Molecular Nutrition

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

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.

Cite this article

CHEN Xiaoling, WANG Huan, HUANG Zhiqing, ZHOU Bo, JIA Gang, LIU Guangmang, ZHAO Hua . Fusion Expression of Porcine Phosphotyrosine Interaction Domain Containing 1 Gene and Preparation, Characterization of Its Polyclonal Antibody[J]. Chinese Journal of Animal Nutrition, 2014 , 26(11) : 3349 -3355 . DOI: 10.3969/j.issn.1006-267x.2014.11.022

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