Construction method and application of porcine encephalomyocarditis virus BD2 strain full-length infectious clone

An encephalomyocarditis virus, infectious cloning technology, applied in application, virus, antiviral agent and other directions, can solve the problem of economic loss in pig industry, achieve the effect of promoting in vitro transcription efficiency, avoiding operation difficulty and easy separation

Active Publication Date: 2017-03-22
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since EMCV was isolated from sick pigs suffering from myocarditis in 1958, there have been reports of the occurrence, prevalen...

Method used

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  • Construction method and application of porcine encephalomyocarditis virus BD2 strain full-length infectious clone
  • Construction method and application of porcine encephalomyocarditis virus BD2 strain full-length infectious clone
  • Construction method and application of porcine encephalomyocarditis virus BD2 strain full-length infectious clone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Construction of full-length infectious clone of porcine encephalomyocarditis virus BD2 strain

[0042] 1.1 Cells and virus strain BHK-21: The cells were kept in the Veterinary Biological Products Laboratory of Hebei Agricultural University and cultured in 10% DMEM medium with high sugar content. EMCV BD2 was isolated and preserved by the Veterinary Biological Products Laboratory of Hebei Agricultural University, and the measured whole genome sequence has been uploaded to NCBI (GenBank serial number: KF709977). The fifth generation virus was used in this study.

[0043] 1.2 Transformation of low-copy vector pWSK29: This test selects low-copy vector pWSK29 ( figure 1 ) as the final vector for the construction of EMCV BD2 wild-type infectious cDNA clones, in order to meet the needs of cloning and full-length correct ligation of each gene fragment, it is an indispensable step to transform the vector. By analyzing the gene sequence of the pWSK29 vector, it is fou...

Embodiment 2

[0050] Embodiment 2: Porcine encephalomyocarditis virus BD2 strain infectious cDNA virus rescue

[0051] 2.1 In vitro transcription of the full-length cDNA clone: ​​Plasmid linearization: Take 15 μg of the constructed full-length plasmid pWSKBD2, and linearize the plasmid with restriction endonuclease BamHI. According to the specification of the endonuclease, establish the following 100 μL reaction system, and digest in a water bath at 37°C for 3 hours. Take 5 μL of the digested product and perform 1% agarose gel electrophoresis to detect whether the plasmid is digested completely. Place the remaining digested products that have been completely linearized in a water bath at 65°C for 20 minutes to inactivate the endonuclease BamH Ⅰ, then add 5 μL of 10% SDS to make the final concentration 0.5%, and then pipette 1 μL of 20 mg / Make the final concentration of 200μg / mL proteinase K into 200 μg / mL, blow and suck repeatedly with a pipette, mix well, and act in a 50°C water bath for...

Embodiment 3

[0056] Example 3: Construction of recombinant encephalomyocarditis virus expressing PCV2Cap protein and virus rescue thereof

[0057] 3.1 Fusion PCR amplification of exogenous insert fragments: independent amplification of porcine circovirus type 2 gene fragments to be fused: use the primer pairs in the table below, use the corresponding templates respectively, and establish a 50 μL amplification system according to the conventional PCR method, Independently amplify each target fragment, primers up-F, up-R, ORF2-F, ORF2-R, down-F, down-R sequences such as SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 , SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, high-fidelity enzymes are used for amplification FastPfu Fly DNA Polymerase, see the size of the target band Figure 9. The amplified products were identified by 1% agarose gel electrophoresis and recovered by cutting the gel. The PCR products of each section were purified and recovered with the SanPrep column DNA gel recovery kit. The ope...

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Abstract

The invention discloses a construction method and an application of a porcine encephalomyocarditis virus BD2 strain full-length infectious clone. On the basis of the constructed porcine encephalomyocarditis virus BD2 strain full-length infectious clone, an ORF2 gene of porcine circovirus is interpolated into an L gene encoding virus pilot protein, so that the encephalomyocarditis virus BD2 strain full-length infectious clone, which stably carries the ORF2 gene of the porcine circovirus 2, is successfully constructed. Both the constructed EMCVBD2 strain full-length infectious clone and the encephalomyocarditis virus BD2 strain full-length infectious clone, which stably carries the ORF2 gene of the porcine circovirus 2, can rescue infectious recombinant virus; therefore, the EMCVBD2 strain full-length infectious clone and the encephalomyocarditis virus BD2 strain full-length infectious clone, which stably carries the ORF2 gene of the porcine circovirus 2, can be applied to vaccine preparation and virus researches.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a full-length infectious clone of porcine encephalomyocarditis virus BD2 strain and its construction method and application, and also relates to using the clone to construct porcine encephalomyocarditis virus BD2 stably carrying porcine circovirus type 2 ORF2 gene Strain full-length infectious clones and the application of recombinant clones. Background technique [0002] Porcine encephalomyocarditis (Porcine encephalomyocarditis) is caused by encephalomyocarditis virus (EMCV), a zoonotic infectious disease characterized by encephalitis, myocarditis and reproductive impairment in sows. Since EMCV was isolated from pigs suffering from myocarditis in 1958, there have been reports of the occurrence, prevalence and EMCV infection of the disease in some pig-raising countries in the world, causing certain economic losses to the pig industry. From the aspects of etiology and se...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N7/01C12N15/86A61K39/125A61K39/12A61K39/295A61P31/14A61P31/20
CPCA61K39/00C12N7/00C12N15/86C12N2750/10034C12N2770/32221C12N2770/32222C12N2770/32234C12N2770/32243
Inventor 袁万哲孙继国戚妍李佳暖刘娜
Owner HEBEI AGRICULTURAL UNIV.
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