AIV-HA1and CTB Fusion Protein Expressing On Bacillus Thuringiensis Cell Surface And Analysis Of Fusion Protein Biological Characteristics

Avian influenza is a highly contagious, actue respiratory dieases by Influenza virus A in Orthomyxoviridae, which caused huge losses of poultry husbandry and theeatened the humans health.Vaccination is the preferred strategy for the prevention and control of influenza infections. The traditional vaccines were used to immunity by injection, that was awkward to handle, and the vaccines should be kept at low temperature. Bacillus thuringiensis forms heat-stable spore during a specific phase, thus,that was used to development of AIV vaccine that can be conserve at room temperature.Fusion proteins of Bacillus thuringiensis S-layer protein and Avian viral antigens were expressed on Bacillus thuringiensis (Bt) cell surface (Liu et al.,2007, Liu et al,2008a, Liu et al.,2008b). Chickens were immunized by recombinant Baillus thuringiensis of Avian influenza virus (AIV) hemagglutinin. After immunization, sera of all chickens were tested for HA-specific antibodies by hemagglutinin inhibition(HI) test,3.8log2HI titer of HA-specific Ab was below the standard (4(log2)). In order to improve protective efficacy of Bt-based vector vaccine expressing the H5-HA gene, Cholera toxin B subunit(CTB) was used as a mucosal adjuvant, Here we present a study on coexpression of CTB, HA1and S-layer protein on Bt surface. The reseach had established the foundation for further development of the heat-stable and oral vaccine.1. Cloning of part hemagglutinin gene hal and Cholera toxin B subunit gene ctb, and the construction of recombinant plasmidThe hal and ctb gene fragments were amplified by PCR. Restriction enzyme recognition sites EcoRI and HinⅡ of that were mutated by PCR-based site-directed mutagenesis. After mutation, the sequence of amino acid was no modification. The two connection modes of hal and ctb genes were ctb-hal and ctb-linker-hal, which were amplified by overlap extension PCR.The PCR gene fragments were restricted with HincⅡ and XbaI and ligated into vector, The csaAB gene fragment was digested with EcoRI and also inserted into pBMB982-304cleaved with EcoRI to obtain the expression vectors pCSCK(csa-ctc-ctb-hal) and pCSCLH(csa-ctc-ctb-linker-hal) and the control plasmids pCSCTB (csa-ctc-ctb) and pCSHAl(csa-ctc-hal).2. Construction of recombinant strains and the characterization of HA1-fusions on recombinant strainsThe expression vectors and control plasmids were transformed into Bacillus thuringiensis BMB171by electroporation to produce the four recombinant strains, which were named MICTB (harboring pCSCTB), MIHA1(harboring pCSHAl), MICH (harboring pCSCH), MICLH (harboring pCSCLH). HA1-fusions on recombinant strains were determined by hemagglutinin(HA) assays and HI assays. The results of HA test show that recombinant strains MIHA1, MICH and McICLH have activity of hemagglutinin, and that is able to specifically inhibit by standard positive serum of AIV. Thus, HA1fusion protein can be displayed on the BMB171cell surface of recombinant stains and has the hemagglutinin activity.3. Heredity stability of Recombinant strainsThe recombinant stains MICH and MICLH was continuous cultured in LB broth without antibiotics on fifty generations, after each ten generations, the heredity stability of MICH and MICLH were detected by LB agar plate with Erm. The heredity stability rate of pCSCH in MICH was maintain80%after passed fifty generations. At thirty generatic,86%of MICLH colonies have the plasmid pCSCLH, but98%of that lost it at fifty generatic.4. Immunogenicity of Recombinant strains in chinkenThe stationary phase cultures of MICH and MICLH were collected by centrifugation. At the age of7days chickens were immunized three times by oral administration of109colony forming units (CFU) of MICH and MICLH each, respectively. The samples of blood, lavage fluids from nasal, trachea and gut were collect from all chickens. HA1-specific IgG in sera and HA1-specific IgA in lavage fluids were tested. Direct ELISA was used to detect the total IgA of lavage fluids. The ELISA and HI consequences indcatesd that the recombinant bacteria MICH can induce the level of HA1-specific antibody in sera after the first immunization, the level of the antibody gradually decline after the second and third immunization, the level SIgA-HAl of The MICH group’ tracheal mucosa was markedly higher than that of the control group, but the highest value was0.113. The recombinant bacteria MICH induce more productions of total IgA-nonspecific in nasal, trachea and gut mucosas, and that in nasal have a significant difference. The results of animals showed CTB didn’t function effect of the immune adjuvant, that hard to practical application.