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[Construction of the eukaryotic coexpression vector containing Mycobacterium tuberculosis heat shock protein 70 and green fluorescent protein].
- Source :
-
Nan fang yi ke da xue xue bao = Journal of Southern Medical University [Nan Fang Yi Ke Da Xue Xue Bao] 2009 Mar; Vol. 29 (3), pp. 433-6. - Publication Year :
- 2009
-
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.<br />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.<br />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.<br />Conclusion: The eukaryotic coexpression vector PCMV-mtHSP70-IRES-EGFP has been established to allow further investigation of the role of mtHSP70 gene in tumor immunotherapy.
- Subjects :
- Animals
Base Sequence
Cancer Vaccines
Cell Line, Tumor
Cytomegalovirus genetics
Cytomegalovirus metabolism
Genetic Vectors biosynthesis
Green Fluorescent Proteins genetics
HSP70 Heat-Shock Proteins genetics
Mice
Molecular Sequence Data
Recombinant Fusion Proteins biosynthesis
Recombinant Fusion Proteins genetics
Sequence Analysis, DNA
Genetic Vectors genetics
Green Fluorescent Proteins biosynthesis
HSP70 Heat-Shock Proteins biosynthesis
Mycobacterium tuberculosis metabolism
Subjects
Details
- Language :
- Chinese
- ISSN :
- 1673-4254
- Volume :
- 29
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Nan fang yi ke da xue xue bao = Journal of Southern Medical University
- Publication Type :
- Academic Journal
- Accession number :
- 19304518