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Development of an indirect ELISA for serological detection of reticuloendotheliosis virus using the gp90 protein expressed in Pichia pastoris

, : Development of an indirect ELISA for serological detection of reticuloendotheliosis virus using the gp90 protein expressed in Pichia pastoris. Journal of Virological Methods 180(1-2): 43-48

The present study was undertaken to express the gp9 protein of reticuloendotheliosis virus (REV) inP. pastorisand evaluate its potential use as a diagnostic antigen in ELISA. The full-length gp9 gene of REV was cloned into pPIC9k vector and then integrated into the chromosome ofP. pastorisfor induced expression. SDS-PAGE and western blot assay demonstrated that gp9 protein was expressed and secreted into the culture medium at about 1 mg/L of culture under optimized condition. An indirect ELISA was then established by using the recombinant gp9 protein as the coating antigen. The optimal concentration of coated antigen was .1 ?g/well at a serum dilution of 1:2 and the optimal positive threshold value of the assay was .49. Cross-reactivity assay showed that this antigen was REV specific. The reproducibility experiment displayed good consistency. Furthermore, the gp9 protein based indirect ELISA showed good correlation rates of 96.3% and 97.5% with virus neutralization test and a commercially whole virus based indirect ELISA, respectively. This study demonstrates the efficacy of recombinant gp9 protein as an antigen in ELISA for seroepidemiological study of REV infection on a large scale.The gp9 protein of reticuloendotheliosis virus (REV) was expressed in P. pastoris The expressed gp9 protein was secreted into the medium with good antigenicity An novel indirect ELISA was established using the gp9 protein as coating antigen The gp9-ELISA displays good sensitivity, specificity and reproducibility The gp9-ELISA shows advantages over virus neutralization and the whole virus based ELISA..


PMID: 22207082

DOI: 10.1016/j.jviromet.2011.12.008

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