福建农业学报2025，Vol.40Issue(4)：361-369,9.DOI:10.19303/j.issn.1008-0384.2025.04.005
红嘴鸥髓样分化因子88基因序列分析、蛋白表达及其多克隆抗体的制备
Sequencing,Protein Expression,and Polyclonal Antibody Preparation of MyD88 in Larus ridibundus
摘要
Abstract
[Objective]Gene sequence and polyclonal antibodies of myeloid differentiation factor 88(MyD88)in Larus ridibundus were studied to reveal the protein structure and functions for further research on the innate immune signaling pathway.[Method]Total RNA was extracted from the peripheral blood lymphocytes of L.ridibundus using the trizol method.MyD88 was amplified by RT-PCR.The similarity,genetic relationship,physiochemical properties,secondary and tertiary structures of the gene were analyzed using bioinformatics software.Prokaryotic expression strain of MyD88 recombinant protein was obtained by transformation with the induction conditions optimized and purified by Ni-column,ultrafiltration and gradient dialysis followed by emulsification with Freund's adjuvant to immunize New Zealand white rabbits in preparing the polyclonal antibody serum.Titer of the obtained polyclonal antibody was determined by two-way agar diffusion assay,and specificity against MyD88 recombinant protein identified by SDS-PAGE and western blotting.[Result]The CDS region of MyD88 was 921 bp encoding 306 amino acids.The similarity of the gene with that of Rissa tridactyla was the highest at 98.8%,while the lowest at 84.6%with that of Anas platyrhynchos.It had a molecular size of approximately 34.5 kDa that contained one N-glycosylation site and 24 phosphorylation sites.There was no signal peptide and transmembrane structure.The secondary structure of the MyD88 protein was mainly random coil,while the tertiary structure,consistent with the secondary structure.The hydrophilic protein consisted of a complete death domain and TIR domain.The expression of the 54 kDa prokaryotic protein peaked in 4 h at 37℃in 1.0 mmol·L-1 IPTG.The prepared polyclonal antibody serum had a titer of 1∶16 that could effectively recognize the recombinant MyD88 protein of L.ridibundus indicating high in specificity.[Conclusion]A hydrophilic protein,MyD88 encoded rich in phosphorylation sites with a complete death domain and TIR domain.The constructed recombinant protein could be expressed in large quantities in vitro exerting strong immunogenicity,and the prepared polyclonal antibodies were highly specific.关键词
红嘴鸥/髓样分化因子88/序列分析/蛋白原核表达/多克隆抗体Key words
Larus ridibundus/myeloid differentiation factor 88/sequence analysis/protein prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
张宏莉,王一涵,马俊蕊,段纲,常华..红嘴鸥髓样分化因子88基因序列分析、蛋白表达及其多克隆抗体的制备[J].福建农业学报,2025,40(4):361-369,9.基金项目
云南省重大科技专项计划项目(202302AA310020、202202AE090024) （202302AA310020、202202AE090024）
云南农业大学校级一流本科课程项目(A1010103001、2021YLKC017) （A1010103001、2021YLKC017）
