Recombinant proteins in horses refer to proteins that are genetically engineered and produced through recombinant DNA technology. This involves inserting the gene encoding the desired protein into a host cell, which then expresses and produces the protein. In equine research, recombinant proteins are utilized for various applications, including the development of vaccines, therapeutic agents, and diagnostic tools. These proteins can mimic natural proteins, allowing researchers to study their functions, interactions, and potential uses in treating or preventing equine diseases. This page compiles peer-reviewed research studies and scholarly articles that explore the production, application, and impact of recombinant proteins in equine health and medicine.
Yamaguchi K, Sawada T, Naraki T, Igata-Yi R, Shiraki H, Horii Y, Ishii T, Ikeda K, Asou N, Okabe H, Mochizuki M, Takahashi K, Yamada S, Kubo K....The prevalence of Borna disease virus (BDV)-specific antibodies among patients with psychiatric disorders and healthy individuals has varied in several reports using several different serological assay methods. A reliable and specific method for anti-BDV antibodies needs to be developed to clarify the pathological significance of BDV infections in humans. We developed a new electrochemiluminescence immunoassay (ECLIA) for the antibody to BDV that uses two recombinant proteins of BDV, p40 and p24 (full length). Using this ECLIA, we examined 3,476 serum samples from humans with various diseases ...
Saxena A, Ashani Y, Raveh L, Stevenson D, Patel T, Doctor BP.To understand the role of glycosylation in the circulation of cholinesterases, we compared the mean residence time of five tissue-derived and two recombinant cholinesterases (injected intravenously in mice) with their oligosaccharide profiles. Monosaccharide composition analysis revealed differences in the total carbohydrate, galactose, and sialic acid contents. The molar ratio of sialic acid to galactose residues on tetrameric human serum butyrylcholinesterase, recombinant human butyrylcholinesterase, and recombinant mouse acetylcholinesterase was found to be approximately 1.0. For Torpedo ca...
Kheyar A, Martin S, St-Laurent G, Timoney PJ, McCollum WH, Archambault D.To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner pr...
Shao H, Robek MD, Threadgill DS, Mankowski LS, Cameron CE, Fuller FJ, Payne SL.The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages. Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis. Specific activities...
Kato H, Ohashi T, Matsushiro H, Watari T, Goitsuka R, Tsujimoto H, Hasegawa A.Equine interleukin-1 receptor antagonist (IL-1ra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse. cDNA clones encoding the whole coding sequence of equine IL-1ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS). The equine IL-1ra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues. The predicted amino acid sequence of equine IL-1ra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and ra...
Chopineau M, Martinat N, Troispoux C, Marichatou H, Combarnous Y, Stewart F, Guillou F.Horse (Equus caballus) luteinizing hormone (eLH) and chorionic gonadotrophin (eCG), which have the same amino acid sequence, are unusual in that, although they express only LH activity in equids, they express dual LH and FSH activities in all other species tested. Donkey (Equus asinus) LH (dkLH) and CG (dkCG), which also share an identical peptide backbone, have been less well characterized and conflicting results concerning their FSH activity in heterologous species have appeared in the literature. In order to assess and compare the intrinsic LH and FSH activities of the horse and donkey LHs ...
Kong XG, Pang H, Sugiura T, Sentsui H, Onodera T, Matsumoto Y, Akashi H.Equine infectious anemia virus (EIAV) core proteins were obtained from a baculovirus expression system. Recombinant baculoviruses (rBVs) highly expressed the Gag precursor and p26 antigens in an rBV-infected Sf21 cell culture supernatant. Enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) were conducted using the expressed proteins to detect antibodies from experimentally infected horses. The expressed antigens showed low background levels, high specificity and sensitivity in ELISA and AGID. The results of the serological tests using the expressed antigens were ident...
Kasza A, Korpula-Mastalerz R, Rose-John S, Dubin A.The horse leucocyte elastase inhibitor (HLEI), present in neutrophils, monocytes and bone marrow cells, is apparently a cytoplasmic protein which is not released from cells even in response to stimulation with lipopolysaccharide, phorbol ester, tumour necrosis factor alpha, interleukin-1 or elastin degradation products. Although no expression of the inhibitor was detected in neutrophils, both monocytes and bone marrow cells were efficient in its synthesis. Using a new expression vector pREST5d, recombinant inhibitor was produced in a large quantity in a soluble form, with a yield of 88 mg per ...
Whalley JM, Love DN, Tewari D, Field HJ.A series of recombinant baculoviruses containing genes for glycoproteins C, D, H and L of equine herpesvirus 1 (EHV-1) have been constructed, and the EHV-1 products characterised by gel electrophoresis and immunoblotting. The EHV-1 glycoproteins expressed in insect cells were similar but not identical in apparent sizes to those expressed in EHV-1 infected mammalian cells. Each of the EHV-1 products was recognised by convalescent equine sera, indicating that they were all targets for an equine immune response. Mice immunised with baculovirus-expressed EHV-1 gD and gC acquired an enhanced abilit...
Chirnside ED, Francis PM, de Vries AA, Sinclair R, Mumford JA.A recombinant glutathione-S-transferase fusion protein expressing amino acids 55-98 of equine arteritis virus (EAV) GL (rGL 55-98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1...
Marquardt J, Heymer J, Heinz H, Adolf GR, Deegen E.Recombinant equine interferon-beta 1 (reqIFN-beta 1) induces an antiviral state in blood mononuclear cells (BMC) of horses. Maximal protection against replication of vesicular stomatitis virus is achieved 6 hours after treatment with IFN in vitro and in vivo. Duration of the protective effect depends on the dose of IFN in vitro and in vivo. Availability of reqIFN-beta 1 in cultures of BMC for up to 48 hours does not prolong the antiviral state. The protective effect on BMC after treatment with IFN has similar duration in vivo and in vitro. Monitoring of the effect of IFN in vivo is, thus, simp...
Lloyd E, Mauk AG.Expression of recombinant horse heart myoglobin in Escherichia coli has been found to result in the production of both native and variable amounts (approximately 16-17% total) of two sulphmyoglobin isomers. The recombinant sulphmyoglobin produced consists primarily of the A and B isomers as identified by 1H NMR spectroscopy with no evidence for production of the C isomer. Conversion of recombinant sulphmyoglobin to the native protein can be achieved by reconstitution with protohaem IX. The possible relationship of this observation to recombinant expression of other heme proteins is discussed.
Jaussaud P, Audran M, Gareau RL, Souillard A, Chavanet I.A plasma kinetic study of erythropoietin (EPO) was carried out in 4 horses after subcutaneous administration (30 IU/kg bwt) of recombinant human erythropoietin (rhEPO). At standardized intervals for 48 h before injection and for 60 h post-administration, the EPO plasma levels were determined with an immunoradiometric assay based on a sandwich technique. The peak plasma concentration (30-113 mIU/ml) was observed after a delay ranging from 6 to 9 h post-administration and the drug levels reached a physiological value around 60 h following rhEPO injection. Moreover, reference values for plasma EP...
Thomas LM, Huntington PJ, Mead LJ, Wingate DL, Rogerson BA, Lew AM.The use of the bacterial expression vector, pGex, to produce an abundant, soluble fusion protein of gp45 from equine infectious anaemia virus is described. Purification of the recombinant protein was achieved by one step affinity chromatography on immobilized glutathione using competitive elution so no harsh conditions were required. This provides a readily available antigen that is defined, plentiful and cheap. Yields of 3.5 mg of purified soluble protein/litre of bacterial culture were obtained. This antigen was found to be suitable for ELISA. Background reactivity to either the glutathione-...
McKinnon AO, Brown RW, Pashen RL, Greenwood PE, Vasey JR.THE name inhibin was first used around 60 years ago for a water-soluble. non-steroidal, gonadal factor that would regulate follicle-stimulating hormone (FSH) secretion through negative feedback McUullagh 1930. Inhihin is now defined as a glycoprotein hormone, consisting of two dissimilar, disulphide-linked, subunits termed at and 13 1 Burger and Igarashi 1988). Effective methods for blocking inhibin production could provide useful means by which FSH secretion, and therefore ovarian function and fertility, could be improved in the female. Increased ovulation rates have been demonstrated in shee...
LaPolt PS, Tilly JL, Aihara T, Nishimori K, Hsueh AJ.The actions of gonadotropins on ovarian differentiation are associated with dynamic changes in gonadotropin receptor content, presumably due to modulation of receptor gene expression. The present studies used a reverse transcription-polymerase chain reaction to obtain a rat FSH receptor cDNA fragment, followed by synthesis of a labeled cRNA probe to examine the regulation of FSH receptor mRNA levels during follicular maturation, ovulation, and luteinization. Northern blot analysis of ovarian RNA with the FSH receptor probe revealed two predominant hybridization signals of 7.0 and 2.5 kilobases...
Xu J, Cai J, Anderson B, Wagner B, Albrecht R, Peek SF, Suresh M, Darien BJ.The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Further...
Pessoa VC, Branco-Ferreira M, Jónsdóttir S, Marti E, Tilley P.Thirty controls (C) and 30 IBH-affected (T) Lusitano horses were evaluated. T horses were included based on anamnesis and physical examination, supported by questionnaires. All horses were submitted to skin tests, Intrademal (IDT) and Skin Prick Tests (SPT), on the neck with 14 specific allergens, 13 recombinant proteins (r-proteins) from Culicoides nubeculosus (Cul n) and Culicoides obsoletus (Cul o) salivary glands and Culicoides nubeculosus Whole Body Extract (Cul n WBE). Addicionally, a cluster of six T and six C horses were also tested with Cul n 3 and Cul n 4 produced in insect cells and...
Ichikawa Y, Iinuma Y, Okagawa T, Shimbo R, Enkhtuul B, Khurtsbaatar O, Kinoshita Y, Niwa H, Aoshima K, Kobayashi A, Batbaatar V, Ohashi K, Kimura T.Glanders is a World Organization for Animal Health (WOAH)-notifiable equine disease caused by the infection of Burkholderia mallei, and is endemic in Mongolia, South Asia, Africa, and South America. While the complement fixation test (CFT) has been widely used for serodiagnosis of glanders and is considered a standard serological test, it has several limitations. These limitations include poor specificity, labor intensive techniques, variability in antigen and protocol. Consequently, indirect enzyme-linked immunosorbent assays (iELISAs) based on recombinant proteins have been developed as alte...
McKinnon AO, Brown RW, Pashen RL, Greenwood PE, Vasey JR.THE name inhibin was first used around 60 years ago for a water-soluble. non-steroidal, gonadal factor that would regulate follicle-stimulating hormone (FSH) secretion through negative feedback McUullagh 1930. Inhihin is now defined as a glycoprotein hormone, consisting of two dissimilar, disulphide-linked, subunits termed at and 13 1 Burger and Igarashi 1988). Effective methods for blocking inhibin production could provide useful means by which FSH secretion, and therefore ovarian function and fertility, could be improved in the female. Increased ovulation rates have been demonstrated in shee...
Bailly-Chouriberry L, Pinel G, Garcia P, Popot MA, Le Bizec B, Bonnaire Y.Equine growth hormone (eGH) has been available since 1998 as an approved drug (EquiGen-5, Bresagen) containing recombinant eGH (reGH). It is suspected of being illegally administered to racehorses in order to improve physical performance and to speed-up wound healing. Thus it may be considered a doping agent which would require a sensitive and reliable method of identification and confirmation in order to regulate its use in racehorses. reGH differs from the native eGH by an additional methionine at the N-terminal (met-eGH) and has never been unambiguously detected in any type of biological ma...
Pietra M, Guglielmini C, Forni M, Cinotti S.The viscosity of the mucus, its DNA concentration and the size range of the DNA were determined on tracheobronchial samples from 11 horses with lower airway diseases before and after incubation with recombinant human deoxyribonuclease (rhDNase). The horses were divided into two groups on the basis of the cytology of the samples: group A (five horses) with more than 60 per cent neutrophils and group B (six horses) with fewer than 50 per cent neutrophils. The mean mucus viscosity and DNA concentration in the preincubation samples were significantly higher in group A than in group B, and there wa...
Kato H, Ohashi T, Matsushiro H, Watari T, Goitsuka R, Tsujimoto H, Hasegawa A.Equine interleukin-1 receptor antagonist (IL-1ra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse. cDNA clones encoding the whole coding sequence of equine IL-1ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS). The equine IL-1ra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues. The predicted amino acid sequence of equine IL-1ra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and ra...
Dai CM, Zhang XY, Zhang RR, Shao YM, Shen RX.To develop a novel vaccine candidate of Equine infectious anemia virus(EIAV). Methods: env genes of EIAV Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virus strain (EIAV LN) were expressed using the BAC-To-BAC system, and Env proteins were confirmed by SDS-PAGE and Western blot. BALB/c mice were immunized with recombinant vaccinia viruses containing env genes of EIAV alone or boosted with Env proteins expressed by recombinant baculovirus. Both protective humoral and cellular immune responses were detected. Results: Recombinant baculovirus could express complete Env pro...
Kumar S, Rakha NK, Goyal L, Goel P, Kumar R, Kumar A, Kumar S.Theileria equi merozoite surface antigens have been an important candidate for development of diagnostics. We developed ELISA based on EMA-1 recombinant antigen, so as to widen our diagnostic confidence in detection of antibodies against T. equi in sero-surveillance studies. The 547 bp EMA-1 gene fragment encoding high hydrophilic antigenic region was expressed with glutathione-S-transferase tag in prokaryotic system and purified protein (43 kDa) was used for development of ELISA (EMA-1t/ELISA). The EMA-1t/ELISA clearly differentiated T. equi-infected from Babesia caballi-infected horse sera o...
de Val N, Herschbach H, Potier N, Dorsselaer AV, Crichton RR.An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the me...
Rotkina AS, Pronina IV, Lazarev VN, Akhaev DN, Baskova IP.The purpose. Identifying the capacity of the medicinal leech novel original recombinant thrombolytic preparation Destabilase-Lysozyme-2 to inhibit the blood platelet aggregation. Gene of destabilase-lysozyme. ds2 (mlDL-Ds2 ), was cloned in E.coli cells. Recombinant protein was isolated in denaturing conditions using metal-chelate chromatography followed by denaturation of the polypeptide by rapid dilution in exact accordance with the procedure described by Kurdyumov A.S. et al. ( 2016, Russian Journal of Bioorganic Chemistry, v.42, s. 42-52). Blood was collected from the jugular vein of 18 hor...
Kasza A, Korpula-Mastalerz R, Rose-John S, Dubin A.The horse leucocyte elastase inhibitor (HLEI), present in neutrophils, monocytes and bone marrow cells, is apparently a cytoplasmic protein which is not released from cells even in response to stimulation with lipopolysaccharide, phorbol ester, tumour necrosis factor alpha, interleukin-1 or elastin degradation products. Although no expression of the inhibitor was detected in neutrophils, both monocytes and bone marrow cells were efficient in its synthesis. Using a new expression vector pREST5d, recombinant inhibitor was produced in a large quantity in a soluble form, with a yield of 88 mg per ...
Marquardt J, Heymer J, Heinz H, Adolf GR, Deegen E.Recombinant equine interferon-beta 1 (reqIFN-beta 1) induces an antiviral state in blood mononuclear cells (BMC) of horses. Maximal protection against replication of vesicular stomatitis virus is achieved 6 hours after treatment with IFN in vitro and in vivo. Duration of the protective effect depends on the dose of IFN in vitro and in vivo. Availability of reqIFN-beta 1 in cultures of BMC for up to 48 hours does not prolong the antiviral state. The protective effect on BMC after treatment with IFN has similar duration in vivo and in vitro. Monitoring of the effect of IFN in vivo is, thus, simp...
Velineni S, DeNegri R, Artiushin SC, Timoney JF.Streptococcus zooepidemicus (Sz) and its clonal derivative Streptococcus equi (Se) share greater than 96% DNA identity and elicit immune responses to many shared proteins. Identification of proteins uniquely targeted by the immune response to each infection would have diagnostic value. Objective: The aim of the study was to compare serum antibody responses of horses infected by Se or Sz. Methods: Antibody levels were measured to panels of recombinant proteins of Sz and Se in sera of horses and ponies before and after experimental and naturally occurring invasive infections by these organisms. ...
Cheung HW, Wong KS, Tam JCL, Farrington AF, Bond AJ, Wan TSM, Ho ENM.Erythropoiesis-stimulating agents (ESAs) continue to be a significant threat to the integrity of human and equine sports. Besides conventional direct testing, monitoring the biomarkers associated with the effects of ESAs may provide a complementary approach via indirect detection to enhance doping control. In this study, we applied label-free proteomics to discover plasma protein biomarkers in Thoroughbred geldings after administration with a long-acting form of recombinant human erythropoietin (rhEPO), methoxy polyethylene glycol epoetin beta, Mircera. Increased haematocrit, haemoglobin and r...
Abreu MC, Conrad NL, Gonçalves VS, Leite FPL.Probiotic microorganisms can stimulate an immune response and increase the efficiency of vaccines. For example, Bacillus toyonensis is a nonpathogenic, Gram-positive bacterium that has been used as a probiotic in animal supplementation. It induces immunomodulatory effects and increases the vaccine response in several species. This study aimed to evaluate the effect of B. toyonensis supplementation on the modulation of the immune response in horses vaccinated with recombinant Clostridium tetani toxin. Twenty horses were vaccinated twice, with an interval of 21 days between doses, and equally di...
