To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. ...To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. pallidum by polymerase chain reaction (PCR) and subcloned into appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzyme digestion, the recombinant plasmid was transfected into HeLa cells using liposome, and the expressed protein was identified by immunocytochemistry and Western blotting. After verifying that the Tp92 antigen gene fragment could be expressed in HeLa cells, 100?μg of recombinant plasmids [pcDNA3.1(+)-Tp92], 100 μg of control plasmids [pcDNA3.1(+)] or 0.5 ml PBS buffer were administered in 3 groups of New Zealand white rabbits (6 rabbits/group), and the booster immunizations were employed at 2-week interval for 3 times. ELISA assay was used for the quantitative detection of the specific antibody in the sera of rabbits, and the proliferation response of spleen cells was detected by MTT assay. It was found that the target gene Tp92 segment about 2103 bp was obtained, and the DNA sequence of Tp92 gene constructed in pcDNA3.1 (+) vector was consistent with the published nucleotide sequence. The homologies of the nucleotide and putative amino acid sequences of Tp92 gene between T.pallidum subsp. pallidum Nichols and various pathogenic treponeme strains were 95.5%-100%. The analysis of immunocytochemistry and Western blotting showed that Tp92 gene segment constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77 kDa in HeLa cells and the expressed protein could react with positive blood serum from syphilis patient. The specific antibody IgG titers were observed and the highest titer was 1∶1024 in rabbits after 3 times with pcDNA3.1(+)-Tp92. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1(+) ( P <0.05). The successful expression of the eukaryotic expression plasmid of Tp92 gene from T. pallidum was obtained in eukaryotic system and strong responses of humoral and cellular immunity was evoked by DNA vaccine of pcDNA3.1(+)-Tp92 in rabbits thus establishing a solid basis for the future studies in the biological activities and for the development of the syphilis DNA vaccine.展开更多
基金This work was supported by Hunan Provincial Education Department (002A046) and Department of Public Health of Hunan province (B2003 085)
文摘To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. pallidum by polymerase chain reaction (PCR) and subcloned into appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzyme digestion, the recombinant plasmid was transfected into HeLa cells using liposome, and the expressed protein was identified by immunocytochemistry and Western blotting. After verifying that the Tp92 antigen gene fragment could be expressed in HeLa cells, 100?μg of recombinant plasmids [pcDNA3.1(+)-Tp92], 100 μg of control plasmids [pcDNA3.1(+)] or 0.5 ml PBS buffer were administered in 3 groups of New Zealand white rabbits (6 rabbits/group), and the booster immunizations were employed at 2-week interval for 3 times. ELISA assay was used for the quantitative detection of the specific antibody in the sera of rabbits, and the proliferation response of spleen cells was detected by MTT assay. It was found that the target gene Tp92 segment about 2103 bp was obtained, and the DNA sequence of Tp92 gene constructed in pcDNA3.1 (+) vector was consistent with the published nucleotide sequence. The homologies of the nucleotide and putative amino acid sequences of Tp92 gene between T.pallidum subsp. pallidum Nichols and various pathogenic treponeme strains were 95.5%-100%. The analysis of immunocytochemistry and Western blotting showed that Tp92 gene segment constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77 kDa in HeLa cells and the expressed protein could react with positive blood serum from syphilis patient. The specific antibody IgG titers were observed and the highest titer was 1∶1024 in rabbits after 3 times with pcDNA3.1(+)-Tp92. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1(+) ( P <0.05). The successful expression of the eukaryotic expression plasmid of Tp92 gene from T. pallidum was obtained in eukaryotic system and strong responses of humoral and cellular immunity was evoked by DNA vaccine of pcDNA3.1(+)-Tp92 in rabbits thus establishing a solid basis for the future studies in the biological activities and for the development of the syphilis DNA vaccine.
