Bacillus Subtilis As A Live Bacterial Carrier Expression Of HA And PB1-F2 Proteins And Its Immunization Efficiency

Recently,the frequent outbreaks of avian influenza have resulted in significant economic losses to the poultry industry,which caused great public concern.More seriously,the avian flu virus spread from time to time and leads to death.Until now,the best method to prevent avian influenza in China is to use inactivated viruses as a vaccine.However,the influenza virus mainly infects the mucosal surface of the respiratory and digestive tracts,where the traditional vaccine may not effective in.Mucosal immunity,due to its advantages such as simplicity,safety and convenience,has received much attention.Mucous membrane is a main entrance point for many pathogens,and once an oral vaccine enters the gastrointestinal tract,the intestinal mucosal immunity can be effectively stimulated.Compared with the traditional intramuscular vaccine,oral vaccine is more stable and convenient to use.However,the mucosal immune also has some shortcomings,as oral vaccines may get degraded when passing through the stomach.The suitable solution is to use live bacterial carriers as oral vaccines.Bacillus subtilis,as antigen delivery carrier,have outstanding advantages.The genetic manipulation methods of bacillus subtilis are well established.The bacillus subtilis can induce mucosal immune responses and it is relatively resistant to high temperature and harsh enviroment.Therefore,this study constructed the recombinant Bacillus subtilis to evaluate the mucosal immune efficiency in animals.We first cloned and inserted the fragment of hemagglutinin HA from H5N1 avian influenza,PB1-F2 from H9N2 avian influenza as well as the green fluorescent protein GFP into the integrated vector pInAmyECotG.The recombinant vector was named pInCotGHP and then identified by digestion with Nhe1 and Xho1.After sequence,the recombinant vector pInCotGHP was transformed into Bacillus subtilis by electric method.western blot result walidated that the target protein was expressed correctly by the recombinant.In order to investgate the immune effect of pInCotGHP recombinant Bacillus subtilis on animal.We first isolated chicken bone marrow dendritic cells and evaluated the stimulation effect of our recombinated Bacillus subtilis(pInCotGHP).Flow cytometry results showed that the recombinant Bacillus subtilis can significantly induce chicken DCs to become mature.Then the mice and avians were used and oral vaccinated with our recombinant Bacillus subtilis for two times.In the mice immunization experiment,the changes of T cells in the spleen and lymph nodes were detected by flow cytometry,as well as the immune antibody levels in mouse serum were detect by hemagglutination inhibition.Results showed that the recombinant Bacillus subtilis can significantly induce the proliferation of CD3+CD4+ and CD3+CD8+ T cells.To some extent,the recombinant Bacillus subtilis which can express the avian influenza hem agglutinin can stimulate the body to produce effective avian influenza-specific antibodies.In chicken immunization experiments,we also studied the changes in systemic antibody levels in chickens,the efficacy of the vaccine against avian influenza was also compared with that of the avian influenza vaccine.Results showed that the avian influenza specific antibody produced by the chickens was lower than the subcutaneous injection of avian influenza vaccine group.But our chickens oral immuned with recombinate Bacillus subtilis could produce avian influenza specific specific antibody when compared with oral PBS group and wild type Bacillus subtilis group.What a pity,,the avian influenza specific antibody in recombinate Bacillus subtilis groups was lower than the critical value of the effective hemagglutination inhibition titer.All the above results suggest that the pInCotGHP recombinant Bacillus subtilis can effectively enhance the mucosal immune response of the organism.It can also stimulate the body to produce lower levels of avian influenza specific antibodies.Therefore,it provides a new train of thought for prevents poultry bird flu and immunity by oral immunization recombinant Bacillus subtilis.