Construction Of A Bivalent Membrane-anchored Expression DNA Vaccine Bearing Mycobacterium Tubeculosis Ag85B And Hsp65 Genes And Study On Immunogenicity Of DNA Vaccine PIRES-Sj26 In Mice

Objective To construct a eukaryotic membrane-anchored co-expression plasmid encoding human Mycobacterium tuberculosis Heat shock protein 65(Hsp65) and Antigen 85B (Ag85B) and study their expression in vitro.Methods The Ag85B and Hsp65 gene of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from M. tuberculosis H37Rv genome and plasmid pBCG-SP-Hsp65 respectively by polymerase Chain reactions(PCR). These two genes were cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmid pVAC-Hsp65 and pVAC-Ag85B.Then two modified gene fragments were respectively amplified by PCR with templates of plasmid pVAC-Hsp65 and pVAC-Ag85B,which encode a nucleotide sequence of human interleukin-2(IL-2) signal peptides in their 5'ends and that of membrane-anchored peptides of carboxyl-terminal of placental alkaline phosphatase(PLAP) in 3'ends. After that, the two modified genes were altogether inserted into a eukaryotic co-expression plasmid pIRES, resulting in another new recombinant plasmid pIRES-MTHsp65-Ag85B. Then, their expression was identified through RT-PCR when the plasmid pIRES-MTHsp65-Ag85B was transfected into eukaryotic Hela cells.Results Restriction enzyme analysis,PCR and sequencing results showed that the recombinant plasmid pIRES-MTHsp65-Ag85B were successfully constructed and the expression of modified Hsp65 and Ag85B genes could be detected by RT-PCR.Conclusion A bivalent membrane-anchored expression DNA vaccine bearing Hsp65 and Ag85B genes was acquired, which layed foundations for later study of its immunogenicity and anti-affection efficiency. Objective To construct a eukaryotic membrane-anchored expression plasmid containing Sjc26GST genes and determine its immunogenicity on BALB/c mice.Methods Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worm as the template and were modified with a nucleotide sequence of human interleukin-2 (IL-2) signal peptides in their 5'ends and that of membrane-anchored peptides of carboxyl-terminal of placental alkaline phosphatase (PLAP) in 3'ends. After that, they were cloned into eukaryotic expression plasmid pIRES to construct a recombinant plasmid pIRES-Sj26.Then, the expression of modified gene was identified through RT-PCR and IFA when the recombinant plasmid pIRES-Sj26 was transfected into eukaryotic HeLa cells. After immunization of BALB/c mice with pIRES-Sj26 vaccine, total IgG in serum and the level of IFN-γwere measured by ELISA. The lymphocyte stimulating index(SI) by MTT , the subgroups of splenocyte by FCM were tested also.Results Restriction enzyme analysis, PCR and sequencing results showed that the recombinant plasmid pIRES-Sj26 were constructed successfully. After the transfection, the expression of modified Sj26 genes could be detected by RT-PCR and IFA. The IgG level and the INF-γlevel in the pIRES-Sj26 group was significantly higher than those in the control group and the vector group(P<0.01),while the SI level and the percentages of CD8+is higher too(P<0.05).Conclusion A membrane-anchored express DNA vaccine was acquired and Sj26 membrane-anchored proteins were proved to be expressed out in vitro. The pIRES-Sj26 vaccine induce stronger immune response in BALB/c mice.