人FL基因高效真核表达载体的构建及其在COS-7细胞中的表达  

Construction of a human Flt3-ligand gene containing eukaryotic expressing vector and its expression in COS-7 cells

作　　者：王冠军[1] 马俐君[1] 李梁[2] 裴雪涛[2] 

机构地区：[1]白求恩医科大学第一临床医学院血液肿瘤科,长春130021 [2]北京军事医学科学院输血研究所,北京100850

出　　处：《中国免疫学杂志》2001年第6期305-308,共4页Chinese Journal of Immunology

基　　金：国家重点基础研究发展规划 ( 973 )项目!(G19990 5 3 90 3 )资助

摘　　要：目的 :构建人FL真核表达载体 pIRES1neo/hFL ,观察其在COS 7细胞中的表达。方法 :采用RT PCR方法自人白血病细胞系TF 1中克隆可溶型FL(Flt3 ligand)cDNA片段 ,测序鉴定正确后 ,插入真核表达载体 pIRES1neo中的EcoRI和BamHI位点 ,构建hFL真核表达载体 pIRES1neo/hFL。脂质体介导法将其转染COS 7细胞 ,72h以RT PCR检测转染细胞中外源hFL基因的转录、ELISA法及脐血CD34 + 细胞增殖实验测定转染细胞上清中hFL的含量和活性。结果 :酶切鉴定表明成功构建了重组真核表达载体 pIRES1neo/hFL ;外源hFL基因能在转染细胞中有效转录 ;ELISA法测得 72h后培养上清中的hFL含量为每 2 4小时 2 5 1ng/ 10 6cells,并且分泌的hFL具有良好的生物学活性。结论 :构建hFL真核表达载体在COS 7细胞中具有良好的表达活性。Objective:To construct an eukaryotic expressing vector pIRES1neo/hFL and express hFL in COS 7 cell.Methods:The cDNA encoding soluble human Flt3 ligand(FL) was obtained by RT PCR from the TF 1 cell lines and was inserted into eukaryotic expressing vector pIRES1neo between EcoR I and BamH I sites after sequencing,and then COS 7 cells were transfected with the recombinant by liposome.The transcription and expression of hFL in the transfected COS 7 cells were assayed by RT PCR,ELISA and the experiment of the human umbilical blood CD34 + cell multiplication,respectively,at 72 hours after transfection.Results:It showed that hFL cDNA cloned in our laboratory was 546 bp in length encoding soluble human FL which was in accordance with the report previously.FL gene was transcripted in transfectants,and FL protein with obvious biological activity was highly expressed with 251 ng/(10 6 cell·d) in the supernatant of the transfectants.Conclusion:Human FL in the recombinant vector is proved to be expressed in COS 7 cells and obvious biological activity of hFL in the supernatant of the transfectants was detected.

关 键 词：FL 真核表达载体 COS-7细胞 表达活性 生物学活性 专职抗原递呈细胞 APC 

分 类 号：R392.12[医药卫生—免疫学]