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

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

本研究旨在克隆牛干扰素调节因子9(bovine interferon regulatory factor 9,BoIRF9)基因,分析其生物学信息,并表达其抗原优势区。从感染水疱性口炎病毒(vesicular stomatitis virus,VSV)的EBK细胞中提取总RNA,以反转录得到的cDNA第一条链为模板,参考GenBank中牛IRF 9基因序列设计特异性引物进行BoIRF 9基因的扩增,对克隆得到的BoIRF 9基因进行分子特征、高级结构与分子进化等生物学分析。根据BoIRF9的亲水性、抗原指数和表面可及性,设计1对特异性引物克隆BoIRF9的抗原优势区域片段,将其连接至pET30a(+)载体,构建重组表达质粒pET30a-IRF9-Part。将鉴定正确的质粒转化大肠杆菌Rosetta感受态细胞,经IPTG诱导表达抗原优势区域蛋白,运用SDS-PAGE和Western blotting方法进行蛋白质鉴定。结果显示,本研究克隆得到BoIRF 9基因全长1684 bp,CDS区长1320 bp,编码439个氨基酸;BoIRF9氨基酸序列与人、马、猪、山羊相似性为80.0%~96.2%;进化树分析结果显示,荷斯坦奶牛与山羊亲缘关系最近,其次为猪、马、非洲象和人;BoIRF9蛋白的分子式为C 2139 H 3342 N 594 O 658 S 18,分子质量为48.5 ku,理论等电点(pI)为5.71;BoIRF 9基因编码蛋白不存在跨膜结构,无信号肽,存在1个潜在的N-糖基化位点和多个磷酸化位点;BoIRF9蛋白二级结构含有115个α-螺旋、22个β-转角、68个延伸链和234个无规则卷曲;BoIRF9蛋白包含2个保守结构域,选择包含第一个保守结构域的第12-221位氨基酸作为抗原优势区域,克隆出648 bp的基因片段,并表达出分子质量为27 ku的重组蛋白。本研究结果可为干扰素调节因子的深入研究提供参考。

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 BoIRF 9 gene according to bovine IRF 9 gene sequence in GenBank.The molecular characteristics,advanced structure and molecular evolution of the cloned BoIRF 9 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 BoIRF 9 gene was 1684 bp,the length of BoIRF 9 gene CDS was 1320 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 C 2139 H 3342 N 594 O 658 S 18,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.