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Porcine circovirus 2 type ELISA antibody detection kit

A porcine circovirus and antibody detection technology, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems of low expression level, difficult virus antigen diagnosis, and common Escherichia coli cannot express, and achieve high sensitivity and specificity strong effect

Inactive Publication Date: 2015-06-24
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because PCV2 does not produce cytopathic changes in vitro, the virus proliferation ability is low, and it is difficult to obtain high-yield virus antigens for diagnosis
Due to the presence of rare codons at the amino terminus of the Cap protein, the expression system of common Escherichia coli (BL21) cannot express
Researcher Tong Guangzhi and others used Escherichia coli to study the expression of truncated Cap protein, but the expression level was very low, and the full-length Cap protein could not be obtained

Method used

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  • Porcine circovirus 2 type ELISA antibody detection kit
  • Porcine circovirus 2 type ELISA antibody detection kit
  • Porcine circovirus 2 type ELISA antibody detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: In vitro expression of Cap protein

[0022] 1. Construction of recombinant expression plasmids

[0023] Design specific primers for Cap protein according to the gene sequence of PCV2:

[0024] Upstream primer P1: 5'-C GAGCTCA TGACGTATCCAAGGAGGC-3'; (Sac I restriction site)

[0025] Downstream primer P2: 5'-CCC AAGCTT TTAGGGTTTAAGTGGGGG-3' (Hind III restriction site)

[0026] PCR method was used to amplify the complete sequence of PCV2—ORF2 gene. ORF2 amplification system: Taq enzyme 0.5 μL, Taq enzyme 10×buffer 5 μL, upstream and downstream primers 1 μL each, PCV2 whole genome template 2 μL, 2.5 mmol / L dNTP 2 μL, Add deionized water to 50 µL. Denaturation at 94°C for 3min, cycle parameters: 94°C for 30s, 60°C for 30s, 72°C for 1min, a total of 30 cycles, and extension at 72°C for 10min.

[0027] Cut out the gel block containing the target DNA fragment under the long-wave ultraviolet lamp, put it into an EP tube, and recover the target DNA fragment with...

Embodiment 2

[0043] The preparation of other solutions of embodiment 2 kit:

[0044] ①Washing solution: PBST, add 0.5ml Tween-20 to 1L phosphate buffered saline (PBS);

[0045] ②Sample diluent: 1% skimmed milk powder, PBS with pH=7.4;

[0046] ③Enzyme substrate solution: 3.3'-5,5'-tetramethylbenzidine solution (TMB): Substrate color development solution A: 13.6g sodium acetate, 1.6g citric acid, 0.3ml 30% hydrogen peroxide, add distilled water to 500ml. Substrate Chromogenic Solution B: 0.2g disodium edetate, 0.95g citric acid, 50ml glycerin, 0.15g TMB dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) and add distilled water to 500ml

[0047] ④Stop solution: add 54.3ml of concentrated sulfuric acid and dilute to 1000ml with deionized water.

[0048] ⑤ Horseradish peroxidase (HRP)-goat anti-pig IgG enzyme conjugate: the HRP-goat anti-pig IgG monoclonal antibody used in the present invention was purchased from Proteintech.

Embodiment 3

[0049] The establishment of embodiment 3 PCV2 antibody detection kit ELISA method

[0050] 1. Establishment of ELISA method for PCV2 virus antibody detection

[0051] The optimal antigen coating concentration of Cap protein and the dilution of the serum to be tested were determined by square array titration, and the optimal reaction program was determined at the same time. According to the ratio P / N value of OD630 value of positive and negative serum wells as an index, determine the best coating antigen degree, serum dilution multiple, the best working dilution of enzyme-labeled secondary antibody, and the best reaction time of each step. Dilute the purified recombinant Cap protein with pH = 9.6 carbonate buffer, dilute according to the ratio of 1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000 respectively, add a 100 μL per well, make two parallel samples respectively, incubate at 37°C for 2 hours, then overnight at 4°C. Wash 5 times with PBST solution, 2min each time; block with 5...

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Abstract

The invention discloses a preparation method of a porcine circovirus 2 type (PVC2) total-length Cap protein and an ELISA antibody detection kit taking the Cap protein as an antigen. The ELISA antibody detection kit contains an elisa plate coated with the porcine circovirus 2 type nucleocapsid protein, and also contains sample diluent, a confining liquid, a washing liquid, an enzyme conjugate, enzyme substrate solution, stop solution and negative and positive control serum. The ELISA antibody detection kit has strong specificity and high sensitivity when being used for detecting PCV2 and can be popularized and applied in a large scale.

Description

technical field [0001] The invention relates to a detection kit, in particular to a porcine circovirus type 2 ELISA antibody detection kit, which belongs to the technical field of animal virus detection. Background technique [0002] Porcine circovirus (PCV) is the smallest animal virus that has been discovered so far. According to its antigenicity, nucleotide sequence and pathogenicity, it can be divided into two serotypes: porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2). Among them, PCV1 is non-pathogenic and widely exists in pigs and pig-derived cell lines. PCV2 can cause postweaning multisystemic wasting syndrome (Postweaning Multisystemic Wasting Syndrome, PMWS), piglet congenital tremor, porcine dermatitis nephritic syndrome, porcine respiratory syndrome, sow reproductive disorders and other diseases. Among them, PMWS has the greatest impact on the pig industry, and is mainly characterized by slow growth, progressive emaciation and multisystem p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01G01N33/68G01N33/569
CPCC07K14/005C12N2750/10031G01N33/569G01N33/68
Inventor 杨克礼时代田永祥周丹娜段正赢郭锐袁芳艳刘泽文刘威
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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