[Construction of the eukaryotic coexpression vector containing Mycobacterium tuberculosis heat shock protein 70 and green fluorescent protein].
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.
Zeng
Shu-Guang
SG
Department of Maxillofacial Surgery, Guangdong Provincial Stomatological Hospital/The Affiliated Stomatological Hospital of Southern Medical University, Guangzhou, China. sunrisez@tom.com
Zhang
Ji-Ren
JR
Zhang
Jin-Cai
JC
Wu
Shi-Qing
SQ
Liu
Qi-Cai
QC
Ai
Wei-Jian
WJ
Xue
Guo-Chu
GC
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
D019496:Cancer Vaccines; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins