摘要
目的:构建人snail基因真核表达载体并鉴定。方法:使用RT-PCR法获取人snail基因全长c DNA,经Bam H I、Eco R I双酶切、连接,插入pc DNA3.1(+)真核表达载体,转化TOP10感受态细胞,用含氨苄青霉素的LB培养基筛选阳性克隆,提取质粒双酶切电泳及测序鉴定,瞬时转染siha细胞Western-blot从蛋白水平鉴定重组质粒在真核细胞内的表达。结果:pc DNA3.1-snail重组质粒经酶切电泳符合预期片段,测序鉴定插入片段与NCBI Gen Bank文库中人snail序列一致,重组质粒瞬时转染后snail蛋白表达量明显增高。结论:成功构建pc DNA3.1-snail重组质粒载体,为进一步探讨snail基因生物学功能奠定了基础。
Objective： To establish pc DNA3.1-snail eukaryotic recombinant expression plasmid. Methods： Human Snail gene c DNA was amplified by reverse transcription polymerase chain reaction（RT-PCR）. After digested by Bam H I,Eco R I and ligation,Snail was inserted into pc DNA3. 1（＋）eukaryotic expression vector. The positive colonies were screened and identified by single,double enzyme digest,agarose gel electrophoresis and DNA sequence analysis. pc DNA3. 1-snail plasmid was then transfected into siha cell line with Turbo Fect Transfection Reagent. Western-blot was used to detect the protein expression of snail. Results： The pc DNA3.1-snail recombinant plasmid was verified by enzyme digestion electrophoresis showed the expected fragment. The inserted sequence in pc DNA3.1（＋）-snail was the same as the sequence of snail c DNA published in NCBI Gen Bank. Western blot results validated the recombinant plasmid expressed in siha cell line efficiently. Conclusion： The eukaryotic recombinant expression plasmid of pc DNA3.1（＋）-snail was successfully constructed.
出处
《现代生物医学进展》
CAS
2017年第3期410-413,共4页
Progress in Modern Biomedicine
基金
陕西省自然科学基础研究计划项目(2014JM2-8182)
关键词
SNAIL
重组质粒
载体构建
Snail
Recombinant plasmid
Vector construction
