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Recombinant adenovirus and construction method thereof

A technology of recombinant adenovirus and adenovirus, applied in the direction of virus, virus/phage, double-stranded DNA virus, etc., can solve the problems of independent enhancement, virus reversion, attenuated swine fever vaccine and attenuated PRRS vaccine can not be immune at the same time. , to achieve strong resistance, solve the problem of not being able to immunize at the same time, and reduce the number and cost of vaccinations

Inactive Publication Date: 2017-06-23
SHAANXI SUYUAN AGRI DEV
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  • Claims
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Problems solved by technology

[0007] In order to solve the problems that the attenuated swine fever vaccine and the attenuated PRRS vaccine cannot be immunized at the same time, and the phenomenon of virus reversion and independent enhancement caused by long-term inoculation of the attenuated vaccine, and the recombination of the attenuated vaccine strain and the wild virus, the invention provides a Recombinant adenovirus expressing classical swine fever virus E2 gene and PRRS virus GP5 gene in the same adenovirus and its construction method

Method used

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  • Recombinant adenovirus and construction method thereof
  • Recombinant adenovirus and construction method thereof
  • Recombinant adenovirus and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0026] A. Amplification of classical swine fever virus E2 gene and PRRS virus GP5 gene

[0027] (1) Design of PCR amplification primers

[0028] Add Kpn I and Bgl II restriction endonuclease sites respectively in the upstream and downstream primers of the classical swine fever virus E2 gene, and the upstream and downstream primers of the classical classical swine fever virus E2 gene are designed as follows:

[0029]Upstream primer P1: 5'-AAAGGTACCATGAGGGGACAGATCGTGC-3'

[0030] Downstream primer P2: 5'-CCCAGATCTACCAGCGGCGAGTTGTTCTG-3'

[0031] In the upstream and downstream primers of the PRRS virus GP5 gene, respectively add Bgl II and Not I and a restriction endonuclease cutting site, the upstream and downstream primers of the PRRS virus GP5 gene are designed as follows:

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

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

[0034] (2) In vitro amplification of classical swine fever virus E2 g...

Embodiment 2

[0124] 1. rAd-E2-CMV-GP5 immune protection test

[0125] (1) Grouping of experimental animals

[0126] 24 pigs were randomly divided into 4 groups, the first group was rAd-E2-CMV-GP5 immunized group, 8 pigs. The second group is the immunization group of swine fever spleen drenching vaccine and PRRS attenuated vaccine, with 8 animals. The third group is the non-recombinant adenovirus immunization group, with 4 heads. The fourth group is the blank control group with 4 heads.

[0127] (2) Immunity and attack

[0128] The immunization doses of the recombinant adenovirus group and the non-recombinant adenovirus group were both 1.2×109pfu (about 2mL), which were injected into the neck muscle and abdomen subcutaneously at multiple points; The dosage was carried out according to the instructions of the vaccine; the blank control was immunized with the DMEM medium cultured with the recombinant adenovirus, 2 mL / head. All test pigs were challenged by intramuscular injection, eye dro...

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Abstract

The invention belongs to the field of biotechnology, and particularly discloses a recombinant adenovirus and a construction method thereof. Hog cholera virus E2 genes are inserted into a downstream multiple cloning site Bgl II and an Sal I digestion site of a CMV promoter of an adenovirus, and porcine reproductive and respiratory syndrome virus (PRRSv) GP5 genes are inserted into downstream Kpn I and Not I digestion sites of the CMV promoter of the adenovirus. The recombinant adenovirus can express both hog cholera virus E2 protein and PRRSv GP5 protein in the same adenovirus, immunized hogs can acquire hog cholera virus antibodies and PRRSv antibodies at the same time through one time of immunization only, vaccination frequency and cost are reduced, the protective effect of allied attack on the hog cholera virus and the PRRSv is 87.5%, the problem that hog cholera vaccine and PRRS vaccine can not be immunized at the same time is solved, the problem of virulence enhancement of attenuated vaccine after immunization is solved, and the problem of homologous recombination is also solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a recombinant adenovirus, in particular to a recombinant adenovirus capable of separately expressing the E2 gene of swine fever virus and the GP5 gene of PRRS virus in the same adenovirus and a construction method thereof. Background technique [0002] Swine fever is one of the main infectious diseases that harm the pig industry. At present, immunization is still the main measure for the prevention and treatment of swine fever, and it is a necessary means to eradicate the disease and prevent the spread of wild virus to pigs without swine fever. No matter in the past, at present or in the future, swine fever vaccine plays an extremely important role in controlling and eliminating swine fever. Scholars from various countries have never given up on the research and development of swine fever vaccine. Traditional swine fever vaccines include inactivated vaccines and attenuated vaccines, b...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/861C12N15/40
CPCC12N7/00C12N2710/10043
Inventor 黄光东孙永科舒建洪敬晓棋李河林郭小参黄广争李俊生
Owner SHAANXI SUYUAN AGRI DEV
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