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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.
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Recombinant proteins are gaining enormous importance these days due to their wide application as biopharmaceutical products and proven safety record. Various recombinant proteins of therapeutic and prophylactic importance have been successfully produced in microbial and higher expression host systems. Since there is no specific antiviral therapy available against dengue, the prevention by vaccination is the mainstay in reducing the disease burden. Therefore, efficacious vaccines are needed to control the spread of dengue worldwide. Dengue is an emerging viral disease caused by any of dengue virus 1-4 serotypes that affects the human population around the globe. Dengue virus is a single stranded RNA virus encoding three structural proteins (capsid protein, pre-membrane protein, and envelope protein) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). As the only licensed dengue vaccine (Dengvaxia) is unable to confer balanced protection against all the serotypes, therefore various approaches for development of dengue vaccines including tetravalent live attenuated, inactivated, plasmid DNA, virus-vectored, virus-like particles, and recombinant subunit vaccines are being explored. These candidates are at different stages of vaccine development and have their own merits and demerits. The promising subunit vaccines are mainly based on envelope or its domain and non-structural proteins of dengue virus. These proteins have been produced in different hosts and are being investigated for development of a successful dengue vaccine. Novel immunogens have been designed employing various strategies like protein engineering and fusion of antigen with various immunostimulatory motif to work as self-adjuvant. Moreover, recombinant proteins can be formulated with novel adjuvants to enhance the immunogenicity and thus conferring better protection to the vaccinees. With the advent of newer and safer host systems, these recombinant proteins can be produced in a cost effective manner at large scale for vaccine studies. In this review, we summarize recent developments in recombinant protein based dengue vaccines that could lead to a good number of efficacious vaccine candidates for future human use and ultimately alternative dengue vaccine candidates.
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Vacunas contra el Dengue , Virus del Dengue , Dengue , Proteínas Virales , Dengue/genética , Dengue/inmunología , Dengue/prevención & control , Vacunas contra el Dengue/genética , Vacunas contra el Dengue/inmunología , Vacunas contra el Dengue/uso terapéutico , Virus del Dengue/genética , Virus del Dengue/inmunología , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/uso terapéutico , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéutico , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z' value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.
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Cisteína Endopeptidasas/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteasas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Virus Chikungunya/enzimología , Virus Chikungunya/fisiología , Chlorocebus aethiops , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , Células Vero , Acetato de Zinc/farmacologíaRESUMEN
West Nile virus (WNV) is an emerging mosquito-borne virus which is responsible for severe and fatal encephalitis in humans and for which there is no licensed vaccine or therapeutic available to prevent infection. The envelope domain III protein (EDIII) of WNV was over-expressed in Escherichia coli and purified using a two-step chromatography process which included immobilized metal affinity chromatography and ion exchange chromatography. E. coli cells were grown in a bioreactor to high density using batch and fed-batch cultivation. Wet biomass obtained after batch and fed-batch cultivation processes was 11.2â¯g and 84â¯g/L of culture respectively. Protein yield after affinity purification was 5.76â¯mg and 5.81â¯mg/g wet cell weight after batch and fed-batch processes respectively. The purified WNV EDIII elicited specific antibodies in rabbits, confirming its immunogenicity. Moreover, the antibodies were able to neutralize WNV in vitro. These results established that the refolded and purified WNV EDIII could be a potential vaccine candidate.
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Proteínas del Envoltorio Viral/inmunología , Virus del Nilo Occidental/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
INTRODUCTION: Dengue is an emerging viral disease that affects the human population around the globe. Recent advancements in dengue virus research have opened new avenues for the development of vaccines against dengue. The development of a vaccine against dengue is a challenging task because any of the four serotypes of dengue viruses can cause disease. The development of a dengue vaccine aims to provide balanced protection against all the serotypes. Several dengue vaccine candidates are in the developmental stages such as inactivated, live attenuated, recombinant subunit, and plasmid DNA vaccines. Area covered: The authors provide an overview of the progress made in the development of much needed dengue vaccines. The authors include their expert opinion and their perspectives for future developments. Expert opinion: Human trials of a live attenuated tetravalent chimeric vaccine have clearly demonstrated its potential as a dengue vaccine. Other vaccine candidate molecules such as DENVax, a recombinant chimeric vaccine andTetraVax, are at different stages of development at this time. The authors believe that the novel strategies for testing and improving the immune response of vaccine candidates in humans will eventually lead to the development of a successful dengue vaccine in future.
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Vacunas contra el Dengue/inmunología , Dengue/prevención & control , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Dengue/patología , Dengue/virología , Virus del Dengue/metabolismo , Humanos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/inmunología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Dengue, a mosquito borne viral disease caused by dengue virus has emerged as a major health problem during the last few decades. The envelope domain III (DIII) protein of dengue virus is highly immunogenic and capable of inducing neutralizing antibodies against wild-type dengue virus. The envelope domain III protein is a potential subunit vaccine candidate as well as a diagnostic reagent for dengue. This report describes the high yield production and immunogenicity of recombinant DIII proteins of dengue virus type 1 and 2. The subunit DIII proteins were produced in Escherichia coli using batch and fed-batch fermentation process. Immobilized metal affinity chromatography was used to capture DIII proteins of dengue virus type 1 and 2. The purified proteins were refolded by diafiltration to achieve biologically active proteins. After fed-batch fermentation, the recombinant E. coli resulted in purified DIII proteins of about 10.06 mg and 47.70 mg per gram of dry cell weight for recombinant dengue virus type 1 and 2 respectively with more than 95% purity. Biological function of the purified DIII proteins were confirmed by their ability to generate DIII specific antibodies in mice. The DIII antigens in combination with adjuvant resulted antibody endpoint titers of 1:64,000 and 1:1,28,000 for recombinant dengue virus type 1 and 2 respectively. These findings establish that the DIII proteins in combination with adjuvant are immunogenic, which suggests that refolded and purified DIII proteins can be a potential vaccine candidates.
RESUMEN
Chikungunya fever, a re-emerging infection, is an arthropod-borne viral disease prevalent in different parts of the world, particularly Africa and South East Asia. Chikungunya virus envelope 2 protein is involved in binding to host receptors and it contains specific epitopes that elicit virus neutralizing antibodies. A highly immunogenic, recombinant Chikungunya virus envelope 2 protein was produced by bioreactor in Escherichia coli for development of a suitable diagnostic and vaccine candidate. This protein was refolded and further purified to achieve biologically active protein. The biological function of refolded and purified recombinant envelope 2 protein of Chikungunya virus was confirmed by its ability to generate envelope 2 specific antibodies with high titers in animal models. These findings suggest that recombinant envelope 2 protein of Chikungunya virus in combination with compatible adjuvant is highly immunogenic. Thus, recombinant envelope 2 protein can be a potential diagnostic reagent and vaccine candidate against Chikungunya virus infection.
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Anticuerpos Antivirales/biosíntesis , Fiebre Chikungunya/prevención & control , Virus Chikungunya/química , Escherichia coli/metabolismo , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Fiebre Chikungunya/inmunología , Virus Chikungunya/inmunología , Medios de Cultivo , Escherichia coli/genética , Análisis Factorial , Fermentación , Adyuvante de Freund/administración & dosificación , Expresión Génica , Replegamiento Proteico , Conejos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Vacunas Virales/inmunologíaRESUMEN
Chikungunya, a mosquito-borne viral disease caused by Chikungunya virus (CHIKV), has drawn substantial attention after its reemergence causing massive outbreaks in tropical regions of Asia and Africa. The recombinant envelope 2 (rE2) protein of CHIKV is a potential diagnostic as well as vaccine candidate. Development of cost-effective cultivation media and appropriate culture conditions are generally favorable for large-scale production of recombinant proteins in Escherichia coli. The effects of medium composition and cultivation conditions on the production of recombinant Chikungunya virus E2 (rCHIKV E2) protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed-batch process was also carried out for high cell density cultivation of E. coli expressing rE2 protein. Expression of rCHIKV E2 protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG) at ~23 g dry cell weight (DCW) per liter of culture and yielded an insoluble protein aggregating to form inclusion bodies. The final DCW after fed-batch cultivation was ~35 g/l. The inclusion bodies were isolated, solubilized in 8 M urea and purified through affinity chromatography to give a final product yield of ~190 mg/l. The reactivity of purified E2 protein was confirmed by Western blotting and enzyme-linked immunosorbent assay. These results show that rE2 protein of CHIKV may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rE2 protein in E. coli with high yield may also offer a promising method for production of other viral recombinant proteins.
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Virus Chikungunya/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Cromatografía de Afinidad , Medios de Cultivo/química , Cuerpos de Inclusión , Isopropil Tiogalactósido/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
The spread of dengue fever depends mainly on the availability of favourable breeding sites for its mosquito vectors around human dwellings. To investigate if the various factors influencing breeding habitats can be mapped from space, dengue indices, such as the container index, the house index and the Breteau index, were calculated from Ministry of Public health data collected three times annually in Phitsanulok, Thailand between 2009 and 2011. The most influential factors were found to be temperature, humidity, rainfall, population density, elevation and land cover. Models were worked out using parameters mostly derived from freely available satellite images and fuzzy logic software with parameter synchronisation and a predication algorithm based on data mining and the Decision Tree method. The models developed were found to be sufficiently flexible to accommodate additional parameters and sampling data that might improve prediction of favourable breeding hotspots. The algorithm applied can not only be used for the prediction of near real-time scenarios with respect to dengue, but can also be applied for monitoring other diseases influenced by environmental and climatic factors. The multi-criteria model presented is a cost-effective way of identifying outbreak hotspots and early warning systems lend themselves for development based on this strategy. The proposed approach demonstrates the successful utilisation of remotely sensed images to map mosquito breeding habitats.
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Aedes , Clima , Ecosistema , Aedes/fisiología , Aedes/virología , Animales , Ciudades/epidemiología , Minería de Datos , Dengue/epidemiología , Brotes de Enfermedades/estadística & datos numéricos , Ambiente , Lógica Difusa , Modelos Estadísticos , Reproducción , Imágenes Satelitales , Tailandia/epidemiologíaRESUMEN
Japanese encephalitis virus (JEV) is the most important cause of encephalitis in most Asian regions. JEV envelope domain III (JEV EDIII) protein is involved in binding to host receptors, and it contains specific epitopes that elicit virus-neutralizing antibodies. A highly immunogenic, recombinant JEV EDIII protein was expressed in Escherichia coli. In order to take this vaccine candidate for further studies, recombinant JEV EDIII protein was produced employing a pilot-scale fermentation process. Recombinant JEV EDIII protein expressed as inclusion bodies (IBs) was solubilized in 8 M urea and renatured by on-column refolding protocol in the presence of glycerol. A three-step purification process comprising of affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. About ~124 mg of highly purified and biologically active EDIII protein was obtained from 100 g of biomass. Biological function of the purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant JEV EDIII protein in combination with compatible adjuvant is highly immunogenic and elicit high-titer neutralizing antibodies. Thus, recombinant JEV EDIII protein produced at large scale can be a potential vaccine candidate.
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Virus de la Encefalitis Japonesa (Especie)/inmunología , Escherichia coli/genética , Expresión Génica , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Biotecnología/métodos , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Virus de la Encefalitis Japonesa (Especie)/genética , Fermentación , Ratones , Pruebas de Neutralización , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/aislamiento & purificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificación , Vacunas Virales/genética , Vacunas Virales/aislamiento & purificaciónRESUMEN
Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freund's complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.
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Virus del Dengue/química , Virus del Dengue/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/aislamiento & purificación , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Dengue/biosíntesis , Vacunas contra el Dengue/química , Vacunas contra el Dengue/inmunología , Vacunas contra el Dengue/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Vacunas Sintéticas/biosíntesis , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificación , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/químicaRESUMEN
Japanese encephalitis is a major public health problem in South-East Asia and Western Pacific countries. The recombinant nonstructural 1 (rNS1) protein of Japanese encephalitis virus is a potential diagnostic as well as vaccine candidate. Developments of cost-effective and simple culture media as well as appropriate culture conditions are generally favourable for large-scale production of recombinant proteins. The effects of medium composition and cultivation conditions on the production of rNS1 protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed-batch process was also carried out for high cell density cultivation (HCDC) of E. coli expressing rNS1 protein. Isopropyl-ß-d-thiogalactopyranoside (IPTG) was used to induce the expression of rNS1 protein at â¼ 13 g dry cell weight per litre of culture. The final dry cell weight after fed-batch cultivation was â¼ 17 g l(-1) . The Inclusion bodies were isolated and purified through affinity chromatography to give a final product yield of â¼ 142 mg l(-1) . The reactivity of purified protein was confirmed by Western blotting and Enzyme linked immunosorbent assay. These results show that rNS1 protein may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rNS1 protein in E. coli with high yield may also offer promising method for production of other viral recombinant proteins.
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Anticuerpos Antivirales/sangre , Biotecnología/métodos , Pruebas Diagnósticas de Rutina/métodos , Encefalitis Japonesa/diagnóstico , Proteínas no Estructurales Virales/aislamiento & purificación , Asia Sudoriental/epidemiología , Western Blotting , Medios de Cultivo/química , Virus de la Encefalitis Japonesa (Especie)/genética , Encefalitis Japonesa/epidemiología , Encefalitis Japonesa/prevención & control , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Expresión Génica/efectos de los fármacos , Islas del Pacífico/epidemiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Activación Transcripcional , Proteínas no Estructurales Virales/genéticaRESUMEN
Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. The envelope protein of dengue virus is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. Optimization of culture media was carried out for enhanced production of recombinant dengue virus type 3 envelope domain III (rDen 3 EDIII) protein in E. coli. Further, batch and fed-batch cultivation process were also developed in optimized medium. After fed-batch cultivation, the dry cell weight was about 22.80 g/L of culture. The rDen 3 EDIII protein was purified using immobilized metal affinity chromatography. This process produced â¼649 mg of purified rDen 3 EDIII protein per liter of culture. The purity of the protein was determined by SDS-PAGE analysis and the reactivity was checked by Western blotting as well as ELISA. These results show that the purified protein may be used for the dengue diagnosis or further prophylactic studies for dengue infection.
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Virus del Dengue/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Bioingeniería/métodos , Virus del Dengue/genética , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Proteínas Recombinantes/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
Global incidence of dengue has increased considerably over the past decade. Dengue fever (DF) is a self-limiting disease; however, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are fatal. Since there is no therapy and vaccine against dengue, timely diagnosis is therefore necessary for patient management. Laboratory diagnosis is carried out by virus isolation, demonstration of viral antigen, presence of viral nucleic acid, and antibodies. Further, recombinant dengue envelope protein can be used to detect specific antibodies, both IgG and IgM against all four serotypes of virus using an E. coli vector. The purified protein can then be used for detection of dengue specific IgG or IgM antibodies in patient serum with higher sensitivity and specificity, than that of traditional assays. Molecular detection can be accomplished by a one-step, single-tube, rapid, multiplex, RT-PCR for serotype determination. Despite many advantages of the modern techniques, isolation of virus is still considered as "gold-standard" in dengue diagnosis.
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Antígenos Virales/genética , Virus del Dengue/aislamiento & purificación , Dengue/diagnóstico , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Dengue Grave/diagnóstico , Dengue Grave/inmunología , Proteínas del Envoltorio Viral/genética , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Dengue/inmunología , Virus del Dengue/genética , Virus del Dengue/inmunología , Humanos , Dengue Grave/epidemiologíaRESUMEN
Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l(-1) and 720 mg l(-1) of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.
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Virus de la Encefalitis Japonesa (Especie) , Escherichia coli/genética , Proteínas del Envoltorio Viral/biosíntesis , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Reactores Biológicos , Western Blotting , Cromatografía de Afinidad , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Humanos , Inmunoglobulina M/análisis , Cuerpos de Inclusión/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
Dengue fever, a mosquito-borne viral disease has become a major worldwide public health problem with a dramatic expansion in recent years. Cultivation process for production of recombinant dengue virus type 4 envelope domain III (rDen 4 EDIII) protein in Escherichia coli was developed for its diagnostic use as well as for further studies in immunoprophylaxis. The dissolved oxygen level was maintained at 20-30% of air saturation. The culture was induced with 1mM of isopropyl beta-d-thiogalactoside when dry cell weight was 13.78 g l(-1) and cells were further grown for 4h to reach 17.31 g l(-1) of culture. The protein was overexpressed in the form of insoluble inclusion bodies. The rDen 4 EDIII protein was purified by affinity chromatography and analyzed by SDS-PAGE. The final yield of purified rDen 4 EDIII protein in this method was approximately 196 mg l(-1) of culture. The purified protein was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue infected human serum samples. These results show that the product has the potential to be used for the diagnosis of dengue infection or for further studies in vaccine development. This production system may also be suitable for the high yield of other recombinant dengue proteins.
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Virus del Dengue , Escherichia coli/metabolismo , Proteínas Recombinantes/análisis , Proteínas del Envoltorio Viral/análisis , Reactores Biológicos , Técnicas de Cultivo de Célula , Cromatografía de Afinidad , Medios de Cultivo , Dengue/inmunología , Virus del Dengue/genética , Virus del Dengue/inmunología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Inmunoglobulina M/sangre , Inmunoglobulina M/química , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Pruebas de Neutralización , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Dengue virus infections have recently undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Early detection of dengue infection based on the identification of antibodies has emerged as a practical and reliable means of diagnosis of dengue fever. The recombinant dengue multiepitope (rDME-M) protein specific to IgM in E. coli was produced in a 5-L fermentor for use in diagnostic purpose. After fermentation, dry cell weight was approximately 11.8 g/L of the culture. The rDME-M protein was purified under denaturing conditions using single-step nickel nitrilotriacetate (Ni-NTA) affinity chromatography. The final yield of purified rDME-M protein from this method was approximately 68.5 mg/L of the culture. The purity of rDME-M protein was checked by SDS-PAGE analysis, and the reactivity of this protein was further checked by Western blotting and enzyme-linked immunosorbent assay (ELISA). The purified protein was used as an antigen in the development of an in-house dipstick ELISA and evaluated with a panel of 80 patient sera, characterized using commercially available tests for detection of dengue antibody. The results were in excellent agreement with those of IgM capture ELISA (Pan-Bio) and rapid immunochromatography (IC) test (Pan-Bio). These results show that the in-house dipstick ELISA using rDME-M protein can be used as a promising kit because of its comparable sensitivity, specificity, field applicability, and low cost.
Asunto(s)
Virus del Dengue/inmunología , Dengue/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina M/inmunología , Proteínas Recombinantes/inmunología , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Dengue/inmunología , Virus del Dengue/genética , Epítopos/genética , Epítopos/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Inmunoglobulina M/genética , Inmunoglobulina M/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Virales/aislamiento & purificaciónRESUMEN
Dengue is an acute mosquito-borne viral disease of humankind. Dengue fever, dengue haemorrhagic fever and dengue shock syndrome have become global public health problems in recent years. rDME-G (recombinant dengue multiepitope protein that can specifically detect IgG) was produced in a 5-litre fermenter in Escherichia coli for use in diagnosis. The culture was induced with 1 mM isopropyl beta-D-thiogalactoside and cells were further grown for 4 h before harvesting. After fermentation, dry cell weight resulted in approx. 16.2 g/l. The rDME-G protein was purified from inclusion bodies using affinity chromatography. The final yield of purified rDME-G protein from fermentation resulted in approx. 168 mg/l of pure biologically active rDME-G protein. The purity of rDME-G protein was checked by SDS/PAGE analysis and the reactivity of this protein was further determined by Western blotting. The purified protein was used to develop an in-house dipstick ELISA and tested using a panel of 60 patient sera characterized using the commercially available tests for detection of dengue antibody. We compared our results with IgG-capture ELISA (Pan-Bio, Windsor, QLD, Australia) and rapid IC (immuno-chromatography) test (Pan-Bio). By using rDME-G protein as an antigen, in the dipstick ELISA, the results were in excellent agreement with commercial rapid IC test and IgG capture ELISA. These results show that the product has a promising potential to be used for diagnosis of dengue in both laboratory- and field-based detection systems with minimum cost and a high degree of sensitivity and specificity.
Asunto(s)
Virus del Dengue/metabolismo , Epítopos/inmunología , Epítopos/aislamiento & purificación , Escherichia coli/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Virales/inmunología , Proteínas Virales/aislamiento & purificación , Virus del Dengue/genética , Epítopos/química , Epítopos/genética , Escherichia coli/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
An outbreak of viral encephalitis occurred in Gorakhpur, India, from July through November 2005. The etiologic agent was confirmed to be Japanese encephalitis virus by analyzing 326 acute-phase clinical specimens for virus-specific antibodies and viral RNA and by virus isolation. Phylogenetic analysis showed that these isolates belonged to genogroup 3.