In each animal, a cross-shaped pattern was shaved into the fur on the right side of the neck. significant boost of anti-rOMP40 antibodies in the IgG1 (and after the second immunization (rOMP40 induced a humoral response in cattle with broad cross-reactivity with related antigens of additional varieties of and family members and the delayed-type hypersensitivity reaction. The acquired results encourage further study to evaluate the protective effect of the produced protein like a subunit vaccine in cattle. Supplementary Info The online version contains supplementary material available at 10.1186/s12917-022-03515-x. Keywords: Recombinant OMP 40?kDa protein, Antibody cross-reactivity, family, including and strains [1], and to the family, including and strains [2]OM is composed of a wide range of proteins, probably the most abundant of which is designed as the major outer membrane protein (MOMP) and called OMP 40?kDa (OMP40) [5]. It has been shown the molecular mass of the MOMP assorted from 43 to 33?kDa; however, most of the strains have a protein about 40?kDa. The characteristics of the MOMP could be one of the markers of virulence. The molecular mass of the MOMP and its reactivity with selected LRP1 anti-MOMP monoclonal antibodies (mAbs) could be used for initial grouping of strains [6]. It was suggested that several epitopes of the MOMP are surface-exposed, and some of these epitopes are conserved as well as others variable among strains [3]. In the secondary structure model of MOMP, putative 16 transmembrane -strands and eight QNZ (EVP4593) loops of variable sequence and size facing the exterior side of the OM have been recognized. The comparison of the MOMP derived from six unrelated strains of to that of strain 8025 exposed identities in the nucleotide sequences in 65.4C99.9% QNZ (EVP4593) and in amino acid sequences in 62.4C99.7%. The lowest sequence similarity was demonstrated for the MOMP of strain 129Pt isolated from your prepuce of healthy bovine service providers [5]. It was demonstrated that sera from cattle, swine, dogs, horses and poultry vaccinated with whole cells exposed a strong immune response against selected antigens, among others those against MOMP [7]. MOMP appeared immunogenic in rabbits and calves. The cross-reactivity of rabbit antiserum with whole-cell antigen of different/selective strains exposed the reaction with proteins having a molecular mass of ~?40?kDa [6]. Related results were acquired in immunoblot analysis of reactivity of two anti-MOMP mAbs (no. 59C8-2; 34C2-1) [8]. OMP40 was identified as a porin with homology between its N-terminal amino acid sequence and the sequences of porins of additional gram-negative bacteria [3, 5]. The porins showing high similarity to OMP40 are OMP39, OmpH, P2 [5] and OmpC [3]. The aim of this study was to produce recombinant OMP40, optimize the harvest conditions and evaluate its immunogenic properties in calves. The cross-reactivity of anti-rOMP40 antibodies was checked by evaluation of antigen acknowledgement in selected gram-negative bacteria, while their influence on bacterial phagocytosis and growth inhibition was also identified. Results Manifestation and purification of recombinant protein The assessment of amino acid sequences of the acquired create with sequences available in the BLAST database exposed a 99% sequence conformity for rOMP40 originating from (the test sequence was compared to sequences of accession figures “type”:”entrez-protein”,”attrs”:”text”:”BBC21771.1″,”term_id”:”1317818457″BBC21771.1). Analysis of the amino acid sequence exposed a difference in the amino acid integrated at positions 2, 304 and 328 compared to the QNZ (EVP4593) research sequence (G was added; E changed to R; E changed to G, respectively). rOMP40 was produced using different tradition conditions and manifestation cells. The majority of host strains, such as BL21, C43 and R. gami, did not express or indicated a very low amount of rOMP40. The highest overexpression of protein was shown by C41 strain in all tested culture conditions (Fig.?1; lines: 3; 5; 22; 31; 40). Open in a separate window Fig. 1 rOMP40 manifestation using numerous manifestation strains and tradition conditions. Story: SDS-PAGE pattern of selected manifestation strains (3??107?cfu/lane) producing rOMP40 under different cultured conditions. a. – e. subsequent stained gels; A. Cells after induction of protein manifestation; B. Cells before induction of protein manifestation. C41 [C41 (DE3)], C43 [C43 (DE3)], BL21 [BL21 (DE3)], R.g. [Rosetta-gami (DE3) pLysS] C manifestation cells; gel a.- the rOMP40 production was induced using 0.5 or 1?mM.