Based on the antigenic analysis of Reticuloendotheliosis virus （REV） envelop glycoprotein （env） protein, an alternative indirect enzyme-linked immunosorbent assay （ELISA） for detection of REV antibody was develo...Based on the antigenic analysis of Reticuloendotheliosis virus （REV） envelop glycoprotein （env） protein, an alternative indirect enzyme-linked immunosorbent assay （ELISA） for detection of REV antibody was developed using a truncated env protein of REV produced in Escherichia coli. This assay was validated by comparison with an indirect immunofluorescence assay （IFA） and a REV-based ELISA. The diagnostic sensitivity （DSN）, specificity （DSP） and accuracy of the env indirect-ELISA （ienv-ELISA） were 93.7, 93.3 and 93.6% compared with IFA on 1 380 field serum samples, and 84.8, 95.2 and 91.7% compared with the REV-based ELISA on 96 field sera, respectively. Cross-reactivity assay showed that this assay was REV-specific. Repeatability test revealed that the coefficients of variation of positive sera within and between runs were less than 15%. This method is simpler to produce and perform, time-saving and suitable for large scale survey of REV antibody at low cost.展开更多
The regulator of expression of virion(Rev)protein binds specifically to the Rev-responsive element(RRE)RNA in order to regulate the expression of the human immunodeficiency virus(HIV)-1 genes.Fluorescence indicator di...The regulator of expression of virion(Rev)protein binds specifically to the Rev-responsive element(RRE)RNA in order to regulate the expression of the human immunodeficiency virus(HIV)-1 genes.Fluorescence indicator displacement assays have been used to identify ligands that can inhibit the ReveRRE interaction;however,the small fluorescence indicators cannot fully replace the Rev peptide or protein.As a result,a single rhodamine B labeled Rev(RB-Rev)model peptide was utilized in this study to develop a direct and efficient ReveRRE inhibitor screening model.Due to photon-induced electron transfer quenching of the tryptophan residue on the RB fluorophore,the fluorescence of RB in Rev was weakened and could be dramatically reactivated by interaction with RRE RNA in ammonium acetate buffer(approximately six times).The interaction could reduce the electron transfer between tryptophan and RB,and RRE could also increase RB fluorescence.The inhibitor screening model was evaluated using three known positive ReveRRE inhibitors,namely,proflavin,6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine(ICR 191),and neomycin,as well as a negative drug,arginine.With the addition of the positive drugs,the fluorescence of the ReveRRE decreased,indicating the displacement of RB-Rev.This was confirmed using atomic force microscopy(AFM)and the fluorescence was essentially unaffected by the addition of arginine.The results demonstrated that RB-Rev can be used as a fluorescent probe for recognizing small ligands that target RRE RNA.The ReveRRE inhibitor screening model offers a novel approach to evaluating and identifying long-acting Rev inhibitors.展开更多
基金founded by the Key Technologies R&D Program of China during the 11th Five-Year Plan period (2006BAD06A11)
文摘Based on the antigenic analysis of Reticuloendotheliosis virus （REV） envelop glycoprotein （env） protein, an alternative indirect enzyme-linked immunosorbent assay （ELISA） for detection of REV antibody was developed using a truncated env protein of REV produced in Escherichia coli. This assay was validated by comparison with an indirect immunofluorescence assay （IFA） and a REV-based ELISA. The diagnostic sensitivity （DSN）, specificity （DSP） and accuracy of the env indirect-ELISA （ienv-ELISA） were 93.7, 93.3 and 93.6% compared with IFA on 1 380 field serum samples, and 84.8, 95.2 and 91.7% compared with the REV-based ELISA on 96 field sera, respectively. Cross-reactivity assay showed that this assay was REV-specific. Repeatability test revealed that the coefficients of variation of positive sera within and between runs were less than 15%. This method is simpler to produce and perform, time-saving and suitable for large scale survey of REV antibody at low cost.
基金supported by the Natural Science Foundation of Shaanxi Province of China(Grant No.:202012119)the Start-up Funding of Shaanxi University of Science and Technology(Grant No.:2019BJ-48)the Innovation Capability Support Program of Shaanxi Province of China(Grant No.:2021PT-044).
文摘The regulator of expression of virion(Rev)protein binds specifically to the Rev-responsive element(RRE)RNA in order to regulate the expression of the human immunodeficiency virus(HIV)-1 genes.Fluorescence indicator displacement assays have been used to identify ligands that can inhibit the ReveRRE interaction;however,the small fluorescence indicators cannot fully replace the Rev peptide or protein.As a result,a single rhodamine B labeled Rev(RB-Rev)model peptide was utilized in this study to develop a direct and efficient ReveRRE inhibitor screening model.Due to photon-induced electron transfer quenching of the tryptophan residue on the RB fluorophore,the fluorescence of RB in Rev was weakened and could be dramatically reactivated by interaction with RRE RNA in ammonium acetate buffer(approximately six times).The interaction could reduce the electron transfer between tryptophan and RB,and RRE could also increase RB fluorescence.The inhibitor screening model was evaluated using three known positive ReveRRE inhibitors,namely,proflavin,6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine(ICR 191),and neomycin,as well as a negative drug,arginine.With the addition of the positive drugs,the fluorescence of the ReveRRE decreased,indicating the displacement of RB-Rev.This was confirmed using atomic force microscopy(AFM)and the fluorescence was essentially unaffected by the addition of arginine.The results demonstrated that RB-Rev can be used as a fluorescent probe for recognizing small ligands that target RRE RNA.The ReveRRE inhibitor screening model offers a novel approach to evaluating and identifying long-acting Rev inhibitors.
