PCV3 Cap protein epitope peptide, anti-PCV3 Cap protein monoclonal antibody, and preparation method and application thereof

A monoclonal antibody and protein technology, applied in the field of cellular immunity, can solve the problem of few immunological diagnostic methods

Active Publication Date: 2021-05-18
河南中泽生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of PCV3-specific monoclonal antibodies,

Method used

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  • PCV3 Cap protein epitope peptide, anti-PCV3 Cap protein monoclonal antibody, and preparation method and application thereof
  • PCV3 Cap protein epitope peptide, anti-PCV3 Cap protein monoclonal antibody, and preparation method and application thereof
  • PCV3 Cap protein epitope peptide, anti-PCV3 Cap protein monoclonal antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The selection and preparation of embodiment 1 immunogen

[0048] 1. Selection of Immunogen

[0049] The inventors have long-term, a large number of studies, practices, and experiments have found that: first, among the proteins encoded by the PCV3 genome, the capsid protein encoded by ORF2, that is, the Cap protein, is a highly immunogenic protein; secondly, in the body, the Cap protein The induced immune reactivity is much higher than that of other proteins, so it is easy to obtain high-affinity monoclonal antibodies; again, Cap protein is the only structural protein of PCV3, and is the preferred target for PCV3 immunological detection method research and vaccine development.

[0050] 2. Preparation of Immunogen

[0051] (1) Construct the pGEX6P-1-Cap recombinant vector expressing Cap protein, and use PCR and sequencing to identify whether the recombinant vector is constructed successfully (identification results are as follows: figure 1 shown);

[0052] (2) Transfor...

Embodiment 2

[0056] Screening and identification of embodiment 2 positive hybridoma cell lines

[0057] 1. Animal immunization

[0058] (1) Add immunogen Cap protein to complete Freund's adjuvant for first immunization;

[0059] (2) Immunize 6 female BALB / c mice aged 4 to 8 weeks by multi-point subcutaneous injection on the back, with an immune dose of 25 μg / mouse;

[0060] (3) BALB / c mice were boosted with the same method and dosage after being emulsified with Freund's incomplete adjuvant and immune antigen every 3 weeks;

[0061] (4) After three times of booster immunization, BALB / c mice were super-immunized with immunogen without adjuvant by tail vein injection 3 to 4 days before cell fusion, and the immunization dose was 50 μg / mouse;

[0062] (5) Determination of titer and sensitivity of multiple antiserum:

[0063] One week after the last booster immunization, tail-cut blood was collected from 6 mice respectively, and then the titers of polyantiserum of 6 mice were measured by indi...

Embodiment 3

[0076] Example 3 Preparation, purification and identification of monoclonal antibody ascites

[0077] 1. Preparation of monoclonal antibody by inducing ascites in vivo

[0078] Select multiparous female Balb / c mice, intraperitoneally inject 500 μl of sterilized paraffin, and a week later, intraperitoneally inject the obtained monoclonal hybridoma cells again, the injection volume is 2×10 5 After another week, the ascites was extracted after the abdomen of the mouse was enlarged, and the supernatant was obtained after centrifugation, and the ascites was purified by octanoic acid ammonium sulfate method.

[0079] 2. Antibody Purification

[0080] Antibody purification by saturated ammonium sulfate precipitation method, the operation method is as follows:

[0081] 1). Take 5ml of monoclonal antibody ascites, add 5ml of PBS buffer solution, and then add 2.5ml of saturated ammonium sulfate solution drop by drop to make a final concentration of 20% ammonium sulfate solution. Stir ...

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Abstract

The invention relates to a PCV3Cap protein epitope peptide, an anti-PCV3Cap protein monoclonal antibody, and a preparation method and application thereof. The invention provides the anti-PCV3Cap protein monoclonal antibody, and further provides a heavy chain variable region sequence and a light chain variable region nucleotide and amino acid sequence of the monoclonal antibody. On the basis, the antibody can be prepared by a genetic engineering method; and meanwhile, modification such as addition, deletion and replacement of one or more amino acids can be performed by genetic engineering and protein engineering methods to obtain an active fragment or a conservative variant thereof, thereby laying a foundation for further improving the specificity and affinity of the antibody. The antibody has relatively high specificity, has no cross reaction with PCV1, PCV2 and other porcine viruses such as CSFV, PRRSV and PRV, and has wide research and application values and commercial use values in immunological detection such as antigen/antibody detection kits, antigen/antibody immunochromatographic test paper, IFA, IPMA and Western Blotting.

Description

technical field [0001] The invention belongs to the technical field of cellular immunity, and in particular relates to a PCV3 Cap protein antigenic epitope peptide, an anti-PCV3 Cap protein monoclonal antibody and a preparation method and application thereof. Background technique [0002] Porcine circovirus (PCV) is a non-enveloped, covalently closed circular single-stranded DNA virus, classified by the International Committee on Taxonomy of Viruses (ICTV) as Circoviridae and Circovirus. So far, four genotypes of porcine circovirus have been reported, namely PCV1, PCV2, PCV3 and PCV4. In 1974, PCV1 was first discovered as a pollutant of porcine kidney (PK15) cell line, and subsequent studies generally believed that PCV1 It is non-pathogenic in pig herds. PCV2, discovered in 1997, is associated with a number of major clinical syndromes collectively known as porcine circovirus-associated disease (PCVAD). PCVAD is an economically important swine disease. Considering the huge...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12P21/08G01N33/577G01N33/569
CPCC07K16/10G01N33/577G01N33/56983C07K2317/33C07K2317/56C07K2317/565G01N2333/14G01N2469/10
Inventor 张改平蒋敏王爱萍陈玉梅刘东民刘红亮梁超刘燕凯王彦伟贾蕊
Owner 河南中泽生物工程有限公司
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