检索规则说明:AND代表“并且”;OR代表“或者”;NOT代表“不包含”;(注意必须大写,运算符两边需空一格)
检 索 范 例 :范例一: (K=图书馆学 OR K=情报学) AND A=范并思 范例二:J=计算机应用与软件 AND (U=C++ OR U=Basic) NOT M=Visual
作 者:周晶[1] 向花花 肖正午 彭华[1] 张宏全 郭紫芬[1] ZHOU Jing;XIANG Huahua;XIAO Zhengwu;PENG Hua;ZHANG Hongquan;GUO Zifen(Institute of Pharmacy and Pharmacology, University of South China, ChinaHunan Province Cooperative innovation Center for Molecular Target New Drug Study, Hengyang 421001, Hunan, China)
机构地区:[1]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5期380-383,共4页Chinese Journal of Clinical Laboratory Science
基 金:湖南省自然科学基金(2018JJ2350)
摘 要: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.
关 键 词:宫颈癌 表皮生长因子受体基因 定点突变 重叠PCR
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在载入数据...
正在链接到云南高校图书馆文献保障联盟下载...
云南高校图书馆联盟文献共享服务平台 版权所有©
您的IP:216.73.216.63