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OBJECTIVE@#This study aims to use Arginine-gingipain A gene vaccine (pVAX1-rgpA) to immunize adult Beagle dogs and to evaluate its effect during peri-implantitis progression and development.@*METHODS@#Plasmid pVAX1-rgpA was constructed. The second and third bilateral mandible premolars of 15 adult Beagle dogs were extracted, and the implants were placed immediately. After 3 months, the animals were randomly divided into groups A, B, and C. Afterward, the animals were immunized thrice with plasmid pVAX1-rgpA, with heat-killed Porphyromonas gingivalis, or pVAX1, respectively. IgG in the serum and secretory IgA (sIgA) in saliva were quantitatively analyzed by enzyme-linked immunosorbent assay before and after 2 weeks of immunization. Peri-implantitis was induced with cotton ligatures fixed around the neck of implants. Probing depth (PD) and bleeding on probing were recorded. All animals were sacrificed after ligaturation for 6 weeks. Decalcified sections with thickness of 50 μm were prepared and dyed with methylene blue to observe the bone phenotype around implants.@*RESULTS@#Levels of serum IgG and sIgA in saliva were higher in groups A and B after immunization than before the process (P0.05). At 4 and 6 weeks after ligaturation, PD of the ligatured side in group C was higher than that in groups A and B (P0.05). Bone loss in group A was significantly lower than that of the other groups (P<0.05). Abundant inflammatory cells and bacteria were present in the bone loss area around the implants in the three groups, as identified through hard tissue section observation. However, group C presented the most number of inflammatory cells and bacteria in the bone loss area around the implants.@*CONCLUSIONS@#IgG and sIgA can be generated by immunity with rgpA DNA vaccine, which can significantly slow down bone loss during experimental peri-implantitis in dogs.
Subject(s)
Animals , Dogs , Adhesins, Bacterial , Therapeutic Uses , Alveolar Bone Loss , Arginine , Cysteine Endopeptidases , Therapeutic Uses , Dental Implants , Peri-Implantitis , Porphyromonas gingivalis , Chemistry , Vaccines , Therapeutic UsesABSTRACT
Objective:To investigate the influence of B-lymphocyte chemoattractant on the immune response of CVB3 fusion gene vaccine pcDNA3/C3d3-sVP1.Methods:BALB/c mice were divided into 4 groups randomly, and injected intramuscularly with pcDNA3,pcDNA3/BLC,pcDNA3/C3d3-sVP1 and the combination with the plasmid pcDNA3/BLC and pcDNA3/C3d3-sVP1.At a certain time,they were measured for the titers for neutralizing antibodies,specific CTL cytotoxic activity.The protective efficacy of DNA vaccinations was evaluated by titers of blood viruses and survival rate.Results:The titers for antibodies increased with the time of inoculation.More specifically,the antibody titers (42.17±1.43) and the specific CTL cytotoxic activity (41.3%±3.51%) of the mice in the combination group were remarkably stronger than in the mice with pcDNA3/C3d3-sVP1(P＜0.05),but the virus titers of blood was lower.After lethal CVB3 challenge,the protection of mice from death in the combination group with the plasmid pcDNA3/BLC and pcDNA3/C3d3-sVP1 was 44%.Survival curves indicated that the survive state of combination group was better than others.Conclusion:BLC can strongly enhance the specific immunity induced by C3d3-sVP1.
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Objective:To observe the anticaries effects of the vaccine gene pcDNA3- PAc against dental caries in BALB/c mice by different routes. Methods: BALB/c mice were immunized with the recombinant plasmid pcDNA3- PAc by the submandibular gland- target injection,the quadriceps femoris injection and the intranasal irrigation respectively. All the mice were immunized two times. The immune interval was two weeks. Saliva and serum samples were collected respectively at 0, 1, 2, 4 weeks after immunization. The specific antibodies were detected by ELISA. Results:The specific antibodies were up at one week after immunization in different routes. The peak time of the antibodies level appeared at 4 weeks.The level of salivary specific anti- PAc IgA induced by submandibular gland- target injection and that of serum IgG induced by thigh bone muscle injection was the highest, respectively. The differences of antibodies level between in experiment groups and negative control group or vacant comparison group were significant(P<0.01). Conclusion: The vaccine gene pcDNA3- PAc effectively induce local mucosal immune response and systemic immune response.
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Objective:To observe inhibitory effrects of DNA vaccine co-expressing CEA tandem repeat epitopes and FL on cancer cells in mice.Methods:The encoding sequences for CEA tandem repeats and FL were inserted into plasmid pcDNA3.0 using gene recombinant technique.BALB/c mice were immunized intramuscularly with the co-expressing DNA vaccine.The survival time and tumor size were measured and specific CTL cytotoxicity was detected by ~(125)I-UdR release method.Results:Compared with that of the control,the survival time was prolonged (P＜0.01)and the tumors were significantly inhibited in the mice immunized with the vaccine peDNA-triCEA_(625-667)-sFL(P＜0.01).The splenic cells from mice immunized with the vaccine pcDNA-triCEA_(625-667)-sFL induced strongly cytotoxicity against tumor cells H22-CEA ~+(P＜0.01).Conclusion:The recombinant DNA vaccine co-expressing pcDNA-triCEA_(625-667)-sFL can suppress the growth of tumor expressing CEA in mice and enhance CTL response against CEA antigen.
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Objective: To construct the eukaryotic expression vector of human survivin gene and stably transfect it into mouse melanoma B16 cell line. Methods: The full length human survivin cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pIRES-neo; the restriction enzyme position and 6 his tag were added to obtain recombinant plasmid pIRES-neo-SUR-(his) 6, which was then transfected into B16 cells by Lipofectamine 2000. After screening culture by G418, a stably transfected cell line was established, and the transcription and expression of the human survivin gene were identified by RT-PCR, Western blotting analysis and immunofluorescence assay. Results: The result of restriction enzyme digestion and the sequence analysis showed that the recombinant of pIRES-neo-SUR-(his)6 was successfully constructed. RT-PCR results showed survivin gene (about 530 bp) was amplified from the total RNA in the group stably transfected with pIRES-neo-SUR-(his)6. Western blotting analysis showed the expression of survivin protein in pIRES-neo-SUR-(his)6 transfection group, but not in the control group. FACS and immunofluorescence assay showed high fluorescence signal in pIRES-neo-SUR-(his)6 transfection group, with the fluorescence positive rate being 91.38% when No. 5 monoclonal antibody was used; no fluorescence signal was found in the control group( transfected with pIRES-neo). Conclusion: We have successfully constructed the eukaryotic expression vector of human survivin pIRES-neo-SUR-(his)6, and established a B16 cell line stably and highly expressing human survivin gene.
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Objecticve To study the immunogenicity and protective effecacy of MTB8.4 with signal peptide gene vaccine against TB in mice.Methods C57BL/6N!mice were vaccinated with MTB8.4 with signal peptide(MS) gene vaccine three times at 3 weeks intervals.Four weeks after the final vaccination,three mice were sacrificed to assess cytokine response and CTL induction and the other five mice were challenged intravenously in a lateral tail vein with(1?10~(6)) CFU of virulent M.tuberculosis H37Rv.Spleen and the left lung were harvested from each mouse 4 weeks after(infection) and homogenized in sterile saline.Serial dilutions of organ homogenates were plated on (L-J agar) and incubated(37 ℃) until colonies were visible 4 weeks later. Protective efficacy in each experiment was expressed as(reduced) CFU and was compared with the negative control group.The right lung was obtained from each mouse and(inflated) with and stored in 10% formalin saline immediately.Tissues were embedded in paraffin,sec-tioned(and stained) with hematoxylin and eosin.Results MS gene vaccine induced the secretion of more of Th1 cytokines(IFN-?and IL-2),but not IL-4 and enhanced CTL activity while BCG induced the secretion of both types of cytokines(IFN-?,IL-2 and IL-4).Mice immunized with MS gene vaccine had fewer and smaller tubercles than those immunized with PBS or control plasmid DNA,but had more tubercles than the mice inoculated with BCG.Virable bacterial counts in the lungs and spleens of mice receiving DNA encoding MS were reduced as compared with mice injected with PBS or control plasmid DNA,while increased as compared with mice immunized with BCG.Conclusion MS gene vaccine can induce secretion of Th1 cytokines and the higher protective efficacy against TB in mice.
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Objective To explore the method to develop a gene vaccine of human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) against its infection. Methods HCMV strain AD169 was propagated in WI-38 cell and viral DNA was extracted as a template for polymerase chain reaction (PCR) amplification of UL83 (pp65), the resulting of PCR was subcloned into pUC118HincII/BAP plasmid and DNA sequence analysis conformed the fidelity of the PCR. The vector pcDNA 5.0 was designed to correctly place CMV promoter sequence, pp65 sequence and secret signal sequence (mouse immunoglobulin kappachain for efficient secretion of recombination protein) into its genomic DNA. Exchanged primers of pp65 sequence, CMV promoter sequence and secret signal sequence to confirm the result by PCR screening. The vector pcDNA 5.0 was transfected into CHO cell, supernatants of transfected cells were extracted and purified. Recombination protein from supernatants was detected by gel electrophoresis and dot blot hybridization of Western- ECL system. Results Compared the sequence of pp65 gene with the standard sequence of pp65 from Medline,it was found that the concordant rate between them was 99.99%,only a nonsense mutation occurrences at 1 455 base.A pcDNA 5.0 Eukaryotic expression vector was established, which including CMV promoter sequence,secretion signal sequence and pp65 sequence. PCR screening and the pp65 protein expressed in CHO cell confirmed it. Extraction from supernatants in transfected CHO cells was recombination protein of pp65, which was detected by gel electrophoresis and dot blot hybridization of Western- ECL system and western blotting.Conclusion Subunit vaccine of HCMV is gained,which is a transfer eukaryotic expression vector pcDNA 5.0 constructed by CMV promoter sequence,secretion signal sequence and pp65 gene sequence.
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0.05).Conclusion Immunization with HBV gene vaccines plus HBsAg protein has no better immune responses.
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Objective:For developing the study of MUC1/Y vaccine,constructed th eukaryotic expression vectors expressing MUC1/Y.Used it to modify the DC inducint CTL in order to treat the gastrointestinal cancers.Methods:Obtained the MCU1/Y cDNA full length cloned it into pIRES2 EGFP that expressing green fluorescence protein and pcDAN3 1+ respectively.Selected 8 cases of gastrointestinal cancer whose HLA phenotype was A2,inducing DC using rhIL 4 combined with GM CSF in vitro.Transfected DC with pcDAN3.1 MUC1/Y then co cultured DC with T cell to induce CTL (named as T pcDAN3 1 MUC1/Y).At the same time,used pIRES2 EGFP MUC1/Y to detect transfection efficiency.SW620 (HLA A2+,MUC1/Y+),the gastric cancer cell line was used as specific target cell;Lovo(HLA A2 ,MUC1/Y+)and Raji(HLA A2 ,MUC1/Y )were used as unspecific target cells.The cytolytic of specific CTL was measured by LDH releasing assay.The apoptosis of target cells were detected by ANNEXIN V FITC kit.The ability of IFN ? releasing of T cells was measured by ELISA.Results:The transfection efficiency of plasmid was about 8%.There was significant difference between the cytolytic activity of T pcDNA3 1 MUC1/Y to SW620,Lovo and Raji.The cytolytic activity was about (42 8?6 15),(27 26?1 96)% and (22 73?2 15)% respectively.Compared with T IL 2)CTL induced by PBMC stimulated of IL 2(100 U/ml)) and T pcDNA3 1(CTL induced by DC transfected by pcDNA3 1+),the cytolytic activity of T pcDNA3 1 MUC1/Y against SW620 cell line showed a significant increase.The results of ANNEXIN V-FITC experimences showed that T pcDNA3 1 MUC1/Y could induce apoptosis of specific target cell.Conclusion:The expressing of MUC1/Y mRNA has important sense in gastrointestinal cancer.Constructed eukaryotic vector pIRES2 EGFP MUC1/Y that contains full length MUC1/Y cDNA could be used to study the transfection efficiency and select the positive clone;pcDAN3 1 MYC1/Y could induce powerful CTL immunoresponse.
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Objective: To construct HBV adr gene vaccine pCMVS 2-S and to observe the specific humoral immune responses in C57BL/6 mice after gene immunization. Methods: The HBV gene vaccine was injected into tibialis anterior muscles of C57BL/6 mice. ELISA was used to detected the anti-HBs antibody in mice sera at various time points after gene transfer. Results: Anti-HBs in sera of mice was tested positive 1 week after plasmid injected, the level of antibody peaked 4 weeks later, and kept high titer for at least 2 months. Conclusion: The good response of humoral immunity can be induced by injection of pCMVS 2-S in C57BL/6 mice.
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Objective To construct a novel VP 1 gene vaccine against coxsackievirus B 2 and to evaluate the effect of the cell-mediated immunity induced by it.Methods The immunodominant capsid protein VP 1 gene of CVB 2 was amplified by reverse transcript polymerase chain reaction (RT-PCR) and pcDNA 3-CVB 2VP 1was constructed by molecular cloning.The cytotoxic T lymphocyte (CTL) activity was measured by standard 51Cr-release cytotoxicity assay eight weeks after BALB/c mice were immuned by pcDNA 3-CVB 2VP 1.Results The eukaryotic expression vector was pcDNA 3 and subcloning fragment was CVB 2VP 1.The CTL activity of pcDNA 3-CVB 2VP 1 group was higher than that of the control (P
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To identification the immunization activation of recombinant Leptospira gene vaccine from many-siden MethodsThe guinea pigs were immunized with recombinant Leptospira gene vaccine [plasmid vector pT7-7 was negtive control ,inactivated whole cell vaccine (WCV) was positive control]. Then spleen cells were taken out. Particularity lymphocyte transformation test(LTT),IL-2 and IL-6 activation of these spleen cells were determined by MTT and 3H-TdR respectively. Results1)The Relative transformation index of gene vaccine group was significance higher than pT7-7 group (vaccin group: 2. 19±0. 18, pT7-7 group 1.42±0. 27 ( P＜0. 005 ); 2 ) the activation of IL- 2 and IL- 6 from recombinant gene vaccine group waw significance stronger than pT7-7 group (vaccine group IL-2:34. 8±3.11,IL-6:94. 6±6.03, pT7-7 group IL-2:20. 4±3. 05,IL-6: 67±6.28), (P＜0. 005). Conclusion1) The activation of Th1 and Th2 lymphocyte cell were increased in the guinea pigs with gene vaccine immunized. It suggested the recombinant Leptospira gene vaccine could elicit an extremely strong immunization effect of T-cell cooperate with B-cell and the gene vaccine was equal the WCV(P＞0. 05)but the gene vaccine sideeffect was small and the applying prospect was good.
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0.05);by the end of the experiment that was 24.5?1.05,28. 3?1.29,26.6?1.19,10.2?1.81, 26.3?1.54 and 27.3?1.38 respectively (D vs each of the other groups P0.05). Conclusion: Gene vaccines pcDNA3-pac and pcDNA3-gtfB have no unfavo rable influence on weight of gnotobiotic rats,while the inactive whole cell vac cine has.
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Objective:To study the possibility of dengue virus E gene vaccine.Methods:The recombinant eukaryotic expression plasmid pcDNA3 E was first transfected into NIH3T3 cells by lipofectin SDS PAGE and Western blotting analyzed the expression of E gene Then the recombinant plasmid was intramuscularly injected to BALB/c mice,and the specific humoral and cellular immunity were tested Results:The recombinant plasmid DNA could induce specific immune reactions and the immune response could last a long time Conclusion:The dengue virus E gene vaccine could induce specific immune reaction,which might have provided some material and new experimential data for the further study of dengue vaccines
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Objective:To evaluate the immunogenicity of the recombinant plasmid pcDNA 3.1(+)/kgp_ cd.Methods:BALB/c mice were immunized with recombinant plasmid pcDNA 3.1(+)/kgp_ cd,KGP_ cd protein(control) or vector pcDNA 3.1(+)(control) by quadriceps injection or targeted submandibular gland(TSG) injection. Serum IgG and salivary sIgA levels were assayed by indirect ELISA after immunization. The expression of the protein KGP_ cd in quadriceps and submandibular gland was detected by immunohistochemistry techniques.Results: Serum specific anti-KGP_ cd IgG elicited by pcDNA 3.1(+)/kgp_ cd and KGP_ cd protein was significantly higher in both the quadriceps injection group and the TSG group than that in the pcDNA 3.1(+) group (P
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Objective To prepare Mtb8.4 gene vaccine and to study the cellular immune response induced by the vaccine. Methods The gene encoding Mtb8.4 from Mycobacterium tuberculosis was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.1 (+). C57BL/6N mice were vaccinated three times with Mtb8.4 gene vaccine at 3 weeks interval. Four weeks after the final inoculation, mice were sacrificed to assess cytokine response and CTL induction. Results The IFN-? and IL-2 titers were 787.317?45.586pg/ml and 319.953?57.978pg/ml in Mtb8.4 gene vaccine group, 1 486.540?39.600pg/ml and 767.043?50.269pg/ml in BCG group, respectively. The level of IL-4 in BCG group (90.580?10.998 pg/ml) increased significantly as compared to other groups (P