Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (6): 1204-1214.doi: 10.3864/j.issn.0578-1752.2024.06.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Screening of Mycobacterium Avium Subsp. Paratuberculosis Immunogenic Proteins and Its Evaluation of Immunological Effect

CHEN FanRuo(), ZHANG JiaJun, LU Ping, CUI Ning, CUI YingYing, CUI ZiYin, DANG GuangHui(), LIU SiGuo()   

  1. State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute/Chinese Academy of Agricultural Sciences, Harbin 150069
  • Received:2023-06-05 Accepted:2023-12-31 Online:2024-03-25 Published:2024-03-25
  • Contact: DANG GuangHui, LIU SiGuo

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Abstract:

【Background】 Paratuberculosis (PTB) is a chronic, wasting infectious disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in ruminants. PTB causes huge economic losses to the livestock industry and poses a serious threat to public health safety. Since the current clinical methods for the detection and control of PTB are inadequate, and the PTB vaccine used is ineffective and interferes with the diagnosis of bovine tuberculosis, there is a need for developing a vaccine with strong immunogenicity, good safety, and excellent protection for the prevention and control of PTB. 【Objective】 The immunogenic protein of MAP was screened and its immunoprotective effect was evaluated, so as to provide the data support for the prevention and control of PTB. 【Method】 Five recombinant plasmids were constructed based on six genes of MAP: p22, map1272c, map3531c, map3783, map3701c, and map3527. The five recombinant proteins were combined with MONTANIDE ISA 61 VG adjuvant to immunize mouse by subcutaneous injection, and the best immunogen was screened by IFN-γ ELISPOT assay. The best immunogen was then mixed with the reported 66NC fusion protein. Mouse were immunized by subcutaneous multi-point injection. At 3 weeks after the second immunization, mice were immunized with 1×108 CFU of the MAP K-10 strain intraperitoneally. The immunogenicity and immunoprotective effect of the candidate subunit vaccine were comprehensively evaluated by IFN-γ ELISPOT assay, monitoring antibody titers and serum cytokines, as well as detecting weight changes, liver pathological and histopathological observations and charge count differences of infected mouse. 【Result】 Five recombinant proteins, such as 58F, 62F, 69F, 46F, and 52F, were expressed based on the genes p22, map1272c, map3531c, map3783 and map3701c. 58F produced the highest level of IFN-γ after immunization and was the most promising candidate immunogen. The fusion protein combination 66NC+58F induced persistent high titers of IgG, IgM, IgG1 and IgG2a, and also induced specific release of IFN-γ, TNF-α, and IL-17A. In the evaluation of protective effects, the fusion protein combination 66NC+58F resisted the weight loss caused by MAP infection, significantly reduced pathological damage in the liver, and decreased MAP colonization in the liver. 【Conclusion】 The fusion protein combination 66NC+58F induced Th1 and Th17-type immune responses in mouse, provided immune protection against MAP infection and was an important candidate subunit vaccine for PTB.

Key words: Mycobacterium avium subsp. Paratuberculosis, subunit vaccine, 58F protein, immune evaluation

Table 1

Specific primers and probes"

引物
Primer		 序列
Sequence (5′-3′)
p22-F
p22-R		 TGGACACCGCCGAATTCTGCTCGTCGGGCTCCAAGCAG
GCGGCGGTGCAAGCTTGCGAGCTCACCGGGGGCTT
map1272c-F
map1272c-R		 GTGGACACCGCCGAATTCGACCCGGGCGTCAGGGCGAT
GGCGGCGGTGCAAGCTTGCCGGGTGAGTCCGGCGCC
map3531c-F
map3531c-R		 CGTGGACACCGCCGAATTCTTCTCCAAGCCGGGGCTTCCG
GGCGGCGGTGCAAGCTTGGGTGGCGGGCTGGGCCGG
map3783-F
map3783-R		 CGTGGACACCGCCGAATTCAGCATGTTGGACGCTCACATT
TGGCGGCGGTGCAAGCTTGGAACGCGGTGTAGCCGGAC
map3701c-F
map3701c-R		 CGTGGACACCGCCGAATTCAGCAACCTCGCATTGTGGACA
GCGGCGGTGCAAGCTTGCTTGGTGATCGCGATGCGCTT
F Primer
R Primer		 GATCCTCACGTCCTACACCTCCTG
CGCGCCAACTCCATTCC
Probe-QSY: TAMRA-CGATTCTCGCCGGTGCCAGC-QSY

Fig. 1

Pattern of recombinant plasmid (a) and affinity chromatographic purification of 58F, 62F, 69F, 46F, and 52F proteins detected by SDS-PAGE (b) M: Protein marker; 1: 58F; 2: 62F; 3: 69F; 4: 46F; 5: 52F; 6: 66NC"

Fig. 2

The expression of IFN-γ in spleen lymphocytes stimulated by antigen was detected by ELISPOT a: The formation of IFN-γ was observed by ELISPOT; b: Statistical map of IFN-γ spots"

Fig. 3

Immunogenicity analysis of fusion protein 58F and 66NC+58F at 3 weeks of secondary immunization a: The formation of IFN-γ was observed by ELISPOT; b: Statistical map of IFN-γ spots; c: Serum cytokine at 3 weeks of second immunization; d: IgG1 at 3 weeks of second immunization; e: IgG2a at 3 weeks of second immunization"

Fig. 4

The monitoring results of serum antibody a: IgG antibody monitoring results; b: IgM antibody monitoring results"

Fig. 5

Serum cytokine assay results a: Serum cytokine levels at 2 weeks of infection; b: Serum cytokine levels at 4 weeks of infection"

Fig. 6

Body weight growth rate of mouse at two weeks of infection"

Fig. 7

Organ pathology and histopathological analysis of mouse a: Liver pathology injury results; b: Liver pathology scoring results; c: Liver histopathological damage; d: Liver histopathological scoring results; e: Colonizaiton of MAP in mouse liver (Bar: 200 μm)"

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