鸭C4BPα的克隆、表达及其与鸭疫里默氏菌的互作

Cloning and expression of duck C4BPα and verification of its interaction with Riemerella anatipestifer

摘要:

为研究鸭C4结合蛋白 (C4b-binding protein，C4BP) 与鸭疫里默氏菌 (Riemerella anatipestifer，RA) 的相互作用，对鸭C4BPα进行克隆、原核表达，免疫小鼠制备多克隆抗体，并利用间接免疫荧光试验及斑点杂交试验验证C4BP与RA的相互作用。结果显示，鸭C4BPα核苷酸序列全长为1 230 bp，与鸡C4BPα的相似性最高 (82.1%)；系统进化树分析发现，鸭C4BPα与鸡C4BPα处于同一系统进化树分支上，两者遗传进化关系最近；C4BPα在大肠杆菌Escherichia coli BL21 (DE3) 中能高效表达，重组蛋白以胞内可溶性形式存在；多克隆抗体效价超过1∶10 000，并且可以与重组蛋白发生特异性反应；间接免疫荧光试验和斑点杂交试验结果显示RA与鸭C4BP可以发生相互作用。研究结果为进一步揭示RA的致病机制奠定了基础。

Abstract:

To study the interaction between C4b-binding protein (C4BP) and Riemerella anatipestifer (RA), we cloned duck C4BPα, conducted prokaryotic expression and prepared the polyclonal antibody by immunizing mice. Then indirect immunofluorescence assay and dot blotting hybridization assay were used to verify the interaction between C4BP and RA. The full length of duck C4BPα nucleotide sequence was 1 230 bp, with the highest similarity to chicken C4BPα (82.1%). Phylogenetic tree analysis showed that duck C4BPα and chicken C4BPα were on the same phylogenetic tree branch and the genetic evolution relationship between them was the closest. C4BPα was efficiently expressed in Escherichia coli BL21 (DE3). The recombinant proteins existed in intracellular soluble form. The titer of polyclonal antibody was more than 1:10 000 and polyclonal antibodies could specifically recognize the recombinant proteins. The results of indirect immunofluorescence assay and dot blot hybridization assay showed that RA could interact with duck C4BP. The results provide a basis to further reveal the pathogenesis of RA.