Pig circular ring virus 2 type antigen subtype identifying test paper card

A test paper card and antigen technology, which is applied in the field of porcine circovirus type 2 antigen subtype identification test paper cards, can solve the problems of difficulty in popularization and promotion, time-consuming, complicated test operation, etc. Easy and fast operation, high sensitivity effect

Active Publication Date: 2009-04-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above technologies and methods can detect PCV2 virus and have achieved certain results in practice, none of them can realize the differential diagnosis of PCV2 antigen typing, and the test

Method used

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  • Pig circular ring virus 2 type antigen subtype identifying test paper card
  • Pig circular ring virus 2 type antigen subtype identifying test paper card
  • Pig circular ring virus 2 type antigen subtype identifying test paper card

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Preparation of PCV2 capsid protein recombinant protein

[0020] Use genetic engineering technology to efficiently express PCV2 capsid protein, and prepare capsid protein recombinant protein. according to 1767 PCV2 isolate HZ0201 (GenBank No.AY188355) complete gene sequence, respectively design PCV2 capsid protein specific primers, the upstream primer is pG1:5’-GC GGATCC AATGGCATCTTCAACAC-3’, with BamHI restriction site, downstream primer pG2: 5’-CCG CTCGAG TTAAGGGTTAAGTGGG-3', containing XhoI restriction site. Using PCV2 virus DNA as a template, amplify a 579bp enucleated localization signal capsid protein gene. The reaction conditions are: 95°C pre-denaturation for 5 minutes, 95°C 30s, 58 or 61°C 30s, 72°C 45s for 30 cycles, Finally, extend at 72°C for another 10 minutes. The PCR product was digested with BamHI and XhoI, respectively connected to the downstream BamHI and XhoI sites of the glutathione-S-transferase gene (GST) of the prokaryotic expression vector ...

Embodiment 2

[0022] Example 2 Preparation and screening of monoclonal antibodies against PCV2 capsid protein

[0023] Recombinant PCV2 capsid protein and purified PCV2 virus were mixed with Freund’s adjuvant in equal amounts, fully emulsified, and BALB / c mice were immunized with 50μg-100μg / mouse 3 times with an interval of 15-30 days; the third time 3-4 days after the booster immunization, the immunized mouse’s eyeballs were bled, necked to death, soaked in 75% alcohol for 5-10 minutes, and the spleen cells were taken aseptically; cut into pieces and filtered through a 100 mesh nylon mesh, centrifuged at 1000 r / min for 10 minutes , Collect spleen cells; add 1×10 8 Spleen cells with 2-5×10 7 Mix the SP2 / 0 myeloma cells, centrifuge at 1000r / min for 10min, discard the supernatant, slowly add 0.7-1ml of 40%-50% PEG4000 (pH8.5-9.0) to the cells in a 37℃ water bath, and incubate After 1 min, slowly add 15ml of serum-free 1640 medium to stop the effect of PEG, 37°C water bath for 5-10min, 1000r / min c...

Embodiment 3

[0024] Example 3 Screening of anti-PCV2 capsid protein monoclonal antibodies

[0025] The 14 strains of anti-capsid protein monoclonal antibodies were screened and identified by indirect immunofluorescence (IFA). Separate PCV1 isolates, 1768 PCV2 isolate, 1767 PCV2 isolates and 1766 PCV2 isolates were inoculated 1:10 with trypsin-digested PK-15 cells without PCV contamination, and the virus cell mixture was added to a 96-well cell culture plate, 100μL per well, 37℃ 5% CO 2 Incubate for 96h, add 100μL of 1:1 methanol-acetone fixing solution, and fix at -20°C for 20min. Discard the fixative and let it dry naturally. Block with 5% skimmed milk powder for 1h, add 100μL of supernatant of hybridoma cells to be tested to each well, incubate at 37℃ for 1h, wash 5 times with PBST, add 50μL of FITC-labeled goat anti-mouse IgG diluted 1:400, incubate at 37℃ for 40min , PBST wash 5 times. Add 50μL of PBS, observe the positive cells with an inverted fluorescence microscope, and screen monoclon...

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Abstract

A PCV2 antigen subtype identification test paper card is applicable to pig PCV2 subtype identification. A reaction reagent carrier absorption layer comprises a fiber layer, a gold-labeled fiber layer, a cellulose membrane layer and a water absorbent material layer; wherein the gold-labeled fiber layer is gold-labeled glass cotton of an anti-capsid protein monoclonal antibody which absorbs a colloidal gold marker and can identify PCV common antigen epitope, detection blots T1, T2 and T3 and a control blot C are printed on the cellulose membrane layer; the detection blot T1 is the blot which is printedby anti-capsid protein monoclonal antibody solution and used for identifying <1767>PCV2 subtype specific antigen epitope, the detection blot T2 is the blot which is printed by the anti-capsid proteinmonoclonal antibody solution and used for identifying <1766>PCV2 and <1767>PCV2 common antigen epitope, the detection blot T3 is the blot which is printed by the anti-capsid protein monoclonal antibody solution and used for identifying PCV2 common antigen epitope, and the control blot C is the blot which is printed by anti-mouse IgG polyclonal antibody solution. The test paper card has strong specificity, high sensitivity, intuitive detection result as well as easy popularization and application.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to a test paper card for identification of porcine circovirus type 2 (PCV2) antigen subtypes. Background technique [0002] Porcine circovirus 2 (PCV2) is the pathogen of porcine circovirus disease (PCVD), which leads to immunosuppression in pigs and causes huge economic losses to the pig industry; it is the smallest animal virus found so far , The diameter of the virus particle is about 17nm, which is a covalently closed, circular, single-stranded DNA virus. At the DNA level, three PCV2 viruses with full genome length of 1768nt, 1767nt, and 1766nt have been found. It is known that PCV2" ORF1 encodes a Rep protein related to virus replication, ORF2 encodes a capsid protein related to PCV2 immunity, and ORF3 encodes a protein that is not related to PCV2 replication but is related to pathogenicity. 1766 PCV2 lacks a nucleotide G at position 1059, causing the C-terminal codon of ORF2 to shift, ma...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/558
Inventor 周继勇颜焰金玉兰
Owner ZHEJIANG UNIV
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