This application provides, in part, pAVEC plasmids and methods of use of such plasmids for the production of polypeptides such as immunoglobulins as well as the generation of recombined versions of such plasmids which contain polynucleotides encoding such polypeptides.
1. An isolated vector having the plasmid map of FIG. 1 which comprises a multiple cloning site that comprises the nucleotide sequence set forth in SEQ ID NO: 1.
2. The vector of claim 1 , comprising the polynucleotide sequence set forth in SEQ ID NO: 2.
3. A method for making a recombined plasmid vector comprising cleaving the vector of claim 1 and ligating the ends of the cleaved plasmid to compatible ends of an introduced polynucleotide such that a closed circular plasmid is produced.
4. The method of claim 3 wherein the introduced polynucleotide encodes an immunoglobulin chain.
5. The method of claim 4 wherein the immunoglobulin chain is a heavy or light chain of a member selected from the group consisting of: Abciximab, Adalimumab, Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab, Certolizumab, Pegol, Dalotuzumab, Daclizumab, Denosumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab, Tiuxetan, Infliximab, Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Robatumumab, Tositumomab, ALD518 and Trastuzumab;
or, wherein the immunoglobulin chain is a heavy or light chain of an antibody or antigen-binding fragment thereof that binds specifically to an antigen selected from the group consisting of: vascular endothelial growth factor (VEGF), vascular endothelial grown factor receptor (VEGFR), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), programmed cell death protein 1 (PD-1), tumor necrosis factor alpha (TNFalpha), tumor necrosis factor beta (TGFbeta), TRAIL-R1, thymic stromal lymphopoietin (TSLP), Nav1.7, Nav1.8, extracellular signal-related kinase (ERK), MEK, TRAIL-R2, interleukin-10 (IL-10), interleukin-6 (IL-6), interleukin-6 receptor (IL-6R), insulin-like growth factor-1 receptor (IGF1R), interleukin-23p19 (IL-23p19), interleukin-23 receptor (IL-23R), proprotein convertase subtilisin/kexin type 9 (PCSK9), CD20, receptor activator of nuclear factor kappa-B ligand (RANKL), receptor activator of nuclear factor kappa-B (RANK), CD33, CD11a, ErbB2, IgE, a G-protein coupled receptor (GPCR), a human immunodeficiency virus (HIV) antigen, a hepatitis C virus (HCV) antigen, and a respiratory syncytial virus (RSV) antigen.
6. A recombined plasmid vector that is the product of the method according to claim 3 .
7. An isolated host cell comprising the vector of claim 1 .
8. The host cell of claim 7 which is a bacterial or mammalian cell.
9. A method for producing a recombinant polypeptide in an isolated host cell, comprising introducing the recombined vector of claim 6 into a host cell; and expressing the polypeptide from the introduced polynucleotide.
10. The method of claim 9 , further comprising purifying the polypeptide.
11. A kit comprising a plasmid vector of claim 1 and one or more additional components.