从江香猪IFN-β基因的序列分析及原核表达

doi:10.16431/j.cnki.1671-7236.2019.01.002

摘要/Abstract

摘要：

试验旨在探明从江香猪β-干扰素（interferon-beta，IFN-β）基因编码区分子序列及原核表达产物特征。以从江香猪为研究对象，提取肝脏总RNA并反转录为cDNA，设计特异性引物扩增IFN-β基因编码区，将目的基因片段克隆至原核表达质粒pET-28a上，获得重组质粒pET28a-CJpoIFN-β，并利用生物学软件对江香猪IFN-β基因编码区进行序列分析；将鉴定正确的重组质粒pET28a-CJpoIFN-β转化大肠杆菌BL21（DE3）感受态细胞，经IPTG诱导表达、SDS-PAGE与Western blotting分析原核表达蛋白。结果表明，从江香猪IFN-β基因编码区长为561 bp，编码186个氨基酸；该蛋白为分泌性蛋白，前21个氨基酸为信号肽序列；二级结构主要以α-螺旋（77.42%）和无规则卷曲（17.74%）为主。从江香猪与其他猪源IFN-β基因核苷酸序列同源性为99.5%~100.0%，与禽的同源性最低（35.2%）；从江香猪与巴马猪、梅山猪IFN-β氨基酸同源性均为100.0%，但与贵州白香猪IFN-β同源性为99.5%，存在E43Q、K73R和C161R 3处氨基酸的差异。Western blotting结果显示，带His标签的重组表达蛋白能被His单抗识别，条带大小约为24 ku。本试验结果为进一步研究IFN-β基因生物学活性及加快从江香猪这一品种资源的有效利用提供参考依据。

关键词: 从江香猪; IFN-β基因; 序列分析; 原核表达

Abstract:

This study was aimed to investigate the molecular sequences and prokaryotic expression product characteristics of interferon-beta (IFN-β) gene coding region in Congjiang Xiang pig.Congjiang Xiang pig was selected as the research object,the total RNA was extracted from liver tissue of Congjiang Xiang pig,and the mRNA was reversely transcribed into cDNA.The encoding region of IFN-β gene was amplified by specific primers,ligated into pET-28a vector,obtained the recombinant plasmid pET28a-CJpoIFN-β,and the CDS sequence of IFN-β gene was analyzed using bioinformatics softwares.The pET28a-CJpoIFN-β was transformed into Escherichia coli BL21(DE3) competent cells.After induction by IPTG,the product of prokaryotic expression was analyzed by SDS-PAGE and Western blotting.The results showed that the CDS sequence of IFN-β gene in Congjiang Xiang pig (CJpoIFN-β) was 561 bp,which encoded 186 amino acids;CJpoIFN-β protein was secretory protein,and the first 21 amino acid residues was predicted to be the signal peptide.The secondary structure of CJpoIFN-β protein was mainly alpha helix (77.42%) and random coli (17.74%).The nucleotide sequence alignment results showed that the IFN-β gene homology was 99.5% to 100.0% between Congjing Xiang pig and others porcine,the lowest homology was 35.2% between Congjing Xiang pig and poultry.Phylogenetic tree result showed that CJpoIFN-β was closely related to that of Meishan pig and Bama pig,the amino acid homology was 100.0% in Congjiang Xiang pig and Bama pig,Meishan pig;And the homology was 99.5% between Congjiang Xiang pig and Guizhou Baixiang pig,there were three amino acid differences in E43Q,K73R and C161R.Western blotting results showed that His-tag recombinant protein could be recognized by His monoclonal antibody,the band was about 24 ku.The results of this experiment provided a reference for further study on the biological activity of IFN-β gene and speeding up the effective utilization of the resources of Congjiang Xiang pig.

Key words: Congjiang Xiang pig; IFN-β gene; sequence analysis; prokaryotic expression

中图分类号:

S813.3

温贵兰, 张升波, 李昌红, 徐丽, 田浪, 文明, 王开功, 程振涛, 赵德刚. 从江香猪IFN-β基因的序列分析及原核表达[J]. 《中国畜牧兽医》, doi: 10.16431/j.cnki.1671-7236.2019.01.002.

WEN Guilan, ZHANG Shengbo, LI Changhong, XU Li, TIAN Lang, WEN Ming, WANG Kaigong, CHENG Zhentao, ZHAO Degang. Sequence Analysis and Prokaryotic Expression of IFN-β Gene in Congjiang Xiang Pig[J]. , doi: 10.16431/j.cnki.1671-7236.2019.01.002.

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