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Serum 4 type aviadenovirus reverse genetic vaccine strain rHN20 as well as construction method and application thereof

A poultry adenovirus and reverse genetic technology, applied in the field of medicine, can solve problems such as the increase of scientific prevention and control, the aggravation of HPS hazards, and the threat to the production of chicken embryo-derived veterinary vaccines

Pending Publication Date: 2021-03-23
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, FAdV-4 infection can lead to immunosuppression of the host, resulting in immune failure, secondary infection, and mixed infection, further exacerbating the harm of HPS
FAdV-4 can infect a wide range of hosts, not only infecting large-scale farmed layer hens, broiler chickens, and broiler breeders, but also infecting ducks, geese, and various wild birds. The diversity of FAdV-4 infection hosts increases the potential for cross-host transmission Risk, which increases the difficulty of scientific prevention and control of the disease
In addition, FAdV-4 can not only be transmitted horizontally through the respiratory tract, but also vertically through chicken embryos, posing a threat to the production of chicken embryo-derived veterinary vaccines. Some human vaccines are also produced through chicken embryos, which also poses a threat to the Human public health security constitutes a certain potential threat

Method used

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  • Serum 4 type aviadenovirus reverse genetic vaccine strain rHN20 as well as construction method and application thereof
  • Serum 4 type aviadenovirus reverse genetic vaccine strain rHN20 as well as construction method and application thereof
  • Serum 4 type aviadenovirus reverse genetic vaccine strain rHN20 as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Isolation and identification of new serotype 4 avian adenovirus virulent strain HLJFAd15

[0051] 1. Materials and Methods

[0052] 1.1 Experimental materials

[0053] 1.1.1 Virus strains and cells

[0054] FAdVs suspected disease materials were collected from affected chicken farms in different provinces in my country, and the virus was isolated from the livers of chickens and ducks with FAdVs suspected symptoms.

[0055] LMH cells were purchased from ATCC and cultured in DMEM medium (Thermo Fisher Scientific) with 1% penicillin and streptomycin and 10% FBS (purchased from Gibco).

[0056] 1.1.2 Main experimental reagents

[0057] Ex Taq premixed enzyme, PrimerSTAR Max premixed enzyme and DL 2,000Marker were purchased from TaKaRa Reagent Company; DNA extraction kit was purchased from Axygen Company; DNase / RNase Deionized Water (dd H2O) was purchased from Beijing Tiangen Co., Ltd.; Agarose was purchased from From Invivogen; primers were synthesized by Jili...

Embodiment 2

[0121] The construction of embodiment 2FAdV-4 reverse genetic vaccine strain rHN20

[0122] 1 Materials and methods

[0123] 1.1 Viruses, cells and strains

[0124] The new serotype 4 avian adenovirus (Fowl adenovirus 4, FAdV-4) HLJFAd15 strain was isolated and identified by Example 1, NCBI accession number: KU991797. Chicken Leghorn Male Hepatocellular Cell (LMH) and DH10B competent strains were preserved by the Poultry Immunosuppressive Disease Team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (hereinafter referred to as the laboratory).

[0125] 1.2 Main reagents

[0126] CopyControl TM Fosmid Library Production Kit was purchased from EpicentreBiotechnologies; ZR BAC DNA miniprep Kit was purchased from ZYMO; Counter-Selection BACModification Kit was purchased from Gene Bridges; β-Sepharose I and FseI restriction enzymes were purchased from NEB; PrimeSTAR HS DNA The polymerase was purchased from Dalian Bao Biological Engineering Co.,...

Embodiment 3

[0146] Example 3 Preliminary Evaluation of the Immune Effect of FAdV-4 Reverse Genetic Vaccine Strain rHN20

[0147] 1 Materials and methods

[0148] 1.1 Cells and viruses

[0149] Chicken Leghorn Male Hepatocellular Cell (LMH) was preserved by the Poultry Immunosuppressive Disease Team of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (hereinafter referred to as our laboratory).

[0150] The new serotype 4 avian adenovirus (Fowl adenovirus 4, FAdV-4) HLJFAd15 strain was isolated and identified in Example 1. The reverse genetic recombinant parental virus rFAdV-4 and the reverse genetic vaccine strain rHN20 were constructed from Example 2.

[0151] 1.3 Test animals

[0152] Specific-pathogen-free (SPF) chickens were purchased from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and kept in the negative pressure isolator of the center.

[0153] 1.4 Virus and vaccine culture

[...

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Abstract

The invention discloses a serum type 4 aviadenovirus reverse genetic vaccine strain rHN20 as well as a construction method and application thereof. According to the invention, infectious cloning cosmid Fos-rFAdV4 with the full length of FAdV-4 recently isolated strain HLJFAd15 genome in China is constructed, and on the basis, the Hexon gene is replaced with the Hexon gene of a foreign attenuated strain ON1 to obtain infectious cloning cosmid Fos-rHN20. The vaccine strain rHN20 is successfully rescued by utilizing the Fos-rHN20. Pathogenicity experiments show that rHN20 does not have pathogenicity on SPF chickens any more (100% of mortality rate exists before mutation). Immune protection experiments prove that the SPF chicken inoculated with the rHN20 can completely resist attacking infection of the FAdV-4 virulent strain, it is proved that the rHN20 has very good immunogenicity and can serve as a vaccine candidate strain, and meanwhile, the attenuated strain greatly reduces the hiddendanger of biosafety of the virulent strain.

Description

technical field [0001] The present invention relates to a serum type 4 poultry adenovirus vaccine strain and its construction method and application, in particular to a serum type 4 poultry adenovirus reverse genetic vaccine strain rHN20 and its construction method and application. The invention belongs to the technical field of medicine. Background technique [0002] Avian adenoviruses (FAdVs) are widely prevalent all over the world, and the popular strains mainly include FAdV-4, FAdV-11, FAdV-1, FAdV-8a, FAdV-8b and other serotypes, which have caused serious economic losses to the poultry industry. . FAdVs infection was first discovered in the Ankara region of Pakistan in 1987, so the disease is also called "Ankara disease", and related reports have been reported in China, Japan, South Korea, India, the United States, Canada and other countries and regions. FAdVs can be divided into three groups according to group-specific antigens: Group I includes traditional adenoviru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/66C12N15/64C12N15/34A61K39/235A61P31/20A61P1/16C12R1/93
CPCC12N7/00C12N15/85C12N15/66C12N15/64C07K14/005A61K39/12A61P31/20A61P1/16C12N2710/10221C12N2710/10222C12N2710/10252C12N2710/10234C12N2710/10262C12N2800/106A61K2039/5254A61K2039/552
Inventor 王笑梅潘青高玉龙崔红玉祁小乐刘长军李凯高宏雷张艳萍高立刘爱晶
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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