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《Chinese Journal of Cellular and Molecular Immunology》 2015-01
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Preparation and identification of the polyclonal antibody against heat shock protein 70(DnaK) from Shigella flexneri 5a M90T

WANG Yu;ZHOU Wei;LIAO Xiang;YUE Jun Jie;SONG Ting;DAI Hongmei;CHEN Xinxin;LIANG Long;Huhe Bateer;College of Veterinary Medicine,Inner Mongolia Agricultural University;Beijing Institute of Biotechnology,Academy of Military Medical Sciences;  
Objective To generate polyclonal antibodies against Shigella flexneri 5a M90 T heat shock protein 70(Dna K)and identify the specificity and titer of antibodies.Methods The sequence of Shigella flexneri 5a M90 T dna K gene was amplified by PCR and inserted into expression vector p ET-24 a to obtain recombinant plasmid p ET24a-dna K.Then the recombinant plasmid p ET24a-dna K was transformed into E.coli BL21(DE3) and expressed under IPTG induction.The recombinant Dna K proteins with His-tag(Dna K-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 polyclonal antibodies were identified by Western blotting,ELISA and immunofluorescent staining.Results The recombinant plasmid p ET24a-dna K was expressed efficiently in E.coli BL21(DE3).The polyclonal antibodies harvested from the mice immunized with Dna K-His were demonstrated being specific to Shigella flexneri 5a M90 T Dna K proteins,and could be efficient in Western blotting,ELISA and immunofluorescent staining.Conclusion The polyclonal antibodies against Shigella flexneri 5a M90 T Dna K protein have been successfully prepared.
【Fund】: 国家自然科学基金(30970122)
【CateGory Index】: R392
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