Avian 4-type adenovirus vector, construction method and attenuated live vaccine and application of avian 4-type adenovirus vector

A technology of live attenuated vaccines and adenoviruses, applied in the direction of viruses/bacteriophages, viruses, vaccines, etc., can solve problems that have not yet been reported on live attenuated vaccines

Active Publication Date: 2021-09-14
中国疾病预防控制中心病毒病预防控制所 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research and development of FAdV-4 vaccines mainly focus on inactiv...

Method used

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  • Avian 4-type adenovirus vector, construction method and attenuated live vaccine and application of avian 4-type adenovirus vector
  • Avian 4-type adenovirus vector, construction method and attenuated live vaccine and application of avian 4-type adenovirus vector
  • Avian 4-type adenovirus vector, construction method and attenuated live vaccine and application of avian 4-type adenovirus vector

Examples

Experimental program
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Embodiment 1

[0053] Embodiment 1, construction of intermediate plasmid pAMS9002

[0054] The schematic diagram of the construction of the intermediate plasmid pAMS9002 is as follows: figure 1 As shown, the specific construction steps include:

[0055] (1) Digest the FAdV-4 genome with MauBI and SpeI (see Genbank accession number: MG547384 for the nucleotide sequence of the FAdV-4 genome), and use the kit after electrophoresis (Zymoclean Large FragmentDNA Recovery Kit, Cat.D4045, Zymo Research company) recovered an 8953bp fragment, which contains the open reading frames of ORF16, ORF17, GAM-1, ORF29, ORF28, ORF43, ORF42, ORF20, ORF20A and ORF22 on the right side of the FAdV-4 genome;

[0056] (2) Under standard polymerase chain reaction (PCR) conditions, using the plasmid vector pMD18-T as a template, use the primers shown in Table 1 below (the following primers are synthesized by BGI) to amplify the ORI of 2326bp - AMP fragment, containing the origin of replication (ORI) of the pBR322 pl...

Embodiment 2

[0062] Embodiment 2, the construction of intermediate plasmid pAMS-XGAM1

[0063] The schematic diagram of the construction of the intermediate plasmid pAMS-XGAM1 is as follows: figure 2 As shown, the specific construction steps include:

[0064] (1) Use BsiW I / Eag I to double digest the pAMS9002 plasmid, and recover a 10014bp fragment by electrophoresis; (2) Use the pAMS9002 plasmid as a template, and perform PCR amplification with the primers in the following Table 2 (the following primers are synthesized by Huada Gene) , the 705bp XGAM-1 fragment (SEQ ID NO: 3) was obtained and recovered by electrophoresis, and about 30bp at both ends of the fragment were identical to the sequence at both ends of the 10014bp fragment recovered above;

[0065] Table 2

[0066]

[0067] Note: Due to the use of DNA assembly technology, the 2010XGAM1f primer may not contain the BsiW I restriction site, and the 2010XGAM1r primer happens to contain the Eag I restriction site.

[0068] (3) ...

Embodiment 3

[0071] Embodiment 3, the construction of recombinant adenovirus plasmid pkFAV4-CXGA

[0072] The schematic diagram of the construction of the recombinant adenovirus plasmid pkFAV4-CXGA is as follows: image 3 As shown, the specific construction steps include:

[0073] (1) Use Pac I to digest the pAMS-XGAM1 plasmid, and recover a large fragment of 8385bp after electrophoresis; the large fragment of 8385bp does not contain an ORI-AMP fragment.

[0074] (2) Digest the pKFAV4-CX19A plasmid with MauB I / Spe I.

[0075] It should be noted that the pKFAV4-CX19A plasmid is a plasmid that has been disclosed in the prior art, and the construction method is described in YAN Bingyu, ZOU xiaohui et al. User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome. Viruses 2020, 12(3), 301.

[0076] Specifically, the pKFAV4-CX19A plasmid comprises the origin of replication of the pBR322 plasmid, the kanamycin resistance gene and the FAdV-4 genome (see...

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Abstract

The invention discloses an avian 4-type adenovirus vector, a construction method and an attenuated live vaccine and application of the avian 4-type adenovirus vector. The avian 4-type adenovirus vector comprises a replication origin nucleic acid sequence of pBR322 plasmid, a nucleic acid sequence of a kanamycin resistance gene and a genomic sequence of avian 4-type adenovirus; and in the genomic sequence of the avian 4-type adenovirus, genes ORF1, ORF1B, ORF2, ORF19A and GAM-1 are all inactivated. The avian 4-type adenovirus obtained by rescuing after the linearized avian 4-type adenovirus vector transfects cells is used as the attenuated live vaccine, and the attenuated live vaccine has the advantages of no pathogenicity, high safety and good immune protection effect, and can be used for preparing drugs for preventing diseases caused by avian adenovirus infection.

Description

technical field [0001] The invention belongs to the field of recombinant vaccines, and in particular relates to a poultry type 4 adenovirus vector, a construction method, an attenuated live vaccine and applications thereof. Background technique [0002] Fowl adenovirus (Fowl adenovirus, FAdV) is a non-enveloped double-stranded DNA virus with an icosahedral symmetrical structure and a diameter of about 70nm to 90nm. It belongs to the genus Aviadenovirus in the family Adenoviridae. . Based on the difference in molecular structure, avian adenoviruses can be divided into 5 groups A, B, C, D, and E; according to the results of cross-neutralization tests, they can be divided into 12 serotypes (FAdV-1 to FAdV- 8a and FAdV-8b to FAdV-11). The icosahedral shell of adenovirus is mainly composed of 240 hexons (hexons) and 12 pentons (pentons) distributed at each apex. Pentons include penton bases and prominent fiber proteins. Most Adenoviruses only encode one fiber protein, but both...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N7/04A61K39/12A61P31/20A61P9/00A61P1/16A61P1/00
CPCC12N15/86C12N7/00A61K39/12A61P31/20A61P9/00A61P1/16A61P1/00C12N2710/10221C12N2710/10234C12N2710/10243A61K2039/5254A61K2039/552
Inventor 邹小辉鲁茁壮刘兴龙章振华赵蕾
Owner 中国疾病预防控制中心病毒病预防控制所
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