Dissertation > Medicine, health > Basic Medical > Medical Immunology

Construction of the Recombinant of HSV-2gD Minotope、 HBsAg、 IL-18 DNA Vaccines and Its Inducing Immune Respons in Mice

Author ZhengZuoZuo
Tutor YuAiLian;WangYu
School Taishan Medical College
Course Pathogen Biology
Keywords Overlap Polymerase Chain Reaction(PCR) HSV-2gD Simulation Antigen Epitope IL18 HBsAg Immunity Effection
CLC R392
Type Master's thesis
Year 2011
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ObjectivePlasmid DNA vaccines encoding the hepatitis B virus(HBV)surface、herpes simplex virus HSV-typeII gD glycoprotein single analog antigen ,and interleukin 18,were constructed by the overlapping PCR method. From the result of the expression of each plasmid in CHO cells,and the immunization of each plasmid in female BALB/C mice, we can get the best vaccine,which provides the new theory and practical information for the nucleic acid vaccine to further research and study.Method1. Construction of pc-IL18-S and pc-SThe coding region of the HBV surface antigen was amplified by PCR. The amplified products connect with the pMD 18 T vector. If the plasmid DNA is right,the PCR products could be digested with EcoRV and BamHI,and then inserted into the pcDNA3.1 vector to gennerate pc-S.The plasmid sT-IL18 was digested with EcoR I and XholI,and the sequence IL18 was inserted into the pcDNA3.1 vector to generate pc-IL18.Then digested pc-IL18 and sT-S with EcoR I and BamH I in the same time ,then the gene of IL18-S was inserted into the pcDNA3.1 vector to generate pc-IL18-S. We can identified these plasmids respectively by digested with Xhol I and EcoR I , EcoR I and BamH I, Xhol I and BamH I, or Xhol、EcoRI and BamH I.2.Get the gene S including some genes of the other templateThe coding region of the HBV surface antigen was amplified by PCR. The amplified products have the overlapping of the other template.3.Construction of plasmid pc-IL18-P6-S, pc-IL18-NP6-S, pc-S-P6-IL18, pc-S-NP6-IL18 by the overlapping PCR.Basing on previous constructed plasmids as following: pc-IL18-P6、pc-IL18-NP6、pc-P6-IL18and pc-NP6-IL18, we can use those plasmids as templates.By overlapping PCR,we can connect S gene with those plasmids,and get the new sequences including IL18-P6-S, IL18-NP6-S,S-P6-IL18, S-NP6-IL18.Then,those products were inserted into the pMD-18 simple T vector,and sequencing by company.If the sequences were right,the plasmids could be digested with XholI and BamHI,and then inserted into the pcDNA3.1 vector to generate pc-IL18-P6-S, pc-IL18-NP6-S,pc-S-P6- IL18, pc-S-NP6-IL18.4. Expression in CHO cells and the best plasmid was selectedBy Lipofectin, plasmid pc-IL18-P6-S, pc-IL18-NP6-S, pc-S-P6-IL18, pc-S-NP6-IL18, pc-S, pc-IL18-S transfet into CHO cells .The expression of each plasmid in CHO cells was confirmed using indirect immunofluorescence (IFA),and choose the best plasmid. The best plasmid was tested by western blot.5. Animals and immunizationFemale BALB/c mice were purchased,and five female mice per group, at 5-6Weeks old,were injected intramuscularly into their tibialis muscle,with 50ug of the best plasmid. Before the first injection,we can use 0.5% lignocaine to deal with per mice.And at the first injection,we mixed those plasmids with Freund adjuvant complete(FCA) by 1:1. Mice received 50ug of the best plasmid DNA in a volume of 50μl via three intramuscular injection,and two-week interval. At the end of the last immunity,we can test by ELISA analysis for serum antibody detection and MTT analysis for antigen specific splenocyte proliferation .Result1.The construction of the plasmid and choose the best one①Get the the gene of IL18-P6-S、IL18-NP6-S、S-P6-IL18、S-NP6-IL18 by the overlapping PCR,and get the the gene of IL18-S by restriction enzyme.②From the restriction enzyme and connection of T4 ligase, we can get the plasmid of pc-IL18-NP6-S、pc-S-P6-IL18、pc-S-NP6-IL18、pc-IL18-S、pc-S.③By indirect immunofluorescence (IFA),we can get the better?plasmids,which are pc-S-P6-IL18、pc-S-NP6-IL18.④The plasmid of pc-S-P6-IL18、pc-S-NP6-IL18 were tested by western blot,and we know the antigen of S can be found,but the antigen of P6 or NP6 cannot be.2. The immune effect observation ? ①Test antibody level of S antigen by elisa,we can get that pc-S-P6-IL18 (0.10)compare with pc-S-NP6-IL18 (0.09), P=0.167>0.05, they have no differences.②Test antibody level of P6 or NP6 antigen by elisa,we can get that pc-S-P6-IL18 (0.09) comparewith pc-S-NP6-IL18 (0.08), P=0.314>0.05, they have no differences.③The MTT method detection spleen cells stimulate index SI, we can get that pc-S-P6-IL18 (1.38)compare with pc-S-NP6-IL18(1.35), P=0.210>0.05, they have no differences.Conclusion1.The overlap PCR method is an effective one,which can get products of the most and shortest sequences in vitro.2.We get the the plasmid of pc-IL18-P6-S、pc-IL18-NP6-S、pc-S-P6-IL18、pc-S-NP6-IL18、pc-IL18-S、pc-S,and choose pc-S-P6-IL18、pc-S-NP6-IL18 as the better one.3.Advantage plasmid pc-S-P6-IL18、pc-S-NP6-IL18 immune BALB/c mice,which can effectively increase the cellular immune effection and humoral immune effection.And pc-S-P6-IL18 compare with pc-S-NP6-IL18, they have no differences,so pc-S-P6-IL18 can be as the candidate vaccine instead of pc-S-NP6-IL18.4.This study further verified that carrying multiple of heterologous gene cloning has good express activity.And we can find that different position will bring different result,which provides more candidate material and theoretical support for the new vaccine research.

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