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Porcine circus-virus 2 type recombinant adenovirus and its construction method and uses

A technology of recombinant adenovirus and porcine circovirus, applied in the fields of application, virus/phage, recombinant DNA technology, etc., can solve the problems of undiscovered recombinant adenovirus of porcine circovirus, etc., and achieve enhanced immune protection and stable expression , the effect of stable potency

Inactive Publication Date: 2006-05-10
广东省农业科学院兽医研究所
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  • Claims
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Problems solved by technology

No recombinant adenoviruses constructed into porcine circovirus (PCV2) have been reported yet

Method used

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  • Porcine circus-virus 2 type recombinant adenovirus and its construction method and uses

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Experimental program
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Effect test

Embodiment 1

[0029] 1. Construction of porcine circovirus type 2 recombinant adenovirus plasmid (pAdCMV-ORF2)

[0030] (1) Primer

[0031] According to the gene sequence of porcine circovirus type 2 (PCV2) in GenBank, a pair of specific primers targeting the ORF2 gene of PCV2GD strain (GenBank accession number AY613854) were designed, and the primers were synthesized by Shanghai Boya Biotechnology Co., Ltd.

[0032] Upstream primer P1: 5'-TTAGGTACCATGACGTATCCAAGGAG-3'

[0033] Downstream primer P2: 5'-TTAAAGCTTAGGGGTTAAGTGGGG-3'

[0034] The 5' ends of the primers contain restriction endonuclease sites.

[0035] (2) Cloning of PCV2 ORF2 gene

[0036] The DNA of PCV2 cultured in pig kidney cells PK-15 cells was extracted as a template to amplify the ORF2 gene of PCV2 GD strain (domestic isolate). Annealing at ℃ for 1 min, extension at 72 °C for 1.5 min, 30 cycles, and finally extension at 72 °C for 10 min. The PCR product was recovered with a kit and ligated with the pMD18-T vector; th...

Embodiment 2

[0064] Porcine Circovirus Type 2 (PCV2) Recombinant Adenovirus Used to Prepare Vaccine

[0065] The purified porcine circovirus type 2 recombinant adenovirus was continuously passaged on 293 cells for 30 times, and the expression of porcine circovirus type 2 ORF2 protein was detected, which proved that the expression of PCV2 ORF2 protein was stable, and the virulence of the recombinant adenovirus was stable at 10 4.56 TCID 50 / 1.0mL.

[0066] During the expanded culture process, the purified and preserved PCV2 recombinant adenovirus was used for 10 4 8TCID 50 Seed to grow as a monolayer of 293 cells. When the cell lesion reaches 70-90%, the virus is harvested, and the PCV2 recombinant adenovirus vaccine can be obtained after repeated freezing and thawing once.

Embodiment 3

[0068] Porcine Circovirus Recombinant Adenovirus as Vaccine to Immunize Pigs

[0069] The recombinant adenovirus containing the ORF2 gene of PCV2 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 than 98%, the effective rate of...

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Abstract

The invention provides a restructing adenovirus of 2-type pig-ring virus and constructing method thereof, and the preventing appliances of pigs' relevant diseases caused by PCV2, including: augment and clone of PCV2 ORF2 genes; construction of recombinant shuttle vectors containing ORF2 genes; construction of recombinant adenovirus plasmid vectors; acquirement of recombinant adenovirus. Said recombinant adenovirus could determinately grow inside the bodies and continueously express ORF2 proteins to stimulate the organisms which generate neutralizing antibodies for ORF2 proteins, and offer endurance protection to pigs with promising future in the prevention and cure of PCV2, which could prevent efficiently poor quality of male-pig sperms, abortion, dead fetus, mummy and low production of female-pigs to increase the uniformity of pigs greatly.

Description

technical field [0001] The invention belongs to the high-tech field of biotechnology, in particular to a recombinant adenovirus of porcine circovirus type 2 (PCV2) and its construction method and application. Background technique [0002] Recombinant adenovirus rAdCMV-ORF2 belongs to Adenoviridae (Adenoviridae), mammalian adenovirus (Mastadenovirus), human adenovirus (Human adenovirus) in classification, and the recombinant adenovirus can effectively express the target protein. Recombinant adenoviruses do not appear clinical symptoms after infecting animals, 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 a...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K39/235C12N1/21C12N15/70C12N15/861
Inventor 宋长绪蒋智勇王贵平黄忠
Owner 广东省农业科学院兽医研究所
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