CAJ | 학술논문

构建与增强型绿色荧光蛋白基因相连的糖基化磷脂酰肌醇(glycosyl phosphatidylinositol,GPI)序列的真核表达质粒,并检测其在A549细胞中的表达.分离人外周血淋巴细胞,提取总RNA,以RT-PCR法扩增CD24基因的243 bp GPI锚定序列,双酶切后定向克隆入pEGFP-C1质粒中,构建并鉴定pEGFP-C1-GPI质粒.经脂质体介导转染A549细胞后,在荧光显微镜下观察目的蛋白在真核细胞内的表达情况.经酶切和测序鉴定证实,所克隆的CD24 GPI序列正确,荧光显微镜观察pEGFP-C1-GPI质粒转染A549细胞可见围绕细胞膜的强绿色荧光,而对照pEGFP-C1质粒转染A549细胞仅见胞内均匀荧光.成功构建与EGFP相连的GPI真核表达质粒,且能在A549细胞膜上锚定表达EGFP-GPI融合蛋白,为构建锚定表达型肿瘤疫苗奠定基础.
구건여증강형록색형광단백기인상련적당기화린지선기순(glycosyl phosphatidylinositol,GPI)서렬적진핵표체질립,병검측기재A549세포중적표체.분리인외주혈림파세포,제취총RNA,이RT-PCR법확증CD24기인적243 bp GPI묘정서렬,쌍매절후정향극륭입pEGFP-C1질립중,구건병감정pEGFP-C1-GPI질립.경지질체개도전염A549세포후,재형광현미경하관찰목적단백재진핵세포내적표체정황.경매절화측서감정증실,소극륭적CD24 GPI서렬정학,형광현미경관찰pEGFP-C1-GPI질립전염A549세포가견위요세포막적강록색형광,이대조pEGFP-C1질립전염A549세포부견포내균균형광.성공구건여EGFP상련적GPI진핵표체질립,차능재A549세포막상묘정표체EGFP-GPI융합단백,위구건묘정표체형종류역묘전정기출.
It was to construct a eukaryotic expression plasmid of CD24 GPI gene linked with EGFP gene at its N terminal,and observe its expression in A549 cells. Lymphocytes were isolated from human peripheral blood and total RNA was extracted. The gene fragment encoding GPI of CD24 was amplified by RT-PCR using the obtained RNA as template, which was inserted into the pEGFP-Cl plasmid to construct recombinant plasmid pEGFP-Cl -GPI. Then the plasmid was transfected into A549 cells by Lipofectamine 2000, and its expression function was detected by observing the expression of enhanced green fluorescent protein ( EGFP) through fluorescent microscope. The results of restriction endonuclease analysis and DNA sequencing proved that the GPI gene fragment was correctly inserted into pEGFP-Cl plasmid. The enhanced green fluorescence was observed around the cell membrane of A549 cells transfected with pEGFP-Cl-GPI. As the pEGFP-Cl control cells,the green fluorescence was observed in the cytoplasm uniformly. Therefore,the eukaryotic expression plasmid pEGFP-Cl-GPI was constructed successfully and could express anchored EGFP around the cell membrane, which provides a basis for the construction of anchored tumor vaccine.