RESUMEN
Several diagnostic measures have been employed to precisely detect the SARS-CoV-2 viral infection using viral antigens, nucleic acids, and other serological approaches. The sensitivity and specificity of the serological tests remain a challenging need. Here, we describe the detection of human anti-SARS-CoV-2 IgG and IgM antibodies qualitatively through two optimized in-house ELISA and lateral flow immunoassay. Both approaches are based on the prokaryotic expression of 50 kDa SARS-CoV-2 recombinant nucleocapsid protein. This SARS-CoV-2rN-6×His was used either to coat ELISA plates or to be conjugated to gold nanoparticles followed by colorimetric detection of bound human IgG or IgM. In the LFA, we show the optimization of nanoparticle size, protein-binding capacity, membrane treatment, and finally testing the potential capacity of using either the optimized ELISA or LFA in detecting antibodies raised against viral infection. Assessment of both methods was carried out using human sera-positive and negative SARS-CoV-2 antibodies. The ELISA and LFA tests showed 86%, 96.5% sensitivity, 92%, 93.75% specificity, 97%, 98.2% PPV, and 64%, 88.2% NPV, respectively. In conclusion, both approaches were able to successfully detect human antibodies against SARS-CoV-2 nucleocapsid protein. The importance of both protocols cannot be overstated in the detection and diagnosis of viral infections, especially in developing countries.
Asunto(s)
COVID-19 , Nanopartículas del Metal , Humanos , COVID-19/diagnóstico , Oro , Egipto , SARS-CoV-2 , Ensayo de Inmunoadsorción Enzimática/métodos , Sensibilidad y Especificidad , Inmunoglobulina G , Inmunoglobulina M , Anticuerpos AntiviralesRESUMEN
Ticks have harmful impacts on both human and animal health and cause considerable economic losses. Leucine aminopeptidase enzymes (LAP) play important roles during tick infestation to liberate vital amino acids necessary for growth. The aim of the current study is to identify, express and characterize the LAP from the hard tick Hyalomma dromedarii and elucidate its biochemical characteristics. We cloned an open reading frame of 1560 bp encoding a protein of 519 amino acids. The LAP full-length was expressed in Escherichia coli BL21 (DE3) and purified. The recombinant enzyme (H.d rLAP- 6×His) had a predicted molecular mass of approximately 55 kDa. Purification and the enzymatic characteristics of H.d rLAP- 6×His were studied. The purified enzyme showed maximum activity at 37 °C and pH 8.0-8.5 using Leu-p-nitroanilide as a substrate. The activity of H.d rLAP- 6×His was sensitive to ß-mercaptoethanol, dl-dithiothreitol, 1,10- phenanthroline, bestatin HCl, and EDTA and completely abolished by 0.05 % SDS. In parallel, the enzymatic activity was enhanced by Ni2+, Mn2+ and Mg2+, partially inhibited by Na+, Cu2+, Ca2+ and completely inhibited by Zn2+.
Asunto(s)
Secuencia de Aminoácidos , Clonación Molecular , Leucil Aminopeptidasa , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/metabolismo , Leucil Aminopeptidasa/genética , Animales , Especificidad por Sustrato , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Cinética , Estabilidad de Enzimas , Temperatura , Filogenia , Ixodidae/enzimología , Ixodidae/genéticaRESUMEN
Hepatocellular carcinoma and bacterial resistance are major health burdens nowadays. Thus, providing new therapies that overcome that resistance is of great interest, particularly those derived from nature rather than chemotherapeutics to avoid cytotoxicity on normal cells. Venomous animals are among the natural sources that assisted in the discovery of novel therapeutic regimens. L-amino acid oxidase Nh-LAAO (140 kDa), purified from Egyptian Naja haje venom by a successive two-step chromatography protocol, has an optimal pH and temperature of 8 and 37 °C. Under standard assay conditions, Nh-LAAO exhibited the highest specificity toward L-Arg, L-Met and L-Leu, with Km and Vmax values of 3.5 mM and 10.4 µmol/min/ml, respectively. Among the metal ions, Ca+2, Na+, and K+ ions are activators, whereas Fe+2 inhibited LAAO activity. PMSF and EDTA slightly inhibited the Nh-LAAO activity. In addition, Nh-LAAO showed antibacterial and antifungal activities, particularly against Gentamicin-resistant P. aeruginosa and E. coli strains with MIC of 18 ± 2 µg/ml, as well as F. proliferatum and A. parasiticus among the selected human pathogenic strains. Furthermore, Nh-LAAO exhibited anti-proliferative activity against cancer HepG2 and Huh7 cells with IC50 of 79.37 and 60.11 µg/ml, respectively, with no detectable effect on normal WI-38 cells. Consequently, the apoptosis % of the HepG2 and Huh7 cells were 12 ± 1 and 34.5 ± 2.5 %, respectively, upon Nh-LAAO treatment. Further, the Nh-LAAO arrested the HepG2 and Huh7 cell cycles in the G0/G1 phase. Thus, the powerful selective cytotoxicity of L-amino acid oxidase opens up the possibility as a good candidate for clinical cancer therapy.
Asunto(s)
Antineoplásicos , Venenos Elapídicos , L-Aminoácido Oxidasa , L-Aminoácido Oxidasa/farmacología , L-Aminoácido Oxidasa/química , Animales , Humanos , Antineoplásicos/farmacología , Venenos Elapídicos/farmacología , Venenos Elapídicos/química , Células Hep G2 , Naja naja , Línea Celular Tumoral , Pruebas de Sensibilidad Microbiana , Antiinfecciosos/farmacología , Egipto , Antibacterianos/farmacología , Apoptosis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
BACKGROUND: The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. The urgent need to control the spread of the virus and rapid application of the suitable health measures raised the importance of available, rapid, and accurate diagnostic approaches. OBJECTIVE: The purpose of this study is to describe a rapid in-house optimized ELISA based on the expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in a prokaryotic system. METHODS: We show the expression of the 30 kDa recombinant SARS-CoV-2 RBD-6×His in four different E. coli strains (at 28∘C using 0.25mM IPTG) including the expression strain E. coli BL21 (DE3) Rosetta Gami. SARS-CoV-2 rRBD-6×His protein was purified, refolded, and used as an antigen coat to assess antibody response in human sera against SARS-CoV-2 infection. RESULTS: The assessment was carried out using a total of 155 human sero-positive and negative SARS-CoV-2 antibodies. The ELISA showed 69.5% sensitivity, 88% specificity, 78.5% agreement, a positive predictive value (PPV) of 92.3%, and a negative predictive value of 56.5%. Moreover, the optical density (OD) values of positive samples significantly correlated with the commercial kit titers. CONCLUSIONS: Specific human antibodies against SARS-CoV-2 spike protein were detected by rapid in-house ELISA in sera of human COVID-19-infected patients. The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications, particularly in developing countries. Future studies are planned for the use of the generated SARS-CoV-2 rRBD-6×His protein in vaccine development and other diagnostic applications.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Humanos , Glicoproteína de la Espiga del CoronavirusRESUMEN
This study reported the purification and characterization of a cytotoxic, neurotoxin-like protein derived from the venom of the Egyptian cobra Naja haje haje, Elapidae family, and explored their mechanistic role in the cell death. The protein purification was performed through ion-exchange chromatography and gel-filtration chromatography and was characterized by SDS-PAGE, amino acid sequencing, and mass spectrum analysis. The antitumor activity of Naja haje venom (NHV) and its fractions (NHVI, NHV-Ia, NHV-Ib, NHV-Ic, NHV-II, NHV-III, and NHV-IV) were tested against different human cancer cell lines. The molecular cascade of cell death was explored through evaluation of apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase (HDAC) activity, mitochondrial transmembrane potential (Δψ(m)), cytochrome c release, total caspases, caspase-3, caspase-9, and cell cycle analysis by flow cytometry. Most of the separated fractions possessed variable cytotoxic effect against different cancer cells. The most potent antitumor fraction was NHV-Ic due to its ability to induce DNA damaging and fragmentation that was associated with a significant induction of apoptosis via mitochondrial pathway and disturbed cell cycle phases as well as an inhibition of HDAC activity. NHV-Ic induced the mitochondrial pathway initially by the impairment of Δψ(m) besides the DNA damage and in response to that the mitochondria-released cytochrome c that may in turn activated total caspases, caspase-3 and caspase-9 in lymphoblastic leukemia 1301 cells. The partial amino acid sequencing of NHV-Ic revealed 100, 95.65, and 91.3% homology with the Long neurotoxin 1 from Naja haje anchietae (Angolan cobra), Naja haje haje (Egyptian cobra), and Boulengerina annulata annulata (banded water cobra), respectively.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Venenos Elapídicos/farmacología , Neurotoxinas/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Venenos Elapídicos/química , Electroforesis en Gel de Poliacrilamida , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/aislamiento & purificación , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/efectos de los fármacos , Histona Desacetilasas/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neurotoxinas/química , Neurotoxinas/aislamiento & purificaciónRESUMEN
Echinococcosis/hydatidosis is one of the most important parasitic zoonotic diseases in the world. Cystic echinococcosis increases public health and socio-economic concern due to considerable morbidity rates that give rise to elevated economic losses both in the public health part and in the farm animal field. The enzyme linked immunosorbent assay (ELISA) is consider the more accurate tool for diagnosis of hydatidosis in camels. In the present study, affinity purified Echinococcus granulosus (E. granulosus) antigens (APA) were purified from crude hydatide E. granulosus germinal layer proteins for detection of E. granulosus antibodies in infected camels, using affinity matrix (camel IgGs coupled to CNBr-activated Sepharose). The electrophoretic profile of the APA showed that it was separated into two bands; one major band of 130 kDa and one minor band at 55 kDa. These antigens were used successfully as specific coating antigenic proteins in detection of echinococcosis in camel. In a trial to prepare an anti-camel IgGs peroxidase conjugate; peroxidase enzyme was purified from turnip roots (TPOD) using ammonium sulfate precipitation and affinity chromatography on phenyl Sepharose CL-4B. The purified TPOD showed a major band at 35 kDa. Rabbit anti-camel IgG antibodies (AC IgGs) were prepared then purified using affinity chromatography on Protein G-Sepharose. The TPOD, and commercial HRP for comparison, enzymes were conjugated to AC IgGs using 1%, 5% and 10% glutaraldehyde. The results revealed that the HRP was much better than TPOD in conjugation with AC-IgG antibodies and the 10% glutaraldehyde concentration was the most efficient concentration with ELISA titer 1:50.
RESUMEN
A full-length cDNA of a glutathione S-transferase (GST) was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. The 672 bp cloned fragment was sequenced and showed an open reading frame encoding a protein of 223 amino acids. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the sequence is closely related to the mammalian mu-class GST. The cloned gene was expressed in E. coli under T7 promotor of pET-30b vector, and purified under native conditions. The purified enzyme appeared as a single band on 12% SDS-PAGE and has a molecular weight of 30.8 kDa including the histidine tag of the vector. The purified enzyme was assayed upon the chromogenic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the recombinant enzyme showed high level of activity even in the presence of the beta-galactosidase region on its 5' end and showed maximum activity at pH 7.5. The Km values for CDNB and GSH were 0.57 and 0.79 mM, respectively. The over expressed rBaGST showed high activity toward CDNB (121 units/mg protein) and less toward DCNB (29.3 units/mg protein). rBaGST exhibited peroxidatic activity on cumene hydroperoxide sharing this property with GSTs belonging to the GST alpha class. I50 values for cibacron blue and bromosulfophthalein were 0.22 and 8.45 microM, respectively, sharing this property with the mammalian GSTmu class. Immunoblotting revealed the presence of the GST molecule in B. annulatus protein extracts; whole tick, larvae, gut, salivary gland and ovary. Homologues to the GSTmu were also detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. while in Ornithodoros moubata, GSTmu homologue could not be detected.
Asunto(s)
Clonación Molecular , Regulación Enzimológica de la Expresión Génica , Glutatión Transferasa/genética , Garrapatas/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN Complementario/química , Biblioteca de Genes , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Cinética , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
AIM: In view of various peroxidase applications, the searching for new sources of unique peroxidase properties is highly desirable. The present study aims to evaluate the efficiency of the peroxidase of locally grown sycamore latex (POL) for conjugation with antibodies and to study the conjugate optimal conditions, storage stability, and affinity toward different substrates as compared with commercial horseradish peroxidase (HRP). MATERIALS AND METHODS: Anti-mouse antibodies were prepared in rabbits and purified by protein A sepharose affinity column chromatography. The POL and HRP conjugates were prepared by one-step glutaraldehyde coupling method. The reactivity of the prepared conjugates was examined using the enzyme-linked immunosorbent assay (ELISA). The optimal enzymatic conditions, storage stability, and affinity toward substrates were also determined for both the conjugates. RESULTS: The POL showed higher percent recovery (98%) than HRP (78%) over the initial activity after conjugation process. The POL and HRP conjugates showed ELISA titers of 1:120 and 1:80, respectively, demonstrating high binding affinity of POL-conjugate. The POL-conjugate showed high thermal stability up to 70°C compared with HRP-conjugate up to 40°C. After conjugation, POL had wide pH stability (5.0-8.0) compared with HPR (4.5-6.0). Both of the prepared conjugates had a high affinity toward the substrates used in immunoassays with lower Km values. The POL-conjugate showed high storage stability for its enzymatic activity and ELISA titer compared with HRP-conjugate after 1 year at -20°C. CONCLUSION: The POL of Ficus sycomorus latex is an efficient source for labeling antibodies and could be utilized in immunodiagnostic kits.
RESUMEN
A thermostable metallo-collagenase enzyme (150â¯kDa), recently identified in a newly isolated actinomycestes strain (Nocardiopsis dassonvillei NRC2aza), has been purified from natural source, characterized to have application in wound healing. A simple 3 step purification procedure gave an increase of purity by 6.23 fold with a specific activity of 387.2â¯Uâ¯mg-1. The enzyme activity showed stability across a range of pH (7.0-8.5) and temperature (40-55⯰C) with optima at pHâ¯8.0 and 60⯰C, respectively. Activators include Mg+2, Ca+2, Zn+2, Na+, K+ and Ba+2, while Mn+2, Co+2, Ni+2and Ag+ ions gave partial inhibition. Full inhibition was given by other tested ions and metalloproteinase inhibitors. Broad substrate specificity was demonstrated including activity against a native collagen. The Km and Vmax of the enzyme using azocollagen were 5.5â¯mg/ml and 1280â¯U, respectively. The purified collagenase enhanced wound closure in vitro and in vivo and the repair process was dose dependent. Topical application of the purified collagenase (either of 25 or 50â¯U) to cutaneous wounds significantly accelerated the rate of wound healing and the formation of granulation tissue. Hence, the purified collagenase has a great potential as a therapeutic agent in wound care and collagen related diseases.
Asunto(s)
Actinobacteria/enzimología , Proteínas Bacterianas , Colagenasas , Fibroblastos/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/tratamiento farmacológico , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Células Cultivadas , Colagenasas/química , Colagenasas/aislamiento & purificación , Colagenasas/farmacología , Fibroblastos/patología , Humanos , Ratas , Ratas Wistar , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patologíaRESUMEN
A homodimeric l-amino acid oxidase enzyme (Cv-LAAOI) was isolated from the venom of Cerastes vipera (Egyptian Sand viper) using gel filtration followed by anion exchange chromatography. The molecular mass of Cv-LAAO is 120â¯kDa in its native form and 60â¯kDa in its monomeric form. The optimum enzyme activity was achieved on l-Leucine as a substrate in 50â¯mM buffer pH 7.5â¯at 50⯰C. The Cv-LAAOI activity was significantly reduced by increasing the temperature over 40⯰C, lost 75% of its activity at 60⯰C and inhibited completely at 80⯰C. The Cv-LAAOI attains the highest substrate specificity towards L-Met. The results have also indicated that Mn2+ enhances the enzyme activity by 10%, while Cu2+, Hg2+, Ni2+, Co2+ have suppressive effects on the Cv-LAAOI activity. On the other hand, EDTA has no significant effect on the enzyme activity. The kinetic parameters of Cv-LAAOI activity (Km, Kcat and Vmax) estimated on l-Leucine at pH 8 and 37⯰C were found to be 2â¯mM, 12â¯S-1 and 16.7⯵mol/min/ml, respectively. In addition, the results have shown that Cv-LAAOI exhibits a significant bactericidal activity against gram-positive and gram-negative bacteria, particularly Staphylococcus aureus and Escherichia coli with MIC values of 20⯵g/ml. Moreover, Cv-LAAOI has exhibited a considerable cytotoxic activity against breast cancer cell line (MCF-7) with IC50 value 2.75⯱â¯0.38⯵g/ml compared with different tumor cell lines (liver HepG2, lung A549, colon HCT116 and prostate PC3). Furthermore, Cv-LAAOI has triggered antiproliferative activity via extensive H2O2 generation as indicated by the increase in H2O2 and TBARS levels accompanied by the depletion in the catalase activity (CAT) in MCF-7 treated cells compared to the untreated ones. Thus, these findings clearly indicate that Cv-LAAOI has a selective cytotoxic effect on breast cancer cell line, demonstrating a great prospective for future use in cancer therapy.
Asunto(s)
Antibacterianos/farmacología , Antineoplásicos/farmacología , Bacterias/efectos de los fármacos , L-Aminoácido Oxidasa/metabolismo , Venenos de Víboras/enzimología , Viperidae/metabolismo , Animales , Antibacterianos/química , Antineoplásicos/química , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , L-Aminoácido Oxidasa/química , Pruebas de Sensibilidad Microbiana , Especificidad por Sustrato , Temperatura , Venenos de Víboras/químicaRESUMEN
The present study was conducted to identify new target immunogenic molecules from the larval stage of the cattle tick, Boophilus annulatus (Acari: Ixodidae). Two specific larval glycoproteins (GLPs) were isolated by two-step affinity chromatography. The larval immunogens were first purified with CNBr-Sepharose coupled to rabbit anti-larval immunoglobulins, and the glycoproteins were then purified with Con-A Sepharose. These glycoproteins have molecular weights of approximately 32 and 15 kDa with isoelectric points between 6.8 and 7.2. Antibodies against the two GLPs, labeled I and II, were detected in the anti-whole tick, -whole larval, and -gut antigens through immunoblot analysis. These results suggest that these GLPs are good immunogens and can be useful in the vaccination of cattle against tick infestation.
Asunto(s)
Enfermedades de los Bovinos/parasitología , Glicoproteínas/aislamiento & purificación , Ixodidae/inmunología , Infestaciones por Garrapatas/veterinaria , Secuencia de Aminoácidos , Animales , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/prevención & control , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/inmunología , Immunoblotting , Focalización Isoeléctrica , Ixodidae/química , Masculino , Peso Molecular , Conejos , Análisis de Secuencia de Proteína , Infestaciones por Garrapatas/inmunología , Infestaciones por Garrapatas/parasitología , Infestaciones por Garrapatas/prevención & controlRESUMEN
A serine metallokeratinase enzyme (30 kDa) produced by a newly isolated Bacillus strain (Bacillus pumilus NRC21) cultivated under optimized conditions in medium containing chicken feather meal was purified and characterized in a set of biochemical assays. The purification was carried out using two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-100 columns. The purified enzyme showed a specific activity of 2000 units/mg protein against 170 units/mg protein for crude extract with 12 fold purification. The enzymatic activity of the keratinase stimulated by (Na(+), K(+), Mg(2+)), Hg(+2) had no effect, and inhibited by entire tested cations, serine and metalloproteinase inhibitors, therefore it can be considered as a serine metalloenzyme. The optimum pH and temperature for the purified enzyme were (7.5, 8.5) and (50, 45 °C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60 °C), respectively and its thermoactivity and thermostability were enhanced in the presence of 5 mM Mg(+2). These results suggest that the purified keratinase may be used in several industrial applications.
Asunto(s)
Bacillus pumilus/enzimología , Fraccionamiento Químico/métodos , Péptido Hidrolasas/aislamiento & purificación , Serina/metabolismo , Bacillus pumilus/clasificación , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Hidrólisis , Cinética , Metales/farmacología , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Proteínas/química , Proteínas/metabolismo , SolubilidadRESUMEN
Infestation of cattle by ticks of Rhipicephalus spp. results in severe veterinary and economical losses. Identification of novel proteins from tick salivary glands will enhance our understanding of several aspects of tick physiology and will aid in the development of anti-tick vaccines. Small heat shock proteins (HSPs) have important roles in infection and immunity, especially between invertebrate vectors and mammalian hosts while initially performing their molecular chaperone activity. Here, we report the identification of a small HSP gene from the salivary glands of Rhipicephalus annulatus ticks through immunoscreening of the corresponding cDNA expression library. The identified cDNA contained a 742bp sequence with 543bp open reading frame. It was subsequently cloned, expressed and successfully purified under both native and denaturing conditions. Sequence analysis and functional investigations showed that the protein belongs to the HSP20 family, hence the annotated name Ra-sHSPI. Indeed, recombinant Ra-sHSPI showed two typical in vitro activities of holdase chaperones, including thermal protection of bacterial cellular extracts and the recombinant HindIII at elevated temperatures. Moreover, the recombinant Ra-sHSPI showed strong immunogenic effect in animal model. These results pave the way toward further investigation of Ra-sHSPI role in ticks feeding and its potential use as protective antigen.
Asunto(s)
Clonación Molecular , Proteínas del Choque Térmico HSP20/genética , Rhipicephalus/genética , Animales , Bovinos/genética , Bovinos/parasitología , ADN Complementario/genética , Glándulas Salivales , Análisis de Secuencia de ADNRESUMEN
Novel keratinolytic enzyme (32kDa) secreted by a newly isolated Bacillus strain (Bacillus subtilis NRC3) cultivated in medium containing chicken feather meal was purified and partially characterized in a set of biochemical assays. The purification was carried out by applying a protocol of two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-75 columns. The purified enzyme showed a specific activity of 5233units/mg protein against 169units/mg protein for crude extract with 31 fold purification. The enzymatic activity of the purified keratinolytic enzyme stimulated by Na(+), K(+), Mg(2+), Ba(2+), Ca(2+), and inhibited by entire tested cations and metalloproteinase inhibitors, indicating that it belongs to metallo-keratinase enzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.0) and (50, 40°C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60°C), respectively. These results suggest that this keratinase may be a useful alternative and ecofriendly route for handling the abundant amount of waste feathers.
Asunto(s)
Bacillus subtilis/metabolismo , Péptido Hidrolasas/metabolismo , Bacillus subtilis/clasificación , Bacillus subtilis/genética , Bacillus subtilis/aislamiento & purificación , Activación Enzimática , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/química , Péptido Hidrolasas/aislamiento & purificación , Filogenia , ARN Ribosómico 16S , TemperaturaRESUMEN
The control of Rhipicephalus annulatus ticks in Egypt and other countries relies principally on the application of acaricides which have many drawbacks. Recently, cattle vaccination against ticks showed a potential unconventional approach to control ticks. As a target, salivary glands contain various proteins that may play specific roles during attachment, feeding and may modulate the immune system of the host. We have performed immunoscreening on expression normalized cDNA library to identify unique R. annulatus proteins from salivary gland (RaSal) that are particularly expressed during engorgement. We also present the cloning and sequencing of four novel cDNAs (RaSal1-4) from salivary glands that are expressed during feeding. RaSal4 shows 13 cysteine amino acid residues forming 6 potential disulfide bonds. We detected the expression level of the four genes during embryogenesis in eggs collected at 6, 12 and 18 days after oviposition. RT-PCR analysis detected these proteins at days 12 and 18 while slight amplification was detected at day 6 for only RaSal2. The expression of these salivary genes may put forward new vaccines to control tick infestations and tick-borne diseases.
Asunto(s)
Proteínas de Artrópodos/genética , Enfermedades de los Bovinos/parasitología , Rhipicephalus/genética , Proteínas y Péptidos Salivales/genética , Infestaciones por Garrapatas/veterinaria , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/inmunología , Bovinos , Enfermedades de los Bovinos/prevención & control , Datos de Secuencia Molecular , Filogenia , Conejos , Rhipicephalus/química , Rhipicephalus/clasificación , Rhipicephalus/inmunología , Proteínas y Péptidos Salivales/química , Proteínas y Péptidos Salivales/inmunología , Alineación de Secuencia , Infestaciones por Garrapatas/parasitología , Infestaciones por Garrapatas/prevención & control , Regulación hacia Arriba , Vacunas/química , Vacunas/genética , Vacunas/inmunologíaRESUMEN
The middle capsid protein of rotavirus, VP6, constitutes approximately 51% of the virion by weight. The high degree of identity (>87-99%) in the primary amino acid sequences of VP6 proteins from mammalian rotaviruses suggests VP6-based vaccines could potentially provide heterotypic protection. For this reason, significant effort has been directed toward producing recombinant rotavirus VP6 vaccines. We have cloned and expressed 155bp of the VP6 most frequent in Egyptian rotavirus sewage isolates, encoding the VP6 protein in the pET30b vector having an apparent molecular mass of 12.5kDa. The recombinant VP6 expressed by the transformants was detected by SDS-PAGE analysis. Rabbits immunized with the recombinant rotavirus expressed VP6 elicited VP6-specific IgG antibodies that provided significant reductions in the infectious units of the Egyptian rotavirus sewage isolate and human reference rotavirus Wa strain in vitro assay.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Proteínas de la Cápside/genética , Clonación Molecular , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus/inmunología , Rotavirus/genética , Animales , Antígenos Virales/inmunología , Cápside/química , Proteínas de la Cápside/inmunología , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Vectores Genéticos , Humanos , Inmunización , Masculino , Peso Molecular , Conejos , Rotavirus/inmunología , Rotavirus/aislamiento & purificación , Infecciones por Rotavirus/sangre , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/virología , Vacunas contra Rotavirus/administración & dosificación , Vacunas contra Rotavirus/genética , Aguas del Alcantarillado/microbiología , Vacunas SintéticasRESUMEN
In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.
Asunto(s)
Glicoproteínas/inmunología , Ixodidae/inmunología , Conejos/inmunología , Infestaciones por Garrapatas/veterinaria , Animales , Antígenos/inmunología , Antígenos/aislamiento & purificación , Argasidae/inmunología , Cromatografía de Afinidad/veterinaria , Electroforesis en Gel de Poliacrilamida/veterinaria , Femenino , Glicoproteínas/aislamiento & purificación , Immunoblotting/veterinaria , Inyecciones Intramusculares/veterinaria , Ixodidae/crecimiento & desarrollo , Estadios del Ciclo de Vida , Masculino , Conejos/parasitología , Reproducción , Especificidad de la Especie , Infestaciones por Garrapatas/inmunología , Infestaciones por Garrapatas/prevención & controlRESUMEN
Immunoscreening of a cDNA expression library of the Rhipicephalus (Boophilus) annulatus tick with purified rabbit anti-R annulatus salivary glands antigens polyclonal antibodies led to the identification of a 661bp sequence. The sequence includes an open reading frame of 543bp encoding a protein of 180 amino acids with calculated molecular weight of 20.51kDa, isoelectric point of 9.071 and with no signal sequence. Comparison of the deduced amino acids with protein data bank showed that the identified polypeptide belongs to the alpha crystallin small heat shock proteins superfamily and shows sequence similarity of 62% and 55% to Ixodes scapularis fed tick salivary gland protein and Ornithodoros parkeri alpha-crystallin protein, respectively. Accordingly, this protein was called Ra-sHSPII. The Ra-sHSPII protein was expressed in E. coli under T7 promotor of the pET-30b vector, purified under denaturation conditions and the immunogenicity and cross-reactivity of the recombinant Ra-sHSPII were evaluated. Direct ELISA showed that the Ra-sHSPII is a strong immunogen. In immunoblotting assay the anti-rRa-sHSPII antisera reacted specifically with purified rRa-sHSPII, with several proteins in R. annulatus whole tick, larval and gut protein extracts in addition to Hyalomma dromedarii and Ornithodoros moubata whole tick protein extracts, as examples of hard and soft tick species, respectively. The rRa-sHSPII protein confers thermal protection to other proteins in vitro as found in other sHSPs. E. coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100°C, whereas extracts from cells not expressing the protein were heat-sensitive at 60°C.