Construction And Immunogenicity Evaluation Of Recombinant Bacillus Subtilis Expressing HA1 Protein Of H9N2 Avian Influenza Virus

H9N2 subtypenvirus（AIV）is one of the main subtypes in low pathogenic AIV.Infected animals often have low incidence,but they are widely spread globally due to difficulty control,which seriously affects the development of the breeding industry.At present,H9N2AIV is widely spread in China,so it is urgent to control its popularity.At this stage,in addition to biological safety,vaccination is still one of the main strategies for China to control H9N2 avian influenza.In recent years,with the advocacy of the"One Health"concept,the development of green,safe,and non-toxic vaccines has become the focus of people’s attention.The feed for probiotics has the advantages of safe and non-disease,no toxins,etc.,and mucous membrane immunity can effectively stimulate the immune response of the macaropathy of the mucosa,but ordinary oral mucosa immunohic vaccines may be affected by the gastrointestinal environment after oral administration.Reduce immune effects,and Bacillus Subtilis（B.subtilis）can adapt to extreme environments.Its biological characteristics and their ability to interact with cells with antigens and stimulate cytokine release make B.subtilis a heterogeneous antigen or any powerful carrier delivered to the gastrointestinal tract by any biological active molecule.Therefore,this study uses B.subtilis to build a reorganization bacteria,expressing HA1 protein based on B.subtilis as a delivery carrier,and evaluating its immune protection effect,providing effective reference for the R&D of the new H9N2 AIV vaccine.This study analyzed the current 66 epidemic strains released by Genbank H9N2 AIV HA1 protein,and determined that the current major popular strains in China are located in the h9.4.2.5 branch.Referring to the amino acid sequence of the HA1 protein of the H9N2 isolate MW548848.1 located in this popular branch,optimize the nucleic acid sequence encoding HA1 protein,to optimize the codon preferences to express it in the B.subtilis expression system.After that After the enzyme cutting and sequencing verification are correct,the electric shock is converted to B.subtilis WB800N to obtain PHT43-HA1/B.subtilis recombinant strain;the immunization analysis（Western-Blot）testing protein expression was verified that the HA1 protein positive serum could It reacts with the bacteria at 36 kda and cultivated the prime protein,indicating that the restructuring bacteria can successfully express HA1 protein and secrete and express it.In order to evaluate the reorganized bacteria PHT43-HA1/B.subtilis’s immune protection effect on animals,the obtained reorganization bacteria will be tested.This study used gavage to immunize mice,with a total of 2 immunizations,each lasting for three days.Recombinant bacteria were induced by 1 mmol/L IPTG and the bacterial concentration was adjusted.On days 0,7,14,21,28,and 35,3-5 mice from each group were randomly selected for blood collection,and small intestine lavage was also collected;Detect whether mice can produce antibodies using hemagglutination inhibition test;Indirect ELISA was used to detect the levels of HA1 protein specific Ig G antibodies in serum and protein specific s Ig A antibodies in small intestine fluid;Detection of cytokines IL-2 and IFN-γin mouse serum using a reagent kit;Pathological sections were used to observe the pathological characteristics of the lungs,spleen,and intestines in mice.On the 14th day after the second immunization,each mouse received nasal drops of 50μL H9N2 virus(10^6.0 ELD₅₀/50μl).On the 7th day after the virus attack,the lungs,spleen,and liver of each mouse were collected,and the viral load in each tissue was detected using RT-q PCR method.The results of this study show that PHT43-HA1/B.subtilis reorganized bacteria oral mice in the serum of HA1 protein specific Ig G level and the HA1 protein specific s Ig A level in small intestinal solution,which was significantly improved.The difference between the ratio is very significant（P<0.01）;the hemorrhage inhibitory is high（HI=4）,which can cause mice to specially perform an immune response;cytokine detection IFN-γ（P<0.05）and IL-2（P<0.01）Significant expression of expression;the virus load of the mouse’s lungs after attack is about 4 times compared with the control group（P<0.01）,the spleen（P<0.01）and the liver（P<0.05）virus load is also compared with the control group,there is a significant decrease;pathological slices show that the PBS group and the experimental group show obvious differences.The PBS group can see the thickening of the alveolar wall,the alveolar wall is invasive,and the occasional lymphocytes are infiltrated by occasional lymphocytes around the bronchium.The capillaries of alveolar walls are irregularly arranged in epithelial cells.It can be seen that a large area of bleeding stoves.There may be more red blood cells in the alveolar cavity and bronchies.There is no obvious lesion in the test group.The PBS group of the spleen tissue shows that the red marrow is congested in large areas,and a small amount of granulocytes can be seen in the red marrow.There is no obvious lesion in the test group.The intestinal tissue PBS group can see a small amount of intestinal velvet top-ended,and the test group has not seen obvious lesions.In summary,this study has successfully constructed PHT43-HA1/B.subtilis reorganization bacteria,which efficiently expresses the current major popular strains in my country,HA1 protein.A preliminary evaluation of immunogenicity found that the reorganized bacteria PHT43-HA1/B.subtilis had good immunogenicity and can effectively induce mice to produce body fluid immunity and cellular immunity.All in all,the candidate vaccine of oral Bacillus HA1 reorganized vaccine provided theoretical data and technical reference for creating prevention and preventing H9N2 AIV-type live carrier vaccines.