重叠PCR法构建宫颈癌相关的EGFR基因G719S和T790M融合重组载体

摘要

Objective To construct the recombinant pMD19-exon18-exon20 plasmid containing locus G719S and T790M of EGFR gene associated with cervical cancer,which may provide a template for preparing the mutant recombinant vector of EGFR gene.Methods Using the healthy human genome DNA as templates,the segments of exon 18 and exon 20 of EGFR gene were amplified by two pairs of specific primers which were designed based on the sequences of overlapping and complementary area.The amplified segments were linked by overlap PCR.The products of linked exon18-exon20 were further inserted into the vector pMD19-T.The constructed recombi nant pMD19-exon18-exon20 plasmid was finally transformed into competent cells E.coli DH5α and then identified by PCR with bacterial solution and genome sequencing.Results The amplified fragments of exon18 and exon20 were clearly appeared at 778 bp and 596 bp and the fused product of exon18-exon20 was showed at 1 374 bp on agarose gel electrophoresis.The recombinant plasmid of fusion EGFR gene was consistent with the expected results via bacterial PCR assay and DNA sequencing.Conclusion We successfully fused the segments of exon18 with exon20 and constructed the recombinant expression vector of EGFR gene by using overlap PCR method.%目的 构建包含与宫颈癌相关的EGFR基因G719S和T790M位点重组pMD19-exon18-exon20载体,为制备突变型EGFR基因重组载体提供模板.方法 以健康人全血基因组DNA作为模板,设计2对有重叠互补区的特异性引物,对EGFR基因的exon 18和exon20两片段分别进行扩增,用重叠PCR技术将exon 18和exon20片段进行连接,再将连接产物exon1 8-exon20片段插入pMD19-T质粒,构建成野生型pMD19-exon1 8-exon20载体,转入至大肠埃希菌感受态细胞DH5α中进行表达,利用菌液PCR和基因组测序进行鉴定.结果 琼脂糖凝胶电泳结果显示,exon18和exon20两位点扩增片段在778 bp和596 bp处有清晰的目的条带,两片段的融合产物exon1 8-exon20片段可在1 374 bp处见一清晰目的条带;经菌液PCR和基因组测序结果鉴定,EGFR基因融合重组质粒均与预期结果一致.结论 利用重叠PCR法将exon 18和exon20片段进行融合,且成功构建了EGFR基因重组表达载体.