志贺菌福氏5a M90T热休克蛋白70(DnaK)多克隆抗体的制备和鉴定  

Preparation and identification of the polyclonal antibody against heat shock protein 70(DnaK) from Shigella flexneri 5a M90T

作　　者：王羽[1,2] 周围[2] 廖翔[2] 岳俊杰[2] 宋婷[2] 戴红梅[2] 陈欣欣[2] 梁龙[2] 呼和巴特尔[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]内蒙古农业大学兽医学院,内蒙古呼和浩特010018

出　　处：《细胞与分子免疫学杂志》2015年第1期105-109,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30970122)

摘　　要：目的制备小鼠抗志贺菌福氏5a M90T热休克蛋白70(Dna K)的多克隆抗体,并鉴定其特异性和效价。方法 PCR扩增M90T中的基因dna K插入到原核表达载体p ET-24a中,获得p ET24a-dna K重组质粒,然后转化到大肠杆菌BL21(DE3)中表达,通过亲和柱纯化融合蛋白Dna K-His。将纯化的融合蛋白作为抗原免疫BALB/c小鼠,采集抗血清。用免疫印迹法、ELISA和免疫荧光技术鉴定抗血清的特异性和效价。结果重组质粒p ET24a-dna K在大肠杆菌BL21(DE3)中可以高效表达。用纯化的融合蛋白Dna K-His免疫小鼠制备得到的多克隆抗体能特异性低检测志贺菌福氏5a M90T Dna K蛋白,能有效地用于免疫印迹法、ELISA和免疫荧光等试验。结论成功制备了抗志贺菌福氏5a M90T Dna K蛋白的小鼠多克隆抗体。Objective To generate polyclonal antibodies against Shigella flexneriSa M90T heat shock protein 70 （DnaK） and identify the specificity and titer of antibodies. Methods The sequence of Shigella flexneri 5a M90T dnaK gene was amplified by PCR and inserted into expression vector pET-24a to obtain recombinant plasmid pET24a-dnaK. Then the recombinant plasmid pET24a-dnaK was transformed into E. coli BL21 （DE3） and expressed under IPTG induction. The recombinant DnaK proteins with His-tag （DnaK-His） were then purified by affinity purification. The polyclonal antibodies were prepared by immunizing the mice with the purified recombinant proteins and then harvesting mouse sera. The specificity and titer of the polycional antibodies were identified by Western blotting, ELISA and immunofluorescent staining. Results The recombinant plasmid pET24a-dnaKwas expressed efficiently in E. coli BL21（DE3）. The polyclonal antibodies harvested from the mice immunized with DnaK-His were demonstrated being specific to Shigella flexneriSa M90T DnaK proteins, and could be efficient in Western blotting, ELISA and immunofluorescent staining. Conclusion The polyclonal antibodies against Shigella flexneri 5a M90T DnaK protein have been successfully prepared.

关 键 词：志贺菌 福氏5a M90T 伴侣蛋白Dna K 热休克蛋白70 多克隆抗体 

分 类 号：R392-33[医药卫生—免疫学] R392.11[医药卫生—基础医学]