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Cloning and Expression of Human RNP-70 Gene and ELISA Application of Recombinant SSA and SSB

Author: HouYingNa
Tutor: ZhaoXiaoZuo；LiuGuoZhen
School: Hebei University
Course: Biochemistry and Molecular Biology
Keywords: Autoimmune diseases ENA U1RNP-70 SSA SSB Cloning and Expression
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 7
Quote: 0
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Abstract


Extractable nuclear antigen (Extractable nuclear antigen, ENA) is a eukaryotic cell a class of small nuclear RNA and protein complexes , including RNP, Sm, SSA, SSB, Jo-1, Scl-70 , with its antibody termed for the anti -ENA antibodies , ENA polypeptide antibody is a variety of autoimmune diseases immunological markers : anti- U1-RNP antibodies are mixed connective tissue disease (MCTD) iconic antibody, anti- SSB antibodies is Sjogren's syndrome (SS) and systemic lupus erythematosus (SLE) and other iconic antibodies. Autoantibodies in autoimmune disease diagnosis has important guiding role. Currently, the hospital detect anti-ENA antibodies peptide ELISA mainly (ELISA), and for this purpose is required to prepare high purity antigens. Therefore, the purpose of this experiment is that the nature of genetic engineering methods to obtain a single and specific immune recombinant proteins. In this experiment, human brain cDNA library as a template obtained by PCR U1RNP-70 polypeptide DNA fragment and cloned into the MBP ( maltose binding protein ) fusion system vector pMALTM-c and in the pMALTM-c-2X . Positive recombinants by double enzyme digestion, PCR and DNA sequencing to obtain identification has been verified. SDS-PAGE shows , U1RNP-70 fusion protein was highly expressed . Purified by affinity chromatography , Western-blotting and ELISA proved that the fusion protein has U1RNP-70 immunogenicity and immune specificity. Factor Xa cutting with MBP-U1RNP-70 obtain a single U1RNP-70 antigen protein , once again detected by ELISA , the results showed that the recombinant fusion protein and non- protein antigen was no significant difference in sensitivity and specificity , indicating that the fusion protein of MBP tag resistance . Immunogenicity little impact. Also in our laboratory on the basis of previous work , Expression, purification of soluble fusion protein SSA, SSB. SDS-PAGE that SSA, SSB expression of the fusion protein obtained . After purification by Western-blotting and ELISA experiments show that expression of protein respectively SSA, SSB immunogenicity and immune specificity, which SSB immunological specificity and sensitivity with considerable natural biochemical products .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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