Method for producing antibody using “naked” expression vector expressing type II transmembrane fusion protein
View Patent ↗Methods are disclosed for generating antibodies and an expression vector used to express protein(s) which provoke the antibody response. The expression vector may be useful in generating an antibody directed to an antigen, comprising a gene in operable linkage with a promoter, which gene encodes upon expressing a fusion protein comprising (i) CD134L, a fragment or homologous protein thereof as N-terminal moiety of the fusion protein; and (ii) all or part of an antigenic protein as C-terminal moiety of the fusion protein. To generate the antibodies, the vector is injected into a subject animal, which produces a fusion protein, against which antibodies are generated.
1. An expression vector for generating an antibody directed to an antigen, the expression vector comprising a polynucleotide in operable linkage with a promoter, wherein the polynucleotide encodes a fusion protein comprising:
(i) the polypeptide of SEQ ID NO:2, or a polypeptide having at least 95% amino acid identity to said polypeptide of SEQ ID NO:2 which anchors in a cell membrane as the N-terminal moiety of the fusion protein;
(ii) an antigenic protein or an antigenic fragment thereof as the C-terminal moiety of the fusion protein; and
(iii) a linker peptide interposed between (i) and (ii).
2. The expression vector of claim 1 , wherein said N-terminal moiety comprises or consists of the amino acid sequence of SEQ ID NO: 2.
3. The expression vector of claim 1 , wherein the linker peptide is (G 4 S) 3 .
4. A recombinant cell, transformed or transfected with the expression vector of claim 1 .
5. The recombinant cell of claim 4 , wherein said cell is a eukaryotic cell.
6. An expression vector for generating an antibody directed to an antigen, the expression vector comprising a polynucleotide encoding a fusion protein comprising an antigenic protein or an antigenic fragment thereof in operable linkage with a promoter, wherein said polynucleotide encoding said fusion protein comprises a nucleic acid sequence encoding said antigenic protein or said antigenic fragment thereof as a C-terminal moiety of said fusion protein and a nucleic acid sequence encoding a polypeptide which anchors in the cell membrane as an N-terminal moiety of said fusion protein, wherein said nucleic acid sequence encoding a polypeptide which anchors in the cell membrane comprises a nucleic acid sequence selected from the group consisting of:
the polynucleotide of SEQ ID NO:1;
a polynucleotide having at least 95% nucleic acid identity to said polynucleotide of SEQ ID NO:1, wherein said polynucleotide encodes a polypeptide which anchors in the cell membrane;
a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 1, wherein said polynucleotide encodes a polypeptide which anchors in the cell membrane,
wherein the expression vector further comprises a nucleic acid sequence encoding a linker peptide interposed between the nucleic acid sequence encoding said antigenic protein or antigenic fragment thereof and the nucleic acid sequence encoding said polypeptide which anchors in the cell membrane.
7. The expression vector of claim 1 , wherein said promoter is selected from the group consisting of SV40 early and late promoters, CMV promoter and promoters of retroviral LTRs, promoter of Rous sarcoma virus (“RSV”), metallothionein promoters, and mouse metallothionein-I promoter.
8. The expression vector of claim 6 , wherein the linker peptide is (G 4 S) 3 .
9. An expression vector for generating an antibody directed to an antigen, the expression vector comprising a polynucleotide in operable linkage with a promoter, wherein the polynucleotide encodes a fusion protein comprising:
(i) a full length mammalian CD134L or a polypeptide having at least 95% amino acid identity to said full length mammalian CD134L which anchors in a cell membrane as the N-terminal moiety of the fusion protein;
(ii) an antigenic protein or an antigenic fragment thereof as the C-terminal moiety of the fusion protein; and
(iii) a linker peptide interposed between (i) and (ii).
10. The expression vector of claim 9 , wherein the linker peptide is (G 4 S) 3 .
11. An expression vector for generating an antibody directed to an antigen in a subject animal, the expression vector comprising a polynucleotide in operable linkage with a promoter, wherein the polynucleotide encodes a fusion protein comprising:
(i) CD134L, or a polypeptide having at least 95% amino acid identity to CD134L which anchors in a cell membrane as the N-terminal moiety of the fusion protein;
(ii) an antigenic protein or antigenic fragment thereof as the C-terminal moiety of the fusion protein; and
(iii) a linker peptide interposed between (i) and (ii);
wherein the said CD134L or said polypeptide having at least 95% amino acid identity to CD134L is from a species different from the species of the subject animal.
12. The expression vector of claim 11 , wherein the linker peptide is (G 4 S) 3 .