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Objective To explore whether a DNA immunization approach targeting the major haemorrhage molecule of a prothrombin activator-like metalloproteinase from Echis ocellatus (E. ocellatus) venom could be conceived to inspire antibodies with more prominent specificity and equal adequacy to current conventional antivenoms systems. Methods The isolated DNA EoMP-6 was used as the template for PCR amplification using the EoDC-2-specific forward and reverse primers. A PCR product of approximately 700 bp was obtained and cloned into pSecTag-B expression vector where anti-EoDC-2 antibodies were generated and analysed for their efficacy to neutralise local haemorrhage in vitro and in vivo. Results Our results suggest that the generated anti-EoDC-2 showed a remarkable efficacy by (a) interfering with the interaction of the recombinant disintegrin “EoDC-2” isolated from the E. ocellatus as well as other viper species to the α
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To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 microg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5x10(4) sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control.
Subject(s)
Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/genetics , Cell Proliferation , Chickens , Coccidiosis/immunology , Disease Models, Animal , Eimeria/genetics , Injections, Intramuscular , Lymphocytes/immunology , Protozoan Vaccines/administration & dosage , Vaccination/methods , Vaccines, DNA/administration & dosageABSTRACT
Objective To study on the expression of the eukaryotic recombinant vector carrying HlyX gene of Leptospira serovar Lai in mammalian cell and explore the humoral immune response in BALB/c mice immunized with the recombinant plasmid. Methods The HlyX gene was amplified from Leptospira serovar Lai genomic DNA by PCR and inserted into pcDNA3.1 vector. After transformed into E. coli DH5α,the recombinant plasmid was assayed for identification by PCR analysis,restriction nuclease enzyme digestion and sequencing. The recombinant plasmid was transfected into COS-7 cells,then RT-PCR and Western blot were performed to test the expression of the target gene. The recombinant plasmid was injected intramuscularly into BALB/c mice for three times at intervals of two weeks,and the antibody titer was measured by ELISA. Results PCR showed the full length HlyX gene was about 1100 bp. PCR analysis,restriction nuclease enzyme digestion and sequencing indicated the recombinant vector was constructed successfully. After the plasmid Was transfected into COS-7 cells,a fragment about 1100 bp was found by RT-PCR and a specific band relative molecular mass(Mr)about 40×103,which was consistent with the expected size of the target proteins was showed by Western blot. ELISA showed the antibody titer in BALB/c mice immunized by the HlyX gene of Leptospira serovar Lai can elicit high-titer antibody in BALB/c mice,which has laid the foundation for the application of the DNA vaccine.
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Objective: FF456 is a new gene cloned in our lab, which belongs to c-type G protein-coupled receptors and has high homology to the olfactory receptor family. Northern blotting and RT-PCR showed that the expression of FF456 was exclusively restricted in liver and was significantly down-regulated in hepatoma. In an effort to study the function of FF456 gene, high titer antibody is indispensable. Methods:Full-length cDNA of FF456 was reconstructed into pcDNA3.1(-) vector, which was used for immunizing 6- to 8- weeks old female BALB/c mice. The effects of different injection manners, solvents and dosages on the antibody production have been compared. For detecting the titer and specificity of the antisera produced by DNA immunization, a peptide containing FF456 specific sequence was expressed by E.coli expression system. Results:Finally specific antisera with titers as high as 1 ∶ 50 000 was obtained. Conclusion:Immunofluorescence assays showed FF456 was expressed in hepatocytes with high tissue specificity. The FF456 expression in hepatoma cells was decreased dramatically.
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Objective IFN-? is a pleiotropic cytokine with potent immunomodulatory effects and antiviral activity. To study the mechanism of IFN-? clearing duck hepatitis B virus (DHBV) in ducks, it is essential to establish a method to quantify expression of DuIFN-? in immune response. In the present study,a semi-quantitative competitive RT-PCR was developed to quantify expression of duck IFN-?(DuIFN-?) mRNA by PBMCs. Methods Based on ?-actin consensus sequence, fishing the ?-actin gene as house keeping gene from duck PBMC by RT-PCR. A competitive internal control was constructed and the competitive RT-PCR system could be used to quantify the transcription of DuIFN-? mRNA. Results After duck PBMCs were stimulated in vitro with PHA, the peak of DuIFN-? expression was at 24-36h. Then RT-PCR method was applied to detect DuIFN-? mRNA transcription by PBMCs from DHBV infected ducks immunized with DuIFN-? plasmid plus DNA vaccine or DNA vaccine alone. Results showed that expression of DuIFN-? in ducks co-immunized with DuIFN-? plasmid were higher than other groups immunized without DuIFN-? plasmid as adjuvant. Conclusions The results indicated that DuIFN-? gene could be a useful adjuvant to develop vaccines. The semi-quantitation of DuIFN-? mRNA by competitive RT-PCR provides the basis for future study of the mechanism of IFN-? in duck hepatitis B virus persistent infection.
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Objective:In the past study, a plasmid of pcDNA3 hCG? C3d3 had been constructed. It was suggested that the fusing protein be expressed by the pcDNA3 hCG? C3d3 plasmid both in the transient expression system in COS 7 cells, and the stable expression system in CHO cells. In the present research, it would be testified that the C3d molecular adjuvant could improve the immunogenicity of the hCG? DNA immunization or not. Methods:BALB/C mice of 6 weeks old were immunized intramuscularly two times at interval of 3 weeks by the plasmid pcDNA3, pcDNA3 hCG?, pcDNA3 hCG? C3d3 at dosage of 5, 10, 20 pmol, respectively. The anti hCG? antibody titers were determined by indirect ELISA in 6 weeks of last immunization. Results:The result showed that the C3d molecular adjuvant could increase significantly the titer of anti hCG? antibody in a dose dependent manner after DNA immunization.Conclusion:The C3d molecular adjuvant does improve the humoral immunity of the hCG? DNA immunization, which would be helpful for technical progress and clinical trial of the contraceptive vaccine. [
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Objective To observe the specific humoral and cellular immune response in BALB/c mice injected with pS and p18S. Methods pS and p18S were constructed separately by inserting HBsAg gene fragment and the fusion gene fragment of HBsAg and mouse interleukin 18(IL 18) into the reading frame of pcDNA3.1+. Mice were injected with either plasmid intramuscularly in a total dose of 300 ?g per mouse. Every serum sample was detected for anti HBs using enzyme linked immunosorbent assay(ELISA). Furthermore, HBsAg specific cytotoxic T lymphocytes activity was measured. Results The expression of HBsAg was demonstrated by ELISA in p815 cells transfected with pS and p18S. pS can stimulate a positive antibody response. The average level was 135 mIU/ml, with the highest level of 530 mIU/ml. p18S could elicit relatively lower antibody response which was 20 mIU/ml. HBsAg specific CTL activities were 37.1% and 34% separately in pS and p18S immunized mice. It was only 13.2% when detected in pcDNA3.1+ immunization. Conclusion pS is effective to stimulate a humoral and cellular response in H 2d mice. IL 18 gene can not enhance the immune response when fused with HBsAg gene. Conversely, it seems to inhibit an immune response.
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Objective: To induce an immune response by intramuscular immunization with HER2/neu oncogene extracellular do- main (ECD) DNA and observe the effect in vivo by an abortion model in mice.Methods: Human and rat HEH2/neu ECD gene were cloned and inserted into expressive vector pCDNA3. The DNA immunization, expression of HER2/neu ECD, antibody de- tection and abortion in immunized mice were studied. Results: HER2/neu ECD protein expressed on muscle cells in the injected site, specific antibody to HER2/neu was induced by DNA immunization. The average of the offspring per delivery from immu- nized mice was significantly lower than that from control group, and the abortive embryos were found in uterus from immunized mice.Conclusion: The results show that both human and rat HER2/neu ECD gene can induced effective immune response by DNA immunization.
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Objective: To study the cellular immune response and the anti-tumor effects induced by intramuscular DNA immu- nization with HER2/neu extracellular domain gene. Methods: HEH2/neu oncogene extracelltilar domain gene was isolated and inserted to pCDNA3 expression vector. The cellular immune response and its anti-tumor effects were analyzed after intramuscular DNA immunization. Results: A specific cytotoxic activity by spleen cells from HER2/neu ECD gene immunized mice was found in vitro. Tumor growth was inhibited after tumor challenge in vivo in immunized mice. Conclusion: Specific cellular immune re- sponse and anti-tumor effect were elicited by HEB2/neu extracellular domain gene immunization.
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Study the immunological adjuvant function of biodegradable microspheres for DNA immunization. Empty poly (D, L-lactide-co-glycolic acid) microspheres were prepared using the water- inoil-in-water (w-o-w) technique; A plasmid DNA pRc-CMV encoding hepatitis B virus S antigen was constructed; The mixture of the microspheres and the plasmid DNA was prepared by incubation method. The mixture was administered to Balb/c mice by intramuscular injection. Result: The high antibody titer(1:1600) of intramuscular injection of the mixture of microspheres and the plasmid DNA was obtained, similar to that of intramuscular injection of the mixture of AL(OH)3 and hepatitis B virus S antigen; while intramuscular injection the plasmid DNA elicited no serum antibody respones. Conclusion: biodegradable microspheres may be used as an good adjuvant for DNA immunization.
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Objective To construct a fusion minigene expression vector of seven HCV NS3 epitopes and express the fusion gene in the eukaryotic cell line, in order to form a foundation for the investigation of the DNA immunization for HCV prevention. Methods Three pairs of complementary oligonucleotides primers covering all seven HCV NS3 epitopes were synthesized. Overlapping extension PCR were performed to construct the fusion minigene and cloned in pEGFP-N3 and pBuDCE vector respectively. The fusion protein was detected by Western blotting and flow cytometric analysis. The transfection efficiency was assessed with by flow cytometric analysis. Results The HCV NS3 epitope fusion minigene was obtained and inserted into pEGFP-N3 and pBuDCE vector respectively. The recombinant plasmid pEGFP-DR4 and pBuDCE-DR4 were expressed in 293T and 046W cell lines respectively. The Western blot result indicated that the expected 36 kDa minigene/EGFP fusion product was expressed from pEGFP-DR4, while 13kDa product from pBuDCE-DR4 respectively. Conclusion The HCV NS3 epitope fusion minigene was expressed in the eukaryocyte expression system.