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Preparation and use of antibacterial peptide PMAP-23 monoclonal antibody

A technology of PMAP-23 and monoclonal antibody, which is applied in the direction of anti-animal/human immunoglobulin, instruments, analytical materials, etc., and can solve the problems of low content, small molecular weight of natural antimicrobial peptides, and difficulty in obtaining antimicrobial peptide immunogens, etc.

Inactive Publication Date: 2009-07-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Due to the small molecular weight and low content of natural antimicrobial peptides, the purity of directly extracted and purified products is very low, and the cost is high, so it is difficult to obtain high-purity antimicrobial peptide immunogens, and there is a lack of specific monoclonal antibodies to antimicrobial peptides on the market, resulting in the The research on the expression level of antimicrobial peptides stays at the gene transcription level, and it is difficult to conduct research on the translation level

Method used

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  • Preparation and use of antibacterial peptide PMAP-23 monoclonal antibody
  • Preparation and use of antibacterial peptide PMAP-23 monoclonal antibody
  • Preparation and use of antibacterial peptide PMAP-23 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Embodiment 1, the preparation of artificial immunogen:

[0012] Artificially synthesized fragments containing amino acid residues of porcine antimicrobial peptide PMAP-23: Arg-Ile-Ile-Asp-Leu-Leu-Trp-Arg-Val-Arg-Arg-Pro-Gln-Lys-Pro-Lys-Phe-Val -Thr-Val-Trp-Val-Arg. The immunogen PMAP-23-OVA was prepared by the carbodiimide (EDC) method, and the protein concentration of the coupled PMAP-23-OVA was measured by a full-wavelength ultraviolet / visible light scanning spectrophotometer, and then stored in -20°C. The artificially coated original PMAP-23-BSA was prepared by the same method, and stored at -20°C in aliquots.

[0013] Healthy female Balb / c pure-line mice aged 8-12 weeks were immunized by direct intraperitoneal injection with two basic immunizations and one booster immunization. On the first day, 100 μl PMAP-23-OVA+100 μl Freund’s complete adjuvant was used for basic immunization; on the 16th day, 100 μl PMPA-23-OVA+100 μl Freund’s incomplete adjuvant was used for ...

Embodiment 2

[0020] Embodiment 2, the clone of positive hybridoma cell:

[0021] Use a pasteurized tube to gently blow and blow the cells in the target well to be cloned to prepare a cell suspension. Prepare a series of double-ratio cell suspensions in a 96-well cell culture plate, 0.2ml per well, cell pellet for 2-3 hours, and select under an inverted microscope Wells with the appropriate number of cells. Pick 80-100 cells / well for the first clone, and 60-80 cells / well for the second clone. Transfer the cells in the selected wells to a cell culture flask, then add 10ml of HT medium, and inoculate 0.1ml / well into a 96-well cell culture plate, that is, each well contains about 1 cell on average. After 9-10 days of cloning, single-cell cloning lines were selected for positive detection, and the obtained positive cloning wells were further cloned for the second and third times until the positive rate of the supernatant was 100% after cloning and the degree of reaction was basically the same ...

Embodiment 3

[0042] Example 3, application of PMAP-23 monoclonal antibody: establishment of an inhibitory ELISA experimental method to detect the effect of lactoferrin on the level of PMAP-23 secreted by bone marrow cells:

[0043] The culture supernatant of porcine bone marrow cells contains the natural antimicrobial peptide PMAP-23. Taking the culture supernatant of bone marrow cells as the inhibitory object, the prepared positive supernatant was used to establish an inhibitory ELISA to detect the effect of lactoferrin on PMAP-23 in the culture supernatant of bone marrow cells. level of impact. Extract 30ml of bone marrow from the femur of a 30-day-old piglet by puncture, place it in RPMI 1640 culture medium (containing 10% fetal bovine serum), use a pipette tip to separate the bone marrow cells into single cells, and then spread them on the same volume with a specific gravity of 1.077g Centrifuge at 2000rpm for 30min, take the granulocyte layer into a new centrifuge tube, wash twice wit...

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Abstract

The invention discloses a preparing method of an antibacterial peptide PMAP-23 monoclonal antibody. Amino fragment of chemically synthesized antibacterial peptide PMAP-23 is used for immunizing Balb / c mouse. Then, spleen cells of the immunized mouse are used to be integrated with Sp2 / 0 myeloma cells. After screening and cloning, hybridoma cell strains for secreting anti-pig antibacterial peptide PMAP-23 monoclonal antibody are obtained. And finally, the required antibody is obtained. The monoclonal antibody of the invention can be used for constructing an enzyme linked immunosorbent assay method for detecting influences of lactoferrin on the PMAP-23 secreting level of the myeloma cells.

Description

technical field [0001] The invention relates to a preparation method of an antibacterial peptide PMAP-23 monoclonal antibody and an ELISA detection method established by using the monoclonal antibody. Background technique [0002] Due to the small molecular weight and low content of natural antimicrobial peptides, the purity of directly extracted and purified products is very low, and the cost is high, so it is difficult to obtain high-purity antimicrobial peptide immunogens, and there is a lack of specific monoclonal antibodies to antimicrobial peptides on the market, resulting in the The research on the expression level of antimicrobial peptides stays at the gene transcription level, and it is difficult to carry out the research on the translation level. Contents of the invention [0003] The technical problem to be solved by the present invention is to provide a method for preparing monoclonal antibodies, by which a large amount of specific monoclonal antibodies can be ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/08C07K16/18G01N33/577
Inventor 汪以真刘倚帆单体中郭佳张艳芳高彦华王爱苹
Owner ZHEJIANG UNIV
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