Raccoon poxvirus expressing genes of porcine virus

A technology of pox virus and pox virus vector, which is applied in the direction of virus, antiviral agent, genetic engineering, etc.

Inactive Publication Date: 2010-06-09
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In an example, the patent only shows how to prepare for the insertion of foreign DNA into the HindIII "U" genomic region

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  • Raccoon poxvirus expressing genes of porcine virus
  • Raccoon poxvirus expressing genes of porcine virus
  • Raccoon poxvirus expressing genes of porcine virus

Examples

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example

[0114] The following examples demonstrate certain aspects of the invention. It should be understood, however, that the examples are for illustration only and are not intended to limit the invention fully to the conditions and scopes set forth. It is understood that where typical reaction conditions (eg, temperature, reaction times, etc.) are given, conditions above and below the stated ranges can also be used, although generally less expedient. The examples were performed at room temperature (about 23°C to about 28°C) and atmospheric pressure. All parts and percentages referred to herein are by weight and all temperatures are in degrees Celsius unless otherwise indicated.

example 1

[0115] Example 1: Construction of rRCNV-PRRS (blue-to-white plaque pathway)

[0116] rRCNV-PRRS btw It is constructed through the following five main steps: (1) cloning ORF5, ORF6, ORF7 or ORF3 of PRRSV ISU12 (Iowa State) and Olot / 91 (Spanish isolate) bacterial strains into plasmid pFD2000A and / or pFD2003SEL; ( 2) Construct plasmid pFD2000A PRRS ORF7 or ORF3 (P 11 )-ORF5(Olot)(P SEL )-ORF6(P SEL )-ORF5(P SEL ); (3) create a clone library by three aspects of infection / transfection: COS7 cells, plasmids and rRCNV-FeLV in step 2; (4) implement clone screening by limiting dilution and blue-to-white plaque approach; and (5) Determination of rRCNV-PRRS by PCR, ELISA and western blot method btw molecular features.

[0117] The blue-to-white plaque screening pathway uses FeLV P27 +Phenotype (blue in FeLV P27 ELISA) of the parental virus rRCNV-FeLV. Through homologous recombination of RCNV HA flanking sequence between rRCNV-FeLV and pFD200A PRRS ORF7-ORF5(Olot)-ORF6-ORF5, the p...

example 2

[0118] Example 2: Construction of rRCNV-PRRS (white-to-blue plaque pathway)

[0119] rRCNV-PRRS wtb It is constructed through the following five main steps: (1) cloning ORF5 and ORF6 and FeLV P27 of PRRSV ISU12 and Olot / 91 strains into plasmid pFD2000A and / or pFD2003SEL; (2) constructing plasmid pFD2000A FeLV P27 (P 11 )-PRRS ORF5(Olot)(P SEL )-ORF6(P SEL )-ORF5(P SEL ); (3) create a clone library by three-way infection / transfection: COS7 cells, plasmids in step 2, and RCNV; (4) implement clone screening by limiting dilution and FeLV P27ELISA or LacZ; and (5) by PCR , ELISA and western blot method to determine rRCNV-PRRS wtb molecular features.

[0120] The white-to-blue plaque screening pathway uses FeLV P27 + or LacZ phenotype (in FeLV P27ELISA or LacZ + blue in phenotype) recombinant virus rRCNV-PRRS wtb , while wild-type RCNV appeared white in FeLV P27ELISA or LacZ staining.

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Abstract

The present invention relates to a new recombinant raccoon poxvirus vector vaccine in which the vector expresses one or more antigenic proteins encoded by multiple open reading frames, preferably the ORF5, ORF6 and / or ORF3 / ORF4 / ORF7, of one or more porcine reproductive and respiratory syndrome virus strains alone or in combination with an open reading frame of porcine circovirus type 2 (PCV-2), preferably ORF2, at the hemagglutinin (ha) and / or thymidine kinase (tk) loci.

Description

technical field [0001] The present invention relates to novel recombinant raccoonpox virus vectors expressing porcine reproductive and respiratory syndrome virus (PRRSV) genes alone or in combination with porcine circovirus (PCV) genes, and said vectors as vaccines against infections caused by PCV and / or PRRSV Uses in the prevention of diseases caused. Background technique [0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-strand RNA virus that is a member of the genus Arterivirus family of the Arteriviridae family. PRRSV infects pigs, causing a "mystery swine disease" first detected in North America in 1987, which subsequently spread to and throughout Europe in 1990. Since 1990, PRRSV has become a major global threat to the swine industry, causing severe production losses in swine herds. [0003] The mysterious swine disease now known as porcine reproductive and respiratory syndrome (PRRS) (also known as blue ear disease, porcine epidemic a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86
CPCA61K2039/5256C12N2710/24043C12N2750/10034C12N2770/10034C12N9/1211C12N15/86A61P11/00A61P15/00A61P31/12A61P37/00C12N15/11C12N15/863
Inventor 斯蒂芬·奇图·吴迈克尔·A·吉尔信觉·朱
Owner WYETH LLC
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