RESUMO
The ORFK8.1 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant 0RFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed: the concentration of antigen was 5 ug/mL, the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein's specificity, the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.
RESUMO
Aim To discuss whether specific Egg yolk antibody(IgY) can be used for snake venom antigens detection.Methods Chickens(white Leghorn) were immunized with detoxicated king cobra venom by formaldehyde.Egg yolk antibody were isolated from egg yolk,and labeled with horse radish peroxidase(HRP).Snake venom antigens samples,including king cobra venom,cobra venom,bungrarus fasciatus venom,bungrarus multicinctus venom,agkistrodon actus venom and Guangdong viper venom,were detected using ELISA,and sensitivity,precision and specificity were tested,respectively.Results At about 32 ?g?L~(-1),the king cobra antigens were detected using the method;Linear relation was better(r=0.963) when the concentration of kingcobra venom was within 32～750?g?L~(-1).The method had good specificity and no cross reactivity was observed among the reagents and agkistrodon acutus guenther venom and vipera russelli siamensis smith venom;little cross reactivity was shown with bungarus multicinctus blyth venom and bungarus fasciatus chmeider venom;cross reactivity was obvious with cobra venom;the average intra-assay coefficient of variation(CV) was 1%～3%,and the inter-assay CV was within 8%.Conclusions The study indicates that IgY can be good reagents for snake venom antigens detecton,and the study provides foundation for the development of the diagnosis kits of snakebites.