摘要
目的:构建一个包含人皮层肌动蛋白(cortactin)反义基因的全部功能区的重组真核表达载体pcDNA3.0/cortac-tin,为进一步研究人皮层肌动蛋白对人肝癌细胞的转移影响的研究奠定基础.方法:Trizol试剂法提取体外培养的人肝癌高转移细胞株MHCC97总RNA,经逆转录聚合酶链式反应(RT-PCR)扩增目的片段,PCR产物及真核表达载体分别双酶切后进行连接,并转化入大肠杆菌Ecoli.Dh5α扩增以获得重组载体.结果:RT-PCR获得预期大小的特异性DNA片段,经PCR、双酶切鉴定及测序证实已将人皮层肌动蛋白基因cDNA片段正确的反向插入真核表达载体中.结论:成功获得反义pcDNA3.0/cortactin重组质粒.
AIM： To construct a recombinant eukaryotic expression vector-pcDNA3. 0/cortactin containing functional region of human anti-sense cortical actin-associated protein gene （cortactin）, so as to provide a tool for the further study on the impact of cortactin in bepatocellular carcinoma （HCC） metastasis. METHODS： Total RNA was extracted by using Trizol one-step method from cell line MHCC97. CDNA was amplified using reverse transcriptase-polymerase chain reaction （RT-PCR）. Product of PCR, amplified by being transformed into E. coli Db.Sct, was inserted reversely into the eukaryotic expression vector after digestion and ligation using restriction endonucleases and ligase. RESULTS： The correct cortactin cDNA clone was obtained and it was confirmed that cortactin eDNA was inserted reversely into the eukaryotic expression vector correctly using PCR, digestion identification and sequencing. CONCLUSION： The recombinant eukaryotic expression vector pcDNA3. 0/ cortactinis successfully constructed.
出处
《第四军医大学学报》
北大核心
2006年第8期720-722,共3页
Journal of the Fourth Military Medical University
