The Construction Of NM23-pIRES2-EGFP Plasmids And Identification

Objective: To construct eukaryotic expression vector NM23-p IRES2-EGFP,and to identify the accuracy of enzyme-cut and DNA sequencing respectively,to futher explore NM23 gene and provide experimental basis for the apoptosis and metastasis of gastric carcinoma cells.Methods: 1.The TRIZOL method is used to extract total RNA from human nor mal gastric mucosa,and the NM23 gene is amplified by RT-PCR,and the CDs se quence of NM23 gene is obtained.Agarose gel electrophoresis identification,the re covery of NM23 genes after tapping purified.2.Connection with p MD18-T-simple cloning carrier,obtained p MD18-T-simple-NM23,bacterial clone after sequencing,t he sequencing results are consistent with the NM23 gene sequences reported by Ge nebank.3.NM23 gene and the carrier pl RES2-EGFP were connected with enzymecut processing,and the expression vector NM23-p IRES2-EGFP was constructed,an d the accuracy of recombinant plasmid was detected by PCR,enzyme-cut and DN A sequencing.Results: 1.NM23 gene was amplified from human normal gastric mucosa.Agarose gel electrophoresis showed RT-PCR products in the strip 500-750 bp,with the theoretical value of the literature 534 bp.2.PMD18-T-simple connection with the clone carrier,the sequencing identification is identical to the CDs region sequence of the NM23 gene recorded by the Genebank database.3.Using gene cloning technology to build NM23-p IRES2-EGFP eukaryotic expression vector,through PCR technology,enzyme-cut reaction and DNA sequencing confirmed that the inserted fragment and the theoretical value of the literature,the true nuclear expression vector NM23-p IRES2-EGFP build successfully.Conclusion: 1.A 534bp-size NM23 gene can be amplified from human normal gastric mucosal tissues.2.The plasmid NM23-p IRES2-EGFP was successfully const ructed and the accuracy of the recombinant plasmid was confirmed.3.The plasmid NM23-p IRES2-EGFP was successfully constructed and the accuracy of recombinan t plasmid was Confirmed by PCR,Xho I/Bam HI double enzyme-cut and DNA sequ encing.