title: Prokaryotic expression, identification and bioinformatics analysis of fbpB-esxA fusing gene from Mycobacterium tuberculosis.
abstract: OBJECTIVE
  To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis (MTB), express the encoded fusing protein in Escherichia coli (E. coli), identify protein acquired, and predict the structure and function of the protein utilizing methods of bioinformatics.
  
  
  METHODS
  fbpB and esxA gene were amplified from genome of MTB H37Rv by PCR. The fbpB-esxA fusing gene ligated by (Gly(4)Ser)(3) linker was gained by means of Gene Splicing by Overlapping Extension PCR (SOE-PCR), and fusing gene was cloned into expression vector pET-30a. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3). The protein was identified by Western blot using anti-HIS antibody. Secondary structure and antigenic epitopes of the protein were predicting using tools of bioinformatics.
  
  
  RESULTS
  The DNA sequences of fbpB-esxA were identical with that published by GenBank. The Ag85B-ESAT-6 fusion protein about 50 kDa comprised 485 amino acids was efficiently produced from expression system in E. coli BL21(DE3) under the induction of IPTG. Bioinformatics analysis showed the protein contained one transmembrane region and fourteen potential antigenic epitopes.
  
  
  CONCLUSIONS
  The Ag85B-ESAT-6 fusion protein is successfully expressed with N-terminal HIS-tag. Gel filtration demonstrated that it exists as insoluble inclusion bodies mainly. The existence of linker doesn't affect immunogenicity of Ag85B and ESAT-6. It will allow for characterization in vitro and establish a foundation of further function research such as vaccine or diagnostic reagent.
authors.affiliation: Institute of Tropical Bioscience and Biotechnology/Analysis & Testing Center, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, PR China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wu
authors.forename: Qiongyao
authors.initials: Q
authors.lastname: Fu
authors.forename: Qian
authors.initials: Q
authors.lastname: Chen
authors.forename: Qunfang
authors.initials: Q
authors.lastname: Cai
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Fan
authors.forename: Zhinong
authors.initials: Z
authors.lastname: Zhan
authors.forename: Lina
authors.initials: L
authors.lastname: Niu
authors.forename: Hua
authors.initials: H
authors.lastname: Pei
authors.forename: Peng
authors.initials: P
authors.lastname: Zhou
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000276:immunology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C525005:ESAT-6 antigen, Mycobacterium leprae; D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis