摘要
目的:构建肝肠钙黏连蛋白(CDH17)基因pcDNA3.1(-)真核表达质粒,为进一步研究胃癌发病的分子机制和生物学行为以及寻找新的抗转移措施奠定理论基础。方法:用Trizol Reagent抽提胃腺癌组织中总RNA,采用逆转录巢式PCR方法扩增CDH17目的片段,双酶切纯化PCR产物及pcDNA3.1(-),再将CDH17基因片断插入pcDNA3.1(-)线性质粒,即构建成CDH17/pcDNA3.1(-)真核细胞表达质粒,将质粒转染感受态细胞DH5a,筛选阳性克隆行双酶切鉴定及DNA测序鉴定。结果:DNA测序结果与预期目的片段序列一致。结论:CDH17/pcDNA3.1(-)真核细胞表达质粒构建成功。
Objective： To construct Eukaryotic expression Plasmid ofpcDNA3.1 （-）/CDH17. Methods： Trizol reagent was used to extract total RNA in gastric adenocarcinoma of human. The proper nested primers of CDH17 were choosed and synthesized. RT-nest-PCR and gene recombinant techniques were used to construct the fragment of CDH17.After purification, The PCR products of CDH 17 and eukaryotic expressing plasmid pcDNA3.1 （-） were cut and CDH 17 were inserted reversely into pcDNA3.1 （-）,and then transferred into DH5α strain and positive clone was selected. Enzyme-cutting and PCR indentification and DNA autosequencing were used to prove the successful construction of CDH 17 eukaryotic expressing plasmid. Results： Sequence analysis showed the same sequence as expected. Conclusion： Construction of CDH 17/pcDNA3.1 （-） eukaryotic expressing plasmids was successful.
出处
《现代生物医学进展》
CAS
2010年第6期1103-1105,1109,共4页
Progress in Modern Biomedicine
