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.
出处
《牙体牙髓牙周病学杂志》
CAS
北大核心
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)
