET28a(+)-pPID1 was constructed and then transformed into Escherichia coli (E. coli) BL21 to express pPID1. The recombinant pPID1 was induced by the addition of isopropyl β-D-thiogalactopyranoside (IPTG). To optimize the expression of recombinant pPID1, IPTG concentration, temperature and time after induction by IPTG were varied. The recombinant protein was purified by Ni2+-iminodiacetic acid(IDA) affinity chromatography and was identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MSMS). To prepare polyclonal antibody against pPID1, the purified fusion protein was used to immunize Sprague Dawley (SD) rats and the antibody titer was determined. The results showed that pPID1 was expressed at a high level when the recombinant E. coli BL21 was induced with 0.1 mmo/L IPTG for 4 h at 30℃; MALDI-TOF-MSMS analysis confirmed that the purified fusion protein was pPID1; the specific polyclonal antibody against pPID1 with the titer of 1:20 480 was obtained. The highly purified recombinant pPID1 protein and its polyclonal antibody were successfully prepared in the present study.

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