[Construction of the fusion gene of Hsp65 of Mycobacterium tuberculosis and EGFP and preparation of dendritic cell vaccine against tuberculosis].
OBJECTIVE
To construct the fusion gene of Hsp65 of Mycobacterium tuberculosis H37Rv and enhanced green fluorescence protein (EGFP) and prepare dendritic cell (DC) vaccine.


METHODS
Hsp65 DNA amplified by PCR was cloned into eukaryotic expression vector EGFP-C1. The recombinant plasmid pEGHsp65 was subsequently transfected into Hela cells, and the transfection rate was determined under confocal laser scanning fluorescence microscope at different times. RT-PCR was used to detect the expression of Hsp65 mRNA in Hela cells. The GM-CSF and IL-4 induced DCs from mouse bone marrow cells were transfected with recombinant plasmid pEGHsp65. Proliferation of unprimed splenocytes activated by transfected DCs was detected by MTT colorimetry.


RESULTS
Restrictive enzyme digestion analysis (EcoR I, Bgl II) confirmed that Hsp65 DNA had been inserted into the vector pEGFP-C1. The recombinant plasmid pEGHsp65 was transfected into Hela cells and the expression of the fusion gene reached peak at the 48 hours after transfection. Expression of Hsp65 mRNA was detected in Hela cells by RT-PCR. DCs transfected with pEGHsp65 could stimulate the proliferation of unprimed splenocytes.


CONCLUSIONS
The pEGHsp65 fusion gene was successfully constructed and DCs transfected with the pEGHsp65 might be a candidate vaccine for tuberculosis.
Xiao
Ling
L
Department of Immunology, Medical College, Wuhan University, Wuhan 430071, China.
Wei
Ya-Zhi
YZ
Xia
Fei
F
Liu
Sheng-Wu
SW
D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods*; D001426:Bacterial Proteins / Q000235:genetics*; D001428:Bacterial Vaccines / Q000235:genetics*; D049109:Cell Proliferation; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D004587:Electrophoresis, Agar Gel; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000166:cytology / Q000276:immunology; D014162:Transfection; D014376:Tuberculosis / Q000276:immunology*
D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D018834:Chaperonin 60; D004274:DNA, Recombinant; C485184:enhanced green fluorescent protein; C074119:heat-shock protein 65, Mycobacterium; D049452:Green Fluorescent Proteins; D004262:DNA Restriction Enzymes; D018833:Chaperonins