title: [Construction of the eukaryotic coexpression vector containing Mycobacterium tuberculosis heat shock protein 70 and green fluorescent protein].
abstract: OBJECTIVE
  To construct an eukaryotic coexpression vector containing Mycobacterium tuberculosis heat shock protein 70 (mtHSP70) and enhanced green fluorescent protein (EGFP) controlled by cytomegalovirus promoter using pIRES-EGFP vector.
  
  
  METHODS
  The mtHSP70 gene fragment was amplified by PCR from pVAX-mtHSP70-HSV2gD using specific primers. The PCR product was cloned into the vector pMD 18-T vector, and the correct clone was selected according to DNA sequence analysis. The interested mtHSP70 gene fragment was subcloned into pCMV-IRES-EGFP vector with XhoI and EcoR I digestion. The recombinant plasmid was transfected into mouse melanoma B16 cell line, and the green fluorescent cells were detected by fluorescence microscopy and mtHSP70 expression was detected by Western blotting.
  
  
  RESULTS
  The recombinant plasmid obtained was confirmed by enzyme digestion. The transfected mouse melanoma B16 cells exhibited green fluorescence under fluorescence microscopy and expressed mtHSP70 protein as demonstrated by Western blotting.
  
  
  CONCLUSIONS
  The eukaryotic coexpression vector PCMV-mtHSP70-IRES-EGFP has been established to allow further investigation of the role of mtHSP70 gene in tumor immunotherapy.
authors.affiliation: Department of Maxillofacial Surgery, Guangdong Provincial Stomatological Hospital/The Affiliated Stomatological Hospital of Southern Medical University, Guangzhou, China. sunrisez@tom.com
authors.forename: Shu-Guang
authors.initials: SG
authors.lastname: Zeng
authors.forename: Ji-Ren
authors.initials: JR
authors.lastname: Zhang
authors.forename: Jin-Cai
authors.initials: JC
authors.lastname: Zhang
authors.forename: Shi-Qing
authors.initials: SQ
authors.lastname: Wu
authors.forename: Qi-Cai
authors.initials: QC
authors.lastname: Liu
authors.forename: Wei-Jian
authors.initials: WJ
authors.lastname: Ai
authors.forename: Guo-Chu
authors.initials: GC
authors.lastname: Xue
mesh_terms: D000818:Animals; D001483:Base Sequence; D019496:Cancer Vaccines; D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D019496:Cancer Vaccines; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins