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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  • Abstract
  • Description
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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 gen

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

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Experimental program
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[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, using the peGFP-N1 vector as a template, the amplified green fluorescent protein (enhanced green fluorescent protein, eGFP) gene. The two genes were connected by fusion PCR method to obtain the eGFP screening expression cassette gene fragment, named p72-eGFP-SV40 polyA, and the expression cassette sequence contained the SV40 polyA termination sequence.

[0041] 1.2 Construction of homologous recombination transfer vector

[0042] Using pUC57 vector as a backbone vector, a homologous recombination transfer vector for MGF-360-9L gene knockout was constructed. The specific steps are as follows: Design 1.5 kb of the upstream and downstream sequence...

Example Embodiment

[0047] Example 2 Titration of virus titer

[0048] The titration of African swine fever virus adopts 50% haemadsorption (HAD) 50 ) Method to operate. HAD 50 The trial was based on the 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 PBMC in a 96-well cell culture plate, and 10 times the sample to be tested Inoculate 0.02ml in each well. Virus infection can be judged according to the rosette formed by red blood cells gathering around the infected cells. Observation for 7 days, according to Reed and Muench method (Reed, L. and H. Muench, A simple method of estimaing fifty percent endpoints. American Journal of Epidemiology 1938.27: p.493-497) calculates the ...

Example Embodiment

[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 injected intramuscularly into piglets to evaluate its pathogenicity. Specifically, in this example, 10 healthy Landrace piglets negative for the African swine fever antigen antibody were divided into 2 groups, each of five groups was used to attack the parental strain (wild African swine fever virus strain) and the deleted strain respectively. After that, the body temperature changes were measured every day, peripheral blood and saliva were collected, and the blood content of ASFV virus was determined by fluorescent quantitative PCR method. The observation was terminated on 19 days.

[0052] Results: The pathogenicity evaluation showed that the parent strain was injected intramuscularly with 10 HAD 50 Later, the typical symptoms of ASFV appeared: high temperature, and all died in the later period; while the miss...

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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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