Construction Of Vector PGEX-6p-1-Ag85A And Immune Responses To Ag85A DNA Vaccine Using Attenuated Salmonella Typhimurium As Carrier Administered Orally

IntroductionTuberculosis(TB) is a chronic respiratory infectious disease that has afflicted humans for thousands of years.despite the use of BCG,TB remains a global epidemic with one-third of the world population being infected and an annual rate of 8 million new cases and 3 million deaths.Regardless of its protection from severe forms of childhood TB,BCG fails to confer protection from adult TB.Thus,there is an urgent need for developing safe and effective TB vaccines that are able to confer potent protection at the mucosal site.So far for the protective antigen of Mycobacterium tuberculosis is still not very clear,extra cellular secretion or cell wall-related protein is estimated to the important antigen recognized by protective immune responses.Antigen 85A is one of the components of the Antigen 85 complex.This protein is expressed by a wide range of bacteria of genus Mycobacterium.Ag85A DNA vaccine mainly causes Th1 type immune response after intramuscular injection,while mainly induces Th2 type immune response after the gene gun injection.Attenuated Salmonella typhimurium can present antigen effectively,play a role as immune adjuvant,as well as the advantages of stimulating mucosal immunity has been widely used in new type of bacterial vector DNA vaccine.We plan to construct vector pGEX-6p-1-Ag85A used for the detection of Ag85A-specific antibodies and preparation of monoclonal antibodies.And oral immunization of mice with attenuated Salmonella typhimurium expressing Ag85A, detect the immune response and lay a foundation for further research about Ag85A DNA vaccine. Materials1.AnimalFemale C57BL/6 mice,6-8 weeks old(Academia Sinica Shanghai experimental animal center).2.AgentsRPMI1640 medium,E.coli BL21,plasmid pGEX-6p-1,Axyprep Plasmid Miniprep Kit,Axyprep DNA Gel Extraction Kit,Easy Taq DNA Polymerase,High Pure dNTPs,T4 DNA Ligase,IPTG,BamHâ… ,EcoR Iand Bglâ…¡restriction endonuclease,FN-γand IL-4 ELISA kit.Methods1.The construction of Vector pGEX-6p-1-Ag85APut the attenuated Salmonella typhimurium carrying plasmid V1Jns.tPA-Ag85A out of -70℃fridge,extracted and digested plasmid with Axyprep Plasmid Miniprep Kit and restriction enzymes Bglâ…¡,PCR amplification of target gene Ag85A with V1Jns.tPA-Ag85A as a template.digest Ag85A PCR product and pGEX-6p-1 vector with restriction endonuclease BamHâ… and EcoRâ… ,using T4 DNA ligase to connect both of them.The recombinant plasmid transformed into E.coli BL21,expression of protein was inducted by IPTG and detected by SDS-PAGE gel electrophoresis and Western Blotting.2.Immune responses to Ag85A DNA vaccine using attenuated Salmonella typhimurium as carrier administered orally(1)Prepared oral vaccine and immune animals.Put the attenuated Salmonella typhimurium carrying plasmid V1Jns.tPA-Ag85A out of -70℃fridge,extracted and digested plasmid with Axyprep Plasmid Miniprep Kit and restriction enzymes Bglâ…¡,10-fold diluted the bacteria harvest and counted colonies.C57BL/6 mice were randomly separated 3 groups:(1)saline control group,(2) empty plasmid vector group,(3)recombinant plasmid group.The mice are immuned every 3weeks,three times altogether.respectively 2w,4w,6w,8w after the initial immunization executed 3-4 mice in each group.(2)Determination of contents of Ag85A-specific antibody in serum. Blood were harvested from eyes,serum were tested in duplicate using ELISA for Ag85A-specific antibody.(3)Determination of contents of IFN-γand IL-4 in spleen cells culture suspension by double antibody sandwich ELISASpleen cells were harvested from mice and adjusted to a final concentration of 10⁷cells/ml.Aliquots(500μl/well) of the cell suspension were incubated in 24-well plat-bottom tissue culture plates in triplicate for 48 hours at 37℃in a humidified 5% CO₂ incubator.Then the 24-well plates were centrifuged at 1000g for 10 min at RT, Supernatants above were tested in duplicate using ELISA kits for IFN-γand IL-4.Result1.The construction of Vector pGEX-6p-1-Ag85AThe nucleotide sequencing of the gene encoding Ag85A was identical and there were no undesired mutations,the cloning gene was correctly inserted into the vector pcDNA3.1⁺as confirmed by restriction endonuclease digestion of BamH I and EcoR I and sequence analysis.We obtain E.coli BL21 which can express the Ag85A protein with molecular mass about 58KD by using SDS-PAGE gel electrophoresis and Western Blotting.2.Determination of contents of Ag85A-specific antibody in serumAfter the initial immune the contents of Ag85A-specific antibody in serum slightly higher and antibody titer was 1:50;After the second plasmid immunized the levels of Ag85A-specific antibody increased rapidly,while in empty vector control group and saline control group the antibody titer is always negative.3.The levels of IFN-γin spleen cell culture suspension at different times after oral immunizationAfter the initial immunization,levels of IFN-γin spleen cell culture supernatant in Ag85A DNA plasmid group is significantly increased(P <0.01);the levels of IFN-γin empty plasmid control group and normal control group also increased,but there is no significant difference between the two groups(P> 0.05).4.The levels of IL-4 in spleen cell culture suspension at different times after oral immunizationAfter the third oral immunization,levels of IL-4 in spleen cell culture supernatant were no significant difference between three groups(P> 0.05). DiscussionAg85A is one of the components of Ag85 complex,a kind of protein ingredient secreted by BCG,Mycobacterium tuberculosis and other genus Mycobacterium.The Antigen 85A protein is coded by the gene called fibronectin-binding protein-A(fbpA) gene.In Mycobacterium tuberculosis,this fibronectin-binding protein-A(fbpA) gene is 1014bp long and has a molecular weight of 32KD.Recent animal studies have shown that vaccination with recombinant Antigen 85A DNA or the protein has powerful immunological properties.In our experiment we immunized mice with attenuated Salmonella typhimurium carrying antigen Ag85A eukaryotic expression vector,and observed the contents of specific antibodies in serum,IFN-γand IL-4 in spleen cells culture supernatant.The results suggest that the levels of specific antibodies in serum increased after the initial immunization has and growth increased after booster immunization,after the last immunization antibody titer reached 1:400 to prove that oral administration of attenuated Salmonella typhimurium carrying Ag85A DNA vaccine effective induced systemic humoral immune response in mice.Levels of IFN-γin Spleen cell culture supernatant significantly improved,and compared to the empty plasmid control group and normal control group there is significant difference(P <0.01),proved that oral administration of attenuated Salmonella typhimurium carrying Ag85A DNA vaccine in mice resulting in Th1-type immune responses which is necessary by anti-TB immune.The results are consistent with reports which injected the Ag85A DNA vaccine in mice by Huygen.Conclusion1.Constructed vector of pGEX-6p-1-Ag85A and expressed the Ag85A protein successfully.2.Oral administration of attenuated Salmonella typhimurium carrying Ag85A DNA vaccine resulted in the contents of Ag85A-specificantibody in serum increased.3.Oral administration of attenuated Salmonella typhimurium carrying Ag85A DNA vaccine increased the levels of IFN-y in spleen cell culture supernatant significantly.