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A type 3 duck hepatitis A virus mutant gene isa-a117c and its construction method

A technology of ISA-A117C and duck hepatitis A virus, applied in the field of type 3 duck hepatitis A virus mutant gene ISA-A117C and its construction, can solve the problems of lack of high-efficiency DHAV-3 live vaccine

Active Publication Date: 2021-07-06
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the prevention and control of DHAV in the market mainly uses DHAV-1 attenuated vaccine, and there is still a lack of efficient DHAV-3 live vaccine. It is imminent to develop a new molecular marker vaccine suitable for the prevalence of DHAV-3 in my country; Basic research on viral genes and key sites can provide a theoretical basis for the prevention and treatment of duck hepatitis, and there is an urgent need to obtain virus strains with virulence changes

Method used

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  • A type 3 duck hepatitis A virus mutant gene isa-a117c and its construction method
  • A type 3 duck hepatitis A virus mutant gene isa-a117c and its construction method
  • A type 3 duck hepatitis A virus mutant gene isa-a117c and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of Type 3 Duck Hepatitis A Virus Mutant Gene ISA-A117C "Infectious Subgenomic Replicon" and Virus Rescue

[0035] 1.1. Design and synthesis of primers

[0036] According to the whole genome sequence of duck hepatitis A virus type 3 in GenBank, 7 pairs of primers were designed to amplify the whole genome sequence of the virus, pCMV and SV40pA sequences. The specific sequence information is shown in Table 1. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0037] Table 1 Construction of primers for infectious subgenomic replicon of type 3 duck hepatitis A virus mutant gene ISA-A117C

[0038]

[0039]

[0040] 1.2. Virus extraction

[0041] According to the instruction manual of the TaKaRa MiniBEST Universal RNA Extraction kit, the total viral genome RNA of the type 3 duck hepatitis A virus isolate was extracted from the allantoic fluid of duck embryos, and its content was determined using a nucleic acid protein detec...

Embodiment 2

[0047] Example 2 Identification and characteristics of type 3 duck hepatitis A virus mutant gene ISA-A117C virus strain

[0048] 2.1. Identification of genetic markers and mutation sites in rescued viruses

[0049] In order to exclude the possibility that the rescued virus may come from the contamination of the parental virus or the wild strain during the transfection or subculture process, the reverse genetics method was used to mutate the 3403rd base of the mutant gene genome from the mutation G to T, which does not change 2A The corresponding amino acid composition of the protein, as a molecular genetic marker site, can be used to distinguish mutant gene virus strains from parental strains and wild strains by PCR method combined with DNA sequencing. The rescued virus was passaged and purified 5 times on duck embryos by limiting dilution. Total RNA was extracted from the allantoic fluid, and after reverse transcription, the DNA fragment containing the mutation site was ampl...

Embodiment 3

[0061] Example 3 Application and Effect Evaluation of Type 3 Duck Hepatitis A Virus Mutant Gene ISA-A117C Virus Strain in the Preparation of Inactivated Vaccine

[0062] According to the mutant gene virus strain determined in embodiment 2, there is higher propagation efficiency and virus titer than parental strain on duck embryo; Can also propagate on chicken embryo; Immunogenicity and genetic stability are good; To duckling The pathogenicity has been significantly reduced, and the mutant gene virus strain can replicate in ducklings but is not pathogenic to ducklings, indicating that the mutant gene ISA-A117C virus strain is an ideal vaccine candidate strain and can be used to prepare type 3 ducks Hepatitis A virus vaccine.

[0063] 3.1 Preparation method of vaccine

[0064] The type 3 duck hepatitis A virus mutant gene ISA-A117C virus strain was diluted 100 times with sterilized saline as the seed virus, and 20 9-day-old duck embryos were inoculated into the allantoic cavity...

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Abstract

The present invention disclosed a type 3 duck armor virus mutation gene isa‑a117c and the construction method. The 3403 -bit nucleotide of the mutant gene genome is T to T. The 117 nucleotide of 5'UTR from A and Wild strains is mutated from A to C. The mutant gene virus strain has the same antigenicity as the pro -based strain, and has higher proliferation efficiency and virus titer than the pro -pro -base strains on the duck embryo, and can be proliferated on the chicken embryo. The immunogenicity and genetic stability are good. The pathogenic force of mutant gene virus strains has fallen significantly to ducky ducks. It can be replicated in the ducky duck, but it does not cause disease to the duck. This has laid an important foundation for the virus pathogenic mechanism and vaccine development. In summary, the type 3 duck noreal virus mutation gene isa‑a117C virus strains can be used as a vaccine candidate or cultivated strain and can be used for the research of type 3 duck nail virus hosts and weak mechanisms. The application prospects are broad.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a type 3 duck hepatitis A virus mutation gene ISA-A117C and a construction method. Background technique [0002] Duck viral hepatitis (DVH) is an acute, highly contagious disease caused by ducklings infected by duck hepatitis virus (DHV). At present, the disease exists in the main duck breeding areas in the world, and has the characteristics of intermittent outbreaks and local epidemics. It is one of the main diseases that endanger the duck industry. The disease mainly affects ducklings less than four weeks old, and has the characteristics of acute onset, rapid spread, short course of disease and high mortality; the main clinical manifestations are convulsions before the duckling dies, and the head is tilted back to the back, showing "opistonus" , The pathological changes were mainly enlarged and inflamed liver and a large number of hemorrhagic spots at a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/41C12N7/01A61K39/125A61P31/14A61P1/16
CPCA61K39/12A61K2039/552A61P1/16A61P31/14C07K14/005C12N7/00C12N2770/32421C12N2770/32422C12N2770/32434
Inventor 程安春文兴建汪铭书
Owner SICHUAN AGRI UNIV
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