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African swine fever virus CD2v protein as well as kit and antibody prepared from African swine fever virus CD2v protein

An African swine fever virus and kit technology, applied in the direction of antiviral immunoglobulin, virus/phage, virus, etc., can solve problems such as blank effect evaluation, and achieve good response characteristics, rapid detection, and good specificity.

Pending Publication Date: 2022-05-03
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the detection of African swine fever is mainly focused on the detection of antigens, and the evaluation of the effect of the vaccine after immunizing pigs is still blank

Method used

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  • African swine fever virus CD2v protein as well as kit and antibody prepared from African swine fever virus CD2v protein
  • African swine fever virus CD2v protein as well as kit and antibody prepared from African swine fever virus CD2v protein
  • African swine fever virus CD2v protein as well as kit and antibody prepared from African swine fever virus CD2v protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Preparation of ASFV CD2v fusion protein

[0075] 1.1 Construction of recombinant plasmid expressing CD2v gene and mouse Fc fragment fusion

[0076] 1.1.1 Optimization and synthesis of CD2v gene and mouse Fc fragment

[0077] Referring to the CD2v gene sequence encoded in the ASFV SY18 genome (accession number MH766894.1) registered in GenBank, it was analyzed with TMHMM software, and the extracellular region was selected for fusion with the mouse Fc fragment (ie, mFc). The codons of CD2v were optimized according to the preferred codons of the CHO expression system (see SEQ ID No.1 for details), and pUC57-CD2v plasmids and pCDNA3.4-mFc plasmids were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0078] 1.1.2 Primer design

[0079] The best signal peptide SP (sequence in italics in Table 1) selected for screening was used for the expression of the target protein. Respectively add restriction sites XbaI and HindIII (underlined sequence in Table 1) u...

Embodiment 2

[0102] Example 2 Preparation and Application of ASFV CD2v ELISA Antibody Detection Kit

[0103] 2.1 Preparation of the kit

[0104] Antigen-coated plate: Dilute the fusion protein mFc-CD2v prepared in Example 1 to 0.5-0.8 μg / mL with carbonate buffer (pH 9.6, 0.05 mol / L) for coating, 100 μL / well, 2- Stand at 8°C for 16-24 hours, wash with washing solution, add blocking solution at 200 μL / well (weigh 50 g of sucrose, add 200 mL of newborn bovine serum, 0.5 mL of Proclin300, add PBS (0.01 mol / L, pH 7.4 ) to 1000mL), sealed at 2-8°C for 16-24 hours, discarded the blocking solution, dried, sealed and stored at 2-8°C for later use.

[0105] Enzyme-labeled reagent: Use enzyme-labeled diluent (measure 200mL newborn bovine serum, 0.5mL Proclin 300, 0.5mL Tween 20, 0.04g AM dye, add PBS (0.01mol / L, pH value 7.4) to make up to 1000mL ) Dilute the commercially available enzyme-labeled goat anti-pig IgG or enzyme-labeled rabbit anti-pig 50,000 times as an enzyme-labeled reagent, and stor...

Embodiment 3

[0132] Example 3 Preparation and Identification of ASFV CD2v Protein Monoclonal Antibody

[0133] 3.1 Preparation of monoclonal antibodies

[0134] Five female BALB / c mice aged 4-6 weeks were immunized with the fusion protein mFc-CD2v at a dose of 100 μg / mouse (volume 400 μL) every 3 weeks by subcutaneous multipoint immunization. For the first immunization, the complete Freund's adjuvant was used to emulsify the same volume of 100 μg protein for immunization, followed by immunization with the Freund's incomplete adjuvant and the same volume of 100 μg protein emulsified for a total of 3 times. After the third immunization, the mouse serum was collected and the serum titer was determined by ELISA method.

[0135] Among them, the kit B is an indirect ELISA method constructed with the fusion protein gD-CD2v as the coating source, prepared according to Example 2.1, and the coating volume is 0.5 μg / mL.

[0136] The detection method is as follows: 1) Adding samples: Dilute the serum ...

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Abstract

The invention relates to an African swine fever virus CD2v protein as well as a kit and an antibody prepared from the African swine fever virus CD2v protein. The gene sequence of the African swine fever virus CD2v protein provided by the invention comprises a nucleotide sequence as shown in SEQ ID No.1 or a nucleotide sequence which is at least 90% homologous with the sequence as shown in SEQ ID No.1. According to the invention, codon optimization is carried out on an African swine fever virus CD2v protein gene sequence according to preferred codons of a CHO expression system, and then fusion protein mFc-CD2v and fusion protein gD-CD2v are prepared through the CHO expression system. An ELISA antibody detection kit or a monoclonal antibody prepared from the fusion protein mFc-CD2v can be used for detecting the African swine fever virus antibody, and the detection is rapid and sensitive and has good specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CHO expressing African swine fever virus CD2v protein, an ELISA antibody detection kit prepared by using the protein, a preparation method and application thereof, and a monoclonal antibody prepared by using the protein and application thereof. Background technique [0002] African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious infectious disease of pigs caused by African swine fever virus (ASFV). Extensive hemorrhage is the main feature, the course of the disease is short, and the case fatality rate can be as high as 100%. The clinical symptoms and pathological changes are similar to acute swine fever, and it is easy to be misdiagnosed during diagnosis, showing high fever, skin hyperemia and cyanosis, miscarriage, edema and organ hemorrhage. The disease is listed as a first-class animal disease in my country, and belongs to the legally ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/34C12N15/85C12N5/20C07K14/01C07K19/00C07K16/08G01N33/569G01N33/577C12R1/91
CPCC07K14/005C07K16/081C12N15/85G01N33/56983G01N33/577C12N2800/22C12N2710/12022C07K2319/00C07K2319/30G01N2333/01G01N2469/20
Inventor 田克恭郝丽影张超林邓均华
Owner LUOYANG PULIKE WANTAI BIOTECH
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