Expression Of Human Ro52 Antigen In Pichia Pastoris And Establishment Of Dot Immunogold Filtration Assay For Detecting Its Antibody

【Objective】A rapid dot immunogold filtration assay (DIGFA) for serodiagnosis of human anti-Ro52 antibody was developed using purified recombinant Ro52 antigen induced by methanol in recombinant yeast which was constructed with Molecular biology techniques.The DIGFA we had estabished was consistent with the dot enzyme immunoassay in diagnosis efficiency.【Methods】1. Construction of Ro52 eukaryotic expression vector of yeast: The full-length human Ro52 gene was amplified by PCR from MPN cells cDNA library of human leukemia. The PCR products were TA cloned and Sequenced; The correctly suquenced TA clone and pPIC9K plasmid were digested by both NotⅠand SnaBⅠand connected subsequently. The linearized recombinant plasmid was transferred into Pichia Pastoris SMD1168 strain by electroporation for construct the recombinant Ro52 yeast eukaryotic expression vector.2. Induced expression of recombinant Ro52: High-copy transformants were selected using G418 and identified by PCR.Ro52 secretory expression was induced by Methanol in yeast and precipitated by ammonium sulfate. And then, protein products were analyzed by SDS-PAGE.3. The establishment of DIGFA for serodiagnosis of human anti-Ro52 antibody: The purified recombinant protein Ro52 was coated onto the nitrocellulose membrane in order to establish the dot immunogold filtration assay (DIGFA) which can detect serum anti-Ro52 antibodies. The DIGFA we had developed was compared with the dot enzyme immunoassay and carried on clinical evaluation.【Results】1. Human Ro52 gene was amplified by PCR using MPN human leukemia cell cDNA library as template. PCR product was collected and inserted into TA clone. Positive clones were sequenced by company. The sequence was consistent with reported documents.2. We had successfully constructed Ro52 protein eukaryotic expression vector of yeast.The extraction of pPIC9K-Ro52 recombinant plasmid was digested by both SnaBⅠand NotⅠand sequenced by company.The result showed that the reading frame and insert fragment were in right direction.3. The targeted gene fragment was transferred into Pichia Pastoris strain SMD1168 by electroporation. Recombinant protein was induced by methanol in yeast. Subsequently, the protein product was analyzed by SDS-PAGE: an apparent bind was located nearby 50 KD.4. Compared with dot enzyme immunoassay, theⅹ2 test results showedⅹ2 = 0.44, p> 0.05. Results of DIGFA showed that the positive coincidence rate of two methods was 95.2%,the negative coincidence rate was 94% and the overall coincidence rate was 95%.【Conclusions】We had successfully constructed recombinant protein Ro52 eukaryotic expression vector of yeast and obtained recombinant Ro52 protein from the vector induced by methanol. The recombinant Ro52 protein was attached on NC membrane in order to detect anti-Ro52 antibodies with established DIGFA. The results of the DIGFA and the dot enzyme immunoassay were consistent. With further study, DIGFA for serodiagnosis anti-Ro52 antibodies would be larger value in clinical application.