System and method for distinguishing marker of secretion type protein

System and method for distinguishing marker of secretion type protein

  • CN 101,093,226 B
  • Filed: 06/23/2006
  • Issued: 10/09/2013
  • Est. Priority Date: 06/23/2006
  • Status: Active Grant
First Claim
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1. the method for the cDNA of an identifier number secreted protein or transmembrane protein is characterized in that described method comprises:

  • (1) the cDNA fragment with external source is inserted in the expression vector that contains the invertase gene that lacks burst;

    Described cDNA fragment derives from the cDNA library, and the cDNA fragment of 5 '"'"' end cDNA that described cDNA fragment has been enrichment, described cDNA library is the cDNA library of the cell or tissue of disease association, deducted the cDNA of some non-these disease specific, the method that subtracts hybridization by difference is carried out described deduction;

    (2) expression vector that step (1) is obtained imports in the yeast cells of the secreting type invertase of not expressing function, and cultivates the yeast cells that acquisition can be grown as the nutrient culture media of sole carbon source at sucrose;

    (3) information of the external source cDNA fragment of discriminating from the yeast cells that step (2) obtains is based on the cDNA of this information acquisition coding secreted protein or transmembrane protein;

    Wherein, described disease is lung cancer;

    Wherein, described enrichment the cDNA fragment of 5 '"'"' end cDNA be prepared as follows;

    the 3 '"'"' end of the specific strand cDNA of lung cancer cell line H125 through subduction is added the dC-tail;

    Then will be through the lung cancer cell line H125 specificity cDNA and the primer SEQ ID NO;

    1 annealing that contains Hind III site of subduction;

    Then synthetic second chain;

    The double-stranded cDNA of ultrasonic degradation and passivation then;

    Then connect Not I restriction enzyme site at 3 '"'"' end;

    At last by using only the increase 5 '"'"' fragment in cDNA library of following primer;

    SEQ ID NO;

    2, SEQ ID NO;

    3;

    Based on above-mentioned PCR program, add Hind III restriction enzyme site at 5 '"'"' of every cDNA-end, the 3 '"'"'-terminal Not I restriction enzyme site that adds, after then each cDNA fragment being cut with Hind III/Not I enzyme, gel electrophoresis separates the cDNA fragment of 400-1000bp.

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