title: [The adjuvant effect of chimeric flagellin fliC/esat].
abstract: OBJECTIVE
  To determine the role of ESAT-6 chimeric flagellin in TB immunology.
  
  
  METHODS
  The coding sequences of flagellin of Salmonella typhimurium and ESAT-6 of Mycobacterium tuberculosis were cloned by PCR and identified by sequencing, respectively. Chimeric flagellin gene fliC/esat was constructed by overlap PCR technique. The ESAT-6 coding fragment was inserted to the hypervariable region of Salmonella flagellin gene fliCi. And then prokaryotic exprssion plasmids of pET-fliC/esat, pET-fliC and pBCX-esat were constructed and transformed into E.coli BL21(DE3), followed by induction of IPTG. The expressed proteins fliC/esat and ESAT-6 were identified by Western-blot assay using specific monoclonal antibody (mAb) HYB076-08. Bone marrow dendritic cells (BMDCs) were in vitro stimulated by fliC/esat and ESAT-6 proteins, and analyzed for the expression levels of CD40, CD80, CD86 and CD54 molecules. The secreted IL-12p70 was determined by ELISA. Moreover, C57BL/6 mice were immunized intravenously with fliC/esat or ESAT-6 protein. The specific IFN-γ-secreting cells and IL-4-secreting cells from the immunized mice were detected by ELISPOT assay using an ESAT-6 peptide as a stimulus.
  
  
  RESULTS
  The results showed that the proteins of fliC/esat and ESAT-6 were expressed solubly, with the sizes of 64 kD and 39 kD respectively. Western blot analysis showed that both proteins reacted with the specific mAb against ESAT-6. BMDCs maturation was triggered by the chimeric flagellin fliC/esat. In contrast, ESAT-6 protein alone didn't activate BMDCs. IL-12p70 was also detected in the supernatants of BMDCs. The results showed that the chimeric flagellin fliC/esat induced significantly higher level of the secreted IL-12p70 than that of ESAT-6 protein. Furthermore, the chimeric flagellin fliC/esat significantly enhanced the Th1-biased immune responses against ESAT-6 in the immunized C57BL/6 mice.
  
  
  CONCLUSIONS
  The chimeric flagellin we generated exerts Th1 type adjuvant activity for ESAT-6 protein.
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou 225009, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Ke
authors.initials: K
authors.lastname: Wen
authors.forename: Jin-lin
authors.initials: JL
authors.lastname: Huang
authors.forename: Mao-zhi
authors.initials: MZ
authors.lastname: Hu
authors.forename: Jun-song
authors.initials: JS
authors.lastname: Shen
authors.forename: Zhi-ming
authors.initials: ZM
authors.lastname: Pan
authors.forename: Xin-an
authors.initials: XA
authors.lastname: Jiao
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology*; D003713:Dendritic Cells / Q000187:drug effects; D005408:Flagellin / Q000235:genetics / Q000494:pharmacology*; D018664:Interleukin-12 / Q000096:biosynthesis; D015847:Interleukin-4 / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D018417:Th1 Cells / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D011993:Recombinant Fusion Proteins; D005408:Flagellin; D018664:Interleukin-12; D015847:Interleukin-4