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Synthetic peptide coupling antigen and reagent for testing porcine circovurus type 2 specific antibody

A porcine circovirus and type 2 antibody technology, applied in the direction of carrier binding/immobilizing peptides, specific peptides, biological testing, etc., can solve problems such as difficult control

Active Publication Date: 2009-02-04
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] 3. The quality stability of the expressed recombinant PCV2 Cap protein antigen between batches is affected by many factors, and it is not easy to control. It is not as good as the synthetic peptide antigen produced automatically by the instrument.

Method used

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  • Synthetic peptide coupling antigen and reagent for testing porcine circovurus type 2 specific antibody
  • Synthetic peptide coupling antigen and reagent for testing porcine circovurus type 2 specific antibody
  • Synthetic peptide coupling antigen and reagent for testing porcine circovurus type 2 specific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Alternative Short Peptide Synthesis

[0065] Referring to the gene sequence of PCV-2 ORF2 detected at home and abroad, the amino acid sequence of the primary structure of ORF2 was deduced, and its main antigenic epitope was analyzed with computer assistance, and a series of polypeptide amino acid sequences were designed. Including SEQ ID NO: 5~SEQ ID NO: 26.

[0066] SEQ ID NO: 5: Trp Ala Val Asp Met Met Arg pHe Asn Ile Asn Asp pHe Leu Pro Pro Gly Gly Gly Ser

[0067] SEQ ID NO: 6: Trp Ala Val Asp Met Met Arg pHe Asn Ile Asn Asp pHe Val Pro Pro Gly Gly Gly Thr

[0068] SEQ ID NO: 7: Trp Ala Val Asp Met Met Arg pHe Asn Ile Asn Asp pHe Leu Pro Pro Gly

[0069] SEQ ID NO: 8: Trp Ala Val Asp Met Met Arg pHe Asn Ile Asn Asp pHe Leu

[0070] SEQ ID NO: 9: Trp Ala Val Asp Met Met Arg pHe Asn Ile Asn

[0071] SEQ ID NO: 10: Asp Met Met Arg pHe Asn Ile Asn Asp pHe Leu Pro Pro Gly Gly Gly Ser

[0072] SEQ ID NO: 11: Arg pHe Asn Ile Asn Asp pHe Leu Pro Pro Gly Gly Gly Ser

...

Embodiment 2

[0098] Conjugated antigen preparation

[0099] The conjugated antigen was prepared by glutaraldehyde coupling method, the specific method is as follows:

[0100] 1. Take 10mg of carrier protein, fully dissolve it in 0.1mol / L boric acid solution with pH 10, add 15mg of polypeptide and mix well;

[0101] 2. Add freshly prepared 0.3% (V / V) glutaraldehyde solution while oscillating, react at room temperature for 2 hours, and invert the test tube several times;

[0102] 3. Add 1mol / L glycine to block unreacted glutaraldehyde, and continue to react for 30 minutes;

[0103] 4. Dialyze with 0.1mol / L boric acid pH 8.5 overnight, change the dialysate, continue dialysis for 4 hours, and store at -20°C for later use.

[0104] Using this method, the 22 polypeptides of Example 1 were prepared into 22 candidate conjugated antigens.

Embodiment 3

[0106] Screening and preparation of PCV2-Mix conjugated antigen

[0107] Positive serum preparation: 10 SPF test pigs negative for PCV antigen and antibody were taken, divided into 5 pigs / group, and each group was isolated and raised separately. A group of pigs (5 heads) were infected with PCV1 and PCV2 standard strains respectively, and pig serum was collected at 0, 7, 14, 21, 35, and 49 days after infection as serum for antigen evaluation.

[0108] Antigen screening by ELISA test method: Dilute the alternative conjugated antigen to 5 μg / mL with pH 9.6 carbonate buffer, coat ELISA plates respectively, and react overnight at 25°C. Pig serum collected at different times was diluted 5 times with PBST (0.02mol / L, pH 7.2) containing 2% BSA, and 100 μL was added to each well, and added to various ELISA antigen plates, and reacted at 37°C 60 minutes. Wash the plate 5 times with PBST. Dilute the rabbit anti-pig enzyme-labeled antibody 1:10000 with PBST containing 2% BSA, add 100 μ...

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Abstract

The application relates to a synthetic peptide clb-bsa of porcine circovirus type 2 and ELISA kits and immune microsphere detection reagents developed by using synthetic peptide clb-bsa, and is used for specifically detecting porcine circovirus type 2 antibodies.

Description

technical field [0001] The invention relates to animal quarantine, in particular to a preparation of a synthetic peptide coupled antigen of porcine circovirus type 2, and a detection method and detection reagent for porcine circovirus type 2 antibody based on the synthetic peptide coupled antigen. Background technique [0002] Porcine circovirus type 2 (PCV2) is a recently discovered circovirus that causes multisystemic wasting syndrome (PMWS) in weaned piglets. The disease occurs in many countries in the world, and the disease is also prevalent in my country in recent years. PMWS mainly affects piglets aged 6 to 8 weeks. The incidence rate is generally 10% to 20%, and the mortality rate is as high as 50% to 100%. loss. Adult pigs are generally recessively infected and do not show any symptoms, but they can be transmitted to piglets. At present, no commercialized vaccine has been developed against the disease, and the most effective prevention and control measure is to ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/02C07K14/795C07K7/08C07K7/06C07K1/04C07K1/10G01N33/569G01N33/53
Inventor 王传彬田克恭曹振陈西钊金萍
Owner CHINA ANIMAL DISEASE CONTROL CENT
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