鸭干扰素刺激基因的克隆、表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2020.11.035

Abstract

Abstract: The aim of this study was to clone the duck interferon-induced protein with tetratricopeptide repeats 5(IFIT5) gene,express duck IFIT5 protein and prepare the polyclonal antibodies against the duck IFIT5 protein.Primers specific for duck IFIT5 gene were designed according to Mallard duck reference sequence(accession No.:KF956064)available in GenBank.The complete ORF of duck IFIT5 gene was amplified by PCR.Recombinant prokaryotic expression plasmids pET-30a-IFIT5,pET-32a-IFIT5 and eukaryotic expression plasmid pcDNA3.1-IFIT5 were constructed.Recombinant duck IFIT5 proteins were expressed and analyzed by SDS-PAGE.Recombinant duck IFIT5 proteins were purified by Ni-NTA column affinity chromatography.Rabbit anti-duck IFIT5 polyclonal antibodies were prepared from rabbits which had immunized by recombinant protein pET-30a-IFIT5.The titer of polyclonal antibodies was detected by indirect ELISA,and the specificity was identified by Western blotting.Eukaryotic expression recombinant plasmid pcDNA3.1-IFIT5 was transfected into BHK21 cells and the reactivity of polyclonal antibodies was verified by indirect immunoinfluscence assay.The results showed that recombinant duck IFIT5 proteins were expressed in the form of inclusion bodies.The titer of polyclonal antibodies was approximately up to 1:49 600.Rabbit anti-duck IFIT5 polyclonal antibodies could identify recombinant protein pET-32a-IFIT5.Indirect immunofluorescence assay (IFA) confirmed that the purified polyclonal antibodies could identify duck IFIT5 protein by eukaryotic expression system specifically.These results demonstrated that the duck interferon stimulating gene IFIT5 was cloned successfully,rabbit anti-duck IFIT5 polyclonal antibodies could be used in the detection of duck IFIT5 protein,and also provided material support for the further study of duck IFIT5 protein.

Key words: duck; interferon-induced proteins with tetratricopeptide repeats 5(IFIT5); gene cloning; protein expression and detection

CLC Number:

S834

HUANG Huilan, LI Wenjun, XIE Zimin, YANG Huihu, CUI Huiyi, HUANG Shujian, ZHANG Xuelian. Molecular Cloning and Expression of Duck IFIT5 Gene and Preparation of Its Polyclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3713-3721.

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Molecular Cloning and Expression of Duck IFIT5 Gene and Preparation of Its Polyclonal Antibodies

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