Construction And Expression Of PGEX-4T-2-TK Procaryon Recombinant Plasmid

objective : To construct procaryon recombinant plasmid of pGEX-4T-2-TK and detect its expression in BL21(DE3)E coli. We choose the procaryon expression vector pGEX-4T-2 and herpes simplex virus typeⅠthymidine kinase gene as target gene to construct recombinant plasmid, which could catalyze nontoxic prodrugs into cytotoxic substance. Used molecular biology gene engineering methods to construct recombinant plasmid pGEX-4T-2-TK, then transform it into BL21(DE3)E coli to observe its expression and prepare for the latter experiment.method: extract the plasmid of pGEX-4T-2 and pORF-HSV-tk by minipreparation plasmid DNA method and use plasmid pGEX-4T-2 to undertake 1% agarose gel electrophoresis, then the target bands were reclaimed and purified by aceticum kalium precipitation for later use. Used pORF-HSV-tk as palpate to amplicate the TK gene. When designing the PCR primers of TK, the sites of restriction endonuclease BamHⅠand SalⅠsequences were added. The product was about 1100bp. Took 10ul product to confirmed by 1% agarose gel electrophoresis after multiply PCR amplification, took the 1100bp target bands re-collected and purified. pGEX-4T-2 and TK were digested by BamHⅠand SalⅠendonuclease at the same time.After stopping enzyme digestion, pGEX-4T-2 and objective gene TK were connected by T4DNA ligase in thermostatic waterbath overnight. After added the recombinant plasmid to competence BL21(DE3)E coli and heat shock in 42℃waterbath for 2mins, then ice bath at once for 2min,at last added into the LB medium. The clones were picked up from LB agarose containing ampicillin and cultured at 200 rpm in the temperature of 37℃overnight. Selected the monoclone serial subcultivation, extracted and confirmed the recombinant plasmid.then uesd the BL21 ( DE3 ) E coli which contain recombinant plasmid of pGEX-4T-2-TK culture with GCV to obtain the treatment liquid ,add the treatment liquid into CNE-2 cell ,observe the rate of tumor inhibition.Result: The recombinant plasmid was transformed into BL21(DE3)Escherichia coli by chemical method successfully. The recombinant plasmid was extracted and digested by BamHⅠand SalⅠendonuclease, undertaking 1% agarose gel electrophoresis, the product was about 1.1Kb(TK gene) and 4.9Kb (pGEX -4T-2),the two strap were coincided with prediction.Use the recombinant plasmid as template to amplicate the TK gene by PCR.The PCR products was about 1.1Kb,coincided the size of TK gene.Recombinant plasmid GEX-4T-2-TK was sent to shanghai invitrogen company to verificate and corrected by DNA sequence analysis. Product of sepuencing were two section base sequence (854bp and 872bp). After removing sequencing primer and linking, the product was about 1124bp which analyzed by chromas software and coincidence with GENEBANK, there were no open reading frameshift and change , no gene mutation. It was suit to latter experiment . The BL21(DE3)E coli which contain recombinant plasmid of pGEX-4T-2-TK induced by IPTG could express the objective protein .The treatment liquid could inhibited the CNE-2 cells growth ,the CNE-2 cells survival rate is only 33.65%,P<0.05,have statistical significance. Conclusion: we successfully constructed the procaryon recombinant plasmid of pGEX-4T-2-TK, without shifting and changing , without gene mutation. Transfer the recombinant plasmid into BL21(DE3)E coli ,it could express the objective protein . The treatment liquid significantly reduces survival of CNE-2 cells .The products were fit to later expression study.