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Nucleotide sequences of pestivirus strains, polypeptides encoded by these sequences and use thereof for diagnosis and prevention of pestivirus infections

A nucleotide sequence, pestivirus technology, applied in the direction of viral peptides, applications, viruses, etc., can solve the problem of not being able to reliably predict the success of infectious RNA

Inactive Publication Date: 2008-09-03
动物保健研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the application of this technique can pose serious problems, which have been characterized in a review by Boyer and Haenni in 1994 (Virology 198:415-426)
In practice, it cannot be reliably predicted whether the success of constructing a full-length DNA copy of the genome of a positive-strand RNA virus and generating synthetic infectious RNA from this full-length DNA copy cannot be reliably predicted

Method used

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  • Nucleotide sequences of pestivirus strains, polypeptides encoded by these sequences and use thereof for diagnosis and prevention of pestivirus infections
  • Nucleotide sequences of pestivirus strains, polypeptides encoded by these sequences and use thereof for diagnosis and prevention of pestivirus infections
  • Nucleotide sequences of pestivirus strains, polypeptides encoded by these sequences and use thereof for diagnosis and prevention of pestivirus infections

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Molecular cloning and sequencing of embodiment 1C strain genome

[0053] cells and viruses

[0054] Pig kidney cells (SK6-M, EP-A-351901) were cultured on Eagle's minimal medium containing 5% fetal bovine serum (FBS) and antibiotics. FBS was tested for BVDV and antibodies to BVDV as described (Moormann et al. 1990, Virology 177:184-198). Only serum free of BVDV and BVDV antibodies can be used. The "Chinese" vaccine strain (strain C) of classical swine fever virus (CSFV) was adapted to SK6-M cells as described in EP-A-351901. The strain termed "Cedipest" was non-cytopathic and was biologically cloned by three-fold endpoint dilution. Three-step amplification produced titers of 3, 5, 10 6 TCID 50 / ml of the cloned virus stock.

[0055] Isolation of Cytoplasmic RNA from SK-6 Cells Infected with C Strain

[0056] Intracellular RNA from cells infected with strain C was neccessarily isolated as described (Moormann et al. 1990. Virolgy 177:198). Briefly, at 162cm 2 SK6...

Embodiment 2

[0068] The production of the infectious transcript of the full-length DNA copy of embodiment 2C strain genome

[0069] Construction of cDNA clone pPRKflc-113

[0070] according to the image 3 The patterns make up the full-length DNA copy of the C-strain genomic RNA. First, two subclones were constructed, one (pPRc64) containing the cDNA sequence of the 5' part of the genome (1-5,560 nucleotides), and the other (pPRc111) containing the cDNA sequence of the 3' part of the genome (5,463- 12,311 nucleotides). The initial construction of a full-length cDNA clone was attempted in pGEM4z-blue. However, this approach failed due to the instability of the full-length sequence inserted into this vector. To increase the stability of the clones, the 5' and 3' partially cloned inserts were recloned in derivatives of the low copy number vector pOK12 (Vieira and Messing, 1991, Gene 100:189-194), resulting in pPRc108 and pPRc108, respectively. pPRc123. To achieve this result, pOK12 was ...

Embodiment 3

[0093] Immunization of pigs with deletion mutants of E1

[0094] Construction and expression of deletion mutant of CSFV Brescia strain E1

[0095] It has been shown that TMR-deficient E1 of CSFV Brescia strain expressed in insect cells can induce a protective immune response against CSF in pigs (Hulst et al., 1993, J. Virol. 67: 5435-5442). Two distinct antigenic units, A and B+C, capable of inducing neutralizing antibodies against CSFV in the N-terminal half of El have also been identified (Wensvoort, 1989, General Virology. 70:2865-2876 ; Van Rijn et al. 1992. Vet Microbiol. 33: 221-230; Van Rijn et al. 1993. General Virology 74: 2053-2060). Furthermore, neutralizing antibodies against regions A and B+C showed synergy in neutralizing CSFV (Wensvoort 1989. General Virology 70:2865-2876). To evaluate the immunogenicity of E1 mutants with deletions in B+C regions or deletions in A regions, relevant constructs can be prepared on baculovirus vectors and the expressed mutant pro...

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Abstract

The invention provides a nucleotide sequence corresponding to a classical swine fever virus (CSFV) genome or a part or a mutant thereof, which comprises at least a part of the nucleotide sequence of the CSFV C-strain depicted in SEQ ID No. 1, or a complement or RNA equivalent of such nucleotide sequence, or which comprises a nucleotide sequence encoding at least the amino acid sequence 268-494 of the amino acid sequence depicted in SEQ ID No. 1, or a complement or RNA equivalent of such nucleotide sequence. Also provided is a pestivirus polypeptide corresponding to the amino acid sequence 690-1063 of SEQ ID No. 1 or part thereof, which contains a mutation in one of the epitopes within amino acid sequences 691-750 or 785-870, said mutation altering said epitope. Further provided is a method of determining the presence of a test substance capable of specifically binding with a binding site of a binding partner, in a sample, by means of competition of said test substance with a measurable amount of a reference substance capable of specifically binding with the same binding site of said binding partner, comprising: (1) contacting said sample with (a) said reference substance bound to a solid carrier, (b) the binding partner of said reference substance, said binding partner molecule containing at least two identical binding sites for said reference substance, and (c) said reference substance provided with a label; (2) measuring the degree of binding of said label to said carrier.

Description

[0001] The application date is June 16, 1995, the application number is 95193665.4, and the title of the invention is "Nucleotide sequences of pestivirus strains, polypeptides encoded by these sequences and their application in diagnosis and prevention of pestivirus infection" divisional application of the Chinese patent application. field of invention [0002] The present invention discloses a method for constructing a full-length DNA copy of the genome of strain C, a typical vaccine strain of classical swine fever virus, and transcribing its RNA which, after transfection in cells, results in the synthesis of viruses of strain C . The invention also includes vaccines derived from the C strain (pestivirus), and subunit vaccines against pestiviruses, as well as diagnostic means and methods for pestivirus infections. The present invention further provides a method for detecting immunoreactive substances in a sample using a competition assay. Background of the invention [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/33C07K14/005G01N33/53G01N33/68G01N33/569A61K39/00A61K39/187A61P31/12C07H21/04C07K14/18C07K14/185C12N7/01C12N7/02C12N15/00C12N15/09C12N15/40C12P21/02C12Q1/70C12R1/91C12R1/92
CPCA61K39/00C12N2770/24322C07K14/005A61P31/12
Inventor 罗伯特斯·雅各布斯·玛丽亚·莫尔曼彼德鲁斯·安东尼厄斯·范赖恩
Owner 动物保健研究所
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