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CSFV antibody detection system and preparation method thereof

A detection system and technology for swine fever antibodies, applied in the fields of molecular biology and livestock disease diagnosis, can solve problems such as false negatives, and achieve the effects of improving sensitivity and high capture efficiency

Active Publication Date: 2016-04-27
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, for some clinical samples with early infection or low levels of CSF antibodies, false negatives may occur simply by detecting E2 protein antibodies

Method used

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  • CSFV antibody detection system and preparation method thereof
  • CSFV antibody detection system and preparation method thereof
  • CSFV antibody detection system and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The preparation of embodiment 1 swine fever E2 protein

[0053] According to the gene sequence (see sequence table SEQIDNo.1) of the E2 protein gene sequence (see sequence table SEQIDNo.1) of the classical swine fever virus C strain (accession number is AF531433) reported in NCBI (http: / / www.ncbi.nlm.nih.gov) to prepare the classical swine fever virus E2 protein.

[0054] 1.1 Construction of pFastBacHBM-E2

[0055] Design a pair of primers to amplify E2 protein by PCR:

[0056] CE2-F: 5'-CGGCTAGCCTGCAAGGAAGATTAC-3'

[0057] CE2-R: 5'-TCTTTCATCTGATGCATGCACCT-3'

[0058] PCR reaction conditions: pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 1min, 30 cycles, and extension at 72°C for 7min. Use 1% agarose gel to perform electrophoresis on the amplified product. After the electrophoresis product is recovered using a DNA gel recovery kit, the target gene fragment is connected to the pFastBacHBM vector. Th...

Embodiment 2

[0066] The preparation of embodiment 2 swine fever Erns protein

[0067] According to the gene sequence (see sequence table SEQIDNo.3) of the Erns protein in the swine fever virus C strain (accession number is AF531433) reported in NCBI (http: / / www.ncbi.nlm.nih.gov) to prepare the swine fever virus Erns protein.

[0068] 2.1 Construction of pFastBacHBM-Erns

[0069] The designed primer sequences are as follows

[0070] CErns-F: 5'-GAAAATATAACTCAATGGAACCTGA-3'

[0071] CErns-R: 5'-GGCATAAGCGCCAAACCAGGT-3'

[0072] Refer to Example 1.1 to construct pFastBacHBM-E2.

[0073] 2.2 Construction of Bacmid-Erns, acquisition of Erns protein and its purification and quantification

[0074] Referring to Example 1.2-1.4, Bacmid-E2 was sequentially constructed, recombinant E2 protein was obtained, and the recombinant E2 protein was purified and quantified. The protein concentration was 2.1 mg / ml.

Embodiment 3

[0075] The preparation of embodiment 3 swine fever antibody ELISA kit

[0076] 3.1 ELISA plate preparation

[0077] (1) Enzyme plate coating antigen: the swine fever virus E2 and Erns protein prepared in Example 1-2 were coated with the components corresponding to the original numbering of each coating as shown in Table 1 with carbonate buffer solution of pH 9.6 Prepare.

[0078] Table 1 Coating raw components and their contents corresponding to each kit

[0079]

[0080] (2) Blocking: the blocking solution is 10% skimmed milk diluted in PBST, 100 μl per well, and blocked at 37° C. for 1 h. After blocking, wash 3 times with PBST.

[0081] 3.2 Preparation of reagents

[0082] Diluent: PBS solution containing 0.05% (V / V) Tween-20 and 10% (V / V) skimmed milk;

[0083] Washing solution: PBS solution containing 0.05% (V / V) Tween-20;

[0084] Chromogenic solution A: add 10ml absolute ethanol to 20mg TMB, then use ddH 2 O is fixed to 100ml;

[0085] Chromogenic solution B: ...

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Abstract

The invention provides a CSFV antibody detection system and a preparation method thereof. A coating antigen of the detection system contains CSFV E2 protein and Erns protein. The CSFV E2 protein and Erns protein are recombinant proteins expressed by eucaryon, correct spatial conformation and posttranslational modification process can be guaranteed, antigen is capable of effectively combining with the antibody in serum, and specificity, sensitivity and repeatability of detection can be increased. The system and the method can be sued for diagnosis of CSFV antibody in prevention and control of CSFV as well as immunization evaluation of a CSFV vaccine.

Description

technical field [0001] The invention belongs to the fields of molecular biology and livestock disease diagnosis, and in particular relates to a swine fever antibody detection system and a preparation method thereof. Background technique [0002] Classical swine fever virus (CSFV) is a highly contagious viral infectious disease caused by classical swine fever virus (CSFV). It spreads rapidly, is widespread, has high morbidity and mortality, and is extremely harmful to the pig industry in my country. According to clinical symptoms and duration of disease, swine fever can be divided into acute type, chronic type and mild type. Sick pigs and infected sows are the main source of infection of CSFV. The World Organization for Animal Health lists it as a Class A disease, and China's "Animal Epidemic Prevention Law" lists it as a Class I disease. [0003] The classical swine fever virus belongs to the Flaviviridae Pestivirus genus. It is a single-stranded positive-sense RNA virus w...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569
Inventor 田克恭杨凡力
Owner LUOYANG PULIKE WANTAI BIOTECH
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