Purification Of Recombined SSB Antigen Expressed In Pichia Pastoris And Establishment Of Dot Immunogold Filtration Assay To Detect Anti-SSB Antibody

Recombination protein SSB antigen which secretory expressed in eukaryotic expression system of Pichia pastoris, be obtained by purified with ammonium sulfate precipitation and polyacrylamide gel extraction after SDS-PAGE. To establish the dot immunogold filtration assay using SSB antigen for detecting anti-SSB autoantibody.【Methods】1. With methanol induction , SSB recombinant Pichia strain SMD1168 secretes recombination protein SSB antigen in the supernatant.2. Recombinant SSB antigen is concentrated from the supernatant by ammonium sulfate precipitation and purified by polyacrylamide gel extraction after SDS-PAGE.3. Colloidal gold to be synthesized, and labled with SPA. Purified recombinant SSB antigen to be bound to nitrocellulose membrane,to establish the dot immunogold filtration assay(DIGFA) to detect clinical specimens, and compare the results with dot enzyme immunoassay's of EUROIMMUN medical laboratory diagnosis company.【Results】1. The expressed amount of recombinant SSB antigen relatively reach peak at the fifth day of culturing, considering impurity proteins to be relatively lowest.2. Recombinant SSB antigen is concentrated from the supernatant by using 50 %(w/v)ammonium sulfate precipitation, then isolated from polyacrylamide gel after SDS-PAGE according to the differences of proteins'molecular weight. Subsequently treated with electroeluting and dialysis, purified SSB antigen recovers it's antigenic activity.3.Using dot immunogold filtration assay established with recombinant SSB antigen to detect 100 clinical specimens,the results show good coincidence rate (92%) with dot enzyme immunoassay's of EUROIMMUN. With repeating test the same specimens monthly for 3 times,recombinant SSB antigen presents satisfactory stability。【Conclusions】1. Pichia Pastoris eukaryotic expression system can expressed high yield recombinant SSB antigen and low hybridproteins. Purified recombinant SSB antigen can be obtained by simple separation methods combined ammonium sulfate precipitation with polyacrylamide gel extraction, electroeluting and dialysis.2. Purified recombinant SSB antigen possess good antigenicity. The dot immunogold filtration assay established with recombination protein SSB shows high specificity and sensitivity in detecting the anti-SSB autoantibody.