Four-gene-deletion weak-toxin strain for African swine fever viruses and application of four-gene-deletion weak-toxin strain

A technology of African swine fever virus and gene deletion, applied in the field of bioengineering, can solve the problems of dose-dependent side effects, extremely severe and complicated prevention and control situation, and reduce the actual use value of vaccines, etc.

Inactive Publication Date: 2019-12-10
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In early August 2018, researcher Hu Rongliang took the lead in reporting the first African swine fever epidemic in my country. In just one year, the disease spread to 30 provinces and autonomous regions across the country, causing more than 20% of the country's production reduction rate and a loss of more than 10 billion yuan. , the current situation of the prevention and control of the disease is extremely severe and complicated
So far, a number of ASFV gene-deleted vaccine candidate strains have been obtained internationally by means of genetic engineering, and attenuated vaccine candidate strains have been obtained through cell passage attenuation and isolation of attenuated strains from nature, some of which are targeted at Homologous virus strains have a 50% to 100% protection rate, but the protection against heterologous virus attacks is not ideal
In addition, whether it is an artificial gene deletion attenuated strain or a natural deletion attenuated strain, the pigs have dose-dependent side effects after immunization, such as depression, loss of appetite, fever, viremia, hypergammaglobulinemia, pneumonia, joint swelling, Lameness, chronic disease and dead pigs, etc., reduce the practical value of the vaccine
At present, people are generally concerned about the safety of African swine fever gene deletion strains and natural attenuated strains as vaccines.

Method used

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  • Four-gene-deletion weak-toxin strain for African swine fever viruses and application of four-gene-deletion weak-toxin strain
  • Four-gene-deletion weak-toxin strain for African swine fever viruses and application of four-gene-deletion weak-toxin strain
  • Four-gene-deletion weak-toxin strain for African swine fever viruses and application of four-gene-deletion weak-toxin strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction and purification identification of recombinant viruses SY18ΔC and SY18ΔMC4

[0038] 1. Materials and Methods

[0039] 1.1 Biosafety license and African swine fever laboratory activity license

[0040] The Military Veterinary Research Institute has approved the Biosafety Committee of the Military Veterinary Research Institute, the Experimental Animal Ethics Committee, the Biosafety Committee of the Military Medical Research Institute, the National The Army Experimental Animal Ethics Committee and the Biosafety Committee of the Academy of Military Sciences reported it step by step, and obtained the permission from the Health Bureau of the Logistics Support Department of the Military Commission to carry out research on highly pathogenic ASFV pathogens and animals, and it has been filed with the Ministry of Agriculture and Rural Affairs, which meets the requirements of the national biosafety level. .

[0041] 1.2 Cells and viruses

[0042] Primary ...

Embodiment 2

[0061] Example 2 Determination of Growth Characteristics of Recombinant Viruses SY18ΔC and SY18ΔMC4 in PAM Cells

[0062] 1. Determination of virus growth curve

[0063] Spread the primary porcine PAM cells into a 6-well plate, grow into a monolayer, inoculate with 0.1moi virus solution, incubate at 37°C for 2h, and harvest at 0h, 6h, 12h, 18h, 24h, 30h, 36h, and 48h after infection The virus was amplified, frozen and thawed three times, and then subpackaged by high-speed centrifugation. The titers of the virus were measured respectively, and the growth curves of the wild virus and the gene knockout virus were drawn.

[0064] 2. Virus titer titration

[0065] Dilute the above-mentioned recombinant virus solution according to a 10-fold gradient, infect the porcine primary PAM cells that have been spread in a 96-well plate to form a monolayer, fix the infected cells with 4% paraformaldehyde solution 72 hours after infection, and add P30 diluted at an appropriate concentration. ...

Embodiment 3

[0068] Example 3 Safety Evaluation Experiment of Recombinant Viruses SY18ΔC and SY18ΔMC4

[0069] In order to detect whether the above two gene knockout viruses have no pathogenicity at all, in this experiment, at first, different infection doses (10 3 、10 4 、10 5 TCID 50 ) recombinant virus to evaluate the safety of piglets. In this experiment, 30 African swine fever antigen antibody-negative healthy Sanyuan piglets were divided into 6 groups. After immunization, the daily feed intake, body weight and body temperature were measured, and peripheral blood and saliva were collected every 5 days. The blood content of ASFV virus was measured by fluorescent quantitative PCR method, and the observation was terminated after 60 days. The experimental scheme is shown in Table 1.

[0070] Table 1 Safety Evaluation

[0071]

[0072] result:

[0073] Through the safety evaluation, it was found that the SY18ΔC strain was inoculated by intramuscular injection for 10 3 TCID 50 Th...

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Abstract

The invention discloses a four-gene-deletion weak-toxin strain for African swine fever viruses. The weak-toxin strain is the four-gene-deletion weak-toxin strain for an African swine fever virus SY18separation strain, and the following gene function protein is deleted: CD2v gene coding products and three multigene family genes( MGF360-12L, MGF360-13L and MGF360-14L ) coding products. The invention further discloses an application of the weak-toxin strain of the African swine fever viruses to preparation of vaccines for preventing or treating African swine fever. The weak-toxin strain of the African swine fever viruses can provide complete immunoprotection effect on attack of ASFV parent toxin strains, is high in safety, and is suitable for being used as vaccine candidate strains for preventing the African swine fever.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a four-gene-deleted attenuated strain of African swine fever virus and its application. Background technique [0002] African swine fever (ASF) is an acute and severe infectious disease of pigs caused by African swine fever virus (ASFV) infection, characterized by fever and systemic organ hemorrhage, and the fatality rate of domestic pigs can be as high as 100%. The disease first broke out in Kenya in 1921 and then became widespread in domestic and wild pigs throughout Africa, gradually evolving into 24 genotypes. It was introduced into Europe in the 1950s, and it took 40 years to eliminate the disease throughout Europe. However, the disease was introduced to Georgia from East Africa again in 2007, and then spread widely in Eastern Europe, and in 2017, it was introduced to Irkutsk in the Russian Far East. In early August 2018, researcher Hu Rongliang took the lead in rep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/34C12Q1/70C12Q1/686G01N33/569A61K39/12A61P31/20
CPCA61K39/12A61K2039/5254A61K2039/552A61P31/20C07K14/005C12N7/00C12N2710/12021C12N2710/12022C12N2710/12034C12Q1/686C12Q1/701G01N33/56983C12Q2563/107
Inventor 陈鸿军朱鸿飞扈荣良钱莺娟郭晓宇李金祥孙怀昌
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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