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Porcine beta-interferon recombinant adenovirus and its construction method and application

A technology of recombinant adenovirus and interferon beta, which is applied in the fields of application, antiviral agent, virus/bacteriophage, etc., to achieve the effects of improved uniformity, stable titer, and improved time to market

Inactive Publication Date: 2007-05-30
广东省农业科学院兽医研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report on the successful construction of porcine interferon beta recombinant adenovirus

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  • Porcine beta-interferon recombinant adenovirus and its construction method and application

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Experimental program
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Effect test

Embodiment 1

[0028] As shown in Fig. 1, it is a flow chart of the production process of porcine beta interferon (IFN-β) recombinant adenovirus.

[0029] 1. Construction of porcine beta interferon recombinant adenoviral plasmid (pAdCMV-IFN-β)

[0030] (1) Primer

[0031] According to the gene sequence of porcine interferon beta (IFN-beta) in GenBank, a pair of specific primers for IFN-beta gene were designed, and the primers were synthesized by Shanghai Boya Biotechnology Co., Ltd.

[0032] Upstream primer P1: 5'-TTAGGTACC ATGGCTAACAAGTGCATC-3';

[0033] Downstream primer P2: 5'-TTATTCTAGA TGAGTTCCGGAGGTAATC-3';

[0034] Each primer contains a restriction endonuclease site at its 5' end.

[0035] (2) Cloning of porcine IFN-β gene

[0036] Extract DNA from pig liver tissue as a template to amplify the IFN-β gene. The reaction conditions are as follows: initial denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 48°C for 1 minute, extension at 72°C for 1.5 ...

Embodiment 2

[0075] Porcine β interferon (IFN-β) recombinant adenovirus used to prepare vaccine

[0076] The purified porcine interferon-beta recombinant adenovirus was continuously passaged on 293 cells for 30 times, and the expression of porcine interferon-beta IFN-β protein was detected, which proved that the expression of porcine IFN-B protein was stable, and the toxicity of the recombinant adenovirus was stable at 10 4.56 TCID 50 / 1.0mL.

[0077] During the expanded culture process, the purified and preserved porcine interferon beta recombinant adenovirus was used for 10 4 TCID 50 Seed to grow as a monolayer of 293 cells. When the cytopathy reaches 70-90%, the virus is harvested, and the porcine beta interferon recombinant adenovirus vaccine can be obtained after repeated freezing and thawing once.

Embodiment 3

[0079] Immunizing pigs with porcine interferon-beta recombinant adenovirus as a vaccine

[0080] The recombinant adenovirus containing porcine β interferon (IFN-β) gene has a virulence of 10 after artificial cultivation. 4.56 TCID 50 / 1.0mL, freeze-dried in a vacuum, used as a vaccine, has a good preventive effect on various diseases of pigs. The method of use is that sows and boars are immunized 3 times a year, 4-6 ml each time; small pigs are injected with 1-3 ml intramuscularly on 7-10 days, and 1-3 ml is injected intramuscularly after 10 days; medium pigs and large pigs can be Boost the immunization again, and inject 3 to 6 ml intramuscularly each time. It can effectively prevent various diseases of pigs caused by various viruses, prevent boars with poor semen quality, sows with abortion, stillbirth, mummification and small litter size, and the effective rate can reach 90%. The effective rate of systemic failure syndrome (PMWS) can reach more than 98%, the effective rate...

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Abstract

The invention discloses a pig alpha-interferon recombinant adenovirus and constructing method and application to prepare antiviral vaccine, which comprises the following steps: augmentating pig alpha-interferon IFN-alpha gene; cloning; constructing recombinant shuttle vector with IFN-alpha gene; establishing recombinant adenovirus plasmid carrier; obtaining recombinant adenovirus. The invention grows and expresses IFN-beta protein to make body stimulated by IFN-beta protein continuously, which generates neutralizing antibody of IFN-beta protein to protect pig permanently.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a porcine beta interferon (IFN-beta) recombinant adenovirus and its construction method and application. Background technique [0002] The recombinant adenovirus rAdCMV-IFN-β belongs to Adenoviridae (Adenoviridae), mammalian adenovirus (Mastadenovirus), and human adenovirus (Human adenovirus) in classification. The recombinant adenovirus can effectively express the target protein. Animals infected with the recombinant adenovirus have no clinical symptoms, so they can be used to express antigenic proteins. Recombinant adenovirus genetic engineering technology has been widely used in human gene therapy, and many recombinant adenoviruses used in human gene therapy have entered the third phase of clinical trials. Because the recombinant adenovirus has a wide range of infected hosts, it can infect dividing and non-dividing cells; it can express human and non-human proteins, and will not ...

Claims

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Application Information

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IPC IPC(8): C12N15/22C12N15/861C12N7/01A61K39/235A61K38/21A61P31/12
Inventor 宋长绪季芳蒋智勇
Owner 广东省农业科学院兽医研究所
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