China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (4): 1499-1510.doi: 10.16431/j.cnki.1671-7236.2023.04.022

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey

HU Leyu1,2, HU Xinyao2, LI Ying2, ZHAO Shengmiao2, SHEN Fengzhen2, WANG Changfa2, LI Liangliang2, WANG Tongtong2, REN Huiying1   

  1. 1. College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China;
    2. College of Agronomy and Agricultural Engineering, Liaocheng University, Liaocheng 252000, China
  • Published:2023-04-06

Abstract: 【Objective】 The purpose of this study was to clone the heme oxygenase-1 (HO-1) gene in Dezhou donkey,carry out its prokaryotic expression and prepare polyclonal antibody,and detect the expression of HO-1 gene in different tissues of Dezhou donkey,so as to provide materials for functional study of HO-1 gene.【Method】 The specific primers were designed according to HO-1 gene of horse (GenBank accession No.:XM_023631135.1).The HO-1 gene coding sequences (CDS) were amplified in cDNA following RNA extracted from donkey primary alveolar macrophages (DPAMs),the sequences obtained by sequencing were compared with other species for similarity,and the phylogenetic tree was constructed.The encoding protein of HO-1 gene was analyzed by bioinformatics software online.HO-1 gene was cloned into pCold-SUMO vector,the recombinant HO-1 protein was purified by Ni-chelating affinity chromatography,and immunized Zealand rabbits to prepare polyclonal antibodies.The specificity of polyclonal antibody and maximum dilution binding to antigen were identified by ELISA and Western blotting.The expression of HO-1 protein in 4 tissues of Dezhou donkey were detected by immunohistochemical assay.【Result】 The CDS sequence of HO-1 gene in Dezhou donkey was 873 bp in total length,and coding for 290 amino acids.The amino acid sequence of HO-1 gene exhibited the highest similarity and the closest genetic relationship with Equss caballus,and showed the lowest similarity and the farthest genetic relationship with Gallus gallus.The HO-1 protein in Dezhou donkey was an instable and hydrophilic protein without signal peptide,but with one transmembrane domain,its molecular mass was 33.04 ku,it contained alpha helix in the secondary structure.ELISA and Western blotting results showed that the maximum dilution of anti-HO-1 polyclonal antibody binding to HO-1 was 1∶102 400,and the host HO-1 protein could be recognized by the polyclonal antibody.The immunohistochemical assay results showed that HO-1 protein was effectively expressed in 4 tissues of Dezhou donkey.【Conclusion】 The recombinant HO-1 protein in Dezhou donkey and specific polyclonal antibody were obtained successfully,these data would provide reference for the role of HO-1 gene in equids infected with pathogens.

Key words: Dezhou donkey; HO-1 gene; prokaryotic expression; polyclonal antibody; tissue expression

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