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Cell strain for expressing African swine fever virus CD2v protein and application of cell strain

A technology of African swine fever virus and protein, applied in the field of biomedical genetic engineering and immunology, can solve the problems of low African swine fever inhibition, poor African swine fever inhibition, complicated preparation process, etc., and achieve easy mass production, antigenic Good performance and good biosafety

Active Publication Date: 2020-02-07
GUANGZHOU BONIZZI BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation process of this vaccine is extremely complicated, and the yield of the corresponding antigen is low, which ultimately leads to low inhibition of African swine fever
[0006] In summary, it can be seen that in the prior art, there are generally problems such as low antigen yield, complicated detection methods, and poor inhibition of African swine fever.

Method used

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  • Cell strain for expressing African swine fever virus CD2v protein and application of cell strain
  • Cell strain for expressing African swine fever virus CD2v protein and application of cell strain
  • Cell strain for expressing African swine fever virus CD2v protein and application of cell strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction and detection of recombinant cell lines stably expressing African swine fever virus CD2v protein

[0037] 1. Design and preparation of gene sequence encoding African swine fever virus CD2v protein

[0038] 1.1 Design of gene sequence encoding African swine fever virus CD2v protein

[0039] According to the nucleotide sequence of the African swine fever virus CD2v protein (ASFV CD2v) (GenBank: KM609392.1), and the codon preference optimization sequence in mammalian cell expression, add the his tag nucleotide sequence, and when designing the gene , adding Kozak sequence, restriction site and protective base before the start codon, and adding terminator, restriction site and protective base at the end of the CD2v protein gene coding; the nucleoside of the obtained CD2v protein The acid sequence is shown in SEQ ID NO.2, and SEQ ID NO.1 is the translated amino acid sequence of SEQ ID NO.1.

[0040] 1.2 Preparation of gene sequence encoding African sw...

Embodiment 2

[0092] Embodiment 2 recombinant African swine fever virus CD2v protein purification

[0093] 1. Purification of recombinant CD2v protein

[0094] The cell culture supernatant was filtered through a 0.45 μm filter membrane, and after the HisTrapTM HP column was correctly connected to the BioLogic LP protein purifier, 3 times the column volume of the sample buffer (20mM pH7.4 phosphate buffer, 0.5M NaCl), 1mL / min to equilibrate the column, load the pretreated sample at a speed of 1mL / min, and wash it with loading buffer after the end, 1mL / min, 5 times the column bed volume, and then use 20mM pH7.4 phosphate buffer Gradient elution of recombinant protein (containing 100-500mM imidazole) was carried out while monitoring with BioLogic LP. When the baseline was observed to rise, that is, when the elution peak appeared, collection began. Collect the eluate until the elution peak returns to the baseline, continue to equilibrate with loading buffer for 3-5 times the column volume, an...

Embodiment 3

[0097] The establishment of embodiment 3 indirect ELISA detection method

[0098] 1. Determination of antigen coating concentration and serum dilution multiple

[0099] Dilute the purified recombinant CD2v protein with 0.05 mol / L carbonate buffer (pH 9.6) to 1 mg / mL as the coating antigen, and make the purified antigen 1:100, 1:200, 1:400, 1:800, 1:1600 times diluted and then coated with 96-well ELISA plate, added 50 μL of purified recombinant antigen diluted by ratio to each well, repeated two wells for each dilution, coated overnight at 37°C, and used 0.5 mL of Wash 6 times with 0.1mol / L PBS (pH 7.4) (PBST) of / L Tween-20; block with 50g / L skimmed milk powder for 2h, and wash 6 times with PBST; positive and negative sera of African swine fever were washed with 50g / L Skimmed milk powder was diluted 1:50, 1:100, 1:200, added to the blocked microplate, 50 μL per well, reacted at 37°C for 2 hours, washed 6 times with PBST; labeled with horseradish peroxidase Rabbit anti-pig Ig...

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Abstract

The invention belongs to the field of biomedical genetic engineering and immunology, and particularly relates to a cell strain for expressing African swine fever virus CD2v protein and an applicationof the cell strain. The cell strain for expressing the African swine fever virus CD2v protein, provided by the invention, is obtained by optimizing and synthesizing a nucleic acid sequence of the African swine fever virus CD2v protein, constructing a recombinant lentiviral vector containing an ASFV-CD2v gene, packaging lentivirus, infecting HEK-293T cells and screening. The cell strain for expressing the African swine fever virus CD2v protein provided by the invention has biological characteristics similar to those of parent cells and is beneficial to large-scale production of antigen protein;the expression protein can obtain native conformation and modification processing close to virus protein in expression cells, has good antigenicity, and is easy for mass production.

Description

technical field [0001] The invention belongs to the fields of biomedical genetic engineering and immunology, and specifically relates to a cell strain expressing African swine fever virus CD2v protein and application thereof. Background technique [0002] African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious disease of pigs caused by African swine fever virus (ASFV), its clinical symptoms and pathological changes are similar to acute Swine fever manifests as high fever, skin congestion, abortion, edema and organ hemorrhage, etc., and the lethal rate can be as high as 100%. The disease was mainly prevalent in Africa at the beginning, and has gradually spread to countries and regions such as Europe and Asia in recent years. After the first case of sick pigs appeared in my country in 2018, within a few months, Liaoning, Henan, Jiangsu, Zhejiang, Anhui Provinces such as China, Heilongjiang, Inner Mongolia, and Jilin have all reported in successio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34C12N5/10G01N33/68G01N33/569A61K39/39A61K39/215A61P31/14
CPCA61K39/12A61K39/39A61K2039/552A61K2039/55516A61P31/14C07K14/005C12N5/0603C12N5/0686C12N2510/02C12N2710/12022C12N2770/20034G01N33/56983G01N33/6854G01N2333/01G01N2469/20
Inventor 李红卫颜仁和王升启毛莹莹王学军高永新
Owner GUANGZHOU BONIZZI BIOTECH CO LTD
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