A type 3 duck hepatitis A virus mutant gene isa-t1142a and its construction method

A technology of ISA-T1142A and duck hepatitis A virus, which is applied in the field of type 3 duck hepatitis A virus mutant gene ISA-T1142A and construction, and can solve the problems of lack of efficient DHAV-3 live vaccine and the like

Active Publication Date: 2021-07-06
SICHUAN AGRI UNIV
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  • 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-t1142a and its construction method
  • A type 3 duck hepatitis A virus mutant gene isa-t1142a and its construction method
  • A type 3 duck hepatitis A virus mutant gene isa-t1142a and its construction method

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Experimental program
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Effect test

Embodiment 13

[0033] Example 13 Construction of duck hepatitis A virus mutant gene ISA-T1142A "infectious subgenomic replicon" and virus rescue

[0034] 1.1. Design and synthesis of primers

[0035] 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.

[0036] Table 1 Construction of primers for infectious subgenomic replicon of type 3 duck hepatitis A virus mutant gene ISA-T1142A

[0037]

[0038] 1.2. Virus extraction

[0039] According to the instruction manual of the TaKaRa MiniBEST Universal RNA Extraction kit, the virus genome RNA of the type 3 duck hepatitis A virus isolate was extracted from the duck embryo allantoic fluid, and its nucleic acid was determined using a nucleic acid protein detector (BioRad, Smartspe...

Embodiment 23

[0045] Identification and characteristics of embodiment 23 type duck hepatitis A virus mutation gene ISA-T1142A virus strain

[0046] 2.1. Identification of genetic markers in rescued viruses

[0047] 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 w...

Embodiment 33

[0059] Application of Example 33 duck hepatitis A virus mutant gene ISA-T1142A virus strain in the preparation of inactivated vaccine

[0060] According to the mutant gene virus strain measured in embodiment 2, there is higher multiplication efficiency and virus titer than parental strain on duck embryo; Immunogenicity and genetic stability are good; To duckling pathogenicity has obviously declined, shows The mutant gene ISA-T1142A virus strain can be used as a vaccine candidate strain for preparing type 3 duck hepatitis A virus vaccine.

[0061] 3.1 Preparation method of inactivated vaccine

[0062] 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, and place it in a constant temperature incubator at 37 °C to continue Incubation, 24 hours after the inoculation, eggs were illuminated once, dead embryos were discarded, and eggs were illumi...

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Abstract

The invention discloses a type 3 duck hepatitis A virus mutant gene ISA-T1142A and its construction method. The 3403rd nucleotide G of the mutant gene genome is mutated into T, which can be used as a genetic marker for infectious clones and can be used to distinguish rescued strains With the parental strain and the wild strain; the 1142nd nucleotide is mutated from T to A, so that the 164th amino acid of the virus VPO protein is mutated from the tyrosine of the parental strain to asparagine. The mutant gene virus strain has the same antigenicity as the parent strain, and has higher proliferation efficiency and virus titer than the parent strain on duck embryos, and has good immunogenicity and genetic stability. The pathogenicity of the mutant gene virus strain to ducklings has been significantly reduced, which has laid an important foundation for the pathogenic mechanism of the virus and the development of vaccines. In conclusion, the type 3 duck hepatitis A virus mutant gene ISA‑T1142A virus strain can be used as a vaccine candidate or strain and can be used for the study of the attenuation mechanism of type 3 duck hepatitis A virus, with 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-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 spots at ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/41C12N7/01C12N7/04A61K39/29A61P31/14A61P1/16C12R1/93
CPCA61K39/12A61K2039/5254A61P1/16A61P31/14C07K14/005C12N7/00C12N2770/32421C12N2770/32422C12N2770/32434C12N2770/32462
Inventor 程安春文兴建汪铭书
Owner SICHUAN AGRI UNIV
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