Pseudorabies virus (PRV) digene deletion attenuated strain and application thereof

A pseudorabies virus, pseudorabies technology, applied in the direction of viruses, antiviral agents, virus antigen components, etc., can solve the problems of preventing detoxification, and the safety of live mutant vaccines is not high enough

Active Publication Date: 2018-03-23
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the safety of existing variant live vaccines is not high enough
For example, the invention patent with the application number 201510388390.9 discloses the LA-A strain obtained by knocking out the gE gene of the wild pseudorabies virus AH02LA strain. The va

Method used

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  • Pseudorabies virus (PRV) digene deletion attenuated strain and application thereof
  • Pseudorabies virus (PRV) digene deletion attenuated strain and application thereof
  • Pseudorabies virus (PRV) digene deletion attenuated strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1. Construction and identification of pseudorabies virus gE gene deletion strain

[0020] 1. Obtaining transfer vectors

[0021] In order to knock out part of the gE gene of the PRV AH02LA strain of pseudorabies virus, a gene fragment composed of the upstream homology arm ΔgE-H1 of the gE gene, the green fluorescent protein expression cassette and the downstream homology arm ΔgE-H2 of the gE gene was artificially synthesized, and then This fragment is cloned into the pUC57 vector (Nanjing GenScript Biotechnology Co., Ltd.), and the GFP transfer vector pPRV(AH02LA)-GFP(gE-) is obtained, and its structure is as follows: figure 1 shown. The sequence of the homology arm ΔgE-H1 upstream of the gE gene is shown in SEQ ID No: 2, the sequence of the homology arm ΔgE-H2 downstream of the gE gene is shown in SEQ ID No: 3, and the sequence of the GFP expression cassette is the same as the application number 201510388390.9 Patent.

[0022] Then artificially synthesized a...

Embodiment 2

[0033] Example 2, construction and identification of pseudorabies virus gE, TK double gene deletion strain

[0034] 1. Obtaining transfer vectors

[0035] In order to continue knocking out part of the TK gene of PRV AH02LA (ΔgE) strain, Nanjing GenScript Biotechnology Co., Ltd. artificially synthesized the upstream homology arm of TK gene ΔTK-H1, green fluorescent protein expression cassette (GFP) and TK The gene fragment composed of the homology arm ΔTK-H2 downstream of the gene, and then clone the fragment into the pUC57 vector to obtain the GFP transfer vector pPRV(AH02LA)-GFP(TK-), its structure is as follows Figure 4 shown. The sequence of the homology arm ΔTK-H1 upstream of the TK gene is shown in SEQ ID No: 4, the sequence of the homology arm ΔTK-H2 downstream of the TK gene is shown in SEQ ID No: 5, and the sequence of the GFP expression cassette is the same as that of the invention with the application number 201510388390.9 patent.

[0036] Send to Nanjing KingScr...

Embodiment 3

[0058] Embodiment three, the identification of the growth characteristic of PRV LA1206-80 strain

[0059] The highly virulent strains PRV AH02LA, PRV LA-A and PRV LA1206-80 were inoculated on monolayer ST cells at an MOI of 0.004, and placed in 5% CO 2 After incubating at 37°C for 1 h in a constant temperature incubator, the supernatant was sucked off, washed three times with PBS, and replaced with cell maintenance medium (DMEM medium (Gibco) containing 3% fetal bovine serum (FBS)) for culture, and The content of free virus in the supernatant of infected cells and cell-associated virus in infected cells was measured at 0h, 6h, 12h, 24h, 36h, 48h and 72h after inoculation, respectively.

[0060] The free virus content of the infected cell supernatant was operated according to the following method: 1ml of the infected cell supernatant was collected at the designated time point, centrifuged at 376×g for 5min to take 100 μl of the supernatant, added to 900 μl of DMEM medium and mi...

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Abstract

The invention provides a pseudorabies virus (PRV) digene deletion attenuated strain and application thereof, and belongs to the field of vaccines for animal medicine. A preservation number of a PRV LA1206-80 strain is CGMCC (China General Microbiological Culture Collection Center) No.14329. The invention also provides application of the PRV digene deletion attenuated strain in preparation of a pseudorabies vaccine, and a recombinant vector live vaccine. A recombinant vector is obtained by inserting an exogenous protective antigen gene expression cassette into an attenuated strain genome. The live vaccine of the PRV LA1206-80 strain is absolutely safe to one-day-old neonatal piglets, can be used for early immunization of the neonatal piglets and blocks off infection of wild type viruses asearly as possible. The vaccine is inoculated to weaned PRV negative piglets, so that a high-efficiency protecting force can be quickly generated; particularly, the vaccine can prevent attacking and detoxify. By monitoring PRV gE and gB antibodies, purification on a PRV variant strain is greatly facilitated.

Description

technical field [0001] The invention belongs to the vaccine field of veterinary medicine, and in particular relates to an attenuated double gene deletion strain of pseudorabies virus and an application thereof. Background technique [0002] Pseudorabies is an acute and severe infectious disease caused by Pseudorabies Virus (PRV). Pseudorabies in domestic and wild animals such as cattle and sheep often manifests as typical symptoms of severe itching, as well as clinical symptoms such as fever and encephalomyelitis. The symptoms of pseudorabies in pigs are related to age, and sows often suffer from abortion; newborn piglets have neurological symptoms and high mortality; fattening pigs mainly have respiratory symptoms, and adult pigs grow stagnant after being infected with pseudorabies virus, and usually do not die. Due to the invention and large-scale promotion and use of PRV gene deletion strain vaccines (such as the Bartha K61 strain with gE gene deletion, etc.), and the es...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/245A61P31/22C12R1/93
CPCA61K39/12A61K2039/5254A61K2039/552C12N7/00C12N2710/16721C12N2710/16734C12N2710/16741C12N2710/16751
Inventor 王继春宋增财郭容利乔永峰王志胜许梦微刘娅梅郑亚婷范红杰侯继波
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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