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African swine fever virus attenuated strain lacking MGF360-9L gene and application of African swine fever virus attenuated strain

A technology of African swine fever virus and attenuated strains, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve the problems of different effects, elevated body temperature, and loss of immunogenicity or protective effect of weak immune side effects strains, etc. Achieving good safety effect

Inactive Publication Date: 2020-08-28
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this attenuated strain will cause a short-term rise in body temperature at the initial stage of vaccination, which poses certain safety risks, and the period for obtaining immunogenicity after vaccination is relatively long, requiring 28 days or longer
[0006] Up to now, the virulence gene knockout vaccine still has the following problems: different strains lack the same gene to produce different effects, immune side effects caused by insufficient deletion and excessive attenuation caused by multiple gene deletions will cause the attenuated strains to lose their immunogenicity or protective effects; and there are many genes knocked out, the operation is complicated, the cost is high, and it will also lead to a decrease in the success rate of knockout

Method used

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  • African swine fever virus attenuated strain lacking MGF360-9L gene and application of African swine fever virus attenuated strain
  • African swine fever virus attenuated strain lacking MGF360-9L gene and application of African swine fever virus attenuated strain
  • African swine fever virus attenuated strain lacking MGF360-9L gene and application of African swine fever virus attenuated strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction, purification and identification of MGF360-9L gene deletion African swine fever virus

[0039] 1.1 Construction of eGFP screening expression cassette

[0040] The p72 promoter sequence was amplified by PCR, that is, from -97 nt upstream of the p72 gene to the sequence before the ATG start codon; at the same time, the enhanced green fluorescent protein (enhanced green fluorescent protein, eGFP) gene. The two genes were connected by the method of fusion PCR to obtain the gene fragment of the eGFP screening expression cassette, which was named p72-eGFP-SV40 polyA, and the sequence of the expression cassette contained the SV40 polyA termination sequence.

[0041] 1.2 Construction of homologous recombination transfer vector

[0042] Using the pUC57 vector as the backbone vector, a homologous recombination transfer vector for MGF-360-9L gene knockout was constructed. The specific steps are: design the upstream and downstream sequences of MGF-360-9L g...

Embodiment 2

[0047] The titration of embodiment 2 virus titers

[0048] The titration of African swine fever virus adopts the half hematosorbate amount (50% haemadsorption, HAD 50 ) method to operate. HAD 50 The test was based on literature (Borca MV, Ramirez-Medina E, Silva E, Vuono E, Rai A, Pruitt S, Holinka LG, Velazquez-Salinas L, Zhu J, GladueDP. Development of a highly effective African swine fever virus vaccine by deletion of the I177Lgene results in sterile immunity against the current epidemic Eurasiastrain.JVirol. 2020. pii: JVI.02017-19) and make appropriate adjustments: Inoculate primary PBMCs in 96-well cell culture plates, and carry out 10 times Gradual dilution, inoculation of 0.02ml in each well, virus infection can be determined according to the rosettes formed by the aggregation of erythrocytes around the infected cells, observed for 7 days, according to the Reed and Muench method (Reed, L.andH.Muench, A simple method of estimaing fifty percent endpoints. American Jou...

Embodiment 3

[0050] Example 3 Animal challenge experiment

[0051] In order to detect the pathogenicity of the gene knockout ΔMGF-360-9L strain, this example uses 10 HAD 50 The dose was evaluated by intramuscular injection in piglets for its pathogenicity. Specifically, in this example, 10 healthy Landrace piglets negative for African swine fever antigen antibody were used, and they were divided into 2 groups of five piglets to attack the parental strain (wild strain of African swine fever virus) and the deletion strain respectively. Afterwards, the changes in body temperature were measured every day, peripheral blood and saliva were collected, and the blood content of ASFV virus was measured by fluorescent quantitative PCR method, and the observation was terminated after 19 days.

[0052] Results: The pathogenicity evaluation showed that the parental strain was injected intramuscularly with 10 HAD 50 Afterwards, the typical symptoms of ASFV appeared: high temperature, and all died in th...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an African swine fever virus attenuated strain lacking an MGF360-9L gene and an application of the African swine fever virus attenuated strain. The MGF360-9L gene of the African classical swine fever virus has a function of obviously inhibiting the response of a host face, and according to the African swine fevervirus attenuated strain lacking the MGF360-9L gene, the gene MGF-360-9L is knocked out from the whole genome of an ASFV CN / GS / 2018 isolated strain by adopting a genetic engineering means and utilizinga homologous recombination method, so that the aim of reducing the virulence is achieved. Experimental results of target animal piglets show that the African swine fever virus attenuated strain lacking the MGF360-9L single gene has good use safety and can be used as a candidate strain for preparing African swine fever vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an attenuated African swine fever virus strain lacking MGF360-9L gene and application thereof. Background technique [0002] African swine fever (ASF) is a severe contagious disease of swine caused by African swine fever virus (ASFV). OIE) lists it as a statutory reportable animal disease, and my country also lists it as a first-class animal disease. The disease was first reported in Kenya in 1921 and introduced to China in 2018. Subsequently, African swine fever broke out in a large area in my country, causing heavy losses to the pig industry. By December 2019, 32 provinces, municipalities, and autonomous regions had occurred 161 outbreaks (157 domestic pigs, 4 wild boars), culling more than one million pigs, is the "number one killer" of my country's pig industry. At present, the sharp decline in the number of pigs on hand and the soaring price of meat have brought seve...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85C12N15/65C12Q1/70A61K39/12A61P31/20
CPCA61K39/12A61K2039/5254A61K2039/552A61P31/20C07K14/005C12N7/00C12N15/65C12N15/85C12N2710/12021C12N2710/12022C12N2710/12034C12Q1/701
Inventor 郑海学张克山申超超党文李丹刘华南曹伟军田宏茹毅杨帆朱紫祥刘湘涛牛庆丽杨吉飞刘志杰
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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