猪ELF4基因的克隆、蛋白表达及其多克隆抗体制备

doi:10.11843/j.issn.0366-6964.2017.03.005

Abstract

Abstract:

This research aimed to clone and express the porcine transcription factor E74-like factor 4 (pELF4) gene in vitro and to prepare its polyclonal antibody. The pELF4 gene was cloned by RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and the characteristics of its nucleotides and coding amino acids were analyzed by the bioinformatics softwares. The recombinant expression vector pET28a-pELF4 was constructed by inserting the pELF4 gene into the prokaryotic expression vector pET28a. Then pET28a-pELF4 was transformed into E. coli Rosetta(DE3) and to investigate its expression under different temperatures, IPTG concentrations and induction time. The expressed proteins were analyzed by SDS-PAGE and Western blot. The anti-pELF4 immuno-globulin egg yolk antibody(IgY) was prepared by immunizing hens after purification of the recombination pELF4 protein (rpELF4). The results showed that the ORF (Open reading frame) of pELF4 gene (GenBank Accession number: KU097322) was 1 989 bp, coding 662 amino acids. The similarity between pELF4 gene and that of human, cattle and mouse were 89.4%, 90.7% and 81.1%, respectively, therefore it was closer to the animals such as cattle while it was further to human and mouse. The relative molecular weight of rpELF4 was 95 ku, and it existed in the form of protein and inclusion body. The amount of inclusion body reached highest when the expression combination was 37℃, 0.8 mmol•L^-1 of IPTG and 8 h.The chicken anti-rpELF4 antibody titer was above 1︰256 000 fold at the 13rd days post the 4th vaccination. The recombinant protein pELF4 was successfully expressed by prokaryotic expression system, the chicken anti-pELF4 polyclonal antibodies with the wonderful activity and specifity was prepared, which provided basic material for the research of the biofunction of this protein and the related diseases.

CLC Number:

S828
S813.1

WANG Jin，TANG Qing-hai，GU Ya-jing，LI Xiao-nan，LI Yu，WANG Xue-yu，YAO Lun-guang，KAN Yun-chao. Cloning and Expression of Porcine ELF4 Gene and Preparation of Its Polyclonal Antibody[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 425-435.

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Cloning and Expression of Porcine ELF4 Gene and Preparation of Its Polyclonal Antibody

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