title: [Preparation and identification of the polyclonal antibody against heat shock protein 70 (DnaK) from Shigella flexneri 5a M90T].
abstract: OBJECTIVE
  To generate polyclonal antibodies against Shigella flexneri 5a 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 polyclonal antibodies were identified by Western blotting, ELISA and immunofluorescent staining.
  
  
  RESULTS
  The recombinant plasmid pET24a-dnaK was expressed efficiently in E.coli BL21 (DE3). The polyclonal antibodies harvested from the mice immunized with DnaK-His were demonstrated being specific to Shigella flexneri 5a M90T DnaK proteins, and could be efficient in Western blotting, ELISA and immunofluorescent staining.
  
  
  CONCLUSIONS
  The polyclonal antibodies against Shigella flexneri 5a M90T DnaK protein have been successfully prepared.
authors.affiliation: College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018; Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Liao
authors.affiliation: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: JunJie
authors.initials: J
authors.lastname: Yue
authors.affiliation: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Song
authors.affiliation: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Dai
authors.affiliation: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Chen
authors.affiliation: Beijing Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Long
authors.initials: L
authors.lastname: Liang
authors.affiliation: College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China.
authors.forename: Bateer
authors.initials: B
authors.lastname: Huhe
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis / Q000276:immunology*; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012763:Shigella flexneri / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins