重叠PCR法构建宫颈癌相关的EGFR基因G719S和T790M融合重组载体  

Construction of fused recombinant vector of EGFR gene containing G719S and T790M by overlap PCR

作　　者：董巍檑[1] 向花花 彭华[2] 龚咏晴 周晶[2] 张宏全 郭紫芬[2] DONG Weilei1, XIANG Huahua2, PENG Hua2, GONG Yongqing2, ZHOU Jing2, ZHANG Hongquanz, GUO Zifen2(1. Department of Obstetrics and Gynecology, The First Affiliated Hospital of University of South China; Hengyang 421001, Hunan ; 2. Institute of Phar- maW and Pharmacology, University of South China, Cooperative Innovation Center for Molecular Target New Drug Study of Hunan Prov- ince, Hengyang 421001, Hunan, Chin)

机构地区：[1]南华大学附属第一医院妇产科,湖南衡阳421001 [2]南华大学药物药理研究所,湖南省分子靶标新药研究协同创新中心,湖南衡阳421001

出　　处：《临床检验杂志》2018年第2期139-141,147,共4页Chinese Journal of Clinical Laboratory Science

基　　金：湖南省卫计委科研计划课题(C2016083)

摘　　要：目的构建包含与宫颈癌相关的EGFR基因G719S和T790M位点重组p MD19-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exon18和exon20两片段分别进行扩增,用重叠PCR技术将exon18和exon20片段进行连接,再将连接产物exon18-exon20片段插入p MD19-T质粒,构建成野生型p MD19-exon18-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exon18-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exon18和exon20片段进行融合,且成功构建了EGFR基因重组表达载体。Objective To construct the recombinant p MD19-exon18-exon20 plasmid containing locus G719 S and T790 M 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 p MD19-T. The constructed recombinant p MD19-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.

关 键 词：EGFR基因 载体构建 宫颈癌 重叠PCR 

分 类 号：Q782[生物学—分子生物学]