Anti-trypanosomiasis vaccines and diagnostics
A vaccine, trypanosome technology, applied in the field of genetic material
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Embodiment 1
[0136] Example 1: Production of polyclonal antibodies against TcoTS-A1 protein
[0137] TcoTS-A1 protein was produced in Pichia pastoris. To this end, the X33 strain was transformed with a PICZ vector (Invitrogen) containing the sequence encoding the TcoTS-A1 protein lacking the first 29 amino acids. After 4 days of induction of expression in methanol, the protein secreted into the culture supernatant was purified by sequential ion exchange chromatography. First, culture supernatants were dialyzed against 20 mM sodium acetate buffer (pH 4.5) for 16 h, centrifuged at 10,000 g for 30 min, and then chromatographed on a 1 ml HiTrap SP HP column (GE Healthcare). Elution was performed with a linear gradient of 0-1M NaCl. will contain sialidase activity (using the substrate 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid, as described in Montagna et al. (2006) J. Biol. Chem. 281 (45) :33949-58) fractions were pooled and dialyzed against 20 mM Tris-HCl buffer (pH 8) for 16 ho...
Embodiment 2
[0139] Example 2: Production of polyclonal antibodies against peptides derived from sialidase-related sequences
[0140] The following peptides, designated as peptides 1, 2 and 3, respectively: C-RTSIDYHLIDTVAKYSADDG (SEQ ID NO: 23), C-PKNIKGSWHRDRLQLWLTD (SEQ ID NO: 24) and C-PVSAQGQDHRYEAANAEHT (SEQ ID NO: 25), were obtained by N- The terminal cysteine was coupled to a carrier protein (KLH) activated with a maleimide functional group and used to immunize rabbits on a schedule of one 100 μg injection every 20 days for a total of 5 injections. The recombinant protein was mixed in emulsion with complete Freund's adjuvant for the first injection and with incomplete Freund's adjuvant for subsequent injections. The obtained polyclonal sera, designated as anti-peptide 1 antibody, anti-peptide 2 antibody and anti-peptide 3 antibody, were collected at the end of the experiment and their reactivity against their corresponding peptides was verified by indirect ELISA.
Embodiment 3
[0141] Example 3: Demonstration of the expression of TcoTS-A1, TcoTS-A2, TcoTS-A3 and TcoTS-like 2 proteins in T. congo blood types
[0142] 3 ml of rabbit serum or 1 ml of mouse serum was dialyzed against 1 l of 20 mM phosphate buffer (pH 7) for 16 h. Dialyzed serum was centrifuged at 5,000 g for 20 min and passed through a Protein G Sepharose Fast Flow column (GE healthcare) prepared in advance according to the manufacturer's instructions. After washing with 20 mM phosphate buffer (pH 7), IgG bound to the column was eluted with 0.1 M glycine-HCl buffer (pH 2.6). IgG thus purified against 1 l of 0.1 M NaHCO 3 (pH 8.3) / 0.5M NaCl buffer for 16 hours. IgG was then incubated with CNBr-activated agarose gel (Sigma) prepared in advance according to the manufacturer's recommendation for 2 hours at room temperature. After centrifugation at 1,000 g for 1 min, the resin was washed with the aforementioned buffer and then saturated by adding 0.1 M Tris-HCl (pH 8) for 2 h at room tempe...
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