中国实用医刊
中國實用醫刊
중국실용의간
CENTRAL PLAINS MEDICAL JOURNAL
2014年
1期
42-45
,共4页
贺杰峰%赵浩亮%田彦璋%董秀山%李辉宇%王晋喜%赵旭晔
賀傑峰%趙浩亮%田彥璋%董秀山%李輝宇%王晉喜%趙旭曄
하걸봉%조호량%전언장%동수산%리휘우%왕진희%조욱엽
可溶性CD40%绿色荧光蛋白%融合蛋白质类%基因表达%树突状细胞
可溶性CD40%綠色熒光蛋白%融閤蛋白質類%基因錶達%樹突狀細胞
가용성CD40%록색형광단백%융합단백질류%기인표체%수돌상세포
Soluble CD40%Green fluorescent protein%Fusion proteins%Gene expression%Dendritic cells
目的 构建携带鼠可溶性CD40分子(sCD40)及增强型绿色荧光蛋白(EGFP)报告基因的融合蛋白真核表达质粒pEGFP-N1/sCD40并检测其在树突状细胞(DC2.4)中的表达水平.方法 采用基因工程技术构建CD40胞外区与EGFP重组载体质粒pEGFP-N1/sCD40,经酶切及序列分析鉴定,脂质体介导转染DC2.4细胞株,荧光显微镜,荧光分光光度计及SDS-PAGE检测sCD40-EGFP融合蛋白的表达,流式细胞仪检测其活性.结果 融合基因在瞬时转染的树突状细胞中获得表达,并分泌至上清,且sCD40-EGFP融合蛋白中分子标签未影响可溶性sCD40膜外活性区的生物学活性.结论 可溶性CD40分子与EGFP融合基因载体构建成功,在树突状细胞中表达的融合蛋白具有生物活性,为进一步研究基因修饰树突状细胞诱导特异性移植免疫耐受奠定了实验基础.
目的 構建攜帶鼠可溶性CD40分子(sCD40)及增彊型綠色熒光蛋白(EGFP)報告基因的融閤蛋白真覈錶達質粒pEGFP-N1/sCD40併檢測其在樹突狀細胞(DC2.4)中的錶達水平.方法 採用基因工程技術構建CD40胞外區與EGFP重組載體質粒pEGFP-N1/sCD40,經酶切及序列分析鑒定,脂質體介導轉染DC2.4細胞株,熒光顯微鏡,熒光分光光度計及SDS-PAGE檢測sCD40-EGFP融閤蛋白的錶達,流式細胞儀檢測其活性.結果 融閤基因在瞬時轉染的樹突狀細胞中穫得錶達,併分泌至上清,且sCD40-EGFP融閤蛋白中分子標籤未影響可溶性sCD40膜外活性區的生物學活性.結論 可溶性CD40分子與EGFP融閤基因載體構建成功,在樹突狀細胞中錶達的融閤蛋白具有生物活性,為進一步研究基因脩飾樹突狀細胞誘導特異性移植免疫耐受奠定瞭實驗基礎.
목적 구건휴대서가용성CD40분자(sCD40)급증강형록색형광단백(EGFP)보고기인적융합단백진핵표체질립pEGFP-N1/sCD40병검측기재수돌상세포(DC2.4)중적표체수평.방법 채용기인공정기술구건CD40포외구여EGFP중조재체질립pEGFP-N1/sCD40,경매절급서렬분석감정,지질체개도전염DC2.4세포주,형광현미경,형광분광광도계급SDS-PAGE검측sCD40-EGFP융합단백적표체,류식세포의검측기활성.결과 융합기인재순시전염적수돌상세포중획득표체,병분비지상청,차sCD40-EGFP융합단백중분자표첨미영향가용성sCD40막외활성구적생물학활성.결론 가용성CD40분자여EGFP융합기인재체구건성공,재수돌상세포중표체적융합단백구유생물활성,위진일보연구기인수식수돌상세포유도특이성이식면역내수전정료실험기출.
Objective To construct a recombinant plasmid carrying enhanced green fluorescent protein(EGFP) and mouse soluble CD40 gene(sCD 40) and detect its expression levels in dendritic Cells (DC 2.4).Methods The standard cloning technology was employed to construct the EGFP/ VEGF fusion gene which was identified with double enzyme digestion and DNA sequencing,then this recombinant plasmid was transfected into mouse dendritic Cells (DC2.4) with lipofectamine.The expression levels of the fusion gene were detected by fluorescence microscope,fluorescence spectrophotometer,SDS-PAGE,and flow cytomerey (FCM),respectively.Results The fusion gene was observed in transiently transfected dendritic cells.The fusion protein was secreted into supernatant.These data suggested that the EGFP tag did not interfere with the natural assembly and the biological activity of soluble CD40.Conclusions The recombinant plasmid carrying enhanced green fluorescent protein and mouse soluble CD40 gene is successfully constructed and expressed positively in rat DCs with activity.
