人碱性成纤维细胞生长因子真核表达质粒的构建及其在犬牙龈成纤维细胞中的表达  

Construction of hbFGF eukaryotic expression plasmid and its expression in gingival fibroblasts of Beagle

作　　者：陈欣戬[1] 李艳芬[1] 钟泉[1] 赵欣[1] 江一平[2] 闫福华[1] 

机构地区：[1]福建医科大学附属口腔医院牙周科,福建福州350002 [2]福福建医科大学细胞与发育工程研究中心,建福州350004

出　　处：《牙体牙髓牙周病学杂志》2009年第6期318-322,共5页Chinese Journal of Conservative Dentistry

基　　金：福建医科大学科学研究发展基金(XZ04011);福建医科大学附属口腔医院重点学科建设基金(NO2008-24)

摘　　要：目的:构建pIRES2-EGFP-bFGF真核表达质粒并检测其在Beagle犬牙龈成纤维细胞(GFs)中的表达。方法:双酶切获得bFGF片段并克隆到真核表达载体pIRES2-EGFP中,HindⅢ单酶切、序列分析鉴定获得的质粒;脂质体介导转染Beagle犬GFs;免疫组织化学,RT-PCR,ELISA检测bFGF基因的表达。结果:bFGF成功插入真核表达载体pIRES2-EGFP中,转染GFs 12 h后即可观察到转染的GFs表达绿色荧光蛋白,48 h后转染效率达到20%。免疫组织化学,RT-PCR,ELISA证实重组pIRES2-EGFP-bFGF在GFs中的表达。结论:成功构建真核表达质粒pIRES2-EGFP-bFGF,并可在GFs中表达。AIM： To construct a recombinant eukaryotic expression plasmid plRES2 -EGFP- bFGF and screen its expression in GFs of Beagle. METHODS ： Complete sequence of human bFGF gene was cloned into the respective restriction enzyme sites of EcoR I and Xba I of pIRES2 - EGFP named plRES2 - EGFP - bFGF. Recombinant plasmid plRES2 - EGFP - bFGF and vector plRES2 - EGFP were transfected into GFs of Beagle respectively. Expres- sion of bFGF in GFs was identified by RT - PCR, ELISA and immunohistochemistry. RESULTS ： Human bFGF gene was inserted into vector plRES2 -EGFP correctly. 12 h after transfection, green fluorescence could be detected in GFs. The efficiency of transfeetion was nearly 20% in 48 h. RT - PCR, ELISA and immunohistochemistry showed that the bFGF was only expressed in GFs which was transfeeted with pIRES2 -EGFP -bFGF, but not in GFs which was transfected with plRES2 - EGFP. CONCLUSION： Human bFGF gene recombinant eukaryotic expression plasmid was constructed successfully, bFGF gene can express in GFs of Beagle by transfeting recombinant plasmid into cell.

关 键 词：碱性成纤维细胞因子 基因 真核细胞 成纤维细胞 

分 类 号：R780.2[医药卫生—口腔医学]