Porcine circovirus type 4 Cap protein monoclonal antibody as well as preparation method and application thereof

A monoclonal antibody and porcine circovirus technology, applied in antiviral immunoglobulin, virus/bacteriophage, biochemical equipment and methods, etc., can solve problems such as difficult to obtain pure pathogen culture and PCV4 culture, and achieve sensitivity High, stable and specific effects

Pending Publication Date: 2021-11-12
兆丰华生物科技(南京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Since PCV4 is a new pathogen and the complex clinical phenomena caused by PCV4 infection, the diagnosis and vaccine development of porcine circovirus will be more complicated in the future, and PCV4 cannot be cultured in cells, and it is difficult to obtain pure pathogenic cultures in vitro, so it is urgent It is to develop more accurate and comprehensive serological detection and antigen detection methods and antigen quantitative methods
[0007] Since there are no serological diagnostic reagents and corresponding methods for PCV4, an effective serological diagnostic technique is urgently needed

Method used

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  • Porcine circovirus type 4 Cap protein monoclonal antibody as well as preparation method and application thereof
  • Porcine circovirus type 4 Cap protein monoclonal antibody as well as preparation method and application thereof
  • Porcine circovirus type 4 Cap protein monoclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Cloning of PCV4-Cap gene

[0036] (1) Collection, processing and DNA extraction of disease materials

[0037] The disease material was collected from the spleen of weaned piglets in a pig farm in Jiangsu in December 2019. Add 10mL PBS to grind each 1g of tissue, freeze and thaw three times at -20°C and 37°C, shake for 15s, centrifuge at 3000rpm for 2min, and take the supernatant in Store at -20°C for later use. DNA was extracted from disease samples using a blood / cell / tissue genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.) to extract and prepare DNA, which was stored at -20°C for later use.

[0038] (2) Primer design and synthesis

[0039] Design a pair of specific primers F1 and R687 (see Table 1) according to the last conservative region of the PCV4 Cap nucleic acid sequence (GenBank No.: MK986820) published on GenBank, as shown in SEQ ID NO: 6-7, the purpose of amplification The DNA fragment size is 687bp.

[...

Embodiment 2

[0049] Example 2 Preparation and identification of recombinant PCV4-Cap protein and its virus-like particles

[0050] (1) Digestion and purification of PCV4 Cap gene and vector

[0051] The PCR amplified PCV4 Cap gene and its truncated fragments were subjected to agarose gel electrophoresis, and the electrophoresis results were recorded by taking pictures and then purified with a PCR product cleaning kit, followed by a double enzyme digestion reaction (as shown in Table 3). At the same time, the pET28a vector was placed in In the same reaction system (as in Table 4). The digested PCV4 Cap gene and the empty vector were purified and concentrated again with a PCR cleaning kit.

[0052] Table 3 Enzyme digestion system of target gene

[0053]

[0054] Table 4 vector digestion system

[0055]

[0056] (2) Construction and identification of recombinant expression strain BL21(DE3)-pET28a-PCV4 Cap

[0057] Add the target gene and vector purified and concentrated in step (1) ...

Embodiment 3

[0070] Example 3 Preparation and Activity Identification of the Monoclonal Antibody (MAb) of PCV4-Cap Protein

[0071] (1) Immunization program

[0072]The virus-like particles formed by the purified PCV4 Cap protein were emulsified with an equal volume of Freund's complete adjuvant, and intraperitoneally injected into BALB / c mice aged 6-8 weeks at multiple points, with an immunization dose of about 100 μg / mouse; 14 days later, the purified The virus-like particle protein formed by the PCV4 Cap protein was emulsified with Freund's incomplete adjuvant for immunization; 3 days before cell fusion, immunization was boosted, and 500 μg of purified recombinant porcine circovirus type 4 Cap protein was injected intraperitoneally.

[0073] (2) Hybridoma cell fusion

[0074] Splenocytes and splenocytes were fused with PEG1450, splenocytes and SP2 / 0 myeloma cells were mixed at a ratio of 1:5 to 1:2, and the supernatant was discarded after centrifugation. Slowly add 50% PEG1400 for fus...

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Abstract

The invention relates to the field of biological vaccines, in particular to a porcine circovirus type 4 Cap protein monoclonal antibody as well as a preparation method and application thereof. An immunogen for preparing the monoclonal antibody is virus-like particles formed by porcine circovirus type 4 Cap protein; and the porcine circovirus type 4 Cap protein comprises three core antigen epitopes, and the three core antigen epitopes are respectively an epitope 1: RRRSRWRRKNGIFHARFMREVTLSVSSFST, the epitope 2: NAYYRIRKIKVEFLPLI, and the epitope 3: SIQNSNFVQVWTVRFTL. According to the method, the porcine circovirus type 4 Cap protein monoclonal antibody is prepared, a single-molecule imaging enzyme-linked immunosorbent assay iELISA method of the PCV4 Cap protein VLP using the 6D5 monoclonal antibody as the capture antibody is established, and the method has the advantages of high sensitivity, strong specificity, good stability, high throughput and the like.

Description

technical field [0001] The invention relates to the technical field of biological vaccines, specifically a porcine circovirus type 4 Cap protein monoclonal antibody and a single-molecule imaging enzyme-linked immunosorbent assay (iELISA) method for antigen quantification established by using the antibody. Background technique [0002] Porcine circovirus (PCV) is a non-enveloped circular DNA virus belonging to the family Circoviridae and the genus Circovirus. PCV is a minimal DNA animal virus with a genome of about 1.7kb. Since the discovery of PCV1 in the 1970s, PCVs that have been discovered are mainly PCV1, PCV2 and PCV3. Based on gene sequence and antigenic differences, PCV1 has been identified as a renal cell contaminant that does not cause clinical disease in pigs. PCV2 and PCV3 infections cause a variety of diseases in pigs and have a global distribution. PCV2 is a major pathogenic agent that can cause a variety of diseases, such as piglet post-weaning multisystemic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C07K14/01G01N33/577G01N33/569
CPCC07K16/081G01N33/577G01N33/56983C07K14/005G01N2333/01G01N2469/10C12N2750/10022C12N2750/10023C07K2317/56C07K2317/76C07K2317/33C07K2317/94
Inventor 何召庆朱晓玮唐青海周永银尹秀凤王婷高云飞顾玉美吴晓张渊魁
Owner 兆丰华生物科技(南京)有限公司
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