CAJ | 학술논문

目的构建人视网膜母细胞瘤(retinoblastoma 1,RB-1)基因真核表达载体,并在前列腺癌细胞株PC-3中表达,为进一步研究其在前列腺癌发病中的作用和机制打下基础.方法采用PCR方法扩增RB-1基因编码区的全长序列,并加上FLAG标签,利用分子克隆技术将其重组于CMV载体中.利用酶切、序列分析鉴定克隆正确性.转染PC-3细胞,用Western Blot检测其表达,用免疫荧光检测其在细胞中的定位.结果克隆的RB-1真核表达载体完全正确,Western Blot检测到RB-1有表达,免疫荧光检测其主要定位于细胞核内.结论成功地构建了RB-1真核表达载体,其能够在PC-3中高效表达,并主要定位于细胞核内.
목적 구건인시망막모세포류(retinoblastoma 1,RB-1)기인진핵표체재체,병재전렬선암세포주PC-3중표체,위진일보연구기재전렬선암발병중적작용화궤제타하기출.방법 채용PCR방법 확증RB-1기인편마구적전장서렬,병가상FLAG표첨,이용분자극륭기술장기중조우CMV재체중.이용매절、서렬분석감정극륭정학성.전염PC-3세포,용Western Blot검측기표체,용면역형광검측기재세포중적정위.결과 극륭적RB-1진핵표체재체완전정학,Western Blot검측도RB-1유표체,면역형광검측기주요정위우세포핵내.결론 성공지구건료RB-1진핵표체재체,기능구재PC-3중고효표체,병주요정위우세포핵내.
Objective To construct an eukaryotic recombinant expression vector for retinoblastoma 1 gene (RB-1) and investigate the role of RB-1 in prostate cancer. Methods The coding sequence of RB-1 gene tagged with FLAG was amplified from the plasmid CMV-RB by PCR method. The fragment was cloned into CMV expression vector and identified by restriction enzyme digestion and sequence analysis. Western Blotting was used to detect RB-1 expression and immunofluorescence was used to observe RB-1 distribution in PC-3 cells transfected with the recombinant. Results The expression vector CMV-FLAG-RB was successfully constructed as confirmed by PCR, endonuclease digestion and DNA sequence analysis. RB-1 protein was highly expressed and showed a nuclear distribution in PC-3 cells transfected with the recombinant. Conclutions The eukaryotic expression vector for RB-1 has been successfully constructed and can be efficiently expressed in PC-3 cells. The expression of RB-1 is located in the cell nuclei.