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DNA sequences encoding African swine fever virus antigens, compositions of antigens encoded by them and their application in immunological detection

An African swine fever virus, immunological detection technology, applied in the application field of immunological detection methods, can solve the problems of many false negatives, low expression, low accuracy, etc., and achieve the effect of high sensitivity

Active Publication Date: 2021-07-20
SHENZHEN ANIEASY BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most companies develop antigens and antibodies by selecting some fragments of CSFV or recombining the selected fragments, but this method has the characteristics of low expression, low accuracy and many false negatives

Method used

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  • DNA sequences encoding African swine fever virus antigens, compositions of antigens encoded by them and their application in immunological detection
  • DNA sequences encoding African swine fever virus antigens, compositions of antigens encoded by them and their application in immunological detection
  • DNA sequences encoding African swine fever virus antigens, compositions of antigens encoded by them and their application in immunological detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1. Construction of the expression system of African swine fever virus antigen

[0062] According to the conventional technical methods in this field, the DNA sequences SEQ ID No.7, 8, 9, 10, 8, 9, 10, 11 or 12 are respectively added with appropriate restriction sites by primers, and connected to the vector through the restriction sites. Subsequently, the vector added with the target DNA sequence is reintroduced into the corresponding host cells, thereby constructing an expression system for expressing the above-mentioned protein. Examples of combinations of vectors, host cells, expression tags, purification tags and restriction sites specifically used in the present invention are shown in Table 1 below.

[0063] Table 1. Examples of combinations of vectors, host cells, expression tags, purification tags, and restriction sites

[0064] carrier name host cell expression tag Purification label Restriction sites pET-28a E. Coli N-term...

Embodiment 2

[0066] Example 2. Expression and purification of African swine fever virus antigen in prokaryotic expression system

[0067] 1. Experimental plan:

[0068] 1) The prokaryotic expression system constructed in Example 1, that is, Escherichia coli (E. Coli), was revived, inoculated with corresponding resistant LB medium, and cultured on a shaker at 37° C. and 200 rpm for 12-16 hours.

[0069] 2) Inoculate the recovered bacterial solution into the corresponding resistant TB medium at a ratio of 100:1, and culture on a shaker at 37°C and 200rpm for 3-4h.

[0070] 3) When culturing for 3-4 hours and the bacterial concentration OD600 = 0.6-1.0, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Then culture at 37° C. and 200 rpm on a shaker for 4-6 hours (ie induction for 4-6 hours).

[0071] 4) After the induction is completed, centrifuge at 4° C. and 8000 rpm for 10 minutes to collect the bacteria.

[0072] 5) The bacterial cells were resuspended an...

Embodiment 3

[0086] Example 3. Expression and purification of African swine fever virus antigen in 293 cells

[0087] 1. Experimental plan:

[0088] 1) On the day before transfection, take 293 cells in the logarithmic growth phase and press 0.8×10 6 cells / mL density for passage.

[0089] 2) Prepare 2 sterile EP tubes, add 200 μL cell serum-free medium to each, then add 20 μg expression vector DNA containing the target gene (see Example 1) and 60 μL PEI transfection reagent, mix well and incubate at room temperature for 5 minutes.

[0090] 3) Quickly add the PEI transfection reagent dilution to the expression vector DNA dilution containing the target gene, mix gently with a pipette gun, and incubate at room temperature for 15-20 minutes.

[0091] 4) The mixture of the expression vector DNA containing the target gene and the transfection reagent PEI is quickly added to the cell suspension.

[0092] 5) When the cell viability drops to about 60%, the cell supernatant is collected by centrif...

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Abstract

The present invention provides a DNA sequence encoding African swine fever virus antigen, a vector comprising the above DNA sequence, an expression system comprising the above vector, an antigen composition encoded by the above DNA sequence, and the above antigen composition in the detection of African pigs Application of immunological detection method of pestivirus. The present invention optimizes the DNA sequences encoding CP204L, PK205R, PB602L, CP530R, E183L, and PB646L in the main membrane protein and capsid protein of African swine fever virus by eliminating rare codons and other codon optimization methods, and finds that The above-mentioned codon-optimized nucleotide sequence can efficiently express the above-mentioned main membrane protein and capsid protein in a suitable expression system, and the immunological detection method thus established can detect African swine fever quickly, easily and on a large scale Virus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA sequence encoding an African swine fever virus antigen, a composition of the antigen encoded by it, and an application of the composition in an immunological detection method. Background technique [0002] African swine fever is a hemorrhagic, severe infectious disease caused by the African swine fever virus infecting domestic pigs and various wild boars. It can affect pigs of all ages and cause hemorrhagic fever. ASF can manifest in various forms, ranging from hyperacute, acute, subacute to chronic and asymptomatic. African swine fever has the characteristics of short onset and high mortality. Once a pig is infected, it will die within 2-10 days, and the mortality rate can be as high as 100%. In addition, African swine fever virus has strong environmental tolerance, can remain active in the air for several days, can remain active in pig manure for several weeks, and can still...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/34C07K14/01G01N33/569
CPCC07K14/005C12N2710/12022G01N33/56983G01N2333/01G01N2469/20
Inventor 王延轶付辉冉勇朱海石正旺袁克湖张克山林季敏马涛易俊伟杨年松严义勇
Owner SHENZHEN ANIEASY BIOTECHNOLOGY CO LTD
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