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

Abstract

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

CLC Number:

S813.3

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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Sequence Analysis and Prokaryotic Expression of IFN-β Gene in Congjiang Xiang Pig

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