中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2005年
5期
493-496
,共4页
梁羽%梁德生%薛志刚%龙志高%邬玲仟%潘乾%胡艺俏%戴和平%夏昆%夏家辉
樑羽%樑德生%薛誌剛%龍誌高%鄔玲仟%潘乾%鬍藝俏%戴和平%夏昆%夏傢輝
량우%량덕생%설지강%룡지고%오령천%반건%호예초%대화평%하곤%하가휘
杜氏肌营养不良%人源载体%基因治疗%微小肌营养不良蛋白%绿色荧光蛋白
杜氏肌營養不良%人源載體%基因治療%微小肌營養不良蛋白%綠色熒光蛋白
두씨기영양불량%인원재체%기인치료%미소기영양불량단백%록색형광단백
Duchenne muscular dystrophy%human source plasmid vector%gene therapy%minidystrophin%green fluorescent protein
目的构建微小肌营养不良蛋白(minidystrophin)和增强绿色荧光蛋白(enhanced green fluoresce protein, EGFP)融合基因的人源载体,观察该载体在Cos-7细胞中的表达.方法以正常人肌营养不良基因cDNA(GenBank NM004006)为模板,通过PCR克隆的方法构建minidystrophin基因,融合EGFP基因后连接到人源载体pHrneo,大量提取重组质粒,转染Cos-7细胞,通过逆转录聚合酶链反应、荧光显微镜观察等方法检测该载体在细胞内的表达.结果成功构建pHrnDysG载体,转染Cos-7后,逆转录聚合酶链反应可扩增出735 bp特异条带,荧光显微镜观察可见表达蛋白分布于细胞膜上.结论 pHrn载体介导的minidystrophin基因可以在真核细胞表达,并被有效地转运至细胞膜,可望用于杜氏肌营养不良基因治疗的研究.
目的構建微小肌營養不良蛋白(minidystrophin)和增彊綠色熒光蛋白(enhanced green fluoresce protein, EGFP)融閤基因的人源載體,觀察該載體在Cos-7細胞中的錶達.方法以正常人肌營養不良基因cDNA(GenBank NM004006)為模闆,通過PCR剋隆的方法構建minidystrophin基因,融閤EGFP基因後連接到人源載體pHrneo,大量提取重組質粒,轉染Cos-7細胞,通過逆轉錄聚閤酶鏈反應、熒光顯微鏡觀察等方法檢測該載體在細胞內的錶達.結果成功構建pHrnDysG載體,轉染Cos-7後,逆轉錄聚閤酶鏈反應可擴增齣735 bp特異條帶,熒光顯微鏡觀察可見錶達蛋白分佈于細胞膜上.結論 pHrn載體介導的minidystrophin基因可以在真覈細胞錶達,併被有效地轉運至細胞膜,可望用于杜氏肌營養不良基因治療的研究.
목적구건미소기영양불량단백(minidystrophin)화증강록색형광단백(enhanced green fluoresce protein, EGFP)융합기인적인원재체,관찰해재체재Cos-7세포중적표체.방법이정상인기영양불량기인cDNA(GenBank NM004006)위모판,통과PCR극륭적방법구건minidystrophin기인,융합EGFP기인후련접도인원재체pHrneo,대량제취중조질립,전염Cos-7세포,통과역전록취합매련반응、형광현미경관찰등방법검측해재체재세포내적표체.결과성공구건pHrnDysG재체,전염Cos-7후,역전록취합매련반응가확증출735 bp특이조대,형광현미경관찰가견표체단백분포우세포막상.결론 pHrn재체개도적minidystrophin기인가이재진핵세포표체,병피유효지전운지세포막,가망용우두씨기영양불량기인치료적연구.
Objective To construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells. Methods The recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine. Results Restrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane. Conclusion The minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.
