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A kind of pseudorabies virus llt region δintron strain and its construction method and application

A technology of pseudorabies virus and pseudorabies, which is applied in the direction of antiviral agents, viruses/bacteriophages, and methods based on microorganisms, can solve problems such as mutation, the inability of the vaccine Batha-K61 to produce protection, and economic losses in the pig industry. Filter for quick results

Inactive Publication Date: 2018-11-20
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In recent years, the virus has caused huge economic losses to the pig industry, and has begun to mutate under immune pressure. The previous preventive vaccine Batha-K61 has been unable to produce good protection against new PRV strains

Method used

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  • A kind of pseudorabies virus llt region δintron strain and its construction method and application
  • A kind of pseudorabies virus llt region δintron strain and its construction method and application
  • A kind of pseudorabies virus llt region δintron strain and its construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Experimental materials and reagents used in this study:

[0066] 18-T was purchased from Dalian Bao Biological Engineering Co., Ltd. (TaKaRa), product number D101A; pcDNA3.1(+) was purchased from Invitrogen Company, product number V79020.

[0067] GIBCO fetal bovine serum and neonatal bovine serum were purchased from Invitrogen, Cat. No. 10099-141.

[0068] DMEM medium was purchased from Invitrogen, USA. Item No.: 12800-017.

[0069] Opti-MEM (1×) medium was purchased from Invitrogen, USA. Item No.: 11058-021.

[0070] 0.25% Trypsin-EDTA (1×) was purchased from Invitrogen, USA. Item No.: 25200-072.

[0071] Phenol red-free DMEM medium was purchased from HyClone, USA. Item No.: SH3021115.

[0072] AxyPrep DNA Gel Recovery Kit was purchased from Ai Si Jin Biological Co., Ltd. Item No.: AP-GX-50.

[0073] Lipofectamine2000, a liposome transfection kit, was purchased from Invitrogen. Item No.: 11668-500.

[0074] Restriction enzymes were purchased from Dalian B...

Embodiment 2

[0085] PCR amplification of the upstream and downstream homology arms of Intron:

[0086] According to the full-length nucleotide sequence of the PRV genome (Genbank accession number is BK001744.1) published on GenBank (http: / / www.ncbi.nlm.nih.gov / ), design 2 pairs of primers (the sequence of the primer pair is shown in the table 1. All primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.), using the plasmid in the PRV genome library as a template to amplify the upstream and downstream homology arms of Intron respectively. For the detailed sequence of the upstream and downstream homology arms, see SEQ ID NO: 1 and SEQ ID NO: 3 in the sequence listing, and for the detailed sequence after Intron deletion, see SEQ ID NO: 2 in the sequence listing.

[0087] Table 1 Primers used for amplification of the upstream and downstream homology arms of Intron gene and identification of recombinant virus

[0088]

[0089] Table Note: The underlined part of the primer seque...

Embodiment 3

[0096] Construction of the transfection vector:

[0097] The upstream and downstream homology arms of Intron were amplified by PCR, recovered with AxyPrep DNA Gel Extraction Kit (purchased from Axygen), and ligated into pMD18-T vector respectively, named pZF11-81 and pZF11-82 respectively. Use HindIII and EcoRI to double digest the plasmid pZF11-81 and pcDNA3.1(+) vector, recover the upstream homology arm fragment and the vector, connect the upstream homology arm into the digested pcDNA3.1(+), and name the plasmid is pZF11-83. pZF11-82 was double-digested with EcoR I and XhoI, and the downstream homology arms were recovered and inserted into pZF11-83 after digestion with the same restriction enzymes, and the plasmid pZF11-84 containing the upstream and downstream homology arms of Intron was constructed (see figure 1 -A), identified by various restriction endonucleases to construct correctly ( figure 2 -A). In order to facilitate the screening of recombinant viruses, an EGF...

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Abstract

The invention discloses a pseudorabies virus LLT region delta Intron strain as well as a construction method and application. The construction method comprises the following steps: A. PCR amplification of upstream and downstream homologous arms of Intron; B. construction of a transfection vector, so that fragments of upstream and downstream homologous arms of Intron gene are cloned onto a pcDNA3.1 (+) vector by virtue of a molecular cloning technique; C. construction and identification of a PRV-delta Intron recombinant virus; D. in-vitro culture biological characteristics of the PRV-delta Intron recombinant virus, wherein PK-15 cells are inoculated to a prepared pseudorabies virus delta Intron gene-deleted strain; and E. animal experiment. The prepared pseudorabies virus delta Intron gene-deleted strain is infected by Balb / c mice, due to the deletion of the Intron, toxicity is greatly reduced. A regulatory effect on host and autologous protein encoding gene loses with the deletion of a miRNA expression region of the strain, causing drop in toxicity; therefore, the strain has a potential value of being developed as a novel vaccine. The method is easy to implement and is simple and convenient to operate, and the deleted region is stable in heredity and free from recurrence. The strain is used for preparing a novel pseudorabies vaccine for preventing and controlling porcine pseudorabies.

Description

technical field [0001] The invention relates to the technical field of genetic engineering of animal pathogens. It specifically relates to a PRVIntron deletion mutant live strain of pseudorabies virus E A strain, and also relates to a method for constructing a pseudorabies virus LLT region ΔIntron mutant strain, and also relates to a use of a pseudorabies virus LLT region ΔIntron mutant strain. Background technique [0002] Pseudorabies (PR), also known as pruritus and Aujeszky's disease (AD), is a disease characterized by fever, itching (except pigs) and encephalomyelitis caused by pseudorabies virus. A Severe Infectious Disease (Yin Zhen, Liu Jinghua. Animal Virology [M]. 2nd Edition. Beijing: Science Press, 1997: 700-713. McCracken RM, McFerran JB and Dow C. The neural spread of pseudorabies virus in calves. J Gen Virol, 1973,). The pathogen of pseudorabies is pseudorabies virus (PRV) of the family Herpesviridae and Alphaherpesvirinae. [0003] Pseudorabies virus infec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K39/245A61P31/22C12R1/93
Inventor 刘正飞张美美陈焕春王昕
Owner HUAZHONG AGRI UNIV
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