真核表达载体pcDNA3.1(+)-GFP-M2的构建及在小鼠骨髓树突状细胞中的表达  

Construction of Eukaryotic Expression Vector pcDNA3.1(+)-GFP-M2 and its Expression in Mouse Marrow-Derived Dendritic Cell

作　　者：曹荣月[1] 孙翁 陈檬[1] 杨梦琪[1] 宦晓军 肖芳[1] 李盼[1] 龙军[2] 

机构地区：[1]中国药科大学生命科学与技术学院微基因药物实验室,江苏南京210009 [2]南京中医药大学,江苏南京210023

出　　处：《药物生物技术》2014年第4期283-286,共4页Pharmaceutical Biotechnology

基　　金：中国国家自然科学基金委员会(Grant No.31270985);国家基础科学人才培养基金(No.J1030830);国家自然科学基金资助项目(No.81373232);江苏高校优势学科建设工程资助项目

摘　　要：为了构建含有绿色荧光蛋白(Green fluorescent protein,GFP)报告基因的M2真核表达载体pcDNA3.1(+)-GFP-M2(2段热休克蛋白70表位序列407-426,mHSP70407-426,M2),探讨M2基因转染小鼠骨髓树突状细胞(Dendritic cell,DC)的表达情况。采用4轮PCR方法从pcDNA3.1(+)-GFP载体中将GFP编码序列扩增出来,并在其上游引入Kozak序列,下游引入M2序列,使用限制性内切酶HindIII和BamHI将目的片段插入到pcDNA3.1(+)中,利用质粒自带的氨苄青霉素抗性筛选出阳性克隆,获得重组基因的表达载体。重组载体通过转染试剂Lipofectamine2 000转染至DC中,通过倒置荧光显微镜观察绿色荧光蛋白在细胞内的表达。构建的pcDNA3.1(+)-GFP-M2经双酶切、PCR和测序鉴定和预期结果一致。荧光显微镜观察显示在转染pcDNA3.1(+)-GFP-M2基因的树突状细胞中,荧光均匀地分布于整个细胞。成功构建含有绿色荧光蛋白报告基因的M2真核表达载体pcDNA3.1(+)-GFP-M2。M2基因成功转染至树突状细胞中。To construct the eukaryotic expression vector pcDNA3. 1（ ＋）-GFP-M2 containing green fluorescent protein（ GFP） as a report gene and two tandem repeats of 407-426 peptide from microbial heat shock protein 70 （2mHSP70407-426,M2）,and to explore its expression in marrow-derived dendritic cell（ DC）,GFP sequence was amplified by four rounds of polymerase chain reaction （PCR） from pcDNA3.1（＋）-GFP vector with simultaneously being attached Kozak sequence in the upstream and M2 sequence in the downstream. And the interest gene fragment was inserted into eukaryotic expression vector pcDNA3.1（ ＋） through the restriction enzymes HindIII and BamHI,then the recombinant vector was acquired after screening positive clones through selective medium.The positive clones were transformed into DC cells with transfect reagent LipofectamineTM 2000,and the expression was observed under inverted fluorescent microscope. The recombinant vector was identified to be correct by enzyme digestion,PCR and sequencing analysis. Green fluorescence was seen homogeneously distributed throughout the cell transfected by the recombinant vector pcDNA3.1（＋）-GFP-M2. pcDNA3.1（＋）-GFP-M2 was successfully constructed and it could be transfected into DC.

关 键 词：真核表达载体 基因转染 树突状细胞 mHSP70407-426 绿色荧光蛋白 KOZAK序列 

分 类 号：Q812[生物学—生物工程]