title: Over-expression of gene encoding heat shock protein 70 from Mycobacterium tuberculosis and its evaluation as vaccine adjuvant.
abstract: BACKGROUND
  Heat shock proteins (Hsps) are evolutionary ancient and highly conserved molecular chaperons found in prokaryotes as well as eukaryotes. Hsp70 is a predominant member of Hsp family. Microbial Hsp70s (mHsp70s) have acquired special significance in immunity since they have been shown to be potent activators of the innate immune system and generate specific immune responses against tumours and infectious agents.
  
  
  OBJECTIVE
  The present study was aimed to clone express and purify recombinant Hsp70 from the Mycobacterium tuberculosis and characterise it immunologically. The study also aimed at determining the potential of recombinant M. tuberculosis heat shock protein (rMTB-Hsp70) as adjuvant or antigen carrier.
  
  
  METHODS
  Cloning of M. tuberculosis heat shock protein (MTB-Hsp70) amplicon was carried out using the pGEMT-Easy vector although for expression, pProExHTb prokaryotic expression vector was used. Purification of recombinant Hsp70 was carried out by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. For immunological characterization and determining the adjuvant effect of MTB-Hsp70, BALB/c mice were used. The data obtained was statistically analysed.
  
  
  RESULTS
  Hsp70 gene was cloned, sequenced and the sequence data were submitted to National Center for Biotechnology Information (NCBI). Recombinant MTB-Hsp70 was successfully over-expressed using the prokaryotic expression system and purified to homogeneity. The protein was found to be immunodominant. Significant adjuvant effect was produced by the rMTB-Hsp70 when inoculated with recombinant outer membrane protein 31; however, effect was less than the conventionally used the Freund's adjuvant.
  
  
  CONCLUSIONS
  Protocol standardised can be followed for bulk production of rHsp70 in a cost-effective manner. Significant adjuvant effect was produced by rMTB-Hsp70; however, the effect was than Freund's adjuvant. Further, studies need to be carried out to explore its applicability as carrier of antigen.
authors.affiliation: Department of Microbiology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
authors.forename: J
authors.initials: J
authors.lastname: Dhakal
authors.forename: G S
authors.initials: GS
authors.lastname: Brah
authors.forename: R K
authors.initials: RK
authors.lastname: Agrawal
authors.forename: H N
authors.initials: HN
authors.lastname: Pawar
authors.forename: D
authors.initials: D
authors.lastname: Kaur
authors.forename: R
authors.initials: R
authors.lastname: Verma
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage / Q000235:genetics; D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004337:Drug Carriers / Q000008:administration & dosage; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D032581:Tuberculosis Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D004337:Drug Carriers; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D032581:Tuberculosis Vaccines; D014614:Vaccines, Synthetic