Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

DP71L gene deleted recombinant African swine fever virus and preparation method and application thereof

An African swine fever virus, DP71L technology, applied in the research and development and prevention of potential vaccines for African swine fever virus, the recombinant African swine fever virus knocked out of the natural immunosuppressive gene DP71L and its construction field, which can solve the problem of large ASFV genome and difficulty of virus antigen Large and other problems, to achieve the effect of high application value

Active Publication Date: 2020-11-13
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
View PDF6 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problem that multiple viral proteins of the above-mentioned African swine fever virus have immunosuppressive functions and the ASFV genome is large, it is difficult to determine the viral antigens that induce protective immune responses. The present invention selects according to the African swine fever immune response suppression process and characteristics. Screening for virulence genes that suppress innate immune responses

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • DP71L gene deleted recombinant African swine fever virus and preparation method and application thereof
  • DP71L gene deleted recombinant African swine fever virus and preparation method and application thereof
  • DP71L gene deleted recombinant African swine fever virus and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The eukaryotic expression vector construction of embodiment 1DP71L gene

[0035] According to the ASFV / CN / GS / 2018 sequence of the African swine fever wild strain, the primers for PCR amplification of the DP71L gene were designed: upstream primer: 5'-ATGGGGAGGCGGCGCAAAAAACG-3'; downstream primer: 5'-TTACTGCTGCTCCAGTAGCTT-3'. Extract the DNA of the wild strain of African swine fever virus (refer to "Molecular Cloning"), use the designed primers, and use the DNA of the wild strain of African swine fever virus as a template to amplify the DP71L gene. The specific amplification system and procedures are as follows:

[0036] Template: 1 μl

[0037] Upstream primer: 1 μl

[0038] Downstream primer: 1 μl

[0039] Primerstar: 0.5 μl

[0040] Buffer: 10μl

[0041] Add water to a total volume of 50 μl

[0042] According to the program: 95°C for 5 minutes, 1 cycle; 98°C for 30 seconds, 98°C for 10 seconds, 60°C for 30 seconds, 72°C for 30 seconds, 35 cycles, and finally 72°C ...

Embodiment 2

[0050] Embodiment 2 eukaryotic vector expression DP71L gene

[0051] 1) Spread HEK293T cells on a 60mm small dish. After 24 hours, the cells grow to 80-90% density. Transfect the eukaryotic expression vectors of the two DP71L genes obtained in Example 1 into HEK293T cells. After 24 hours, collect Cells were lysed with protein lysate, added to 5×SDS loading buffer, boiled for 10 minutes, identified by SDS-PAGE and western blot detection (using flag tag antibody), the results were as follows figure 1 shown. The results showed that the eukaryotic expression vectors pCDNA3.1 and p3xFLAG-CMV-7.1 containing the target gene DP71L gene could express DP71L gene normally.

[0052] 2) Use DP71L gene eukaryotic expression vector to transfect HEK293 cells (1×10 5 ), while co-transfecting the IFNβ-luc luciferase reporter system, 24h later, transfected with cGAS+MITA, 24h later, lysing the cells and detecting the dual luciferase value, and using different doses of DP71L protein to detect ...

Embodiment 3

[0054] Construction of Example 3 DP71L Gene Knockout Strain

[0055] Materials: ASFV / CN / GS / 2018, the Chinese epidemic strain of African swine fever virus, was isolated and preserved by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. Porcine alveolar macrophages were isolated from SPF pigs aged 30-50 days, and cultured in 1640 medium (containing 10% FBS, from Thermo Scientific Company). Peripheral blood cells were separated using the PBMC separation kit from QIAGEN according to the instructions.

[0056] 1) Homologous recombination vector construction

[0057] The gene sequence group of about 1500bp around the ORF of the DP71L gene (as the left and right homology arms, the sequence position of the left arm is 182567-184067 relative to the full-length sequence, and the sequence position of the right arm is 184280-185780 relative to the full-length sequence) and the PCR fragment of eGFP were cloned into the pUC57 vector with a one-step...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the field of veterinary prevention, and particularly relates to a DP71L gene deleted recombinant African swine fever virus and a preparation method and application thereof. According to the method, the African swine fever virus DP71L protein can inhibit the activity of cGAS + MITA or IFN beta induced by MITA; the invention also provides the DP71L gene deleted recombinant African swine fever virus. The DP71L gene deleted recombinant African swine fever virus is obtained by deleting the DP71L gene of the African swine fever virus. Compared with a wild strain, the gene-deleted African swine fever virus infected cell shows that the toxicity is weakened; after pigs are immunized by the constructed gene-deleted African swine fever virus, clinical symptoms such as death and the like are avoided, the content of blood viruses is remarkably reduced in the later period of infection, and the survival rate is 100 percent so that the recombinant virus is fully weakened and is very safe as a vaccine. Therefore, the DP71L gene deleted recombinant African swine fever virus can be applied to preparation of African swine fever vaccines, and has corresponding social value.

Description

technical field [0001] The invention relates to the field of preventive veterinary medicine, specifically the research and development and prevention of potential vaccines for African swine fever virus. The details relate to a recombinant African swine fever virus knocked out of the natural immunosuppressive gene DP71L and its construction method and application. Background technique [0002] ASFV (African swine fever Virus) is the only member of the African swine fever virus family and the African swine fever virus genus. It mainly infects domestic pigs, wild boars, warthogs and soft ticks (Tulman et al., 2009). It is an acute , Violent, highly contagious infectious diseases, the mortality rate of virus-infected animals can reach 100%. No effective vaccine or drug for prevention or treatment has yet been developed. The virus can be transmitted through various channels, such as contact, feces, tick bites, etc. It is mainly prevalent in many countries such as Africa, Europe,...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N7/00C12N15/85C12N15/34A61K39/12A61P31/20C12Q1/70C12R1/93
CPCC12N7/00C12N15/85C07K14/005A61K39/12A61P31/20C12Q1/701C12N2710/12021C12N2710/12022C12N2710/12052C12N2710/12062A61K2039/552A61K2039/5254Y02A50/30A61K2039/55
Inventor 郑海学马旭升宋锐党文冯涛曾宗波罗志宽莫希丁·乔杜里张克山毛若箐田宏杨帆刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com