牛Ⅰ型干扰素受体α链与其配体结合活性的鉴定

doi:10.16431/j.cnki.1671-7236.2018.09.034

Abstract

Abstract:

In order to study the biological properties between the bovine type Ⅰ interferon receptor alpha (IFNAR1) chain and its binding ligand, the bovine IFNAR1 (BoIFNAR1) was amplified by RT-PCR from primary kidney cells infected with vesicular stomatitis virus (VSV),and the recombinant plasmid pET30a-BoIFNAR1-EC was constructed to express the recombinant protein of BoIFNAR1 extracellular region (rIFNAR1-EC) in Rosetta^TM (DE3) pLysS.Besides,the polyclonal antibody (PAb) against BoIFNAR1 was prepared in rabbits with purified rBoIFNAR1-EC.Then the binding ability of bovine IFNAR1 to its ligands was identified.The results showed that the cloned CDS length of BoIFNAR1 gene was 1 685 bp.Moreover,the open reading frame of BoIFNAR1 encoded 560 amino acids,which consisted of one singal peptide encoded by 24 amino acids,one extracelluar region encoded by 414 amino acids,one transmembrane region encoded by 23 amino acids and one intracelluar region encoded by 99 amino acids.What's more,the homology of amino acid among BoIFNAR1 and other species ranges from 63% to 91%,indicating it was highly conserved in evolution,and especially close to sheep.And then a recombinant expression vector containing the extracellular region of BoIFNAR1 was constructed and the recombinant protein rBoIFNAR1-EC was induced.The results showed that rBoIFNAR1-EC was expressed in E.coli as a soluble form,which could be used as the immnuogen in preparing specific antibody against BoIFNAR1 after purification and dialysis.The titer of PAb obtained here was 1:204 800.Ligand binding experiment showed that rBoIFNAR1-EC could bind with bovine IFN-αA and IFN-ε.The PAb against BoIFNAR1 could block the specific binding area of bovine IFN-αA and IFN-ε to the cell surface IFNAR1, resulted in blocking the antiviral signal transduction of bovine IFN-αA and IFN-ε.

Key words: type Ⅰ interferon receptor alpha chain(IFNAR1); recombinant protein; polyclonal antibody; antiviral activity

CLC Number:

S852.4

WANG Yujiao, GUO Yongli, LUO Xiuxin, GAO Mingchun, WANG Junwei. Identification of Ligand Binding Activity Between Bovine Type Ⅰ Interferon Receptor α Chain and Its Ligand[J]. , 2018, 45(9): 2610-2619.

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Identification of Ligand Binding Activity Between Bovine Type Ⅰ Interferon Receptor α Chain and Its Ligand

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