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Anti-trypanosomiasis vaccines and diagnostics

A vaccine, trypanosome technology, applied in the field of genetic material

Inactive Publication Date: 2013-01-02
波尔多第二大学 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no new molecules have been on the market for at least 30 years, while the past few years have seen new outbreaks of disease due to the emergence of trypanocide resistance and the widespread and occasional inappropriate use of the drug. Reports of selection and amplification of resistance due to use, especially in all regions of Africa affected by the disease

Method used

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  • Anti-trypanosomiasis vaccines and diagnostics
  • Anti-trypanosomiasis vaccines and diagnostics
  • Anti-trypanosomiasis vaccines and diagnostics

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The present invention relates to a novel genetic material coding for trans-sialidase-like proteins belonging to the African trypanosomes, and relates to the use of said genes and proteins for vaccinal, therapeutic and diagnostic purposes. The present invention also relates to the immunization of humans and / or non-human animals against trypanosomiasis.

Description

technical field [0001] The present invention relates to novel genetic material encoding a sialyltransferase-like protein of the African trypanosome parasite, and to the use of said genes and proteins in vaccines, therapeutics and diagnostics. The invention also relates to the immunization of humans and / or non-human animals against trypanosomiasis and trypanosomiasis. Background technique [0002] Trypanosomiasis and trypanosomiasis are caused by several parasitic protozoan species of the genus Trypanosoma, and African trypanosoma generally refers to trypanosomes belonging to the class Salivaria, which itself includes three Major subgenera: Trypanozoon, Duttonella and Nannomonas. [0003] Only the subgenus Trypanosoma contains two species that infect humans, in addition to species that infect animals, and which cause sleeping sickness in humans. Other subgenuses contain species that infect wild and domesticated animals and never infect humans, but may have important indirec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54G01N33/577A61K39/395A61K39/005C12N9/24
CPCC12N9/1081C12Y302/01018C12N9/2402C12N9/24C12Y204/99A61P33/00A61P33/02A61P37/04
Inventor 维尔日妮·柯思图利纳雷斯西奥·巴茨尼古拉斯·普拉佐雷斯
Owner 波尔多第二大学
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