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目的制备针对鸡的高迁移率族蛋白B1(chHMGB1)特异性抗原亲和层析多克隆抗体,并进行初步应用。方法反转录PCR扩增chHMGB1的全长编码基因,通过序列比对分析筛选一段特异性的多肽序列,偶联载体后作为抗原免疫新西兰兔,采集血清,通过蛋白A柱及多肽偶联的Seprose4B柱得到亲和层析纯化的多克隆抗体;并利用Western blot法、间接免疫荧光染色、免疫组化方法进行鉴定。结果成功制备了抗chHMGB1特异性的多克隆抗体。各项实验结果表明该抗体能特异性的识别重组蛋白以及天然的chHMGB1蛋白。结论成功制备了鸡源性高迁移率族蛋白B1亲和层析多克隆抗体。
Objective To prepare polyclonal antibody against chickens with high mobility group box protein B1 (chHMGB1) specific antigen and to conduct preliminary application. Methods The full-length chHMGB1 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). A specific peptide sequence was screened by sequence analysis. After conjugation, the vector was used as an antigen to immunize New Zealand rabbits. Serum was collected and purified by protein A column and Seprose4B Column affinity chromatography purification of polyclonal antibodies; and using Western blot, indirect immunofluorescence staining, immunohistochemical methods for identification. Results The anti-chHMGB1 specific polyclonal antibody was successfully prepared. The experimental results show that the antibody can specifically recognize the recombinant protein as well as the native chHMGB1 protein. Conclusion The polyclonal antibody of chick-derived high-mobility group protein B1 affinity chromatography was successfully prepared.