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Recombination and Expression of Huamn Smith D1 Antigen and Establishing a Method for Detecting Its Antibody

Author: WuWenBing
Tutor: LanXiaoPeng
School: Fujian Medical
Course: Clinical Laboratory Science
Keywords: The prokaryotic expression Eukaryotic expression Smith D1 antigen Dot immunogold filtration assay
CLC: R446.6
Type: Master's thesis
Year: 2007
Downloads: 55
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Abstract


SLE is an autoimmune disease can be detected in the serum of patients with a variety of autoantibodies, one of the most important anti-ds-DNA antibody and anti-Sm antibodies, although anti-ds-DNA antibodies for the diagnosis of SLE and judgments lupus activity reference value of the larger, SLE patients after treatment (such as the use of hormones), anti-ds-DNA antibody positive rate was significantly lower, and anti-Sm antibodies had nothing to do with the SLE disease activity, but anti-Sm antibodies remained positive after treatment anti-Sm antibodies is also known as retrospective antibodies are considered iconic antibody of SLE, and has become the American Rheumatism Association (American College of Rheumatology ARA) was amended in 1997, one of the diagnostic criteria. Sm antigen able to react with anti-Sm antibodies are the Sm B / B and Sm D, Sm E, F, G, and N is almost not react with anti-Sm antibodies. Anti-Sm B / B antibodies and other anti-RNP antibody cross-reactivity exists to reduce the anti-Sm B / B antibody specificity for the diagnosis of SLE, it is anti-Sm D antibodies are considered iconic antibody of SLE, while the the Sm D antigen most The major immunoreactive protein is Sm D1, and therefore the detection of anti-Sm D1 antibody having an important role in the diagnosis of SLE. In this study, the use of molecular cloning technology in human leukemia HL-60 cells cDNA library as a template, PCR amplification of the human the Sm D1 gene encoding sequence, and inserted into the prokaryotic expression vector pGEX-5T, prokaryotic expression vector pGEX-5T- Sm D1, in E.coli BL21 (DE3 plysS) to induce the expression of the Sm D1. The expression product is identified by 12% SDS-PAGE electrophoresis, western blot analyzes. The results show that recombinant the Sm D1 to obtain a higher expression in E.coli BL21 (DE3 plysS) and with a high antigenic activity. Resulting inclusion bodies were denatured, refolded GST column preliminary separation and purification, the high purity of the recombinant protein, which laid the foundation for the detection of specific anti-Sm D1 antibodies. Prokaryotic expression products are more complex in purified inclusion bodies, the study attempts to be expressed in a eukaryotic expression system. Same cDNA library of human leukemia HL-60 cells as a template, PCR amplification of the the Sm D1 gene encoding sequence, and inserted into the eukaryotic expression vector pPIC9K, constructed the eukaryotic expression vector pPIC9K-Sm D1 in Pichia pastoris SMD1168 inducing the secretory expression of the Sm D1. Was concentrated by ammonium sulfate precipitation, the expression product identified by Tricine-SDS-PAGE electrophoresis and Western blot. The results show that the reorganization of Sm D1 for higher expression in Pichia pastoris SMD1168 same. Application of preliminary purification eukaryotic product establish DIGFA law detection of anti-Sm D1 antibodies same sensitivity IB law, the specificity somewhat less, but the method is simple, rapid and economic advantages due to the detection of specimens is limited, the method in clinical on the application of value for further study. In short, our laboratory by molecular cloning techniques preliminary human Sm D1 antigen and its antigenic some research, and the establishment of DIGFA detected by anti-Sm D1 antibodies.

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