Sensitization polystyrene nano microsphere for detection of canine parvo virus structural protein VP2 antibody, and preparation method and application thereof

A polystyrene nano- and canine parvovirus technology, which is used in measurement devices, biological tests, and material testing products, can solve problems such as high price and achieve simple operation, good hydrophilicity, strong specificity and immunogenicity. Effect

Active Publication Date: 2019-01-15
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the spot ELISA kit can be tested on a single serum sample at any time, it is expensive

Method used

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  • Sensitization polystyrene nano microsphere for detection of canine parvo virus structural protein VP2 antibody, and preparation method and application thereof
  • Sensitization polystyrene nano microsphere for detection of canine parvo virus structural protein VP2 antibody, and preparation method and application thereof
  • Sensitization polystyrene nano microsphere for detection of canine parvo virus structural protein VP2 antibody, and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The isolation and identification of embodiment 1CPV

[0054] 1. Method

[0055] 1.1 Virus culture and proliferation

[0056] Disease material treatment: Thaw frozen dog diarrhea suspected to be infected with CPV in a 4°C refrigerator, take 200 μL of disease material and add 800 μL of PBS to prepare a disease material suspension with a volume ratio of 1:4, and add double antibodies (final concentration of penicillin is 100 μg / mL, the final concentration of streptomycin is 50 μg / mL) solution, mixed well, repeated freezing and thawing 3 times, and centrifuged at 10000 r / min at 4 °C for 10 min. The supernatant was sterilized by filtration with a 0.22 μm microporous membrane, the filtrate was collected, and stored at -80°C for later use.

[0057] Cell culture: resuscitate F81 cells, and continue for 2 passages. After reaching the best growth state, fully suspend the digested F81 cells with 10% serum DMEM cell culture medium, and place them in a 5% CO2 incubator at 37°C for...

Embodiment 2

[0131] Cloning and expression of embodiment 2 CPV VP2 recombinant gene

[0132] 1 method

[0133] 1.1 Primer design and synthesis

[0134] Combined with DNAStar-Protein software analysis, two pairs of primers sVP2-F1 / R1 and sVP2-F2 / R2 were designed in the main epitope region encoded by the VP2 gene using the genomic DNA of the CPV isolate as a template, and the 5′ ends of the primers were respectively introduced into BamHI and XhoI restriction site (Table 5), amplified two truncated VP2 genes, named vp2-s1 and vp2-s2.

[0135] Table 5 Primer sequence and product length

[0136]

[0137]

[0138] Note: Restriction enzymes are in brackets; restriction enzyme cutting sites are underlined.

[0139] 1.2 Cloning of VP2 recombinant gene

[0140] Amplification of the target gene: use the genomic DNA of the CPV isolate as a template, and use sVP2-F1 / R1 and sVP2-F2 / R2 as primers to perform PCR amplification respectively. The reaction system and reaction conditions refer to Sec...

Embodiment 3

[0187] The establishment and application of embodiment 3 CPV antibody detection indirect agglutination test

[0188] 1 method

[0189] 1.1 Preparation of sensitized polystyrene nanospheres for detecting antibody to canine parvovirus structural protein VP2

[0190] After mixing the recombinant protein rVP2-S1 (0.1-0.3 mg / L) purified in Example 2, acetate buffer (pH4.0-5.0), EDC and polystyrene nanospheres (25-50 μL), room temperature ( 25°C) on a decolorizing shaker and shake at 100r / min for 2h. After sensitization, place in a centrifuge at 8000r / min, centrifuge for 15min, discard the supernatant, add 1mL ethanesulfonic acid buffer (MES pH4.7), fully suspend, centrifuge for 15min under the same conditions, discard the supernatant, and re-use 500μL acetate buffer Suspend and store at 4°C.

[0191] Condition optimization of recombinant protein sensitized microspheres: use the matrix method to determine the optimal protein amount, EDC amount and buffer type. Firstly, the amoun...

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Abstract

The invention discloses a sensitization polystyrene nano microsphere for detection of a canine parvo virus structural protein VP2 antibody, and a preparation method and application thereof. A surfaceof the sensitization polystyrene nano microsphere is coupled with a canine parvo virus recombinant VP2 protein, the recombinant VP2 protein is a truncated protein of a canine parvo virus VP2 protein and located at the 365-486 digits of the canine parvo virus VP2 protein, and an amino acid sequence of the recombinant VP2 protein is shown as the SEQ ID NO.3. The protein contains a main epitope domain of the VP2 protein and has high antigenicity, excellent hydrophilicity and relatively strong specificity and immunogenicity. With the recombinant protein used as an antigen, a sensitization color polystyrene nano microsphere is prepared, and the sensitization color polystyrene nano microsphere is used for detecting a canine parvo virus serum antibody. Experiments show that the prepared sensitization microsphere cannot generate an autoagglutination phenomenon and has excellent repeatability and stable property. A detection method provided by the invention is suitable for clinical fast detection of single or multiple dog serum samples for pets, and compared with a commercial detection kit, the detection method is simple and convenient to operate, and a detection result has relatively highaccuracy and coincidence rate.

Description

technical field [0001] The present invention relates to a sensitized polystyrene nanosphere for detecting antibody to canine parvovirus and its preparation method and application, in particular to a sensitized polystyrene nanosphere for detecting antibody to canine parvovirus structural protein VP2 Ball and its preparation method and application, the invention belongs to the technical field of virus detection. Background technique [0002] Canine parvovirus disease (Canine parvovirus disease) is a highly contagious infectious disease of dogs caused by canine parvovirus (Canine parvovirus, CPV), with high morbidity and mortality. Clinically, the main manifestations are hemorrhagic enteritis and non-suppurative myocarditis. Dogs of all ages can be infected with the disease, among which puppies and purebred dogs are mostly affected. In recent years, due to the increase in the number of dogs, the increase in circulation, and the lack of scientific vaccination, the epidemic tre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/569
CPCG01N33/54346G01N33/56983G01N33/6857G01N33/686G01N2333/015G01N2333/47
Inventor 师东方唐毓董秀梅苏瑞红张萍
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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