Construction Of Recombinant Bacillus Subtilis Expressing Immunogen Proteins Of Mycoplasma Hyopneumoniae And Application Of Nasal Immunization

Mycoplasmal pneumonia of s2ine(Mps)is a chronic respiratory disease caused by Mycoplasma pneumoniae(Mhp),all age of pigs can be infected.The main symptoms of the disease are cough,asthma,slow growth,reduced feed efficiency,followed by secondary infections,etc.It has caused huge economic losses to the pig industry all over the world.Currently,vaccines are the main measures to prevent and control the disease,but the commercial inactivated or attenuated vaccines have provided partially immunity protection,therefore a new Mhp vaccine needs to be developed urgently.In recent years,with the development of reverse genetics,some immunogenic proteins of Mhp have been discovered,such as P97,P46,and P36.Genetically engineered vaccines have become a research hotspot of new Mhp vaccines.Bacillus subtili.s(B.subtilis)has been used as a mucosal immune delivery vector because of its safety,strong resistance and the function of mucosal immune adjuvant.In order to develop an effective,safe and convenient nasal spray immune live vector vaccine,B.subtilis was used as mucosal immune delivery vector to construct recombinant B.S-P97R1,B.S-P46 expressing Mhp immunogenic protein P97R1,P46.Then,the immunogenicity of recombinant B.S-P97R1 and B.S-P46 were evaluated in mice by intranasal immunization.Moreover,the immune response of piglets after intranasal immunization was evaluated,and determine its protective efficacy in vaccinated pigs following a Mhp challenge infection.The content of this study was divided into three parts:1 Construction of recombinant B.subtilis expressing P97R1 or P46 protein of Mhp In the present experiment,the gene of Mhp 168 strain was used as a template to design specific primers to amplify the P97R1 and P46 genes,and then the P46 gene TGA was mutated to TGG by overlapping PCR to obtain the correctly P46 gene.The gene of EGFP was amplified using the pLJM1-EGFP plasmid as a template.Then,the P97R1,P46 and EGFP target genes were respectively inserted into the linearized secretory expression plasmid pP43NMK of B.subtilis by homologous recombination technique,and obtained the recombinant B.subtilis expression vectors pP43NMK-P97R1,pP43NMK-P46 and pP43NMK-EGFP.Then,the pP43NMK-P97R1,pP43NMK-P46,pP43NMK-EGFP plasmids were electroporated into B.subtilis WB800 strain to obtain recombinant B.S-P97R1,B.S-P46,B.S-EGFP.Finally,the results showed that Mhp P97R1,P46 immunogenic proteins and EGFP could be expressed in B.S-P97R1,B.S-P46 and B.S-EGFP by Western-blot and confocal microscopy analysis.It will provide experimental basis for the further study of B.subtilis expressing P97R1,P46 proteins of Mhp as mucosal vaccines.2 Evaluation of the immunogenicity of the B.S-P97R1,B.S-P46 by intranasal immunization on miceIn the present study,6-week-old SPF female BALB/c mice were intranasal immunized by B.S-P97R1,B.S-P46.The level of specific SIgA antibody in alveolar lavage fluid was first detected by ELISA,and the effect of recombinant B.subtilis on the local mucosal immunity level in the respiratory tract of mice was evaluated.Then,serum-specific IgG antibody and IgG1 and IG2a antibody were measured by ELISA,and the effect of systemic immunity was evaluated.Then,the effect of the activation of CD69+lymphocytes in spleen in intranasal immunized mice by flow cytometry.Moreover,the proliferation of spleen lymphocytes and the secretion of cytokines IFN-y and IL-4 were detected by CCK8 test and qPCR,to evaluate the cellular immunity level on mice.The results showed that mice intranasal immunized with BS-P97R1,BS-P46 induced the secretion of P97R1,P46-specific SIgA antibodies in the alveolar lavage fluid,indicating that BS-P97R1,BS-P46 induced local mucosal immunity of respiratory tract.In addition,we found that P97R1,P46-specific IgG antibodies and IgGl,IgG2a antibodies in serum were significantly increased after intranasal immunization.It suggested that recombinant B.subtilis induced humoral immune response which a Thl and Th2 mixed type immune response.Flow-through detection revealed a significant increase in CD69+lymphocytes in mice after immunization,and,in vitro,CCK8 and cytokine detection showed that it specifically induced the proliferation of spleen lymphocytes and the secretion of IFN-y and IL-4.In general,the recombinant B.subtilis expressing P97R1,P46 proteins of Mhp stimulate the mice to produce specific local mucosal immunity and systemic immunity,which a mixed Th1/Th2.3 Evaluation of the immunogenicity of the B.S-P97R1,B.S-P46 by intranasal immunization on pigletsPigs are the only natural host of Mhp.To further investigate whether recombinant BS-P97R1 and BS-P46 could induce a better immune response in piglets.In the present study,21-day-old SPF piglets were intranasal immunized with B.S-P97R1 and B.S-P46,and specific SIgA antibody in nasal swabs and IgG antibody in serum were detected by ELISA.Then,all piglets were challenged by Mhp at 42 days after immunization,and lung lesions were observed at 28 days after challenged.Compared with the control group(not immunized but challenged),the degree of lung lesions was significantly reduced,and TNF-α.IL-1β and IL-6 were significantly lower than the control group,the loading of Mhp was also significantly lower than that of the control group.These results indicated that recombinant B.subtilis intranasal immunized piglets induced piglets to produce certain immune protective effects against Mhp infection,which provides a theoretical basis and new ideas for the development of Mhp vaccines.