Genetically-engineered vaccine against fowl adenovirus type 4, and preparation method and application thereof

A technology of genetically engineered vaccine and poultry adenovirus, which is applied in the field of type 4 poultry adenovirus genetically engineered vaccine and its preparation, can solve the problems of harsh culture conditions, high production costs, and difficulty in large-scale cultivation, and achieve high protein yield and safe The effect of high sex and strong immunity

Pending Publication Date: 2020-04-21
乾元浩生物股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of whole-virus inactivated vaccines requires the use of sensitive cell lines of chicken liver cancer cells, which are not firmly attached to the wall and are difficult to cultivate on a large scale. The culture conditions are harsh, fetal bovine serum is required, and the production cost is high.
Chicken liver cancer cells are cancer cells, and there are safety risks when injected into chickens

Method used

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  • Genetically-engineered vaccine against fowl adenovirus type 4, and preparation method and application thereof
  • Genetically-engineered vaccine against fowl adenovirus type 4, and preparation method and application thereof
  • Genetically-engineered vaccine against fowl adenovirus type 4, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0040] The preparation method of the vaccine can adopt the conventional preparation method of genetic engineering subunit vaccine in the field.

[0041] Preferably, the preparation method includes:

[0042]The pMD19-T vector containing the target gene was double digested with Bam HI and Hind III to obtain the target fragment, and the target fragment was recovered and purified from the gel. At the same time, the pFastBac 1 vector was digested with double enzymes and recovered by gel. The target gene fragment and pFastBac 1 vector were ligated overnight with T4 DNA ligase. Then it was transformed into DH5α competent cells, picked and cultured and then sent for sequencing. After the sequencing is correct, extract the pFastBac 1 vector containing the target gene.

[0043] The pFastBac 1 vector containing the target gene was transformed into DH10Bac, and the transformed product was cultured in 37°C SOC medium for 5 hours, then diluted and inoculated on a blue-white spot screenin...

Embodiment 14

[0048] Example 1 Preparation of Type 4 Avian Adenovirus Genetic Engineering Vaccine

[0049] 1. Screening and cloning of target genes

[0050] Find the protein coding gene sequence and protein sequence of penton and hexon of avian adenovirus, and perform sequencing comparison on RNA extracted from existing viruses. According to the previous research results, the region with strong antigenicity was selected for splicing, and a linker (Linker) was added between the penton and hexon proteins to ensure its spatial conformation and the formation of virus-like particles (VLPs).

[0051] Check the restriction enzyme cutting sites in the optimized sequence to ensure that it does not contain Bam H I and Hind III cutting sites, and send the spliced ​​sequence to the biological company for synthesis.

[0052] A pair of splicing primers for amplifying avian adenovirus gene fragments were designed with Primer5.0 software, and protective bases and restriction sites were added to the 5' end...

Embodiment 2

[0064] Example 2 Expression characteristics and immunogenicity of type 4 avian adenovirus genetic engineering vaccine

[0065] 1. Expression characteristics

[0066] Sf9 cells were cultured to 5 × 10 in Sf9 serum-free suspension medium 6 cells / ml, dilute the cell density to 2.5×10 with fresh serum-free medium 6 cells / ml, the recombinant baculovirus was inoculated at a ratio of 1‰, and the supernatant was harvested after culturing for 96 hours. The cells at 96h post-infection were significantly larger than the healthy cells. Determine virus titer by plaque method, recombinant baculovirus P1 generation virus titer 10 8.25 TCID 50 / ml.

[0067] Sf9 cells were cultured with Sf9 serum-free suspension medium to 5 × 10 6 cells / ml, dilute the cell density to 2.5×10 with fresh serum-free medium 6 cells / ml, inoculate the recombinant baculovirus at a ratio of 1%-1‰, culture until 168-192h and harvest the culture supernatant. Carry out SDS-PAGE electrophoresis identification prote...

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Abstract

The invention provides a genetically-engineered vaccine against fowl adenovirus type 4, and a preparation method and application thereof. According to the invention, protective antigens, namely pentonand hexon proteins of fowl adenovirus serotype 4 are connected by a linker, and are expressed by an insect cell-baculovirus expression system to form virus-like particles of fowl adenovirus type 4 onspatial conformation; an adjuvant is added; and emulsification is carried out so as to prepare the genetically-engineered vaccine against fowl adenovirus type 4. The preparation method for the vaccine provided by the invention is simple in process, capable of preparing a large amount of fowl adenovirus type 4 antigen protein, short in time consumption, high in expression quantity and beneficial to large-scale production; and the obtained genetically-engineered vaccine is good in immune effect and capable of effectively preventing infection of the fowl adenovirus type 4.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a type 4 poultry adenovirus genetic engineering vaccine and its preparation method and application. Background technique [0002] In 1987, Angola, Pakistan, first reported a large outbreak of hydropericardium hepatitis syndrome (HHS) caused by Fowladenovirus group Iserum type (FAdV-4). , Mumbai and other places broke out one after another, and caused huge economic losses. FAdV-4 is the most pathogenic to chicks, and can be transmitted horizontally and vertically through contact. The main lesions are clear or light yellow watery or jelly-like liquid in the pericardial cavity, occasionally focal necrosis or discoloration of the liver, and basophilic inclusion bodies in liver cells, with a high mortality rate. [0003] FAdV-4 belongs to the adenovirus group A, and the virus particles are spherical, with a diameter of 70-90nm, no envelope, icosahedral symmet...

Claims

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Application Information

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IPC IPC(8): C07K14/075C12N15/34C12N15/866A61K39/235A61P31/20
CPCC07K14/005C12N15/86A61K39/12A61P31/20C12N2710/10222C12N2710/10234C12N2800/105C12N2710/14043A61K2039/552
Inventor 袁野岳建新王飞孙灵睿周蕾蕾陈秋阁向王震张秀美李浩鹏李浩哲朱昊敏魏杰安铁军
Owner 乾元浩生物股份有限公司
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