| Influenza A virus possesses a segmented genome of single-stranded negative-sense RNA and belongs to the family Orthomyxoviridae (Swayne et al., 2000), which is divided into 16 hemagglutinin and 9 neuraminidase subtypes.Avian influenza (AI) has caused enormous economic loss and grand challenges for the public health. Further more, there is new situation that many subtype avian influenza viruses (AIV) are coexistent. More over, It is appears that the viruses can be transmitted directly from birds to humans without the involvement of an intermediate mammalian host. The task of prevent the avian influenza disease becomes more and more hard. Vaccines are still the main means to prevent AI. But traditional vaccines can't play an efficient role to prevent the AI for the mucosal infection. Traditional vaccine injectiong needs special instruments and worker and also many times. And it is unrealistic idea to vaccinate all nature hosts against avian infection. Avian influenza has caused enormous economic loss and grand challenges for the public health. It is an urgent question how to design and develop new vaccines, especially vaccinated through mucosal route.Mucosal immne system which is one of main important ingredient of the system immunological network play vital role in anti-infection. The surface of mucosal contact with outside antigen directly, is the first line of defense. Lots of research proved that vaccines inoculated by mucosal tract were effective against mucosal infection disease.The promoter P43 of Bacillus Subtilis, 426 bp was amplified from the bacillus genome and was sequenced. There are two part stacked promoters in the sequence, one effect at exponential phase of growth and the other at stage of latency. Using bioinformatin software, signal peptide and terminator sequence were selected and optimized and synthesized. Then those gene sequences were cloned into pGJ103 produced expression vector named pBC38C. The green fluorescent protein gene (GFP) gene was cloned into pBC38C and yielding pBC38C-GFP and was tranfered to bacillus subtilis, the recombinant bacteria appear evident fluorescence. The results show that the expression vector can express exogenous gene in bacillus subtilis.The heredity stability of the pBC38C in Bacillus subtilis were detected and results show that after passed 65 generations, the heredity stability rate of pBC38C was maintain 95% under selective pressure. While, without selective pressure, at 65 genetic, 55% of the colonies lost the plasmid. The phade of heredity stability without selective pressure was long enough for industrialized production.Theβ-galactosidas gene was inserted into pBC38C yielding the recombinant plasmid pBC38C-lacZ. The activities of promoter and signal peptide were reflected by detecting the unit of theβ-galactosidase in supernatant and cells pellet. The results show that the highest level after cultivation for 22 h, the enzyme activity reaches 26 milliunits per milliliter in supernatant. The highest level ofβ-galactosidas in cell pellets reached 6 milliunits per milliliter cultivated 14 hours. There was no activity was detected in negative control. Those informations were useful for the further use of the plamid pBC38C.The most important part for vaccine was safty, in order to verification the safty of Bacillus Subtilis, large dose of different bacteria strains were intra-gastric administrated for long times, the body weight, thymus index, spleen index and macrophage active of test mice were detected and organs histopathology slide were also made. All results show that the bacteria were safety for mouse. The feeds utilization rate of mouse inoculated with strain 1A751, 1A751/pBC38C, B.S.0701 and B.S.0702 are elevated about 3.8%, 3.12%, 2.4%, 3.0% than that of control group (p<0.05), but the discrepancy is not quiet interclass. The lymphocyte transformation rate of strain 1A751, 1A751/pBC38C, B.S.0701 and B.S.0702 were higher (1.61, 1.58, 1.51, 1.53) than that of control group (1.31). There is no difference between test groups and control group in tissue structure. For the results of research, the bacillus subtilis 1A751 was be used as the host.Based on the above reseaches, the AIV H5N1 HA gene was cloned into pBC38C, and Cholera toxin B subunit gene was used as adjuvant, the recombinant plasmids pBC38C-H5HA and pBC38C-CTB-H5HA were constructed. BALB/C mice was used as mammal experimental model, were inoculated by recombinant 1A751/pBC38C -CTB-H5HA and 1A751/pBC38C-H5HA. The immunity levels of humoral, cell and mucosal were detected. The results of HI antibody show that of H5 subtype could be generated in group 1A751/pBC38C-CTB-H5HA and 1A751/pBC38C-H5HA, but the level about 2.33 and 2.34 were lower than that of the inactivated vaccine group almost 6.64 after inoculated 5 weeks.The proliferation ability of splenic lymphocyte stimulated by antigen and ConA were detected and the results show that the special and nospecial stimulate index of 1A751/pBC38C-CTB-H5HA(2.4361,2.3210) and 1A751/pBC38C-H5HA(2.0359, 2.011) were higher than that of control group (1.529, 1.3916) and inactivated vaccine group (1.7882, 1.5859) (p<0.05) and the stimulated index of former was higher a little than that of the latter, presumed that it may be the CTB adjuvant effection, which can be enhance the level of cell immunity. The resultsts of ELISPOT detection for number of IFN-γblot showed the number of group 1A751/pBC38C-CTB-H5HA 64 and that of 1A751/pBC38C-H5HA 56 were higher than that of inactivated vaccine groups which was only 37. The results of IgA level in lung and gut lavage fluids shows that recombinant bacteria 1A751/pBC38C-H5HA and 1A751/pBC38C-CTB-H5HA can induce higher level of specific IgA (0.358, 0.334; 0.224, 0.234) were higher than that of inactived vaccine group (0.129,0.136). The total level of IgA in group 1A751/pBC38C-H5HA and 1A751/pBC38C-CTB-H5HA were also higher than that of inactived vaccine group. And the level of total IgA of 1A751/pBC38C was higher than that of pBS, which means the bacillus subtilis can enhance the mucosal immunity and this effection was nonspecificity.The resulults above indicated that the expression vector pBC38C which ware constructed above can expresse exogenous gene in bacillus subtili. The recombinant bacteria 1A751/pBC38C-H5HA and 1A751/pBC38C-CTB-H5HA can induce more cell and mucosal immunity in test mice, and also with low humoral immunity which compared with inactivated vaccine group. The research had estabilised the foundation for the mucosal vaccine of avian influenza viruse. |
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