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

Abstract

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

CLC Number:

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.

References

[1] RYTER S W.Heme oxgenase-1,a cardinal modulator of regulated cell death and inflammation[J].Cells,2021,10(3):515.
[2] NAITO Y,TAKAGI T,HIGASHIMURA Y.Heme oxygenase-1 and anti-inflammatory M2 macrophages[J].Archives of Biochemistry and Biophysics,2014,564:83-88.
[3] XIE J,HE X,FANG H,et al.Identification of heme oxygenase-1 from golden pompano (Trachinotus ovatus) and response of Nrf2/HO-1 signaling pathway to copper-induced oxidative stress[J].Chemosphere,2020,253:126654.
[4] 刘靖松,付京城,温丙言,等.猪肠黏膜保护因子HO-1的短小芽孢杆菌表达系统建立与分析[J].中国畜牧兽医,2022,49(6):2064-2071. LIU J S,FU J C,WEN B Y,et al.Establishment and analysis of expression system of porcine intestinal mucosal protein factor HO-1 in Bacillus pumilus[J]. China Animal Husbandry & Veterinary Medicine,2022,49(6):2064-2071.(in Chinese).
[5] ESPINOZA J A,GONZALEZ P A,KALERGIS A M.Modulation of antiviral immunity by heme oxygenase-1[J].American Journal of Pathology, 2017,187(3):487-493.
[6] LIU D,MO X,ZHANG H,et al.Heme oxygenase-1 (HO-1) alleviates vascular restenosis after balloon injury in a rabbit carotid artery model[J].International Journal of Clinical Experimental Pathology,2018,11(5):2479-2487.
[7] DEVADAS K,DHAWAN S.Hemin activation ameliorates HIV-1 infection via heme oxygenase-1 induction[J].Journal of Immunology,2006,176(7):4252-4257.
[8] HILL-BATORSKI L,HALFMANN P,NEUMANN G,et al.The cytoprotective enzyme heme oxygenase-1 suppresses Ebola virus replication[J].Journal of Virology,2013,87(24):13795-13802.
[9] LU W,SHI L,GAO J,et al.Piperlongumine inhibits Zika virus replication in vitro and promotes up-regulation of HO-1 expression,suggesting an implication of oxidative stress[J].Virologica Sinica,2021,36(3):510-520.
[10] PROTZER U,SEYFRIED S,QUASDORFF M,et al.Antiviral activity and hepatoprotection by heme oxygenase-1 in Hepatitis B virus infection[J].Gastroenterology,2007,133(4):1156-1165.
[11] ZHENG Y,LI Z,YIN M,et al.Heme oxygenase1 improves the survival of ischemic skin flaps (Review)[J].Molecular Medicine Reports,2021,23(4):235.
[12] ZHU Z,WILSON A T,MATHAHS M M,et al.Heme oxygenase-1 suppresses Hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury[J].Hepatology,2008,48(5):1430-1439.
[13] XIAO S,WANG X,NI H,et al.microRNA miR-24-3p promotes Porcine reproductive and respiratory syndrome virus replication through suppression of heme oxygenase-1 expression[J].Journal of Virology,2015,89(8):4494-4503.
[14] WANG T,HU L,LIU M,et al.The emergence of viral encephalitis in donkeys by Equid herpesvirus 8 in China[J].Frontiers in Microbiology,2022,13:840754.
[15] GARVEY M,SUAREZ N M,KERR K,et al.Equid herpesvirus 8:Complete genome sequence and association with abortion in mares[J].PLoS One,2018,13(2):e0192301.
[16] 王彤彤,刘梦媛,张敬文,等.驴源马疱疹病毒8型的分离鉴定与生物信息学分析[J].中国畜牧兽医,2021,48(8):3030-3037. WANG T T,LIU M Y,ZHANG J W,et al.Isolation,identification and genetic characterization of Equine herpesvirus type 8 from donkey[J].China Animal Husbandry & Veterinary Medicine,2022,48(8):3030-3037.(in Chinese)
[17] XIAO S,ZHANG A,ZHANG C,et al.Heme oxygenase-1 acts as an antiviral factor for Porcine reproductive and respiratory syndrome virus infection and over-expression inhibits virus replication in vitro[J]. Antiviral Research,2014,110:60-69.
[18] ZHANG A,WAN B,JIANG D,et al.The cytoprotective enzyme heme oxygenase-1 suppresses Pseudorabies virus replication in vitro[J].Frontiers in Microbiology,2020,11:412.
[19] LI L,XUE B,SUN W,et al.Recombinant MYH9 protein C-terminal domain blocks Porcine reproductive and respiratory syndrome virus internalization by direct interaction with viral glycoprotein 5[J]. Antiviral Research,2018,156:10-20.
[20] WU X,WU D,LU Z,et al.A novel method for high-level production of TEV protease by superfolder GFP tag[J].Journal of Biomedicine and Biotechnology,2009,2009:591923.
[21] 吴丽丽,宫丽荣,赵丁欢,等.HO-1在脂多糖诱导大鼠肺泡巨噬细胞NLRP3炎性小体活化中的作用[J].中华麻醉学杂志,2019,39(8):989-992. WU L L,GONG L R,ZHAO D H,et al.Role of heme oxygenase-1 on lipopolysaccharide-induced activation of NLRP3 inflammasomes in alveolar macrophages of rats[J]. Chinese Journal of Anesthesiology,2019,39(8):989-992.(in Chinese)
[22] DENG S,YU K,JIANG W,et al.Over-expression of Toll-like receptor 2 up-regulates heme oxygenase-1 expression and decreases oxidative injury in dairy goats[J].Journal of Animal Science and Biotechnology,2017,8:3.
[23] LI J,QIAO Z,HU W,et al.Baicalin mitigated Mycoplasma gallisepticum-induced structural damage and attenuated oxidative stress and apoptosis in chicken thymus through the Nrf2/HO-1 defence pathway[J].Veterinary Research,2019,50(1):83.
[24] WANG Y,YANG C,ELSHEIKH NAH,et al.HO-1 reduces heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative stress[J].Aging (Albany NY),2019,11(15):5535-5547.
[25] WANG Y,YUYIN D,FENGYANG C,et al.Heme oxygenase-1 suppresses Duck Tembusu virus replication in vitro[J].Veterinary Microbiology,2020,251:108885.
[26] ZHANG C,PU F,ZHANG A,et al.Heme oxygenase-1 suppresses Bovine viral diarrhoea virus replication in vitro[J].Scientific Reports,2015,5:15575.
[27] 郭玲玲,张晓磊,张进顺,等.刚地弓形虫ROP18基因的克隆及生物信息学分析[J].中国病原生物学杂志,2015,10(5):414-419. GUO L L,ZHANG X L,ZHANG J S,et al.Cloning and bioinformatic analysis of rhoptry protein 18 of Toxoplasma gondii[J].Journal of Pathogen Biology,2015,10(5):414-419.(in Chinese)
[28] ZHANG A,DUAN H,LI N,et al.Heme oxygenase-1 metabolite biliverdin,not iron,inhibits Porcine reproductive and respiratory syndrome virus replication[J].Free Radical Biology and Medicine,2017,102:149-161.
[29] WYSE CA,NIXON C,MCLAUGHLIN M,et al.Evidence for the expression and enzymatic activity of haem oxygenase-1 in the lungs of horses[J].Veterinary Record, 2006,158(23):795-797.
[30] MAESTRELLI P,EL MESSLEMANI AH,DE FINA O,et al.Increased expression of heme oxygenase (HO)-1 in alveolar spaces and HO-2 in alveolar walls of smokers[J].American Journal of Respiratory and Critical Care Medicine,2001,164(8Pt1):1508-1513.
[31] ANDRADE B B,PAVAN K N,MAYER-BARBER K D,et al.Plasma heme oxygenase-1 levels distinguish latent or successfully treated human tuberculosis from active disease[J].PLoS One,2013,8(5):e62618.
[32] PAVAN KUMAR N,ANURADHA R,ANDRADE B B,et al.Circulating biomarkers of pulmonary and extrapulmonary tuberculosis in children[J].Clinical and Vaccine Immunology,2013,20(5):704-711.
[33] SILVA-GOMES S,APPELBERG R,LARSEN R,et al.Heme catabolism by heme oxygenase-1 confers host resistance to Mycobacterium infection[J].Infection and Immunity,2013,81(7):2536-2345.
[34] 孙白荷,王艺婷,陆雨霏,等.血红素加氧酶-1在病毒感染过程中的保护作用研究进展[J].药学学报,2022,57(6):1574-1583. SUN B H,WANG Y T,LU Y F,et al.Progress on the protective effect of heme oxygenase-1 in viral infection[J]. Acta Pharmaceutica Sinica,2022,57(6):1574-1583.(in Chinese)

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

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