CAJ | 학술논문

目的：对大鼠抑癌基因Mob1 a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cDNA。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cDNA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pCDNA3．1－HA载体酶切后进行连接。转染HEK293 T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pCDNA3．1－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Mob1 a基因并且成功在体外表达该蛋白。
목적：대대서억암기인Mob1 a진행기인확증，체외표체재체구건병차체외표체，위료진일보연구대서암증발생분자궤제제공지지。방법채용RNA제취기술획득총RNA후，진행체외반전득도cDNA。이용의거대서Mob1a기인서렬설계합성적일대특이성인물，이대서cDNA위모판，RT－PCR확증득도료예기대소적DNA편단。이용NheⅠ화HindⅢ한제성매절위점장해목적DNA편단여pCDNA3．1－HA재체매절후진행련접。전염HEK293 T세포계，획득총단백후통과Western Blot분석검측해기인재체외시부성공표체。결과통과RT－PCR방법확증료Mob1a기인，병차장해기인극륭지pCDNA3．1－HA。 Western Blot분석현시Mob1a－HA융합단백재체외표체。결론성공극륭료Mob1 a기인병차성공재체외표체해단백。
Objective To amplify the antioncogene Mob 1a,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 Mob 1a,using rat cDNA as a template,Mob1a 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 NheⅠand HindⅢ.Western Blot assay was applied to determining whether Mob 1a was expressed in vitro after HEK293T cell transfection.Results By RT-PCR,Mob1a was amplified and cloned to pCDNA3.1-HA vector.Western Blot assay showed that Mob1a-HA was expressed in vitro.Conclusion Mob1a was successfully cloned and expressed with a HA-tag in vitro.