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Vaccine for treating and/or preventing African swine fever virus and preparation method thereof

A vaccine and sequence technology, applied in the field of vaccines, can solve problems such as economic threats to people's livelihood and the advent of vaccines, and achieve the effects of reducing production costs, rapid research and development, and simple production processes

Active Publication Date: 2021-12-07
绵阳市游仙区创新科技产业技术研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the fatality rate of domestic pigs infected by African swine fever virus is nearly 100%, it poses a major threat to the domestic pig breeding industry and even the people's livelihood and economy. Unit vaccines, peptide vaccines and gene deletion vaccines, etc., but so far no effective vaccines have come out

Method used

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  • Vaccine for treating and/or preventing African swine fever virus and preparation method thereof
  • Vaccine for treating and/or preventing African swine fever virus and preparation method thereof
  • Vaccine for treating and/or preventing African swine fever virus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] In vitro transcription synthesis of embodiment 1 encoding ASFV antigen mRNA

[0040] 1. Reagent source

[0041] Restriction endonucleases BtsaⅠ and EcoRV were purchased from Yubao Bioengineering (Dalian) Co., Ltd., T7 HighYield Transcription Kit and Cap1 Capping System were purchased from Jinan Protein Technology Co., Ltd., and DH5α competent cells were purchased from Nanjing Nuoweizan Biotechnology Co., Ltd., DNA purification and recovery kits, and plasmid extraction kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and nucleotide fragment synthesis and carrier connection were completed by Sangon Bioengineering (Shanghai) Co., Ltd.

[0042] 2. Synthesis of nucleotide fragments and carrier ligation

[0043]Import the nucleotide sequence SEQ ID NO.6 data encoding the P72 protein (its amino acid sequence is SEQ ID NO.7) into the DNAstar software for sequence analysis, and perform homology with the DNA sequence obtained in the gene bank of NCBI ...

Embodiment 2

[0054] Embodiment 2 ASFV mRNA eukaryotic expression

[0055] 1. Reagent source

[0056] The mRNA prepared in embodiment 1, Lipofectamine TM 3000 was purchased from Thermo Fisher Scientific (China) Co., Ltd., ASFV positive serum was purchased from Qingdao Ruier Biotechnology Co., Ltd., HRP-labeled rabbit anti-pig IgG, and highly sensitive ECL luminescent reagent were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

[0057] 2. ASFV mRNA transfection 293T cell operation steps

[0058] The 239T cells that can grow stably were selected for six-well plates for plating, and transfection was performed when the cells grew to 60-70%. First, the mRNA encoding P72 protein and the mRNA encoding CAP80 protein were mixed at a mass fraction ratio of 2:1; then transfected according to the instructions of lip3000. First prepare liquid A and liquid B; liquid A: dilute 4 μg mRNA with 200 μL Opti-MEM; liquid B: dilute 10 μl lipo2000 with 200 μl Opti-MEM, mix liquid A and liquid B ge...

Embodiment 3

[0061] Example 3 LNP-mRNA vaccine production

[0062] 1. Reagent source

[0063] The mRNA prepared in Example 1, DLin-MC3-DMA, cholesterol, DSPC, and polyethylene glycol were purchased from Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd., and protamine was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. Co., absolute ethanol, ammonium citrate buffer.

[0064] 2. The operation process of vaccine preparation

[0065] Dissolve ionizable lipid (DLin-MC3-DMA), cholesterol, auxiliary lipid (DSPC), and polyethylene glycol in absolute ethanol according to the ratio of 50%, 29%, 20%, and 1%, and the total concentration is at 5-7mg / ml, constitute the organic phase; mix the mRNA encoding ASFV P72 protein and the mRNA encoding ASFV CAP80 protein at a mass fraction of 1:2, then add 0.05% protamine and mix evenly, with 0.8-1.2mg The total concentration per ml is dissolved in sodium citrate buffer to form the aqueous phase; the organic phase and the aqueous phase are mix...

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Abstract

The invention provides a vaccine for treating and / or preventing African swine fever virus and a preparation method thereof, and relates to the technical field of vaccines. The vaccine comprises two mRNAs, and the nucleotide sequences are SEQ ID NO. 1 and SEQ ID NO. 2 respectively. When the two mRNAs are transfected into 293T cells at the same time, the mRNAs can be correctly translated to form P72 protein, and the two mRNAs are wrapped with LNP and then delivered into the body to enable the body to generate an immune response.

Description

technical field [0001] The invention relates to the technical field of vaccines, in particular to a vaccine for treating and / or preventing African swine fever virus and a preparation method thereof. Background technique [0002] African swine fever is an acute and severe infectious disease caused by African swine fever virus, characterized by high fever, respiratory disturbance and neurological symptoms. African swine fever virus (African swine fever virus, ASFV) is the only species under the African swine fever virus family. to propagate. ASFV can infect domestic pigs and wild boars of different ages. Clinically, the mortality rate of the most acute type and the acute type ASF is as high as 100%, and the mortality rate of the subacute type can be reduced by 30% to 70%. The transmission routes of ASFV mainly include direct contact with infected animals or soft tick bites, or through contaminated swill, feed and other food, and also through contact with infected pig carcass...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/34A61K39/12A61K39/39A61K9/51A61P31/20
CPCC07K14/005A61K39/12A61K39/39A61K9/5123A61P31/20C12N2710/12022C12N2710/12034A61K2039/53Y02A50/30
Inventor 伍锐江峰孙岩松夏志平焦鹏涛
Owner 绵阳市游仙区创新科技产业技术研究院