摘要
目的对大鼠抑癌基因Mob1a进行基因扩增,体外表达载体构建并且体外表达,为了进一步研究大鼠癌症发生分子机制提供支持。方法采用RNA提取技术获得总RNA后,进行体外反转得到c DNA。利用依据大鼠Mob1a基因序列设计合成的一对特异性引物,以大鼠c DNA为模板,RT-PCR扩增得到了预期大小的DNA片段。利用NheⅠ和HindⅢ限制性酶切位点将该目的 DNA片段与p CDNA3.1-HA载体酶切后进行连接。转染HEK293T细胞系,获得总蛋白后通过Western Blot分析检测该基因在体外是否成功表达。结果通过RT-PCR方法扩增了Mob1a基因,并且将该基因克隆至p CDNA3.1-HA。Western Blot分析显示Mob1a-HA融合蛋白在体外表达。结论成功克隆了Mob1a基因并且成功在体外表达该蛋白。
Objective To amplify the antioncogene Mobla,construct the in vitro expression vector and express in vitro in rats,which can provide the necessary support for further researches on molecular mechanisms of rat carcinogenesis. Methods RNA extracting method was used to obtain the total RNA which was reverse-transcripted in vitro to gain cDNA. Based on a pair of primers designed and synthesized from the gene sequence of rat Mobla,using rat cDNA as a template ,Mobla DNA fragments of the expected size was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. The DNA fragment was ligased to pCDNA3.1-HA vector after being digested by Nhe I and Hind m. Western Blot assay was applied to determining whether Mobla was expressed in vitro after HEK293T cell transfection. Results By RT-PCR, Mobla was amplified and cloned to pCDNA3.1-HA vector. Western Blot assay showed that Mobla-HA was expressed in vitro. Conclusion Mobla was successfully cloned and expressed with a HA-tag in vitro.
出处
《广西医学》
CAS
2014年第11期1522-1525,共4页
Guangxi Medical Journal
