pcDNA6/V5-His A, B, and C are 5.1 kb vectors derived from pcDNA3.1(+) and designed for high-level expression, selection on blasticidin, purification, and detection of recombinant proteins in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells. pcDNA6/V5-His A, B, and C 含有以下元件: ? Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells
? Three reading frames to facilitate in-frame cloning with a C-terminal tag encoding the V5 epitope and a polyhistidine metal-binding peptide
? Blasticidin resistance gene (BSD) from Aspergillus terreus for selection in prokaryotic and eukaryotic cells (Kimura et al., 1994)
? Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS7)
The control plasmid, pcDNA6/V5-His/lacZ, is the pcDNA6/V5-His A vector with a 3.2 kb fragment containing the β-galactosidase gene cloned in frame with the C-terminal peptide (see page 11). It is included for use as a positive control for transfection, expression, purification, and detection in the cell line of choice. 實(shí)驗(yàn)流程 使用pcDNA.6/V5-His 載體表達(dá)目的基因的實(shí)驗(yàn)流程： 1. Consult the multiple cloning sites described on pages 3–5 to determine which vector (A, B, or C) should be used to clone your gene in frame with the C-terminal V5 epitope and polyhistidine tag.
2. Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 100 μg/mL blasticidin or 50 to 100 μg/mL ampicillin.
3. Analyze your transformants for the presence of insert by restriction digestion.
4. Select a transformant with the correct restriction pattern and confirm that your gene is in frame with the C-terminal peptide by sequencing.
5. Transfect your construct into the cell line of choice using your own method of transfection.
6. Test for expression of your recombinant gene by western blot analysis or functional assay. If you do not have an antibody to your protein, Life Technologies offers the Anti-V5 Antibody or the Anti-His(C-term) Antibody to detect your recombinant fusion protein.
7. To purify your recombinant fusion protein, you may use metal-chelating resin such as ProBond.. ProBond. resin is available separately.