CAJ | 학술논문

目的克隆人Importin 8(IPO8)基因,观察IPO8与绿色荧光蛋白(GFP)真核表达载体融合蛋白在宫颈癌细胞HeLa内的表达和定位.方法以HeLa细胞总RNA为模板进行逆转录-聚合酶链反应(RT-PCR),扩增全长IPO8编码序列,经Hind Ⅲ和Xba Ⅰ双酶切后,插入GFP质粒pEGFP-C1,构建pEGFP-IPO8重组表达载体.瞬时转染重组质粒入HeLa细胞,荧光显微镜观察pEGFP-IPO8在细胞内的表达与定位.结果测序结果显示经PCR扩增的IPO8编码序列完全正确,酶切显示重组质粒pEGFP-IPO8构建成功,荧光显微镜显示融合蛋白GFP-IPO8主要在细胞核内分布.结论成功克隆IPO8基因并构建真核表达载体,证实GFP-IPO8蛋白主要定位于HeLa细胞核.
목적 극륭인Importin 8(IPO8)기인,관찰IPO8여록색형광단백(GFP)진핵표체재체융합단백재궁경암세포HeLa내적표체화정위.방법 이HeLa세포총RNA위모판진행역전록-취합매련반응(RT-PCR),확증전장IPO8편마서렬,경Hind Ⅲ화Xba Ⅰ쌍매절후,삽입GFP질립pEGFP-C1,구건pEGFP-IPO8중조표체재체.순시전염중조질립입HeLa세포,형광현미경관찰pEGFP-IPO8재세포내적표체여정위.결과 측서결과현시경PCR확증적IPO8편마서렬완전정학,매절현시중조질립pEGFP-IPO8구건성공,형광현미경현시융합단백GFP-IPO8주요재세포핵내분포.결론 성공극륭IPO8기인병구건진핵표체재체,증실GFP-IPO8단백주요정위우HeLa세포핵.
Objective To clone the coding sequence of Importin 8 (IPO8) cDNA from HeLa cells and construct a eukaryotic expression vector for its green fluorescent protein (GFP)-IPO8 fusion protein expression in HeLa cells.Methods The total RNA was extracted from HeLa cells and the full length cDNA of IPO8 was amplified using reverse transcription-polymerase chain reaction (RT-PCR),and cloned into green fluorescence protein vector phosphorylated enhanced green fluorescent protein (pEGFP)-C1 to construct recombinant expression plasmid pEGFP-IPO8.After recombinant plasmids were transfected into HeLa cells,the expression of IPO8 was observed by fluorescent microscope.Results DNA sequence analysis showed that the amplified rat IPO8 gene was in concordance with that published on Gene Bank.The results of enzyme digestion and sequence analysis demonstrated that the recombinant plasmid pEGFP-IPO8 was constructed successfully.Fluorescence microscopy revealed the fusion protein GFP-IPO8 was mainly located in the nucleus of HeLa cells.Conclusion The IPO8 gene was cloned successfully and the recombinant plasmid were constructed successfully.The fusion protein GFP-IPO8 was demonstrated to be mainly located in the nucleus of HeLa cells,which provides a basis for biological functional study of IPO8 gene.