■ 基本信息
启动子: | RSV/PGK |
复制子: | M13 |
终止子: | SV40 poly(A) signal |
质粒分类: | 病毒系列，慢病毒双表达载体 |
质粒大小: | 8292bp |
原核抗性: | Kan |
真核抗性: | Puro |
克隆菌株: | Stbl3 |
培养条件: | 37℃ |
表达宿主: | 哺乳细胞 |
诱导方式: | 无须诱导，瞬时表达 |
5'测序引物:
| 根据序列设计引物
|
3'测序引物: | 根据序列设计引物 |
■ 质粒属性
质粒宿主: | 哺乳细胞,慢病毒 |
质粒用途: | 蛋白表达 |
片段类型: | 基因CDS
|
片段物种: | 双框空载体
|
原核抗性: | 卡那霉素 |
真核抗性: | 嘌呤霉素
|
荧光标记: |
|
■ 质粒简介
This study was aimed to construct, package and purify the recombinant lentivirus vector carrying the firefly luciferase gene (FLUC) and red fluorescent protein gene (RFP) and to transfect the recombinant lentivirus into HeLa cells, so as to observe the expression levels of these two genes. The FLUC and RFP genes were amplified by RT-PCR and inserted in the lentiviral expression vector (pLenti-Bi-cistronic) to construct the lentiviral vector pLenti-FLUC-RFP. The viral particles were generated by cotransfection of 293T cells with pLenti-FLUC-RFP and three packaging vectors, and the virus titer was determined by calculating the percentage of RFP positive cells. After transfection of pLenti-FLUC-RFP into HeLa cells, the expression of RFP was observed by fluorescent microscopy, and the activity of FLUC was determined by luciferase reporter gene assay kit. The results showed that the inserting orientation of the RFP and FLUC genes in the lentiviral vector pLenti-FLUC-RFP were verified by restriction analysis. Targeted RFP and FLUC sequences were confirmed by DNA sequencing. The final titer obtained was 1×10TU/ml. The expressions of RFP and FLUC were observed in the transfected HeLa cells. It is concluded that the pLenti-III-FLUC-RFP recombinant lentivirus vector carrying RFP gene and FLUC gene with high viral titer is constructed and packaged successfully, and provides experimental basis for studying dynamic distribution of mesenchymal stem cells in vivo.
■ 质粒图谱
■ 质粒序列
质粒序列请下载：
ZK808pLenti-Bi-cistronic慢病毒双框表达质粒.txt
