Construction And Application Of Recombinant Plasmid Containing Exon 19 And Exon 21 Of EGFR Gene

Objective:Construction of quality control products and standards for detection of EGFR gene mutation.Method:From the clone of healthy human lymphocyte genome,four pairs of primers were designed to introduce mutant loci,RNA reverse transcriptional cDNA,and the common PCR method was used to amplify the exon 19 fragment of the exon 19 of the wild type EGFR gene and the EGFR gene containing the deletion mutation(del E746-A750),the wild type EGFR based exon 21 and the EGFR based on the point mutation(L858R).The recombinant plasmid containing exon 19 and exon 21 of EGFR gene was constructed to detect the quality control and standard of EGFR gene mutation due to the exon 21 fragment and cloned into the carrier pEGFP-C1 respectively.Results:The recombinant plasmid containing exon 19 and exon 21 of the EGFR gene was constructed.The wild recombinant plasmid was named pEGFP-C1-EGFR-19 and pEGFP-C1-EGFR-21,the mutant recombinant plasmid pEGFP-C1-EGFR-19(del E746-A750)and pEGFP-C1-EGFR-21(L858R).The recombinant plasmid was successfully constructed by enzyme digestion and sequencing.The concentration,purity and stability reached the quality.The requirements of control and standard products.Conclusion:The positive and negative control plasmids were successfully constructed for subsequent construction of EGFR gene mutation detection methods.