Construction of a eukaryotic expression system of HSP65 gene from Mycobacterium tuberculosis, and anti-HSP65 IgG produced in mice.
The purpose of this study was to express the HSP65 gene of Mycobacterium tuberculosis in eukaryotic cells and study its primary immune effect in animals. The HSP65 gene was amplified from the H37Rv strain of M. tuberculosis by PCR and then inserted into the expression plasmid pcDNA3.1(-). The recombinant plasmid pcHSP65 was transfected into HeLa cells by using the liposome transfection method and also injected into BALB/C mice to accomplish DNA immunization. The inserted gene was demonstrated to be identical to the reported HSP65 gene sequence. The transfected HeLa cells expressed HSP65 protein; Western blot showed the presence of a 65 kDa band of the inclusion body protein and immunofluorescence testing identified the protein expressed in cytoplasm. Specific IgG for the HSP65 protein could be identified in immunized mice. This study shows that recombinant eukaryotic expression plasmid pcHSP65 was constructed successfully, which lays a foundation for further study of gene therapy.
Ju
Wei
W
Department of Immunology, Medical School of Wuhan University, Wuhan 430071, PR China.
Liu
Junyan
J
Department of Immunology, Medical School of Wuhan University, Wuhan 430071, PR China.
Xiao
Wenjun
W
Department of Immunology, Medical School of Wuhan University, Wuhan 430071, PR China.
Liu
Min
M
Department of Immunology, Medical School of Wuhan University, Wuhan 430071, PR China.
Qu
Xueju
X
Department of Immunology, Medical School of Wuhan University, Wuhan 430071, PR China.
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D014162:Transfection; D014376:Tuberculosis / Q000517:prevention & control; D032581:Tuberculosis Vaccines / Q000276:immunology*; D014611:Vaccination; D019444:Vaccines, DNA / Q000276:immunology*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D018834:Chaperonin 60; D007074:Immunoglobulin G; D032581:Tuberculosis Vaccines; D019444:Vaccines, DNA; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins