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Live antenuated parasite vaccine

A technology for parasites and vaccines, applied in protozoa, protozoan antigen components, biochemical equipment and methods, etc., can solve the problems of time-consuming, complicated vaccine development, and not providing local immunity

Inactive Publication Date: 2008-06-11
INTERVET INT BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0023] Disadvantages of inactivated vaccines are: they often do not induce (cellular) immunity, need to be boosted, they do not provide local immunity (important), they are more expensive, and their application is dangerous if the inactivation is less than 100%
It also complicates vaccine development
[0025] Therefore, to date, the development of vaccines against parasitic infections has been difficult, time-consuming and not very successful

Method used

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  • Live antenuated parasite vaccine
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Primers used during the experimental procedure:

[0155] Inserted restriction sites are underlined.

[0156]

[0157] Construction of TubYFP / TR-sagCAT (9332bp)

[0158] The plasmid ptubYFP / TR-sagCAT was stepwise engineered as described below. First, using primers SAG3FW (#1, SEQ ID NO: 5) and TUB5RV (#2, SEQ ID NO: 6), by constructing from ptubYFP / YFP-sagCAT (Llopis, J. et al., PNAS 97 (8): 4363-4368 (2000)) amplified the murine Toxoplasma gondii tubulin A (tub) promoter to make construct ptubCAT / GFP. The PCR product and plasmid pdhfrCAT / GFP were digested with HindIII and BgIII (Striepen, B. et al., Molecular and Biochemical Parasitology 92:325-338 (1998)). and connect them to each other. This generated ptubCAT / GFP in which the dhfr promoter had been replaced with the tub promoter. The resulting plasmid is based on Bluescript pKS+ (Stratagene, La Jolla, CA), and contains the α-tubulin promoter to which the fusion of the chloramphenicol acetyltransferase (CAT) ...

Embodiment 2

[0163] Determination of the Initiation Transcription Site of S13 Ribosomal Protein Gene of Toxoplasma gondii

[0164] To determine the initiation of transcription of the ribosomal protein gene S13, RNA was isolated from murine Toxoplasma gondii RHΔHXGPRT tachyzoites grown in Vero cells. Using GeneRacer Kit (Invitrogen) to obtain gene-specific full-length cDNA from total RNA. Using this kit, oligo RNA is ligated to the end of full-length mRNA, and after reverse transcription with oligo dT, the product is amplified by PCR using GeneRacer primers combined with oligo RNA and gene-specific primers. The start of transcription (STS) can then be determined. For ribosomal protein gene S13, this can be done using the following primers: REV13A (#7, SEQ ID NO: 11) and REV13B (#8, SEQ ID NO: 12). Primer #7 was used with the GeneRacer primer to obtain the product, followed by primer #8 for nested PCR. The PCR product showed 3 bands; 2 weak and 1 strong. The band showing the greatest a...

Embodiment 3

[0166] S13 / LZ construct

[0167] To test the induced expression of the tet repressor, several reporter constructs were made using the lacZ gene under the control of the S13 promoter with and without the presence of a single tetO site. First, plasmid S13 / lacZ was prepared (see Figure 2 for structure and sequence of the final construct), which was then used to insert or replace the tetO site sequence as described below.

[0168]The S13 primer was amplified by PCR from genomic DNA of the murine Toxoplasma gondii RH / ΔHXGPRT strain using primers S13PROMFUS FW (#9, SEQ ID NO: 13) and S13PROMFUS RV (#10, SEQ ID NO: 14) sub-area. The lacZ coding sequence was amplified by PCR from genomic DNA of BL21 bacteria using primers LACZ-AVRII FW (#11, SEQ ID NO: 15) and LACZ-PSTI RV (#12, SEQ ID NO: 16). Subsequently, the S13 PCR product was digested with HindIII and XbaI, while the lacZ PCR product was digested with AvrII and PstI. The plasmid ptubYFP / YFP-sagCAT was used to exchange the ptu...

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Abstract

The present invention relates inter alia to attenuated live parasites of the phylum Apicomplexa and the family of Trypanosomatdae and to the use of such attenuated live parasites in a vaccine and in the manufacturing of such a vaccine. Furthermore, the present invention relates to vaccines comprising such attenuated live parasites and to methods for the production of such vaccines. Finally, the invention relates to specific tet-repressor fusion proteins and to attenuated live parasites according to the invention comprising such tet-repressor fusion proteins.

Description

technical field [0001] The present invention relates to live attenuated parasites of the order Apicomplexa and Kinetoplastida, to the use of such live attenuated parasites in vaccines and in the preparation of such vaccines, to The invention relates to a vaccine against worms, to a method for producing such a vaccine, to a specific tet-repressor fusion protein and to live attenuated parasites comprising such a tet-repressor fusion protein. Background technique [0002] In the kingdom Protozoa, the phylum Apicomplexa and the order Kinetoplastida, more specifically the family Trypanosoma tidae, are known to contain several notoriously pathogenic parasites. [0003] The family Trypanosoma tidae comprises parasites belonging to the genera Leishmania and Trypanosoma. [0004] Leishmaniasis is the term for various disease manifestations caused by Leishmania. The disease occurs most often in dogs and humans. The parasite is transmitted by sandflies to mammalian hosts and is ende...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/11C12N15/30A61K39/002A61K39/005A61K39/008A61K39/012A61K39/015A61K39/018A61P33/02A61P33/04A61P33/06C07K14/245C07K14/435C07K14/45C12N15/63
CPCC07K14/245C07K2319/00A61K2039/52C12N15/635C07K14/43595A61P33/02A61P33/06A61P33/14Y02A50/30
Inventor N·F·J·范泊佩尔A·N·沃梅伦T·C·沙普
Owner INTERVET INT BV
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