牛支原体热休克蛋白 GrpE的表达及黏附功能分析

Investigating the expression and adhesive role of the heat shock protein GrpE in Mycoplasma bovis

摘要:

目的 分析牛支原体( Mycoplasma bovis, Mb)热休克蛋白GrpE的进化保守性与结构特征，解析其亚细胞定位及在介导黏附过程中的生物学特性。 方法 参照Mb PG45株GrpE基因序列(GenBank登录号为CP002188.1)设计引物，构建原核表达载体pET-GrpE。基于基因测序结果，利用生物信息学方法分析GrpE的同源性、遗传进化、理化性质及结构特征。将重组质粒转化后诱导表达，经镍亲和层析纯化获得重组GrpE蛋白，采用SDS-PAGE分析结果。以重组蛋白免疫新西兰白兔制备多克隆抗体，采用ELISA测定抗体效价，通过Western blotting检测其免疫原性。利用间接免疫荧光(indirect fluorescent antibody assay, IFA)、ELISA及Western blotting分析GrpE的亚细胞定位；采用IFA和ELISA结合试验验证GrpE的黏附功能。 结果 成功构建原核表达载体pET-GrpE，生物信息学分析发现GrpE序列在Mb内高度保守(相似性达95%以上)，该序列编码的蛋白由341个氨基酸组成，无信号肽和跨膜结构，含有潜在N-糖基化位点及磷酸化位点。SDS-PAGE分析显示GrpE成功表达且以可溶性形式存在。利用重组蛋白制备多克隆抗体测得其效价为1:16 000，Western blotting结果表明GrpE蛋白具有良好的免疫原性。IFA、ELISA及Western blotting结果显示GrpE定位于菌体胞膜与胞质，但主要分布于膜表面。IFA显示GrpE蛋白多抗血清可抑制Mb对胎牛肺(embryonic bovine lung, EBL)细胞的黏附。结合试验结果表明GrpE蛋白以剂量依赖性方式结合宿主细胞膜蛋白，且该结合可被GrpE蛋白多抗抑制( P<0.001)。 结论 GrpE蛋白是Mb的一种高度保守的新型黏附素，直接参与Mb对宿主细胞的黏附过程，为阐明牛支原体致病机制提供了关键分子靶标。

Abstract:

Objective To analyze the evolutionary conservation and structural characteristics of the heat shock protein GrpE from Mycoplasma bovis, elucidate its subcellular localization, and investigate its biological properties in mediating the adhesion process. Methods Primers were designed based on the GrpE gene sequence (GenBank accession number: CP002188.1) of Mycoplasma bovis PG45, and the prokaryotic expression vector pET-GrpE was constructed. Following gene sequencing, bioinformatics methods were employed to analyze the homology, phylogenetic relationships, physicochemical properties, and structural characteristics of GrpE. Following transformation of the recombinant plasmid and induced expression, the yielded recombinant GrpE protein was purified via nickel affinity chromatography, and then SDS-PAGE was conducted. The purified recombinant protein was used to immunize New Zealand White rabbits to generate polyclonal antibodies, with the antibody titer determined by ELISA and immunogenicity assessed via Western blotting. The subcellular localization of GrpE was examined via indirect indirect fluorescent antibody assay (IFA), ELISA, and Western blotting. The adhesion function of GrpE was validated through integrated IFA and ELISA. Results The prokaryotic expression vector pET-GrpE was successfully constructed in this study. Bioinformatics analysis revealed that the GrpE sequence was highly conserved in Mycoplasma bovis (with identity exceeding 95%). The encoded protein consisted of 341 amino acid residues, with no signal peptide and transmembrane domain but potential N-glycosylation and phosphorylation sites. SDS-PAGE results confirmed the successful expression of GrpE in a soluble form. Polyclonal antibodies generated via the purified recombinant protein exhibited a titer of 1:16 000. Western blotting analysis further verified the strong immunogenicity of the GrpE protein. Localization studies using IFA, ELISA, and Western blotting indicated that GrpE is distributed in both the cell membrane and the cytoplasm, with predominant distribution observed on the membrane surface. Importantly, the anti-GrpE polyclonal antiserum significantly inhibited the adhesion of Mycoplasma bovis to embryonic bovine lung (EBL) cells. Furthermore, binding assays demonstrated that the interaction between GrpE and host cell membrane proteins is dose-dependent, and this binding was inhibited by the polyclonal antibody ( P<0.001). Conclusion GrpE is identified as a highly conserved novel adhesion of Mycoplasma bovis that directly participates in the adhesion to host cells, providing a key molecular target for elucidating the pathogenic mechanism of Mycoplasma bovis.