Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody

Abstract

ABSTRACT. We examined the immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. Genomic DNA from the M5 strain of goat Brucella was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T-1. The expression and immunological characteristics of the fusion protein GST-omp31 were subjected to preliminary western blot detection with goat Brucella rabbit immune serum. The Brucella immunized BALB/c mouse serum was detected using purified protein. The high-potency mouse splenocytes and myeloma Sp2/0 cells were fused. Positive clones were screened by enzyme-linked immunosorbent assay to establish a hybridoma cell line. Mice were inoculated intraperitoneally with hybridoma cells to prepare ascites. The mAb was purified using the n-caprylic acidammonium sulfate method. The characteristics of mAb were examined using western blotting and enzyme-linked immunosorbent assay. A 680-base pair band was observed after polymerase chain reaction. Enzyme digestion identification and sequencing showed that the pGEX-4T-1-omp31 11965-11974 (2015) prokaryotic expression vector was successfully established; a target band of approximately 57 kDa with an apparent molecular weight consistent with the size of the target fusion protein. At 25°C, the expression of soluble expression increased significantly; the fusion protein GST-omp31 was detected by western blotting. Anti-omp31 protein mAb was obtained from 2 strains of Brucella. The antibody showed strong specificity and sensitivity and did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus pyocyaneus. The pGEX-4T-1-omp31 prokaryotic expression vector was successfully established and showed good immunogenicity. The antibody also showed strong specificity and good sensitivity.

Immunization with a combination of recombinant Brucella abortus proteins induces T helper immune response and confers protection against wild‐type challenge in BALB/c mice

“…In addition, ribosomal protein L7/L12 functionally constitutes the 50S ribosome and plays an important role in controlling protein translational accuracy (Kirsebom and Isaksson, 1985). Based on previous reports, L7/L12, OMP22, OMP25 and OMP31 could stimulate strong immunity against Brucella infection (Goel and Bhatnagar, 2012; Zheng et al ., 2015; Du et al ., 2016; Minhas et al ., 2021). Therefore, in this study, a combination of four recombinant proteins L7/L12, OMP22, OMP25 and OMP31 can activate strong immune responses against Brucella infection in RAW 264.7 cells and BALB/c mice.…”

A Multi-Epitope Fusion Protein-Based p-ELISA Method for Diagnosing Bovine and Goat Brucellosis

“…A subunit vaccine comprising BP26 triggers a mixed Th1/Th2 immune response in a mice model ( 7 ), and it has been also used in diagnosis of brucellosis ( 22 ). Animal experiments indicated that Omp31 can not only elicit a strong humoral immune response in mice, but also protects mice against Brucella infection ( 23 , 24 ). Omp2b is another important candidate for brucellosis diagnostics and vaccine research ( 25 , 26 ).…”

Detection of Brucella abortus by immunofluorescence assay using anti outer membrane protein of 19 kDa antibody

“…The OMPs detected by these antibodies were the proteins with a molecular weight of 25-27 kDa and 36-38 kDa (porin) (major proteins), and the proteins with a molecular weight of 10, 16.5, 19, 31-34, and 89 kDa (minor proteins). 26 The production of an mAb against OMPs with a molecular mass of 25-27 kDa (Ba-4, Ba-5), 36-38 kDa (Ba-6) of B. abortus S99 and rOMP31 of goat Brucella was performed by Farshad et al and Zheng et al 36,37 It was proven by Ghasemi et al that the immunized serum was achieved from a rabbit that was inoculated with attenuated B. melitensis. This serum contained antibodies against recombinant OMP31 (rOMP31).…”