牛干扰素调节因子9基因的克隆、序列分析与抗原优势区表达

doi:10.16431/j.cnki.1671-7236.2020.02.002

Abstract

Abstract: The aim of this study was to clone bovine interferon regulatory factor 9 (BoIRF9) gene,analyze its biological information,and express its antigenic dominant region.In this study,total RNA was extracted from EBK cells infected with vesicular stomatitis virus (VSV).Using the first chain of retroviral DNA as template,the specific primers were designed to amplify BoIRF9 gene according to bovine IRF9 gene sequence in GenBank.The molecular characteristics,advanced structure and molecular evolution of the cloned BoIRF9 gene were analyzed.According to the hydrophilicity,antigen index and surface accessibility of BoIRF9,a pair of specific primers were designed to clone the dominant antigen region fragment of BoIRF9 and connected it to pET30a (+) vector to construct the recombinant expression plasmid pET30a-IRF9-Part.The correct plasmid was transformed into E.coli Rosetta competent cells,and the dominant region protein was induced by IPTG.The protein was identified by SDS-PAGE and Western blotting.The results showed that the full-length of BoIRF9 gene was 1 684 bp,the length of BoIRF9 gene CDS was 1 320 bp,incoding 439 amino acids.The amino acids sequence similarity of BoIRF9 was 80.0% to 96.2% with Homo sapiens,Equus caballus,Sus scrofa and Capra hircus.Phylogenetic tree analysis showed that Holstein dairy cows had the closest relationship with Capra hircus,followed by Sus scrofa,Equus caballus,Loxodonta africana and Homo sapiens.The molecular formula of BoIRF9 protein was C₂₁₃₉H₃₃₄₂N₅₉₄O₆₅₈S₁₈,the molecular weight was 48.5 ku,and the theoretical isoelectric point (pI) was 5.71.There was no transmembrane structure and no signal peptide in BoIRF9 protein,and there was a potential N-glycosylation site and multiple phosphorylation sites.The secondary structure of BoIRF9 protein contained 115 alpha helix,22 beta turn,68 extend chain and 234 random coil,respectively.There were two conserved domains of BoIRF9 protein,a 648 bp gene fragment was cloned from the 12 to 221 amino acids containing the first conserved domain,and the recombinant protein with a molecular weight of 27 ku was expressed.This results could provide a reference for the further study of interferon regulatory factors.

Key words: BoIRF9 gene; cloning; prokaryotic expression; bioinformatics analysis

CLC Number:

LU Yingfei, HUANG Chunzheng, DAI Haiyue, GUO Yongli, ZHANG Yilei, ZHANG Boyang, LU Zhaoxiang, XU Weicheng, WANG Junwei, GAO Mingchun. A Cloning,Sequence Analysis and Antigen Dominant Region Expression of Bovine Interferon Regulatory Factor 9 Gene[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 326-335.

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A Cloning,Sequence Analysis and Antigen Dominant Region Expression of Bovine Interferon Regulatory Factor 9 Gene

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