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A kind of porcine circovirus type ii antigen and colloidal gold immunochromatographic test strip for detecting porcine circovirus type ii antibody

A technology of porcine circovirus and immune chromatography, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems of unsuitable promotion, expensive equipment, cumbersome operation methods, etc. The effect of practical value

Active Publication Date: 2021-07-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, ELISA is the most widely used serological detection method for the diagnosis of circovirus type II. ELISA detection mainly uses recombinant expressed Cap protein to coat the plate. ELISA has the advantages of high specificity, good sensitivity, and large-scale detection. There are shortcomings such as high technical requirements, cumbersome operation methods, and expensive instruments, which are not suitable for expansion and promotion at the grassroots level

Method used

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  • A kind of porcine circovirus type ii antigen and colloidal gold immunochromatographic test strip for detecting porcine circovirus type ii antibody
  • A kind of porcine circovirus type ii antigen and colloidal gold immunochromatographic test strip for detecting porcine circovirus type ii antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Expression and purification of porcine circovirus type II virus-like particles

[0028] 1. Construction of recombinant expression plasmid pGAPZα-Cap of porcine circovirus type II virus-like particle gene

[0029]According to the conservation and antigenicity analysis of the porcine circovirus type II Cap protein sequence on the NCBI website, and site mutations based on the known virus-like particles of porcine circovirus type II, the specific Cap protein of porcine circovirus type II was designed and synthesized The gene (any one of SEQ ID NO: 4-6) makes the virus-like particle formed by recombinant expression more stable. Digest the pGAPZα vector with HindⅢ and NotI. The digestion system is 50uL: 1uL each for HindⅢ and NotI, 20uL for the vector fragment, 5uL for the 10X digestion buffer, and 23uL for ddH2O. box recycling. Then the digested vector was ligated with the Cap gene sequence to construct a 10 μL ligation system: 5.5 μL of NG gene fragment, 2.5 μL...

Embodiment 2

[0039] Embodiment 2: Colloidal gold immunochromatography detection test strip for preparing porcine circovirus type II antibody

[0040] 1. Preparation of gold-labeled conjugates of SPA labeled with gold particles

[0041] Take 2ml of 40nm colloidal gold particles and use 0.1mol / L potassium carbonate to adjust the pH to 6.8, add 10ug Staphylococcus aureus protein A (SPA), mix quickly and mix at room temperature on a 3D rotator for 30min, then add the final concentration 1% BSA and mix on a 3D rotary mixer for 30min, centrifuge the gold standard solution at 12000r / min at 4°C for 10min, discard the supernatant carefully, wash the precipitate twice with 0.01MPBS buffer, and centrifuge again to get the precipitate To purify the SPA gold-labeled complex, the prepared colloidal gold-labeled SPA was resuspended in 0.01MPBS and stored at 4°C for later use.

[0042] 2. Nitrocellulose membrane testing line and quality control line spraying and colloidal gold bonding pad preparation

...

Embodiment 3

[0048] Embodiment 3: Colloidal gold immunochromatography test strip specificity test

[0049] Crossover test was carried out on the known porcine foot-and-mouth disease type O positive serum, porcine parvovirus positive serum, porcine rotavirus positive serum, swine fever virus positive serum and porcine circovirus type II positive serum, the results are shown in figure 2 .

[0050] figure 2 The results showed that only the porcine circovirus type II positive serum sample had a positive reaction with a detection line, but when it reacted with several other sera, the detection line had no color development and a negative reaction, indicating that the antigen was used for detection Porcine circovirus type II antibodies are highly specific.

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Abstract

The invention relates to the technical field of immune detection, and discloses a porcine circovirus type II antigen and a colloidal gold immunochromatographic test strip for detecting porcine circovirus type II antibody. The antigen sequence of the present invention is shown in any one of SEQ ID NO: 1-3. The present invention provides a novel porcine circovirus type II antigen. When its self-assembled virus-like particles are used as the detection antigen of porcine circovirus type II antibody, it has extremely high specificity, repeatability and accuracy. It can be applied to related immunochromatographic detection products, and the result is intuitive and can be judged by naked eyes. It can quickly and specifically detect porcine circovirus type II antibody in serum, has high practical value, and is convenient for popularization and use at the grassroots level.

Description

technical field [0001] The invention relates to the technical field of immune detection, in particular to a porcine circovirus type II antigen and a colloidal gold immunochromatographic test strip for detecting porcine circovirus type II antibody. Background technique [0002] Porcine circovirus belongs to the family Circoviridae and the genus Circovirus. It is a class of single-stranded circular DNA viruses with no envelope and an icosahedral spherical structure. There are currently two serotypes of circovirus, circovirus type I and circovirus type II. Pigs infected with circovirus type 1 do not show clinical symptoms, while circovirus type 2 is associated with various diseases of pigs, especially the multisystem wasting syndrome of weaned piglets, which has caused great losses to the pig industry. [0003] Porcine circovirus type II has three main open reading frames, namely ORF1, ORF2, and ORF3, among which ORF1 encodes the virus replication-related protein Rep protein. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01G01N33/569G01N33/558C12N15/81G01N33/68C12R1/84
CPCC07K14/005C12N15/815C12N2750/10022C12N2750/10023G01N33/558G01N33/56983G01N33/6854G01N2333/01
Inventor 孟赓朱文壮顾贵波
Owner CHINA AGRI UNIV
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