P72 fusion protein of African swine fever virus , and preparation method and application thereof

A technology of African swine fever virus and fusion protein, which is applied in the field of African swine fever virus p72 fusion protein and its preparation, can solve the problems of loose virus and limit further application, and achieve the goal of alleviating biological safety problems, alleviating latent infection, and good antigenicity Effect

Active Publication Date: 2020-06-05
天康制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In short, many side effects caused by naturally attenuated strains and potential safety hazards such as the possibility of poison release limit its further application in actual production.

Method used

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  • P72 fusion protein of African swine fever virus , and preparation method and application thereof
  • P72 fusion protein of African swine fever virus , and preparation method and application thereof
  • P72 fusion protein of African swine fever virus , and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 expresses the construction of the recombinant expression vector of ASFV p72 fusion protein

[0062] African swine fever virus B646L (p72) gene synthesis and construction of recombinant plasmids UCOE-Puro-p72 and UCOE-H-p72: 9 nucleotide sequences of SP signal peptide were added at the N-terminus, fused with the recombinant ASFV B646L gene sequence, N The end is then fused to the GCN4 fragment through the first connecting peptide. The ASFV p72 fusion protein consists of signal peptide, GCN4 fragment, first connecting peptide, p72 protein fragment, Strep II tag, second connecting peptide and His tag from N-terminal to C-terminal, such as figure 1 shown. The nucleotide sequence encoding it obtained after optimization of biased codons is shown in SEQ ID NO.8. Insert restriction sites Nhe I and Sal I at both ends of the gene, and send it to the company for gene synthesis. At the same time, this sequence was subcloned into the vector UCOE to obtain recombinant ...

Embodiment 2

[0063] The expression detection of embodiment 2ASFV p72 fusion protein in CHO-K1 cell

[0064] (1) Transfection of CHO-K1 adherent cells: UV sterilization in a biosafety cabinet for 30 min; medium (DME / F-12 containing 2.5% serum and DME / F-12 containing 10% serum) and PBS were preheated to 37°C. Take out the cells (T25 cell culture flask) from the incubator at 37°C, discard the medium, and take 5 mL of preheated PBS to rinse the cells. After discarding the PBS, add 500 μL of 0.25% trypsin-EDTA to each T25 cell culture flask, digest at room temperature for about 2 minutes, until the cells shrink and become round, the gap becomes larger, and a single cell appears. Aspirate the trypsin and terminate the digestion reaction, use 5 mL of DME / F-12 medium containing 10% FBS to terminate the digestion reaction, and blow off the cells. The cell suspension and trypan blue staining solution were mixed at a ratio of 1:1 and counted. When the cell viability is ≥90%, dilute the cells to 1....

Embodiment 3

[0067] Example 3 Positive monoclonal cell strain suspension acclimatization

[0068] (1) Preparation: UV sterilization in a biosafety cabinet for 30 minutes; DME / F-12 (containing 10% serum), Hycell (containing 8mM GlutaMAX) and PBS were placed in a 37°C water bath and preheated to 37°C. DME / F-12 containing 10% serum and Hycell containing 8mM GlutaMAX were used to configure the acclimatization medium according to the ratio of 3:1, 1:1 and 1:3, respectively.

[0069] (2) Take out the cells (T75 cell culture flask) from the 37°C incubator, discard the cell supernatant, and then take preheated PBS to wash the cells. After discarding the PBS, digest the cells with 1mL 0.25% trypsin-EDTA for about 2min until the cells become shrunken and rounded, the gaps become larger, and appear as a single cell, discard the trypsin, and then add 15mL of DME / F-12 containing 10% FBS to culture Stop the digestion reaction, gently blow off the cells with a pipette, mix the cell suspension and trypan...

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Abstract

The invention provides a p72 fusion protein of an African swine fever virus, and a preparation method and an application thereof, and relates to the technical field of biology. The p72 fusion proteinof the African swine fever virus contains a p72 protein fragment and a GCN4 fragment of the African swine fever virus, wherein the GCN4 fragment has an amino acid sequence of SEQ ID NO. 1. The p72 fusion protein of the African swine fever virus can form a trimer and has good immunogenicity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an African swine fever virus p72 fusion protein and a preparation method and application thereof. Background technique [0002] African swine fever (ASF) is an acute, virulent, and highly contagious infectious disease caused by African swine fever virus (ASFV), with a high incidence and a mortality rate of up to 100%. The World Organization for Animal Health (OIE) will It is listed as an animal disease that must be reported, and our country lists it as a first-class animal disease. The disease was first confirmed in Kenya in Africa in 1921. Since 2007, African swine fever has occurred, spread, and spread in many countries around the world. On August 3, 2018, the first case of African swine fever was confirmed in China. my country is a big pig raising and pork consumption country. The number of live pigs slaughtered, the amount of stocks and the consumption of pork are all ranked fir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K16/08C12N15/62C12N15/85C12N5/10G01N33/569A61K39/12A61P31/20
CPCC07K14/005C07K16/081C12N15/85G01N33/56983A61K39/12A61P31/20C07K2319/02C07K2319/21C07K2319/22C12N2710/12022C12N2710/12034G01N2333/01
Inventor 贺笋张国庆潘毅平李延涛郭苗苗孔楚心
Owner 天康制药股份有限公司
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