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

摘要

The aim of this experiment was to construct recombinant protein of porcine phosphotyrosine interac-tion 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 re-combinant 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 isopropylβ-D-thio-galactopyranoside ( IPTG) . To optimize the expression of recombinant pPID1, IPTG concentration, tempera-ture 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 re-sults 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 pu-rified recombinant pPID1 protein and its polyclonal antibody were successfully prepared in the present study.%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∶20480。本试验成功制备了高纯度的重组pPID1及其多克隆抗体。