載體描述 pcDNA4/myc-His A, B, and C are 5.1 kb vectors designed for overproduction of recombinant proteins in mammalian cell lines. Features of the vectors allow purification and detection of expressed proteins (see pages 11-12 for more information). High-level stable and transient expression can be carried out in most mammalian cells. The vectors contain the following elements:
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 peptide encoding the myc (c-myc) epitope and a polyhistidine (6xHis) metal-binding tag
Zeocin resistance gene for selection of stable cell lines (Mulsant et al., 1988) (see page 14 for more information).
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, pcDNA4/myc-His/lacZ is included for use as a positive control for transfection, expression, and detection in the cell line of choice. 實驗流程： Use the following outline to clone and express your gene of interest in pcDNA4/myc-His:
1.Consult the multiple cloning sites described on pages 3-4 to determine which vector (A, B, or C) to use for cloning your gene in frame with the C-terminal myc epitope and the polyhistidine tag.
2.Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 50 to 100 μg/mL ampicillin or 25 to 50g/mL Zeocin in Low Salt LB. For more information.
3.Analyze your transformants for the presence of insert by restriction digestion.
4.Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in-frame with the C-terminal peptide.
5.Transfect your construct into the cell line of choice using your own method of transfection. Generate a stable cell line, if desired.
6.Test for expression of your recombinant gene by western blot analysis or functional assay. For antibodies to the myc epitope or the C-terminal polyhistidine tag.
7.To purify your recombinant protein, you may use metal-chelating resin such as ProBond. ProBond resin is available separately
