■ 基本信息
啟動子: | RSV/PGK |
復(fù)制子: | M13 |
終止子: | SV40 poly(A) signal |
質(zhì)粒分類: | 病毒系列，慢病毒雙表達(dá)載體 |
質(zhì)粒大小: | 8292bp |
原核抗性: | Kan |
真核抗性: | Puro |
克隆菌株: | Stbl3 |
培養(yǎng)條件: | 37℃ |
表達(dá)宿主: | 哺乳細(xì)胞 |
誘導(dǎo)方式: | 無須誘導(dǎo)，瞬時(shí)表達(dá) |
5'測序引物:
| 根據(jù)序列設(shè)計(jì)引物
|
3'測序引物: | 根據(jù)序列設(shè)計(jì)引物 |
■ 質(zhì)粒屬性
質(zhì)粒宿主: | 哺乳細(xì)胞,慢病毒 |
質(zhì)粒用途: | 蛋白表達(dá) |
片段類型: | 基因CDS
|
片段物種: | 雙框空載體
|
原核抗性: | 卡那霉素 |
真核抗性: | 嘌呤霉素
|
熒光標(biāo)記: |
|
■ 質(zhì)粒簡介
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.
■ 質(zhì)粒圖譜
■ 質(zhì)粒序列
質(zhì)粒序列請下載：
ZK808pLenti-Bi-cistronic慢病毒雙框表達(dá)質(zhì)粒.txt
