Method for producing transgene protein medicine of mammary gland expression using gland virus as carrier mammary

Method for producing transgene protein medicine of mammary gland expression using gland virus as carrier mammary

  • CN 1,869,217 A
  • Filed: 05/22/2006
  • Published: 11/29/2006
  • Est. Priority Date: 05/22/2006
  • Status: Active Application
First Claim
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1. , a kind of method of expressing the production transgene protein medicine with the adenovirus for carrier mammary gland, it is characterized in that:

  • this method may further comprise the steps;

    (1), separate, clone the drug target protein gene sequence;

    With pUC18, pUC19, pGEM-3ZF, pShuttle, pcDNA3 series plasmid is a cloning vector, with target protein or cytokine hLTF (human lactoferrin gene), hGH (human growth hormone gene), hEPO (human erythropoietin), t-PA (tissue-type plasminogen activator), IL-15 (Interleukin-15), human Protein C (human protein C), IL-2 (interleukin II), hVEGF (vascular endothelial growth factor), NKSF (natural kill cell stimulating factor), 10 kinds of protein drug genes of HGF (pHGF) have obtained to contain the plasmid serial carrier of target gene respectively by molecular cloning;

    (2), institute clone target protein gene constituted with single label screening carrier or double alternative carrier merges carrier for expression of eukaryon, external eukaryotic cell expression detects target protein, the correct pharmaceutical protein gene of acquisition vivoexpression;

    With COS7 (African green monkey kidney cell), CHO (Chinese hamster ovary cell), HEK293 (human embryonic kidney cell) is that host cell carries out vivoexpression to constructed eucaryon fusion protein expression vector;

    Western Blotting, ELASA detect the target gene expression level, have obtained the correct desirable proteins medicine gene of vivoexpression;

    (3), with the reorganization of required target protein gene and adenoviral gene group molecule of the skeleton, obtain to contain the adenovirus carrier of target recombinant pharmaceutical protein gene;

    Required target gene is inserted shuttle vectors, recombinate in bacterium or eukaryotic cell with adenoviral gene group skeleton plasmid again, and produce the replication-defective adenoviral vector that contains the target recombinant protein gene in adenovirus packaging cell system;

    (4), this target recombinant pharmaceutical protein gene adenovirus carrier is imported animal'"'"'s mammary gland;

    Replication-defective adenoviral with the target recombinant protein gene is a carrier, directly imports animal'"'"'s mammary gland, and the target protein gene is expressed in mammary epithelial cell, utilizes the mammary epithelial cell fundamental characteristics, and target protein is secreted in the animal milk;

    (5), secretion obtains required drug target albumen, the animal of acquisition high expression level target protein medicine in the milk.

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