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African swine fever virus CD2v truncated protein as well as application thereof in preparation of wild virus and natural attenuated virus detection kit

A technology of African swine fever virus and truncated protein, which is applied in the fields of application, virus, and viral peptide, can solve the problems of unclear specificity and antigenic protein without serum, and achieve strong clinical practicability, high protein purity, and expression high volume effect

Active Publication Date: 2021-09-28
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

202110073736.1 discloses the detection of serum antibodies using CD2v truncated protein expressed by sf9 cells. When the antigen coating amount is 100ng / well, it can distinguish positive serum from negative serum infected by wild virus, but immune serum from gene deletion attenuated vaccine and The specificity of serum detection of natural attenuated strain infection is not clear. 202110024959.9 discloses the use of prokaryotic expression of a fragment of 555 nucleotides in the extracellular region of CD2v for the preparation of an African swine fever antibody detection ELISA kit
[0008] As of the date of application, there is still no report on the antigenic protein that can distinguish the serum of natural attenuated African swine fever strains and wild strains infected

Method used

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  • African swine fever virus CD2v truncated protein as well as application thereof in preparation of wild virus and natural attenuated virus detection kit
  • African swine fever virus CD2v truncated protein as well as application thereof in preparation of wild virus and natural attenuated virus detection kit
  • African swine fever virus CD2v truncated protein as well as application thereof in preparation of wild virus and natural attenuated virus detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Obtaining of African swine fever virus truncated protein:

[0029] 1. Obtaining and identification of recombinant plasmids

[0030] The amino acid positions of the N-terminal region and the C-terminal region of the CD2v protein of the African swine fever HLJ strain (GenBank accession number: MK333180.1) were analyzed. Since the expression process of the CD2v N-terminal region requires glycosylation modification, therefore It needs to be expressed in a eukaryotic system, but the CD2vC-terminal region does not require glycosylation modification, so a prokaryotic expression system can be selected for expression. Codon optimization is a key technical means to achieve high-efficiency expression of heterologous proteins, and protein expression is a systematic project, so when performing codon optimization, only replacing codons with the most frequently used codons of a certain species may not be effective. For the best results, it is necessary to consider adjusting GC conten...

Embodiment 2

[0041] African swine fever virus CD2v truncated proteins with different expressions are used to distinguish ASFV wild virus and natural attenuated virus:

[0042] Using the method in Example 1, the applicant expressed and purified the eukaryotic expression protein N1 in the N-terminal region of African swine fever virus protein CD2v and the prokaryotic expression protein in the C-terminal region. The prokaryotic-expressed protein and eukaryotic-expressed protein were respectively used as coating antigens to detect clinically known background clinical sera (10 positive sera from wild strain infection, 10 negative sera, and 10 natural attenuated strain infection sera).

[0043] Wherein the P54 antibody positive control and negative control sample preparation methods:

[0044] Referring to the African swine fever virus indirect ELISA antibody detection kit (application number: Example 2 in 202010700335.X), operate according to Example 3 in the patent document.

[0045] CD2v anti...

Embodiment 3

[0062] Construction of African swine fever virus indirect ELISA antibody detection kit (CD2v coating antigen):

[0063] 1. Preparation of positive control:

[0064] For the preparation method of positive control, refer to the preparation method of CD2v antibody positive serum in Example 1.

[0065] 2. Preparation of negative control:

[0066] For the preparation method of negative control, refer to the preparation method of CD2v antibody negative serum in Example 1.

[0067] 3. Preparation of ELISA plate

[0068] The optimal coating concentration of recombinant antigen N1 was determined by square array method to be 50ng / well, and coated at 2-8°C for 14 hours. Discard the coating solution, add 1% bovine serum albumin (BSA) as a blocking solution, and block for 2 hours at 37°C. Discard the blocking solution, air-dry naturally, and then vacuum-pack it into a finished kit enzyme plate.

[0069] 4. Preparation of other components of the kit

[0070] Coating solution: take sod...

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Abstract

The invention belongs to the technical field of biology, and discloses an African swine fever virus CD2v truncated protein as well as application thereof in preparation of wild virus and natural attenuated virus detection kit Through comparison, it is found that positive serum infected by the African swine fever virus cannot be detected when prokaryotic expression CD2v truncated protein serves as the ELISA coating antigen; however, positive serum infected by the African swine fever virus can be detected when eukaryotic expression CD2v protein serves as the ELISA coating antigen. An indirect ELISA kit prepared by taking the eukaryotic expression CD2v protein as a coating antigen is matched with an indirect ELISA kit taking the African swine fever virus p54 protein as a coating antigen, so that the kit can be used for identifying infection of ASFV wild strains and natural attenuated strains; and moreover, the operation steps are simple, and the result is reliable. Therefore, the African swine fever virus indirect ELISA detection kit prepared from the eukaryotic expression truncated protein provided by the invention is very suitable for clinical large sample detection, and is suitable for large-scale popularization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the CD2v truncated protein of African swine fever virus and its application in the preparation of wild virus and natural attenuated virus detection kits. Background technique [0002] African swine fever (African Swine Fever, ASF) is an acute, severe and highly contagious zoonotic disease of pigs caused by African swine fever virus (ASFV). Severe internal organ bleeding, high morbidity, high mortality, short course of disease. The World Organization for Animal Health (OIE) lists it as a category A animal disease of legally notifiable animals, and this disease is regulated as a first-class animal infectious disease in my country. ASFV was introduced into my country in 2018, causing huge economic losses to the breeding industry in our country. With the spread of the virus, a mutant attenuated strain appeared in the field. This strain has a longer incubation period and caus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34C12N15/85G01N33/569
CPCC07K14/005C12N15/85G01N33/56983C12N2710/12022G01N2333/01G01N2469/20
Inventor 金梅林吕长杰姜丽丽赵亚杨丽赵丽吴超杨永邹忠孙小美
Owner HUAZHONG AGRI UNIV
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