Fusion of EGFP and porcine α 1,3GT genes decrease GFP expression  

作　　者：Yongxiang Zhao Jing Tang Qin Yao Yuan Zhou Huange Zhao Xiaoyun Zeng Jiaqi Shi Guorong Luo Xiaoxun Xie Sufang Zhou Zuguo Liu Xiaoling Lu Donghai Lin Jianming Liu 

机构地区：[1]Hainan Medical University [2]The First Affiliated Hospital,Guangxi Medical University [3]Zhongshan Hospital,Xiaman University [4]Department of Pharmacology,Medical College,Hunan Normal University [5]Preclinical Medical College,Guangxi Medical University [6]Department of Pathology,Arizona Cancer Center,University [7]Ophthalmology Institute,Xiaman University [8]The Key Laboratory for Chemical Biology of Fujian Province,College of Chemistry and Chemical Engneering [9]The Third xiangya Hospital, Central South Cniversity

出　　处：《Asian Pacific Journal of Tropical Medicine》2010年第12期925-929,共5页亚太热带医药杂志（英文版）

基　　金：supported in part by National Natural Scientific Foundation of China(8l072148,30760248,30660185,30000203);Program for New Century Excellent Talents in University of China(NCET-06-0761);International Cooperation Project of The Ministry of Science and Technology of the People's Republic of China(2008DFA31380);Project of Scentific Activity of Ministry of Personnel for Returnee (2007-170);Program of Science and Technology of Hunan Province(06SK4060);Program of Science and Technology of Hainan Province(070210);Program of Science and Technology of Fujian Province(2008-59-08);Project of Medical Innovation of Fujian Province(2007CX18);Program of Science and Technology of Xiamen (3502Z20084012)

摘　　要：Objective:To investigate the effect of fusion proteins expressed by the fused gene of porcineα1,3 galactosyltransierase(α1,3 GT) and enhanced green fluorescent protein(EGFP) on the green fluorescence intensity of EGFP.Methods:The fragment containingα1.3GT was firstly recovered after the pcDNA3.1-α1.3GT recombinant vector were digested with BamHl and EcoRI,and then,the resultant fragment was ligated to the pEGFP-N1 vector which was also digested with the same enzymes.The new recombinant eukaryotic expression pEGFP/a 1,3GT vector was obtained and sequenced.The pEGFP/α1,3GT was used to transfect human lung carcinoma cells A549 and HEKC 293FT,and the expression of EGFP was quantitatively analyzed by fluorescent microscope and flow cytometry.Results:The positive percentage of A549 was 80.5%,and that of 293 FT was 86.5%48 hours after the two cell lines both were transfected by pEGFP-N1.The positive percentage of A549 was 75.8%,and that of 293 FT was 81.2%48 hours after the two cell lines were transfected by pEGFP/α1.3GT.The mean fluorescence intensities of A549 transfected with pEGFP-N1 and pEGFP/α1.3GT were 1.21 and 0.956,respectively when compared with that of A549 without transfection.Meanwhile,the those of the 293FT that were transfected with pEGFP-N1 and pEGFP/αl,3GT were 7.66 and 1.00.respectively when compared with that of 293FT cells without transfection.Conclusions:These results suggested that the expression of EGFP gene fused with porcineα1,3GT gene was partly inhibited.Objective:To investigate the effect of fusion proteins expressed by the fused gene of porcineα1,3 galactosyltransierase(α1,3 GT) and enhanced green fluorescent protein(EGFP) on the green fluorescence intensity of EGFP.Methods:The fragment containingα1.3GT was firstly recovered after the pcDNA3.1-α1.3GT recombinant vector were digested with BamHl and EcoRI,and then,the resultant fragment was ligated to the pEGFP-N1 vector which was also digested with the same enzymes.The new recombinant eukaryotic expression pEGFP/a 1,3GT vector was obtained and sequenced.The pEGFP/α1,3GT was used to transfect human lung carcinoma cells A549 and HEKC 293FT,and the expression of EGFP was quantitatively analyzed by fluorescent microscope and flow cytometry.Results:The positive percentage of A549 was 80.5%,and that of 293 FT was 86.5%48 hours after the two cell lines both were transfected by pEGFP-N1.The positive percentage of A549 was 75.8%,and that of 293 FT was 81.2%48 hours after the two cell lines were transfected by pEGFP/α1.3GT.The mean fluorescence intensities of A549 transfected with pEGFP-N1 and pEGFP/α1.3GT were 1.21 and 0.956,respectively when compared with that of A549 without transfection.Meanwhile,the those of the 293FT that were transfected with pEGFP-N1 and pEGFP/αl,3GT were 7.66 and 1.00.respectively when compared with that of 293FT cells without transfection.Conclusions:These results suggested that the expression of EGFP gene fused with porcineα1,3GT gene was partly inhibited.

关 键 词：Enhanced green FLUORESCENT protein Porcineα1.3 GALACTOSYLTRANSFERASE FUSION gene Fluorescence intensity 

分 类 号：Q78[生物学—分子生物学]