title: [Gene cloning,  expression and polyclonal antibody preparation and application of translocated intimin receptor-cytoskeleton coupling protein].
abstract: OBJECTIVE
  To prepare translocated intimin receptor-cytoskeleton coupling protein (TccP) of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and its polyclonal antibody.
  
  
  METHODS
  TccP was amplified from the genome of EHEC O157:H7 Sakai strain by PCR and used to construct the recombinant prokaryotic expression vector pET28a-TccP. The recombinant vector was transformed into E.coli BL21( DE3) to express the protein in the bacteria under the induction of isopropy-D-thiogalactoside (IPTG). After purification,  the protein was injected into New Zealand rabbits to prepare polyclonal antibody. Then the antibody was tested by ELISA and Western blotting for its sensitivity and specificity. The rabbit anti-TccP polyclonal antibody was then applied in the study on the localization of TccP within the host cells adhered by EHEC O157:H7.
  
  
  RESULTS
  The sequence of TccP cDNA we amplified was the same as reported by GenBank. The recombinant prokaryotic expression vector pET28a-TccP was constructed successfully. Western blotting revealed that M(r); of the target protein expressed in E.coli BL21(DE3) was 37 000 and the rabbit anti-TccP polyclonal antibody had a specific reaction with the target protein,  which demonstrated that the recombinant protein and its polyclonal antibody were prepared successfully. Immunofluorescence detection using rabbit anti-TccP polyclonal antibody showed that TccP aggregated in  the cell membrane of the host cell adhered by EHEC O157:H7.
  
  
  CONCLUSIONS
  We successfully prepared the recombinant vector pET28a-TccP and the anti-TccP polyclonal antibody and applied the antibody to confirm the localization of TccP in EHEC O157:H7 adhesion host cells.
authors.affiliation: Department of Clinical Laboratory,  Wuhan General Hospital,  Guangzhou Military Command Area,  Wuhan 430070,  China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Yang
authors.forename: Jun-hao
authors.initials: JH
authors.lastname: Cao
authors.forename: Jin-ya
authors.initials: JY
authors.lastname: Ding
authors.forename: Qian-chuan
authors.initials: QC
authors.lastname: Huang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis*; D001422:Bacterial Adhesion; D003001:Cloning, Molecular; D004927:Escherichia coli Infections / Q000382:microbiology; D019453:Escherichia coli O157 / Q000235:genetics* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D011817:Rabbits
chemical_list: D000906:Antibodies; D029968:Escherichia coli Proteins; C505370:tccP protein, E coli