奶牛结合珠蛋白生物信息学分析及其多克隆抗体的制备

Bioinformatics analysis of dairy cattle haptoglobin and its preparation of polyclonal antibody

结合珠蛋白(haptoglobin,Hp)是奶牛罹患乳房炎期间一种重要的标志性蛋白,其在血液和乳汁中含量变化与患牛乳房炎病情密切相关。为制备奶牛Hp多克隆抗体及了解其理化性质、结构和功能,本研究通过PCR成功扩增获得奶牛Hp完整CDS区序列,对其进行生物信息学分析和pQE-80L-Hp原核表达载体的构建。将测序成功的重组质粒转化至M15感受态细胞,用1 mmol/L IPTG诱导重组蛋白的表达。重组蛋白表达后用亲和层析法进行纯化。利用纯化后的蛋白免疫动物制备多克隆抗体,利用ELISA方法和Western blot对多克隆抗体进行效价检测和反应性分析。结果显示,Hp是一种不稳定亲水性蛋白,分子式为C_(1936)H_(3049)N_(545O567)S_(19),编码388个氨基酸残基,由α-螺旋、延伸链、β-转角和无规则卷曲4种结构组成,有15个抗原决定族,36个磷酸化位点。在1~42氨基酸位置存在信号肽,无跨膜结构域与CD163、MMP9、CP等蛋白具有较好的互作关系。pQE-80L-Hp在大肠杆菌中经诱导表达的重组蛋白的相对分子质量为42 kDa,主要以包涵体的形式存在,用其免疫兔子制备得到的多克隆抗体效价可达到1∶25600。本研究结果不仅有助于了解Hp的结构,而且为后续进行其功能的研究以及利用其作为标志物进行奶牛乳房炎早期高效快速诊断技术的研发奠定了基础。

Haptoglobin(Hp)is an important signature protein in dairy cattle suffering from mastitis and its content changes in blood and milk are closely related to bovine mastitis.In order to prepare polyclonal antibody against bovine Hp and to understand its physicochemical properties,structure and function.In this study,the complete CDS region sequence of dairy cattle Hp was successfully amplified by PCR,and bioinformatics analysis was conducted on it and the prokaryotic expression vector of pQE-80L-Hp was constructed.The recombinant plasmid was transformed into M15 receptive cells,and the expression of the recombinant protein was induced by 1 mmol/L IPTG.The recombinant protein was purified by affinity chromatography.Polyclonal antibody was prepared by immunizing animals with purified protein.The titer and reactivity of polyclonal antibody were detected by ELISA and Western blot.The results showed that Hp was an unstable hydrophilic protein,whose molecular formula was C_(1936)H_(3049)N_(545)O_(567)S_(19),encoding 388 amino acid residues,consisting ofα-helix,extended chain,β-angle,and random coil structure,with 15 antigen determination groups and 60 phosphorylation sites.Signal peptide exists in the position of 1-42 amino acids,and has good interaction with CD163,MMP9,CP and other proteins without transmembrane domain.pQE-80L-Hp was transformed into E.coli and induced to express the recombinant protein Hp,it had a relative molecular mass of 42 kDa and existed mainly in the form of inclusion bodies.The titer of polyclonal antibody prepared by immunizing rabbit with the recombinant protein Hp was 1:25600.The results of this study not only help to understand the structure of Hp,but also lay a foundation for the subsequent study of its function and the development of an efficient and rapid diagnosis technique using Hp as a marker in the early stage of dairy cow mastitis.