Mammalian expression vector pUHAB
View Patent ↗The present invention relates to the construction and utilization of a new mammalian expression vector that contains a unique multiple cloning site (MCS), designated pUHAB. The pUHAB vector comprises a high copy replication origin (ColE1), a drug resistance gene (TK-Hygromycin), and a human cytomegalovirus promoter operably associated with a unique intron (hCMV/intron). Further, pUHAB comprises a selectable marker conferring resistance to kanamycin in bacterial cells, and a phage f1(+) region. pUHAB can be used to transiently or stably express cloned genes when transfected into mammalian cells. The invention also encompasses kits and host cells and cell lines comprising pUHAB, and methods of producing a recombinant protein using pUHAB.
1. An isolated vector comprising a multiple cloning site comprising the nucleotide sequence set forth in SEQ ID NO: 2.
2. The vector of claim 1 , comprising a promoter located upstream of or within the multiple cloning site.
3. The vector of claim 2 , wherein the promoter is human cytomegalovirus (hCMV) promoter.
4. The vector of claim 2 , wherein said promoter is operably associated with an intron that enhances expression from said promoter.
5. The vector of claim 4 , wherein the nucleotide sequence of said intron comprises the nucleotide sequence of SEQ ID NO: 4.
6. The vector of claim 1 , further comprising at least three elements selected from the group consisting of:
(a) a selectable marker for eukaryotic cells;
(b) a prokaryotic origin of replication;
(c) a bacterial drug resistance marker; and
(d) a phage f1(+) region.
7. The vector of claim 6 , wherein said vector comprises all of said elements.
8. The vector of claim 6 , wherein said selectable marker for eukaryotic cells is a hygromycin (TK-Hygromycin) drug resistance gene operably linked to a thymidine kinase promoter.
9. The vector of claim 6 , wherein said prokaryotic origin of replication is the ColE1 origin of replication.
10. The vector of claim 6 , wherein said bacterial drug resistance marker is a kanamycin resistance gene.
11. The vector of claim 1 , comprising a terminator/polyA addition site.
12. The vector of claim 1 , comprising the nucleotide sequence set forth in SEQ ID NO: 1.
13. The vector of claim 1 , comprising a heterologous DNA sequence encoding a polypeptide, which DNA sequence is operably linked to a promoter and an intron.
14. The vector of claim 13 wherein the polypeptide is an antibody immunoglobulin chain.
15. The vector of claim 14 wherein the antibody immunoglobulin chain is from an antibody that binds specifically to IGFIR.
16. The vector of claim 15 wherein the immunoglobulin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-30.
17. An isolated plasmid vector by having the plasmid map of FIG. 2 .
18. An isolated host cell comprising the vector of claim 1 .
19. A host cell line comprising the host cell of claim 18 .
20. A kit comprising the vector of claim 1 and instructions for use of the vector.