Construction And Immunogenicity Research Of Recombinant Adenovirus Of Mycobacterium Avium Subsp.Paratuberculosis 35kDa Protein Gene

A chronic intestinal inflammatory disease mainly in ruminants was caused by Mycobacterium avium subsp.Paratuberculosis(MAP).Similar to Mycobacterium bovis,primary infection leads to persistent infection under immune control.In some cases,immune control may be compromised,leading to granulomatous ileitis,malabsorption,wasting and death,causing considerable economic loss to the breeding industry.In addition,pathogens have also been suggested as possible causes of Crohn’s disease in humans.So far,no para-tuberculosis vaccine has been developed that can induce protective immunity,and it is urgent to research and develop such a vaccine.In this study,the recombinant adenovirus containing 35kDa protein gene of MAP was constructed with Ad5 as vector,and the immunogenicity of 35kDa protein expressed by recombinant adenovirus was studied by immunizing mice,which laid the foundation for the development of a new paratuberculosis vaccine.Using the MAP genomic DNA as a template,the 35kDa protein gene was amplified by PCR,purified and recovered,and then ligated with the cloning vector pMD19-T to construct the pMD19T-35 recombinant clone plasmid.The recombinant clone plasmid pMD19T-35 was digested with Hind Ⅲ and EcoR Ⅰ,and the target gene was purified and recovered.The recombinant adenovirus expression plasmid pacAd5 CMV35 was constructed by ligating with adenovirus expression vector pacAd5 CMV K-NpA.The recombinant adenovirus expression plasmid pacAd5 CMV-35 and adenovirus cytoskeleton plasmid pacAd5 9.2-100 were linearized by Pac Ⅰ restriction endonuclease,and cotransfected into HEK293 cells by liposome transfection reagent LipoFiterTM.The recombinant adenovirus rAd5 CMV-35 was obtained.The expression of the target gene was verified by viral genomic PCR,the transcriptional level of 35kDa protein gene was verified by reverse transcription PCR(RT-PCR),and the translation level of 3 5kDa protein gene was detected by indirect immunofluorescence.The recombinant adenovirus rAd5 CMV-35 was amplified and purified,and the half tissue infection amount of purified recombinant adenovirus rAd5 CMV-35 and empty virus rAd5 CMV K-NpA was determined by TCID50 method.The recombinant adenovirus rAd5 CMV-35,empty adenovirus rAd5 CMV K-NpA,PBS buffer,inactivated MAP and pET32a-35 prokaryotic expression protein were used to immunize 5-week-old BALB/c male mice by intramuscular injection.The mice were immunized three times every two weeks,and the mice were killed 2 weeks later.Detection of CD4~+and CD8~+T lymphocyte subsets and T lymphocytes secreting IL-2,IFN-y and TNF-α cytokines in mouse spleen by flow cytometry.The level of IgG antibody in serum of mice was detected by enzymelinked immunosorbent assay to evaluate the effect of recombinant adenovirus vaccine on cellular and humoral immunity in mice.Six weeks after the last immunization,the challenge experiment was carried out,and 1 ×108 CFU live MAP was injected intraperitoneally.Eight weeks after challenge,the colon,liver and spleen of mice were taken for bacterial detection and pathological detection to analyze the resistance effect of recombinant adenovirus rAd5 CMV-35 against MAP infection.The results showed that the antibody level of the recombinant adenovirus group was higher than that of the control group,indicating that the recombinant adenovirus vaccine could stimulate the body to produce a high level of specific antibody against 3 5kDa protein.The number of CD4~+and CD8~+T cells in the recombinant adenovirus vaccine group increased significantly,and the levels of IL-2,IFN-γ and TNF-α cytokines in the recombinant adenovirus vaccine group were higher than those in the control group,indicating that the recombinant adenovirus rAd5 CMV-35 can mediate a strong cellular immune response.Eight weeks after challenge,the pathological results of colon,liver and spleen in mice immunized with recombinant adenovirus vaccine showed that recombinant adenovirus vaccine could reduce the degree of pathological injury and slow down the course of disease.In summary,in this study,the adenovirus vector vaccine was constructed by encoding and expressing the 3 5kDa protein gene MAP2121c of MAP.The constructed adenovirus vector vaccine can improve the humoral and cellular immunity of mice to a certain extent,and has a certain resistance to MAP infection,which lays a foundation for the development of a new paratuberculosis vaccine and provides new ideas for the prevention and control of paratuberculosis.