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

A technology of recombinant adenovirus and alpha interferon, which is applied in the fields of application, antiviral agent, virus/phage, etc., to achieve the effect of preventing various diseases, inhibiting virus replication, and stabilizing the titer

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

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

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Embodiment 1

[0033] As shown in FIG. 1 , it is a flow chart of the production process of the recombinant porcine interferon-α adenovirus.

[0034] 1. Construction of porcine alpha interferon recombinant adenoviral plasmid (pAdCMV-IFN-α)

[0035] (1) Primer

[0036] Two pairs of primers were designed according to GenBank porcine interferon alpha IFN-α1 gene sequence (accession number X57191) and related pseudogene sequence (accession number AF350425), wherein primers P1 and primer P2 amplified the first half of the gene (F fragment), primers P3, Primer P4 amplifies the second half (fragment B). There is a 37bp repeat sequence between the two genes of the F segment and the B segment, which can meet the needs of splice overlap extension PCR (splice overlap extension PCR, SOE-PCR). Primer P2 and primer P3 are located in the region where the variation is concentrated within the gene, and their 3' terminal bases are only specifically complementary to the porcine IFN-α gene, but have difference...

Embodiment 2

[0079] Preparation of porcine alpha interferon (IFN-α) recombinant adenovirus vaccine

[0080] The purified porcine interferon-alpha recombinant adenovirus was continuously passaged on 293 cells for 30 times, and the expression of porcine interferon-alpha was detected, which proved that the expression of porcine interferon-alpha was stable, and the toxicity of the recombinant adenovirus was stable at 10 4.56 TCID 50 / 1.0mL.

[0081] During the expanded culture process, the purified and preserved porcine interferon-alpha recombinant adenovirus was used for 10 4 TCID 50 Seed to grow into a monolayer of 293 cells. When the cytopathy reaches 70-90%, the virus is harvested, and the porcine alpha interferon recombinant adenovirus vaccine can be obtained after repeated freezing and thawing once.

Embodiment 3

[0083] Immunizing pigs with porcine interferon-alpha recombinant adenovirus as a vaccine

[0084] The recombinant adenovirus containing porcine alpha interferon (IFN-α) 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 PCV2, prevent poor semen quality of boars, miscarriage, stillbirth, mummification and small litter size of sows, the effective rate can reach 90%, and prevent multi-system failure of pigs after weaning Syndrome (PMWS) the effective rate can reach more tha...

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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.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a porcine alpha interferon (IFN-α) recombinant adenovirus and its construction method and application. Background technique [0002] The recombinant adenovirus rAdCMV-IFN-α belongs to the Adenoviridae family (Adenoviridae), the mammalian adenovirus (Mastadenovirus), and the human adenovirus species (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-huma...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/21C12N7/01A61K39/235A61K38/21A61P31/12
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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