Type 3 duck hepatitis A virus mutation gene ISA-A117C-T1142A and construction method

A technology of ISA-A117C-T1142A and duck hepatitis A virus, which is applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve the problems of lack of high-efficiency DHAV-3 live vaccines, etc., and achieve the goals of short time period, increased production, and reduced costs Effect

Active Publication Date: 2020-01-14
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; at the same time, DHAV host tropism and virulence Basic research on virus genes and key sites with changes in virulence can provide a theoretical basis for the prevention and treatment of duck hepatitis, and there is an urgent need to obtain virus strains with changes in host tropism and virulence.

Method used

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  • Type 3 duck hepatitis A virus mutation gene ISA-A117C-T1142A and construction method
  • Type 3 duck hepatitis A virus mutation gene ISA-A117C-T1142A and construction method
  • Type 3 duck hepatitis A virus mutation gene ISA-A117C-T1142A and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of Type 3 Duck Hepatitis A Virus Mutant Gene ISA-A117C-T1142A "Infectious Subgenomic Replicon" and Virus Rescue

[0036] 1.1. Design and synthesis of primers

[0037] According to the whole genome sequence of duck hepatitis A virus type 3 in GenBank, 8 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.

[0038] Table 1 Construction of primers for infectious subgenomic replicon of type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A

[0039]

[0040]

[0041] 1.2. Virus extraction

[0042] 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...

Embodiment 2

[0048] Example 2 Identification and characteristics of type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A virus strain

[0049] 2.1. Identification of genetic markers in rescued viruses

[0050] 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 amplified by PCR...

Embodiment 3

[0062] Example 3 Application of Type 3 Duck Hepatitis A Virus Mutation Gene ISA-A117C-T1142A Virus Strain in the Preparation of Inactivated Vaccine

[0063] 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-T1142A virus strain is an ideal vaccine candidate strain and can be used for the development of 3 Duck hepatitis A virus vaccine.

[0064] 3.1 Preparation method of inactivated vaccine

[0065] Dilute the type 3 duck hepatitis A virus mutant gene ISA-T1142A virus strain 100 times with sterilized saline, inoculate 20 9-day-old duck embryos in the allantoic cavity, 0.2 mL per embryo, a...

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Abstract

The invention discloses a type 3 duck hepatitis A virus mutation gene ISA-A117C-T1142A and a construction method. According to the mutant gene ISA-A117C-T1142A, 117 nucleotide of a type 3 duck hepatitis A virus virulent strain genome 5'UTR is mutated from A to C, and 1,142<nd> nucleotide is mutated from T to A, so that 164 amino acid of a virus VP0 protein is mutated from tyrosine of a parent strain to asparagine. The mutant gene virus strain has the same antigenicity as the parent strain, and has higher proliferation efficiency and virus titer on duck embryos than the parent strain, and can proliferate on chicken embryos with good immunogenicity and genetic stability. The pathogenicity of the mutant gene virus strain on ducklings is significantly reduced. The mutant gene virus strain can replicate in the ducklings but does not cause disease to the ducklings, and the important foundation is laid for a pathogenic mechanism of the virus and the development of vaccines. To sum up,the type 3 duck hepatitis A virus mutation gene ISA-A117C-T1142A virus strain is an ideal vaccine candidate strain, can be used for the study of the host tropism and attenuated mechanism of type 3 duck hepatitis virus, and has broad application prospects.

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-T1142A 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 spo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/41C07K14/10C12N15/85C12N7/00A61K39/125A61P31/14
CPCA61K39/12A61K2039/5252A61K2039/552A61P31/14C07K14/005C12N7/00C12N15/85C12N2770/32422C12N2770/32434C12N2770/32452C12N2800/106
Inventor 汪铭书文兴建程安春吴莉萍
Owner SICHUAN AGRI UNIV
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