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<p><b>OBJECTIVE</b>To break immune tolerance to prion (PrP) proteins using DNA vaccines.</p><p><b>METHODS</b>Four different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector: PrP-WT expressing wild-type PrP, Ubiq-PrP expressing PrP fused to ubiquitin, PrP-LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome-targeting signal, and PrP-ER expressing PrP locating the ER. Using a prime-boost strategy, three-doses of DNA vaccine were injected intramuscularly into Balb/c mice, followed by two doses of PrP protein. Two weeks after the last immunization, sera and spleens were collected and PrP-specific humoral and cellular immune responses evaluated by ELISA and ELISPOT tests.</p><p><b>RESULTS</b>Higher levels of serum PrP antibodies were detected in mice vaccinated using the strategy of DNA priming followed by protein boosting. Of these, WT-PrP, Ubiq-PrP, and PrP-LII induced significantly higher humoral responses. ELISPOT tests showed markedly increased numbers of IFN-γ-secreting T cells in mice vaccinated using the strategy of DNA priming followed by protein boosting after stimulation with recombinant PrP23-90 and PrP23-231. PrP-ER induced the strongest T-cell response.</p><p><b>CONCLUSION</b>Prion vaccines can break tolerance to PrP proteins and induce PrP-specific humoral and cellular immune responses.</p>
Subject(s)
Animals , Cricetinae , Female , Humans , Mice , Antibodies , Allergy and Immunology , CHO Cells , COS Cells , Chlorocebus aethiops , Cricetulus , Enzyme-Linked Immunosorbent Assay , HeLa Cells , Immune Tolerance , Interferon-gamma , Allergy and Immunology , Lysosomal-Associated Membrane Protein 2 , Genetics , Allergy and Immunology , Mice, Inbred BALB C , Peptide Fragments , Allergy and Immunology , Prions , Genetics , Allergy and Immunology , Receptors, Peptide , Genetics , Allergy and Immunology , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Recombinant Proteins , Allergy and Immunology , Transfection , Ubiquitin , Genetics , Allergy and Immunology , Vaccines, DNAABSTRACT
Cancer/testis(CT) antigens,of which more than 40 kinds have now been identified,are encoded by genes that are expressed normally in the human germ line,and are also expressed in various tumor types,including melanoma,and carcinomas of the bladder,lung and liver.These immunogenic proteins are being vigorously pursued as targets for therapeutic cancer vaccines.In this paper,the classification,expression and function of CT antigens are reviewed.
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0.05).In spinal cord tissues of injury groups,the level of HIF-1? mRNA began to elevate at 3h after injury and reached the maximize on 3d,then it began to decline and returned to the basal level on 14d;the HIF-1? expression showed a time-dependent difference as followed: It began to increase at 3h after injury,peaked on 1d,then gradually decreased,and recovered to the normal level on 14d after injury.Conclusion The result suggested that hypoxia circumstance can enhance the stability of HIF-1? and the transcription of HIF-1? mRNA,and the time-dependent expression of HIF-1? and HIF-1? mRNA can be used to help the diagnosis of SCI and estimation of injury time.
ABSTRACT
Objective:To construct an eukaryotic radiation-inducible expressing vector of the human perforin N-terminal(hPFN-N),and to investigate the distribution and the killing effect of human perforin N-terminal truncated 118 amino acid polypeptide (rhPFN-N,22-139aa) on tumor cells.Methods:The gene hPFN-N was amplified by PCR from the plasmid pcDNA3.1(+)/hPFN and an enkaryotic radiation-inducible expression vector pcDNA3.1(+)/ Egr-hPFN-N was constructed after DNA recombination.After transfecting SPC-A1 cells with this recombination vector via liposome mediation,the expression of the hPFN-N protein was detected by RT-PCR and Immunocytochemical method and the killing effect of hPFN-N protein was assessed by standard MTT chromatometry.Results:DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic radiation-inducible expressing vector pcDNA3.1(+)/ Egr-hPFN-N had been constructed successfully.After the recombinant plasmid being transfected into SPC-A1 cells and being irradiated by X ray,RT-PCR verified the expression of hPFN-N mRNA.The result of Immunocytochemical assay was positive and in MTT assay the killing activity of rhPFN-N on target cells was 29.2%.Conclusion:After being irradiated the hPFN-N gene is expressed on the cell membrane and the killing activity of rhPFN-N on target cells is 29.2%.