Construction And Experimental Immunization Of Recombinant Oral Bacillus Subtilis Expressing Rabies Virus G Protein

Rabies is a highly neurotropic zoonotic infectious disease caused by rabies virus invading the central nervous system of the body.The disease is prevalent all over the world,and its host animals are very extensive,and all warm-blooded animals can be infected.After the onset of human and animals,once clinical symptoms appear,the mortality rate is almost 100%.According to statistics,about 60,000 people die of rabies every year,most of which occur in developing countries,causing great mental pressure and serious economic losses to the affected countries and people.There is no effective treatment for the disease.Vaccination is an effective way to prevent the occurrence and spread of rabies.At present,rabies in humans and companion animals has been effectively controlled by intramuscular vaccination,but the prevalence of rabies in stray dogs,free-range livestock and wild animals has greatly increased the risk of contracting the disease.at the same time,it brings great challenges to our country to realize the"2030 global plan to eliminate dog-borne human rabies".For these animals,increasing vaccination is essential for rabies control,and the most practical method of vaccination for these animals is oral immunization.Therefore,the development of a safe,economical,convenient and efficient oral rabies vaccine is very important to prevent and control the occurrence of rabies.In view of the above problems,this study has carried out the following research:1.Construction and Identification of Recombinant Bacillus subtilis expressing RABV G proteinUsing Bacillus subtilis shuttle plasmid PDG1661 as vector,the capsid protein Cot G gene on the spore surface was selected,and the RABV G protein genes(CVS-11-G and ERA-G)of two different strains were fused with Cot G gene to obtain two kinds of plasmids,named PDG1661-Cot G-C-G and PDG1661-Cot G-E-G,respectively.By optimizing the conditions of electric shock transformation and determining the stable conditions of electric shock transformation,two kinds of recombinant Bacillus subtilis with RABV G protein were obtained,named B.subtilis Cot G-C-G and B.subtilis Cot G-E-G respectively.Verified by genomic PCR extracted from bacteria,two RABVG protein genes were successfully integrated into B.subtilis168 genome.The successful expression of the recombinant protein was identified by Western blot and indirect immunofluorescence.The correct recombinant Bacillus subtilis was continuously passed on for 15 generations,and the genomic PCR was extracted.The results showed that the recombinant foreign gene still existed,indicating that the foreign gene could exist stably in Bacillus subtilis.To sum up,two kinds of recombinant Bacillus subtilis:B.subtilis Cot G-C-G and B.subtilis Cot G-E-G were successfully obtained,and the two kinds of recombinant Bacillus subtilis have good genetic stability,which laid a good foundation for subsequent research in animal experiments.2.Experimental immune study of Recombinant Bacillus subtilis expressing RABV G proteinMice were immunized with recombinant B.subtilis Cot G-C-G and B.subtilis Cot G-E-G orally,and their immune effects were evaluated.The results showed that the body weight of mice immunized with recombinant Bacillus subtilis did not change significantly during the whole immune period,the mental state of mice was good,no adverse reactions such as loss of appetite,diarrhea and dishevelled back hair occurred,and it could effectively stimulate the level of RABV specific Ig G antibody and neutralization antibody.The titer of RABV neutralizing antibody in serum of immunized mice was higher than that of 0.5IU/m L.The level of SIg A antibody in bronchoalveolar lavage fluid,intestinal lavage fluid and feces increased significantly,the number of spleen lymphocytes secreting IFN-γand IL-4 increased significantly,it could promote the proliferation of spleen lymphocytes and the secretion of many kinds of cytokines,and the number of CD3~+T lymphocytes in intestine increased significantly.The above results show that the recombinant B.subtilis Cot G-C-G and B.subtilis Cot G-E-G have good safety in mice,can effectively induce specific humoral and cellular immune response,and can resist the lethal infection of RABV to some extent.3.Effects of immunization with recombinant Bacillus subtilis on intestinal flora diversity in miceIn this study,macrogenomic sequencing was used to analyze the fecal samples of mice immunized with B.subtilis Cot G-C-G and B.subtilis Cot G-E-G.Through the analysis of the structure and potential function of intestinal flora,the results showed that the immune response induced by oral immunization of B.subtilis Cot G-C-G and B.subtilis Cot G-E-G in mice could regulate intestinal flora,increase the abundance of thick-walled bacteria,Lactobacillus,actinomycetes and actinomycetes,and promote the proliferation of lactic acid bacteria and bifidobacteria.These probiotics play an important role in the regulation of cellular and humoral immunity,indicating that B.subtilis Cot G-C-G and B.subtilis Cot G-E-G enhance the immune response of mice by regulating the structure of intestinal flora.4.Evaluation of immunogenicity of recombinant Bacillus subtilis expressing RABV G protein in dogsBeagle dogs were immunized orally with recombinant Bacillus subtilis B.subtilis Cot G-C-G and B.subtilis Cot G-E-G using boiled chicken head as bait.The specific Ig G antibody,neutralization antibody,fecal SIg A antibody and the secretion level of IFN-γand IL-4 in peripheral blood lymphocytes were detected.The results showed that both B.subtilis Cot G-C-G and B.subtilis Cot G-E-G could significantly increase the levels of specific Ig G antibody and RABV neutralizing antibody in serum,the secretion of specific SIg A antibody in feces,and the secretion of IFN-γand IL-4 in peripheral blood lymphocytes of dogs.The antibody seroconversion rate was 100%in the first 3 months after immunization,and the B.subtilis Cot G-E-G seroconversion rate was 80%in the 4th month after immunization.B.subtilis Cot G-C-G seroconversion rate was 60%.And with the extension of immunization time,the level of neutralizing antibody in the immunization group showed a significant downward trend.These results suggest that recombinant B.subtilis Cot G-C-G and B.subtilis Cot G-E-G can effectively stimulate specific humoral and cellular immune responses in dogs.In conclusion,both recombinant B.subtilis Cot G-C-G and B.subtilis Cot G-E-G could effectively stimulate humoral and cellular immune responses,indicating that B.subtilis Cot G-C-G and B.subtilis Cot G-E-G have the potential to be developed as economical and safe oral rabies vaccines,and provide new ideas and research basis for the development of oral rabies vaccines.