德州驴HO-1基因生物信息学分析及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.04.022

摘要/Abstract

摘要： 【目的】克隆德州驴血红素氧合酶-1(heme oxygenase-1,HO-1)基因,对其进行原核表达及多克隆抗体制备,同时检测HO-1基因在德州驴不同组织中的表达情况,为后期研究HO-1基因功能提供支撑材料。【方法】参考GenBank中公布的马HO-1基因序列(登录号:XM_023631135.1)设计特异性引物,从驴原代肺泡巨噬细胞(donkey primary alveolar macrophages,DPAMs)中提取RNA,反转录为cDNA后进行PCR扩增HO-1基因编码区(coding sequences,CDS),对测序所获序列与其他物种进行相似性比对并构建系统进化树;应用多种在线软件对HO-1基因编码蛋白进行生物信息学分析。将HO-1基因序列克隆至pCold-SUMO载体进行原核表达,经镍柱亲和层析纯化后,以重组的HO-1蛋白免疫新西兰大白兔,制备多克隆抗体,利用ELISA和Western blotting检测多克隆抗体特异性及与抗原结合的最大稀释度。利用免疫组化试验检测德州驴4种组织中HO-1蛋白的表达水平。【结果】德州驴HO-1基因CDS区全长873 bp,编码290个氨基酸,其氨基酸序列与马的相似性最高,亲缘关系最近,与鸡的相似性最低,亲缘关系最远。德州驴HO-1蛋白是一种不稳定的亲水性蛋白,分子质量为33.04 ku,无信号肽,存在1个跨膜区,二级结构以α-螺旋为主。ELISA、Western blotting结果显示,针对HO-1制备的多克隆抗体与HO-1结合最大的稀释度为1∶102 400,宿主HO-1蛋白可被制备的多克隆抗体识别。免疫组化结果显示,德州驴4种组织中均有效表达HO-1蛋白。【结论】本研究成功获得德州驴HO-1重组蛋白,并制备特异性多克隆抗体,为后续深入探究HO-1基因在马属动物感染病原过程中的作用提供参考依据。

关键词: 德州驴; HO-1基因; 原核表达; 多克隆抗体; 组织表达

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

中图分类号:

胡乐玉, 胡欣瑶, 李颖, 赵盛淼, 沈凤臻, 王长法, 李亮亮, 王彤彤, 任慧英. 德州驴HO-1基因生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1499-1510.

HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.

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