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  

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

作　　者：陈小玲[1] 王欢[1] 黄志清[1] 周波[1] 贾刚[1] 刘光芒[1] 赵华[1] 

机构地区：[1]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

出　　处：《动物营养学报》2014年第11期3349-3355,共7页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：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

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(p PID1)重组蛋白,并制备p PID1多克隆抗体。将p PID1基因插入p ET28a(+),构建重组p ET28a(+)-p PID1大肠杆菌表达质粒,然后将重组质粒p ET28a(+)-p PID1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p PID1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p PID1融合蛋白免疫SD大鼠,制备p PID1多克隆抗体,并检测抗体效价。结果表明:p PID1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p PID1;特异性的p PID1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p PID1及其多克隆抗体。The aim of this experiment was to construct recombinant protein of porcine phosphotyrosine interaction domain containing 1（ p PID1） by prokaryotic expression system and prepare polyclonal antibody against the recombinant p PID1. p PID1 gene was cloned into the prokaryotic expression vector p ET28a（ ＋）. The recombinant expression plasmid p ET28a（ ＋）-p PID1 was constructed and then transformed into Escherichia coli（ E. coli） BL21 to express p PID1. The recombinant p PID1 was induced by the addition of isopropyl β-D-thiogalactopyranoside（ IPTG）. To optimize the expression of recombinant p PID1,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 p PID1,the purified fusion protein was used to immunize Sprague Dawley（ SD） rats and the antibody titer was determined. The results showed that p PID1 was expressed at a high level when the recombinant E. coli BL21 was induced with0.1 mmo / L IPTG for 4 h at 30℃; MALDI-TOF-MSMS analysis confirmed that the purified fusion protein was p PID1; the specific polyclonal antibody against p PID1 with the titer of 1 ∶ 20 480 was obtained. The highly purified recombinant p PID1 protein and its polyclonal antibody were successfully prepared in the present study.

关 键 词：猪PID1 原核表达 纯化 质谱鉴定 多克隆抗体 

分 类 号：S828[农业科学—畜牧学]