Construction And Immunogenicity Research Of Recombinant Adenovirus Encoding Mycobacterium Bovis Mpb83-ag85b Fusion Gene

Bovine tuberculosis is a chronic expendable infectious disease caused by Mycobacterium bovis.At present,the prevention and treatment of tuberculosis mainly focus on prevention,and BCG vaccine is the only vaccine used to prevent tuberculosis,but its immune effect is not stable.Therefore,the development of new and efficient TB vaccine is still the primary task of bovine TB control.Viral vectors are relatively ideal candidates for construction of recombinant vaccines,among which replication-deficient adenovirus vectors are widely used in tuberculosis vaccine research.In recent years,the immunogenicity of a single protein gene vaccine is often not ideal.Therefore,the fusion of multiple protective antigenic genes into live vector virus vaccine has become a hot research topic.In this study,recombinant adenovirus expressing mpb83 and ag85 b fusion genes of Mycobacterium bovis was constructed using replication-deficient human type 5 adenovirus as expression vector.In this study,plasmid pMD-83-85 bL was used as template to amplify the target gene mpb83-ag85 b and construct the adenovirus shuttle plasmid pacAd5CMV-83-85 b.It was linearized with adenovirus skeleton plasmid pacAd5 9.2-100 by restriction endonuclease of Pac?.By transfection reagent LipoFiter? Co-transfection into HEK293 cells and packaging of recombinant adenovirus.The recombinant adenovirus was named rAd5-CMV-83-85 b and the target gene was identified by PCR.The adenovirus purification kit was used to purify the virus and the titer of the virus was determined.Indirect immunofluorescence assay(IFA),reverse transcription PCR assay(RT-PCR)and Western blot were used to detect the expression of the target gene mpb83-ag85 b at the transcriptional and protein levels.Immunize mice and strengthen the immune system once.The T lymphocyte subsets and cytokines such as IL-2 IFN-? in the spleen of mice immunized with each group were detected by flow cytometry.The specific immunoglobulin IgG in serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay(ELISA).The results of this study are as follows: 1.Recombinant adenovirus shuttle vector pacAd5CMV-83-85 b was successfully constructed,and the recombinant adenovirus rAd5-CMV-83-85 b was successfully packaged in HEK293 cells.2.MPB83-Ag85 B fusion protein was detected by IFA and RT-PCR to express in vitro and have biological activity.3.The serum level of IgG in rAd5-CMV-83-85 b group was lower than that in BCG group(P > 0.05),but higher than that in non-loaded adenovirus group and 0.9% BCG group(P < 0.05).Flow cytometry showed that the number of CD4 T lymphocytes in rAd5-CMV-83-85 b group was higher than that in rAd-CMV-NPA group(P < 0.05),but lower than that in BCG group(P > 0.05).The results of CD8 T lymphocyte analysis showed that rAd5-CMV-83-85 b was significantly higher than that in 0.9 NaCl group and rAd-CMV-NpA group(P < 0.05).but no significant difference between BCG group and BCG group(P > 0.05).Flow cytometry analysis showed that the number of lymphocytes secreting IFN-? in rAd5-CMV-83-85 b group was higher than that in non-loaded adenovirus group and 0.9Nacl group(P < 0.05),Compared with group BCG,the difference was not significant(P > 0.05).The number of lymphocytes secreting IL-2 was significantly higher than that of non-loaded adenovirus group and 0.9% Nacl group(P < 0.05),Slightly lower than the BCG group,the difference was not significant(P>0.05).The results of serological and flow cytometry showed that recombinant adenovirus rAd5-CMV-83-85 b could induce humoral and cellular immune responses in mice,and had the ability of immunogenicity.Through the study of recombinant adenovirus vaccine rAd5-CMV-83-85 b,the foundation for further study of bovine tuberculosis vaccine was established.