Studies On Oriented Drug And Tolerability To Chemotherapeutic Drug Of MDR1 Gene

A recombinant plasmid named pETP-gp was constructed by cloning MDR 1 1kb geneinto prokaryotic expression vector pET-28b(+). E.coli competent host BL21(DE3) andBL21(DE)lyss were transformed with pETP-gp. Both recombinant bacteria could express theMDR 1 1kb gene at high level by IPTG inducing.The expression in BL21(DE3)plyss washigher until to 48% of the total protein of the induced recombinant bacteria. Expressionprotein was shown as specificity P-gp by immunoprint. It was the first report that MDR 1 1kbgene was expressed with high level in E.coli.Indirect ELISA for the detection of serum antibodies against MDR 1 was establishedwith the E.coli high expressed recombinant pETP-gp protein as antigen and the rabbit againsthuman IgG by tagging HRP as the second antibodies. The optimal reactive condition and theconcentration of the second antibodies were confirmed. The assay was characterized by specificity,simplicity, rapidity and economical cost.Two kinds of eukaryotic expressing plasmids, pcDNA3/MDR 1 and pcDNA3/P-gp,were constructed by cloning each of MDR 1 or MDR 1 1kb gene into pcDNA3. K562 cellsthat transferred by these two kinds of plasmid can express MDR 1 protein in vitro. Animalexperimentation displays that MDR 1 specificity immunoreaction was induced with these twokinds of plasmid.Four individual of cross bred cancer cellular MdrA, MdrB, MdrC and MdrD ofexudation against MDR 1 monoclonal antibodies(McAb) were obtained by injection Balb/Cmice with pcDNA3/P-gp plasmid enwrapped by span and glycerin and followed byamalgamation, riddling and cloning. By Western-blotting and ELISA it was shown that thesefour McAb could react with three kinds of recombinant antigen, pcDNA3/MDR 1,pcDNA3/P-gp and pHVaMDR 1. They create advantaged condition for clinic test.A recombinant plasmid pHVaMDR 1 was constructed by inserting MDR 1 gene intoadenovirus expression vector. MDR 1 gene was transferred effectively to umbilical cordblood nucleate cells(CBNC) by the concentrated virus supernatant liquor. The CBNC canexpress P-gp protein and display tolerability to chemotherapeutic drugs such as vincristineand daunorubicin(DNR) etc.Embryonic stem cells of Kunming mice was separated and cultivated in vitro. The miceembryonic stem cells that were transferred by MDR 1 gene were inputted back to samespecies of mice. The receptor mice was enhanced the tolerability to chemotherapeutic drugs.It is confirmed in experimental level that the transfer of MDR 1 gene protects marrow cells invivo. The test offered credible gist for safeguarding malignancy patient marrow in clinicchemotherapy.