Anti-microbial targeting chimeric pharmaceutical
a technology of chimeric constructs and anti-microbials, applied in the direction of peptide/protein ingredients, human health protection, peptide sources, etc., can solve the problems of affecting the ecology of the normal human microbiota, affecting the antimicrobial effect, and reducing the antimicrobial effect of chimeric constructs,
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example 1
Construction and Expression of a Histatin 5 and Dhvar 1 / SWLA3 Chimeric Antibody Fusion Protein with Activity Against S. mutans
[0084]a. Construction of an Expression Vector for an Antibody-Based Fusion Protein
[0085]The construct that is ultimately cloned into an IgG1 expression vector and leads to the expression of the targeted anti-microbial fusion protein was assembled according to the following method (see FIG. 1). The construct was assembled using sequential PCR and restriction enzymes techniques. The recognition sequence of the fusion protein was derived from heavy chain sequences of SWLA3, produced by hybridoma ATCC HB 12558. See Shi, U.S. Pat. No. 6,231,857, the disclosure of which is incorporated herein by reference, and U.S. patent application Ser. Nos. 09 / 378,577 and 09 / 881,823. Sequences encoding histatin 5 or dhvar1 were inserted upstream of the variable region of the heavy chain of SWLA3. The amino acid sequences used for histatin 5 and dhvar 1 are listed below:
Histatin...
example 2
Construction of a Chimeric Construct Containing Minibody and Anti-Microbial Peptide, and its Expression in Yeast
[0110]a. Construction of SWLA3 minibody-PG-1 Peptide Fusion Protein
[0111]A minibody is a modified antibody molecule that comprises of the variable regions of the heavy and light chain (VH and VL) covalently linked via a short linker in a head-to-tail fashion (see FIG. 5). To construct the SWLA3 minibody-PG-1 anti-microbial peptide fusion, PG-1 was linked to the N-terminus of VH via a poly serine-glycine linker peptide, (SGGGG)3 (SEQ ID NO. 17). The C-terminus of VH was connected via a short GS linker, (GGGS)2 (SEQ ID NO. 18), to the N-terminus of VL.
[0112]The starting material for constructing the minibody was the anti S. mutans monoclonal antibody, SWLA3, as described in the U.S. Pat. No. 6,231,857. The anti-microbial peptide was protegrin PG-1 as described in the U.S. Pat. Nos. 5,693,486, 5,708,145, 5,804,558, 5,994,306, and 6,159,936 and Zhao et al., FEBS lett, 1994, 34...
example 3
Construction of Chimeric Construct Containing Surface-Binding Peptide and Anti-Microbial Peptide
[0116]In addition to antibodies, some small peptide can also bind to surface structures of microorganisms or eukaryotic cells. These peptides, which we term “docking moiety”, allow more flexibility for the antimicrobial peptides (the killing moiety) to insert into the cell membrane for killing. These peptides can be selected from phage display libraries that contain random peptide sequences. Phage-display libraries of 8-12 amino acids peptide are commercially available. In this experiment, we have screened these libraries for peptides capable of specifically binding to a target organism, which can be bacteria, yeast, or other fungi. One or more of these peptides will then be fused to the anti-microbial peptide via a peptide linker, and expressed in an appropriate host, or chemically synthesized.
a. Selection of Species-Specific Binding Peptides with Phage Display
[0117]We employed three pha...
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