EGFR基因G719S和T790M突变载体的构建及临床应用研究

Construction of EGFR gene G719S and T790M mutation vector and preliminary clinical application

目的构建与宫颈癌组织表皮生长因子受体(epidermal growth factor receptor,EGFR)基因G719S和T790M位点突变型重组载体,利用其建立分子开关平台检测宫颈癌EGFR基因突变。方法以野生型重组质粒为模板,利用重叠PCR技术,得到突变型融合目的 DNA片段,再将此目的片段连接入p MD19-T质粒中,构建成突变型重组载体,将其转化入大肠埃希菌E.coli DH5α感受态细胞进行表达,用菌液PCR和基因组测序进行鉴定。设计特异性检测引物,建立分子开关检测平台用于临床宫颈癌样本的检测。结果通过基因组测序证实G719S和T790M突变位点成功引入,定点突变载体构建成功。成功建立了分子开关检测平台用于宫颈癌组织DNA的检测。结论利用重叠PCR技术简便、高效地构建了EGFR基因突变重组载体,并建立了分子开关检测平台,为基因定点突变及临床上检测EGFR基因突变提供了新的技术手段。

Objective To construct mutant recombinant vector of epidermal growth factor receptor（ EGFR） gene G719 S and T790 M sites associated with cervical cancer,lay the foundation for the detection of EGFR gene mutation in cervical cancer. And using it to establish a molecular switch platform to detect cervical cancer EGFR gene mutations. Methods Using the wild-type recombinant plasmid as template,the mutant fusion target fragment were amplified by overlap PCR,then connect this target fragment into the vector p MD19-T. The constructed mutant recombinant plasmid was finally transformed into competent cells E.coli DH5α further identified by PCR with bacterial solution and genome sequencing. Establishing the molecular switch for the detection of clinical cervical cancer samples. Results The G719 S and T790 M mutations were successfully certified by genome sequencing,and the site-directed mutant vector was successfully constructed. In addition,a molecular switch detection platform was also successfully established for the detection of cervical cancer tissue DNA. Conclusion We successfully constructed an EGFR gene mutant recombinant vector by overlap PCR technique,which providing a new technical means for gene site-directed mutagenesis. And the molecular switch detection platform was successfully established based on it,which furnishing a new method for clinical detection of EGFR gene mutations.