Monoclonal antibody for identifying PCV2 virus-like particles and application thereof in qualitative and quantitative detection of PCV2 virus-like particles

A monoclonal antibody and polyclonal antibody technology, applied in the field of biomedicine, can solve the problems of inability to specifically identify the structural integrity and purity of VLPs, inability to distinguish non-specific proteins and broken subunits, and expensive VLP particle structure detection instruments. , to achieve the effect of fast and accurate protein content, good linear relationship and good precision

Active Publication Date: 2019-03-29
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the BCA method to measure the protein content of VLPs, the greater the dilution factor, the higher the final protein concentration value; the determination of the UV spectrophotometric value also cannot specifically identify the structural integrity and purity of VLPs, and the absorbance value of each protein varies greatly. large, need t

Method used

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  • Monoclonal antibody for identifying PCV2 virus-like particles and application thereof in qualitative and quantitative detection of PCV2 virus-like particles
  • Monoclonal antibody for identifying PCV2 virus-like particles and application thereof in qualitative and quantitative detection of PCV2 virus-like particles
  • Monoclonal antibody for identifying PCV2 virus-like particles and application thereof in qualitative and quantitative detection of PCV2 virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of monoclonal antibody specifically recognizing porcine circovirus type 2 virus-like particles

[0052] 1. Preparation of PCV2 VLPs

[0053] (1) Clone the PCV2 cap protein gene and connect it to the PET-28a expression vector. After the correct insertion of the clone was confirmed by restriction enzyme digestion, the sequence of the recombinant plasmid was confirmed to be correct by sequencing. The recombinant plasmid was transformed into the expression strain BL21 to induce expression, and the collected cells were broken by a high-pressure homogenizer, redissolved after ammonium sulfate precipitation, and the results of SDS-PAGE electrophoresis showed that there was an obvious band at the size of 27KD, which was consistent with the molecular weight of the cap protein ( figure 1 ).

[0054] (2) The expressed protein was identified by western blot, the bacterial protein was transferred to PEDV membrane by SDS-PAGE, and identified by his tag antibody...

Embodiment 2

[0064] Example 2 Preparation of Double Antibody Sandwich ELISA Quantitative Kit

[0065] 1. Using 3H9 monoclonal antibody as coating antibody, rabbit anti-PCV2 VLPs polyclonal antibody and HRP-labeled goat anti-rabbit polyclonal antibody as detection antibody, a double sandwich ELISA detection method was established to analyze the protein concentration of VLPs.

[0066] 2. The operation process of the double-antibody sandwich ELISA quantitative kit

[0067] (1) Dilute the 3H9 monoclonal antibody with coating solution (50mM carbonate coating buffer, pH9.6) at 1:12800, and add 100 microliters per well to the ELISA plate. Coating overnight at 4°C;

[0068] (2) Wash the coated ELISA plate 4 times with PBST solution (0.1mol / L PBS solution containing 0.5v / v% Tween20, pH 7.6, diluted 10 times when used, the same below), 200 microliters per well , 5 minutes each time. Add 200 microliters of blocking solution (0.01M PBS solution containing 1w / v% BSA, pH 7.6) to the ELISA plate, and ...

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Abstract

The invention discloses a monoclonal antibody for identifying PCV2 virus-like particles and an application thereof in qualitative and quantitative detection of PCV2 virus-like particles, wherein the monoclonal antibody capable of specifically identifying porcine circovirus type 2 virus-like particles is secreted by a hybridoma cell line with the preservation number of CGMCC NO.15793. The specificity of the obtained monoclonal antibody 3H9 is identified by indirect immunofluorescence assay, and the result indicates that the monoclonal antibody can specifically identified the PCV2 virus-like particles and can react with the PCV2 VLPs, but not with an ELISA plate coated with a linear cap protein. Therefore, a simple method for simultaneous qualitative and quantitative analysis of the PCV2 VLPs is established, and the problems of expensive instruments for detecting a structure of PCV2 VLP particles, difficult operation and inability of quantitative methods to distinguish non-specific proteins from broken subunits are solved.

Description

technical field [0001] The present invention relates to a monoclonal antibody that recognizes PCV2 virus-like particles and a hybridoma cell that secretes the monoclonal antibody, and also relates to the application of the monoclonal antibody in the qualitative and quantitative detection of PCV2 virus-like particles (virus-like particles, VLPs) . The invention belongs to the technical field of biomedicine. Background technique [0002] In all links of vaccine research and development and production, such as strain identification and cultivation, separation and purification, and product testing, it is necessary to conduct qualitative and quantitative analysis and testing of the active ingredients of the vaccine. Quantitative detection and analysis of vaccine active ingredient VLPs in the process of strain culture and fermentation, protein purification, and vaccine preparation, and rapid and accurate analysis and characterization of vaccines are useful for establishing and op...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/08G01N33/577G01N33/569
CPCC07K16/081G01N33/56983G01N33/577G01N2333/01G01N2469/10
Inventor 蔡雪辉孙明霞涂亚斌王淑杰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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