RESUMO
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.
Assuntos
Cisteína Endopeptidases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteases/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Vírus Chikungunya/enzimologia , Vírus Chikungunya/fisiologia , Chlorocebus aethiops , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Células Vero , Acetato de Zinco/farmacologiaRESUMO
The resurgence of chikungunya virus in the form of unprecedented explosive epidemic with unusual clinical severity after a gap of 32 years is a point of major public health concern. Definitive diagnosis is critical in differentiating the disease, especially in dengue endemic areas. The immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is widely used for diagnosis of chikungunya infection. However IgM ELISA based on whole virus antigen is associated with biohazard risk. The present study describes the development and evaluation of recombinant capsid protein based indirect IgM antibody capture micro plate enzyme linked immunosorbent assay (ELISA) for rapid and accurate diagnosis of chikungunya infection. The gene coding for capsid protein was cloned in frame with GST tag in pET41a+ vector and expressed in E. coli followed by purification with affinity chromatography. The comparative evaluation of in-house chikungunya IgM ELISA vis-a-vis commercially available SD ELISA kit with 90 chikungunya suspected acute phase human patient serum samples revealed 97% accordance. The overall sensitivity and specificity of the reported capsid protein based IgM ELISA was 100% and 95% respectively with 96% PPV and 100% NPV. These findings clearly demonstrated the usefulness of the recombinant capsid protein based CHIKV IgM ELISA for reliable clinical diagnosis of CHIKV infection in human patient.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais , Proteínas do Capsídeo , Febre de Chikungunya/diagnóstico , Vírus Chikungunya/imunologia , Imunoglobulina M/sangue , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Febre de Chikungunya/imunologia , Vírus Chikungunya/genética , Cromatografia de Afinidade , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Expressão Gênica , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
The hemagglutinin (HA) gene of novel Swine Origin Influenza A/California/04/2009 (H1N1) was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA-synthetic gene having α secretory tag under the control of AOX1 promoter was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having single and multiple copy integrants of the expression cassettes were screened for the expression of full length HA protein in the culture supernatant. In order to completely exploit the expression potential of the P. pastoris expression system, a systematic investigation on the influence of gene copy number on the expression of the recombinant protein was made. A panel of Pichia clones carrying increasing copies of the heterologous gene was selected based on Geneticin resistance and SYBR green-based quantitative real-time PCR approach. Using these strategies, recombinant Pichia transformants carrying up to a maximum of four to six copies of the transgene were identified. After optimising the expression conditions for shaker flask culture, the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the expression level of H1N1HA recombinant protein. Our findings clearly suggest that the gene dosage effect play a vital role in high level expression of the pandemic Influenza HA protein in yeast system.
