牛巴贝斯虫SBP2蛋白生物信息学分析及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.08.033

摘要/Abstract

摘要： 【目的】探究牛巴贝斯虫(Babesia bovis)SBP2蛋白(spherical body protein 2，SBP2)的生物信息学特征并进行原核表达及多克隆抗体制备，为牛巴贝斯虫疫苗和诊断抗原筛选提供理论依据。【方法】对牛巴贝斯虫SBP2基因进行扩增和克隆，运用Mega 7.0构建牛巴贝斯虫SBP2蛋白系统进化树，利用IEDB Analysis Resource等生物信息学方法对牛巴贝斯虫SBP2蛋白的磷酸化位点和B细胞抗原表位进行预测分析；对SBP2蛋白与弓形虫致密颗粒蛋白(GRA)的氨基酸序列比对分析；构建原核表达载体pET-28a-SBP2，诱导表达SBP2重组蛋白并进行蛋白纯化，通过Western blotting验证SBP2重组蛋白反应原性；以SBP2重组蛋白为免疫原，免疫BALB/c小鼠制备多克隆抗体，并利用间接ELISA方法检测其效价。【结果】系统进化树显示，本研究牛巴贝斯虫SBP2蛋白与泰国株(OM46855)的氨基酸序列亲缘关系最近。生物信息学分析显示，SBP2蛋白包含17个B细胞抗原表位和31个磷酸化位点，其中包括12个丝氨酸位点、14个苏氨酸位点及5个酪氨酸位点。牛巴贝斯虫SBP2蛋白氨基酸序列与弓形虫GRA蛋白之间有很强的相关性。成功构建了重组质粒pET-28a-SBP2；Western blotting结果显示，SBP2重组蛋白分子质量大小约为35 ku，具有良好的反应原性；制备的多克隆抗体效价为1∶204 800。【结论】本研究通过预测牛巴贝斯虫SBP2蛋白生物学特性，加深了对SBP2蛋白的认识，利用原核表达系统成功获得牛巴贝斯虫SBP2重组蛋白，并获得其小鼠源多克隆抗体，为进一步研究SBP2功能及其在牛巴贝斯虫致病机制中的作用奠定了基础。

关键词: 牛巴贝斯虫; 球状体蛋白2; 生物信息学分析; 原核表达; 多克隆抗体

Abstract: 【Objective】 The experiment aimed to explore the bioinformatics characteristics of the spherical body protein 2 (SBP2) of Babesia bovis,and conduct prokaryotic expression and prepare polyclonal antibodies,providing a theoretical basis for the screening of Babesia bovis vaccines and diagnostic antigens. 【Method】 SBP2 gene of Babesia bovis was amplified and cloned. The phylogenetic tree of SBP2 protein of Babesia bovis was constructed using Mega 7.0.The phosphorylation sites and B-cell antigenic epitopes of the SBP2 protein in Babesia bovis were predicted and analyzed using bioinformatics methods such as IEDB Analysis Resource.The amino acid sequences of SBP2 protein and dense granules antigen (GRA) of Toxoplasma gondii were compared and analyzed. The prokaryotic expression vector pET-28a-SBP2 was constructed. The recombinant protein of SBP2 was induced to be expressed and purified.The reactivity of the recombinant protein of SBP2 was verified by Western blotting.Polyclonal antibodies were prepared by immunizing BALB/c mice with the recombinant protein SBP2 as the immunogen,and their titers were detected by indirect ELISA method. 【Result】 The phylogenetic tree showed that SBP2 protein of Babesia bovis in this study was most closely related to the amino acid sequence of the Thai strain (OM46855).Bioinformatics analysis revealed that SBP2 protein contained 17 B-cell antigenic epitopes and 31 phosphorylation sites,including 12 serine sites,14 threonine sites and 5 tyrosine sites.There was a strong correlation between the amino acid sequence of SBP2 protein of Babesia bovis and GRA protein of Toxoplasma gondii.The recombinant plasmid pET-28a-SBP2 was successfully constructed.The results of Western blotting showed that the molecular weight of the SBP2 recombinant protein was approximately 35 ku and it had good reactivity.The titer of the prepared polyclonal antibody was 1∶204 800. 【Conclusion】 This study deepened the understanding of SBP2 protein by predicting its biological characteristics.The SBP2 recombinant protein of Babesia bovis was successfully obtained using the prokaryotic expression system,and its polyclonal antibodies derived from mice were also acquired,laying a foundation for further research on the function of SBP2 and its role in the pathogenic mechanism of Babesia bovis.

Key words: Babesia bovis; SBP2; bioinformatics analysis; prokaryotic expression; polyclonal antibodies

中图分类号:

S852.72

冯秀娟, 任冀超, 崔泽云, 赵雪晴, 李佳欣, 张杨, 张伟, 巴音查汗·盖力克, 郭庆勇, 李永畅. 牛巴贝斯虫SBP2蛋白生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2025, 52(8): 3847-3856.

FENG Xiujuan, REN Jichao, CUI Zeyun, ZHAO Xueqing, LI Jiaxin, ZHANG Yang, ZHANG Wei, BAYIN Chahan·Gailike, GUO Qingyong, LI Yongchang. Bioinformatics Analysis and Preparation of Polyclonal Antibodies Against Babesia bovis SBP2 Protein[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3847-3856.

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