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.
Jentsch MC, Lübke S, Schrödl W, Volke D, Krizsan A, Hoffmann R, Kaiser-Thom S, Gerber V, Marti E, Wagner B, Schnabel CL.Severe equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. () is a common mold species in hay and has been described as a major provoking agent of SEA. Unassigned: Aiming to identify disease-relevant antigens, we analyzed using an immunoproteomics approach on two-dimensional ...
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...
El-Sayed SAE, Rizk MA, Baghdadi HB, Ringo AE, Sambuu G, Nugraha AB, Igarashi I.In this study, we designed novel truncated Babesia caballi (B. caballi) recombinant proteins from the previously used B. caballi proteins; 134-Kilodalton Protein (rBC134) and Merozoite Rhoptry 48 Protein (rBC48). Then, we evaluated the diagnostic performance of the newly designed proteins when used as a single antigen or when used as cocktail antigen consists of rBC134 full length (rBC134f) + newly designed rBC48 (rBC48t) or newly designed rBC134 (rBC134t) + rBC48t for the detection of B. caballi infection in horse using indirect enzyme-linked immunosorbent assay (iELISA). We used one dose and...
The protein journalJanuary 18, 2023
Volume 42, Issue 1 24-36 doi: 10.1007/s10930-023-10092-x
Rodríguez MC, Mussio PE, Villarraza J, Tardivo MB, Antuña S, Fontana D, Ceaglio N, Prieto C.Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purifi...
Qiu X, Cao X, Shi N, Zhang H, Zhu X, Gao Y, Mai Z, Jin N, Lu H.Getah virus (GETV) disease is a mosquito-borne infectious disease that causes fever, aseptic meningitis, and abortion in a variety of animals. Currently, the epidemic trend of GETV disease increases seriously worldwide, especially in China, posing a potential threat to animal safety and public health. However, there are few reports about the epidemiological investigation of GETV disease in China as well as a lack of commercial diagnostic kit for GETV antibody. Therefore, the establishment of a rapid, sensitive and suitable GETV antibody detection method for large-scale samples is an urgent req...
Byambaragchaa M, Choi SH, Joo HE, Kim SG, Kim YJ, Park GE, Kang MH, Min KS.Equine chorionic gonadotropin (eCG), produced by the endometrial cups of the placenta after the first trimester, is a specific glycoprotein that displays dual luteinizing hormone (LH)-like and follicle-stimulating hormone (FSH)-like effects in non-equid species. However, in equidaes, eCG exhibits only LH-like activity. To identify the specific biological functions of glycosylated sites in eCG, we constructed the following site mutants of N- and O-linked glycosylation: eCGβ/αΔ56, substitution of α-subunit N-linked glycosylation site; eCGβ-D/α, deletion of the O-linked glycosylation sites ...
Villarraza CJ, Antuña S, Tardivo MB, Rodríguez MC, Mussio P, Cattaneo L, Fontana D, Díaz PU, Ortega HH, Tríbulo A, Macagno A, Bó GA, Ceaglio N....Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we des...
Novotny EN, White SJ, Wilson AD, Stefánsdóttir SB, Tijhaar E, Jonsdóttir S, Frey R, Reiche D, Rose H, Rhyner C, Schüpbach-Regula G....Allergy to bites of blood-sucking insects, including biting midges, can affect both human and veterinary patients. Horses are often suffering from an IgE-mediated allergic dermatitis caused by bites of midges (Culicoides spp). With the aim to improve allergen immunotherapy (AIT), numerous Culicoides allergens have been produced as recombinant (r-) proteins. This study aimed to test a comprehensive panel of differently expressed Culicoides r-allergens on a cohort of IBH-affected and control horses using an allergen microarray. IgE levels to 27 Culicoides r-allergens, including 8 previously unpu...
Aimaletdinov A, Mindubaeva G, Khalikova S, Kabwe E, Salmakova A, Alexandrova N, Rutland C, Rizvanov A, Zakirova E.Tendon injuries are one of the most common causes of orthopedic disorders in horses. Such injuries involve a long course of treatment and recovery. The most promising method of treating these injuries is the use of recombinant proteins and gene therapy. In this work, we evaluated the therapeutic efficacy of plasmid DNA (pDNA) containing two species-specific coding sequences, i.e. vascular endothelial growth factor 164 (VEGF164) and fibroblast growth factor 2 (FGF2), in the treatment of severe damage to the tendon of the superficial digital flexor. A pDNA construct was used to restore the damag...
de la Rosa G, Olvera F, Archundia IG, Lomonte B, Alagón A, Corzo G.Antivenoms are fundamental in the therapy for snakebites. In elapid venoms, there are toxins, e.g. short-chain α-neurotoxins, which are quite abundant, highly toxic, and consequently play a major role in envenomation processes. The core problem is that such α-neurotoxins are weakly immunogenic, and many current elapid antivenoms show low reactivity towards them. We have previously developed a recombinant consensus short-chain α-neurotoxin (ScNtx) based on sequences from the most lethal elapid venoms from America, Africa, Asia, and Oceania. Here we report that an antivenom generated by immu...
Matczuk AK, Chodaczek G, Ugorski M.Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorpo...
Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Kokado H.Getah virus causes fever, skin eruptions, and limb edema in horses. For a high-throughput and time-saving method for serodiagnosis, we explored immunogenic antigens of Getah virus, and established an enzyme-linked immunosorbent assay (ELISA) using a recombinant protein. Western blot analysis using sera from infected horses showed strong reaction with viral antigens around 46 kDa corresponding to E1 or E2 glycoproteins. Recombinant E2 (rE2) protein reacted more strongly with infected horse sera than did rE1 protein in both Western blotting and ELISA. In ELISA using rE2 protein (rE2-ELISA), fo...
Nolan MB, Schulman ML, Botha AE, Human AM, Roth R, Crampton MC, Bertschinger HJ.Commercial and regulatory limitations associated with native porcine zona pellucida (pZP) vaccines formulated with Freund's adjuvants may be overcome by developing effective recombinant ZP vaccines (reZP) and identifying alternative adjuvant formulations. A two-part preparatory study used 15 geldings and identified potentially effective alternative adjuvant formulations based on anti-pZP antibody response following treatment with pZP formulated with Addavax (AddaVax ™, Invivogen), Quil A (Quil-A® Adjuvant, Invivogen), Quil A and Poly (I:C) (HMW VacciGrade™, Invivogen), Pet Gel A (Montanid...
Byambaragchaa M, Lee SY, Kim DJ, Kang MH, Min KS.Previous studies showed that recombinant equine chorionic gonadotropin (rec-eCGβ/α) exhibits both follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-like activities in rat LHR- and FSHR-expressing cells. In this study, we analyzed signal transduction by eelFSHR and eelLHR upon stimulation with rec-eCGβ/α and native eCG. The cyclic adenosine monophosphate (cAMP) stimulation in CHO-K1 cells expressing eelLHR was determined upon exposure to different doses (0-1,450 ng/mL) of rec-eCGβ/α and native eCG. The EC values of rec-eCGβ/α and native eCG were 172.4 and 786.6 ng/mL, resp...
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...
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...
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. ...
Ferra B, Holec-Gąsior L, Kur J.Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the animal husbandry. Commonly used serological tests for diagnosis of toxoplasmosis involve preparation of whole Toxoplasma lysate antigen (TLA) from tachyzoites. The production of this antigen is associated with high costs and lengthy preparation and the possibility of staff infection. There are also some difficulties in the standardization of such tests. One approach in order to improve the diagnosis of T. gondii infection is to use recombinant chimeric anti...
Duarte CG, Bonilla C, Guimarães G, Machado de Avila RA, Mendes TM, Silva W, Tintaya B, Yarleque A, Chávez-Olórtegui C.In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.
Alberca B, Bachanek-Bankowska K, Cabana M, Calvo-Pinilla E, Viaplana E, Frost L, Gubbins S, Urniza A, Mertens P, Castillo-Olivares J.African horse sickness virus (AHSV) is an arthropod-borne pathogen that infects all species of equidae and causes high mortality in horses. Previously, a recombinant modified vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 was shown to induce virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR -/-) against virulent AHSV challenge. This study builds on the previous work, examining the protective efficacy of MVA-VP2 vaccination in the natural host of AHSV infection. A study group of 4 horses was vaccinated twice wit...
Ye C, Yan W, McDonough PL, McDonough SP, Mohamed H, Divers TJ, Chang YF, Yang Z.Leptospirosis, caused by Leptospira spp., is one of the most common zoonotic diseases in the world. We tested four recombinant proteins of Leptospira interrogans, namely, rLipL21, rLoa22, rLipL32, and rLigACon4-8, to evaluate their potential for use as antigens for the diagnosis of equine leptospirosis. We employed equine sera (n = 130) that were microscopic agglutination test (MAT) negative and sera (n = 176) that were MAT positive for the 5 serovars that most commonly cause equine leptospirosis. The sensitivity and specificity of ELISA compared to MAT were 82.39% and 86.15%, respectively, fo...
Sguazza GH, Fuentealba NA, Tizzano MA, Galosi CM, Pecoraro MR.Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for prod...
van der Meide NM, Savelkoul HF, Meulenbroeks C, Ducro BJ, Tijhaar E.Culicoides spp. of the Obsoletus complex belong to the most important species of midge, involved in causing insect bite hypersensitivity (IBH) in horses in The Netherlands. The aim of the current study was to evaluate seven different Obsoletus complex-derived recombinant allergens (Cul o 1-Cul o 7) and to compare these with Obsoletus complex whole body extract (WBE) in an IgE ELISA, using sera of 194 clinically-confirmed cases of IBH and 175 unaffected horses. The highest test accuracy was obtained with WBE, followed by Cul o 2, 3 and 5. Two ELISAs with a combination of recombinant allergens, ...
Singha H, Goyal SK, Malik P, Khurana SK, Singh RK.Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the ...
Peeters LM, Janssens S, Goddeeris BM, De Keyser K, Wilson AD, Kaufmann C, Schaffartzik A, Marti E, Buys N.Insect bite hypersensitivity (IBH) in horses represents an immunoglobulin E (IgE)-mediated hypersensitivity to salivary antigens from biting midges (Culicoides spp.). The aim of this study was to evaluate and compare the performances of IgE ELISAs using recombinant Culicoides spp. Obsoletus group salivary gland antigens or crude whole body extracts ('ObsWBE'), C. nubeculosus recombinant proteins (Culn1, 3, 4, 5, 7, 8 and 10) and Obsoletus group recombinant proteins (Culo1 and 2). IgE levels were measured in plasma of 343 Warmblood horses classified as IBH-affected (n=167) and IBH-unaffected (n...
de Arruda Coutinho LC, de Jesus AL, de Paiva Fontes KF, Coimbra EC, Mariz FC, de Freitas AC, de Cássia Carvalho Maia R, de Castro RS.Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZαA vector after appropr...
Pal V, Kumar S, Malik P, Rai GP.Glanders is a contagious disease caused by the Gram-negative bacillus Burkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently us...
Noronha LE, Harman RM, Wagner B, Antczak DF.The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize ...
Schröper F, Baumann A, Offenhäusser A, Mayer D.During the last decade protein electrochemistry at miniaturized electrodes has become important not only for functional studies of the charge transfer properties of redox proteins but also for fostering the development of sensitive biosensor and bioelectronic devices. One of the major challenges in this field is the directed coupling between electronic and biologically active components. A prerequisite for a fast and reversible electron transfer between electrode and protein is that the protein can be bound to the electrode in a favourable orientation. We examined electrostatic and bioaffinity...
Hornig M, Briese T, Licinio J, Khabbaz RF, Altshuler LL, Potkin SG, Schwemmle M, Siemetzki U, Mintz J, Honkavuori K, Kraemer HC, Egan MF, Whybrow PC....In 1983, reports of antibodies in subjects with major depressive disorder (MDD) to an as-yet uncharacterized infectious agent associated with meningoencephalitis in horses and sheep led to molecular cloning of the genome of a novel, negative-stranded neurotropic virus, Borna disease virus (BDV). This advance has enabled the development of new diagnostic assays, including in situ hybridization, PCR and serology based on recombinant proteins. Since these assays were first implemented in 1990, more than 80 studies have reported an association between BDV and a wide range of human illnesses that i...
Hornig M, Briese T, Licinio J, Khabbaz RF, Altshuler LL, Potkin SG, Schwemmle M, Siemetzki U, Mintz J, Honkavuori K, Kraemer HC, Egan MF, Whybrow PC....In 1983, reports of antibodies in subjects with major depressive disorder (MDD) to an as-yet uncharacterized infectious agent associated with meningoencephalitis in horses and sheep led to molecular cloning of the genome of a novel, negative-stranded neurotropic virus, Borna disease virus (BDV). This advance has enabled the development of new diagnostic assays, including in situ hybridization, PCR and serology based on recombinant proteins. Since these assays were first implemented in 1990, more than 80 studies have reported an association between BDV and a wide range of human illnesses that i...
McDonald RE, Fleming RI, Beeley JG, Bovell DL, Lu JR, Zhao X, Cooper A, Kennedy MW.Horses are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein. The amino acid sequence of latherin, determined from cDNA analysis, is highly conserved across four geographically dispersed equid species (horse, zebra, onager, ass), and is similar to a family of proteins only found previously in the oral cavity and associated tissues of mammals. Latherin produces a significant reduction in water surface tension at low concentrations (< or = 1 mg ml(-1)), and therefore probably acts as a wettin...
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...
de la Rosa G, Olvera F, Archundia IG, Lomonte B, Alagón A, Corzo G.Antivenoms are fundamental in the therapy for snakebites. In elapid venoms, there are toxins, e.g. short-chain α-neurotoxins, which are quite abundant, highly toxic, and consequently play a major role in envenomation processes. The core problem is that such α-neurotoxins are weakly immunogenic, and many current elapid antivenoms show low reactivity towards them. We have previously developed a recombinant consensus short-chain α-neurotoxin (ScNtx) based on sequences from the most lethal elapid venoms from America, Africa, Asia, and Oceania. Here we report that an antivenom generated by immu...
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...
Ferra B, Holec-Gąsior L, Kur J.Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the animal husbandry. Commonly used serological tests for diagnosis of toxoplasmosis involve preparation of whole Toxoplasma lysate antigen (TLA) from tachyzoites. The production of this antigen is associated with high costs and lengthy preparation and the possibility of staff infection. There are also some difficulties in the standardization of such tests. One approach in order to improve the diagnosis of T. gondii infection is to use recombinant chimeric anti...
Toth AM, Geisler C, Aumiller JJ, Jarvis DL.In an effort to produce processed, soluble Western equine encephalitis virus (WEEV) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different WEEV glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. The WEEV glycoprotein constructs encoded full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera. The three different promoters induced gene expression during the immediate early (ie1), late (p6.9), and very lat...
Peeters LM, Janssens S, Goddeeris BM, De Keyser K, Wilson AD, Kaufmann C, Schaffartzik A, Marti E, Buys N.Insect bite hypersensitivity (IBH) in horses represents an immunoglobulin E (IgE)-mediated hypersensitivity to salivary antigens from biting midges (Culicoides spp.). The aim of this study was to evaluate and compare the performances of IgE ELISAs using recombinant Culicoides spp. Obsoletus group salivary gland antigens or crude whole body extracts ('ObsWBE'), C. nubeculosus recombinant proteins (Culn1, 3, 4, 5, 7, 8 and 10) and Obsoletus group recombinant proteins (Culo1 and 2). IgE levels were measured in plasma of 343 Warmblood horses classified as IBH-affected (n=167) and IBH-unaffected (n...
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 ...
Duarte CG, Bonilla C, Guimarães G, Machado de Avila RA, Mendes TM, Silva W, Tintaya B, Yarleque A, Chávez-Olórtegui C.In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.
Tetaz T, Detzner S, Friedlein A, Molitor B, Mary JL.The separation of intact proteins by means of Hydrophilic Interaction Chromatography (HILIC) was demonstrated with human apoA-I, recombinant human apoM, and equine cytochrome C. Five different commercially available HILIC columns were compared. Using one of these columns, different glycosylated isoforms of apoM were separated from each other and from the aglyco-form.
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...
Ellison SP, Omara-Opyene AL, Yowell CA, Marsh AE, Dame JB.A gene encoding a major 29 kDa surface antigen from Sarcocystis neurona, the primary causative agent of equine protozoal myeloencephalitis (EPM), was cloned, sequenced, and expressed as a recombinant protein. A cDNA library was prepared in the expression vector lambda ZAP from polyA+mRNA isolated from S. neurona merozoites cultivated in vitro. Random sequencing of 96 clones identified a clone of an abundant transcript having a translated amino acid sequence with 30% identity to the 31-kDa surface antigen of Sarcocystis muris cyst merozoites. Southern blot analysis indicated that the correspond...
Lewis MJ, Meehan M, Owen P, Woof JM.The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. ...
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...
Villarraza CJ, Antuña S, Tardivo MB, Rodríguez MC, Mussio P, Cattaneo L, Fontana D, Díaz PU, Ortega HH, Tríbulo A, Macagno A, Bó GA, Ceaglio N....Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we des...
Reis JK, Diniz RS, Haddad JP, Ferraz IB, Carvalho AF, Kroon EG, Ferreira PC, Leite RC.Equine infectious anemia (EIA) is an important viral infection affecting horses worldwide. The course of infection is accompanied generally by three characteristic stages: acute, chronic and inapparent. There is no effective EIA vaccine or treatment, and the control of the disease is based currently on identification of EIAV inapparent carriers by laboratory tests. Recombinant envelope protein (rgp90) was expressed in Escherichia coli and evaluated via enzyme-linked immunosorbent assay (ELISA). There was an excellent agreement (95.42%) between the ELISA results using rgp90 and agar gel immunod...
Noronha LE, Harman RM, Wagner B, Antczak DF.The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize ...
Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Kokado H.Getah virus causes fever, skin eruptions, and limb edema in horses. For a high-throughput and time-saving method for serodiagnosis, we explored immunogenic antigens of Getah virus, and established an enzyme-linked immunosorbent assay (ELISA) using a recombinant protein. Western blot analysis using sera from infected horses showed strong reaction with viral antigens around 46 kDa corresponding to E1 or E2 glycoproteins. Recombinant E2 (rE2) protein reacted more strongly with infected horse sera than did rE1 protein in both Western blotting and ELISA. In ELISA using rE2 protein (rE2-ELISA), fo...
Flock M, Karlström A, Lannergård J, Guss B, Flock JI.A mouse model resembling Streptococcus equi subspecies equi infection in the horse, strangles, was used to assess the protective effect of vaccination with selected recombinant proteins from S. equi subsp. equi. After challenge the infection was monitored by weight loss and by nasal colonisation with S. equi subsp. equi. Vaccination with a collagen-binding protein (CNE) and a collagen-like protein (SclC) resulted in protective antibodies, whereas a novel fibronectin-binding protein (FNEB) did not. Co-administration of CNE with EAG, a poorly immunogenic alpha2-macroglobulin-, albumin- and immun...
van der Meide NM, Savelkoul HF, Meulenbroeks C, Ducro BJ, Tijhaar E.Culicoides spp. of the Obsoletus complex belong to the most important species of midge, involved in causing insect bite hypersensitivity (IBH) in horses in The Netherlands. The aim of the current study was to evaluate seven different Obsoletus complex-derived recombinant allergens (Cul o 1-Cul o 7) and to compare these with Obsoletus complex whole body extract (WBE) in an IgE ELISA, using sera of 194 clinically-confirmed cases of IBH and 175 unaffected horses. The highest test accuracy was obtained with WBE, followed by Cul o 2, 3 and 5. Two ELISAs with a combination of recombinant allergens, ...
Novotny EN, White SJ, Wilson AD, Stefánsdóttir SB, Tijhaar E, Jonsdóttir S, Frey R, Reiche D, Rose H, Rhyner C, Schüpbach-Regula G....Allergy to bites of blood-sucking insects, including biting midges, can affect both human and veterinary patients. Horses are often suffering from an IgE-mediated allergic dermatitis caused by bites of midges (Culicoides spp). With the aim to improve allergen immunotherapy (AIT), numerous Culicoides allergens have been produced as recombinant (r-) proteins. This study aimed to test a comprehensive panel of differently expressed Culicoides r-allergens on a cohort of IBH-affected and control horses using an allergen microarray. IgE levels to 27 Culicoides r-allergens, including 8 previously unpu...
Nolan MB, Schulman ML, Botha AE, Human AM, Roth R, Crampton MC, Bertschinger HJ.Commercial and regulatory limitations associated with native porcine zona pellucida (pZP) vaccines formulated with Freund's adjuvants may be overcome by developing effective recombinant ZP vaccines (reZP) and identifying alternative adjuvant formulations. A two-part preparatory study used 15 geldings and identified potentially effective alternative adjuvant formulations based on anti-pZP antibody response following treatment with pZP formulated with Addavax (AddaVax ™, Invivogen), Quil A (Quil-A® Adjuvant, Invivogen), Quil A and Poly (I:C) (HMW VacciGrade™, Invivogen), Pet Gel A (Montanid...
Byambaragchaa M, Lee SY, Kim DJ, Kang MH, Min KS.Previous studies showed that recombinant equine chorionic gonadotropin (rec-eCGβ/α) exhibits both follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-like activities in rat LHR- and FSHR-expressing cells. In this study, we analyzed signal transduction by eelFSHR and eelLHR upon stimulation with rec-eCGβ/α and native eCG. The cyclic adenosine monophosphate (cAMP) stimulation in CHO-K1 cells expressing eelLHR was determined upon exposure to different doses (0-1,450 ng/mL) of rec-eCGβ/α and native eCG. The EC values of rec-eCGβ/α and native eCG were 172.4 and 786.6 ng/mL, resp...
Qiu X, Cao X, Shi N, Zhang H, Zhu X, Gao Y, Mai Z, Jin N, Lu H.Getah virus (GETV) disease is a mosquito-borne infectious disease that causes fever, aseptic meningitis, and abortion in a variety of animals. Currently, the epidemic trend of GETV disease increases seriously worldwide, especially in China, posing a potential threat to animal safety and public health. However, there are few reports about the epidemiological investigation of GETV disease in China as well as a lack of commercial diagnostic kit for GETV antibody. Therefore, the establishment of a rapid, sensitive and suitable GETV antibody detection method for large-scale samples is an urgent req...
Aimaletdinov A, Mindubaeva G, Khalikova S, Kabwe E, Salmakova A, Alexandrova N, Rutland C, Rizvanov A, Zakirova E.Tendon injuries are one of the most common causes of orthopedic disorders in horses. Such injuries involve a long course of treatment and recovery. The most promising method of treating these injuries is the use of recombinant proteins and gene therapy. In this work, we evaluated the therapeutic efficacy of plasmid DNA (pDNA) containing two species-specific coding sequences, i.e. vascular endothelial growth factor 164 (VEGF164) and fibroblast growth factor 2 (FGF2), in the treatment of severe damage to the tendon of the superficial digital flexor. A pDNA construct was used to restore the damag...
Bhaskaran S, Jay CM, Berghman LR, Wagner GG, Waghela SD.Bovine colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is a worldwide problem. Adhesion of ETEC to intestinal cell receptors mediated by the surface protein F5 fimbriae is the initial step in the establishment of colibacillosis. Prevention of ETEC F5(+) adhesion to enterocytes protects newborn calves against collibacillosis. On the enterocytes, the F5 fimbriae bind to a ganglioside that is also found on horse red blood cells. Thus, the presence of F5 fimbriae induces haemagglutination, which is useful as an indicator in a functional assay system. In this study, recombinant ant...
Cooper C, Sears W, Bienzle D.Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine athletes. Recombinant human EPO promotes growth and differentiation of equine erythroid precursor cells, but in some horses repeat administration induces immune interference with endogenous EPO resulting in fatal anemia. Although blood reticulocyte parameters acquire unique changes in humans treated with EPO, with manual enumeration methods, horses were not considered to release reticulocytes from the bone marrow into circulatio...
Xuan X, Igarashi I, Tanaka T, Fukumoto S, Nagasawa H, Fujisaki K, Mikami T.A latex agglutination test (LAT) using recombinant equi merozoite antigen 1 (EMA-1) for the detection of antibodies to Babesia equi was developed. The LAT was able to differentiate very clearly between sera from B. equi-infected horses and sera from Babesia caballi-infected horses or from normal horses. The LAT results were identical to those of a previously developed enzyme-linked immunosorbent assay. These results indicate that LAT using recombinant EMA-1 might be very useful as a routine screening method for the diagnosis of B. equi infection.
