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Blood group A antigen epitope mimic peptide and its filtration method

A technology of antigenic epitope and screening method, applied in the field of polypeptide biology, can solve the problems of low purity, difficult preservation, insufficient source, etc.

Active Publication Date: 2008-01-02
胡丽华 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the ABO antigens currently used in affinity filtration blood type antibodies and antibody detection tests are all natural sources or artificially synthesized oligosaccharide components, these antigens have disadvantages such as insufficient sources, low purity, and difficulty in storage.

Method used

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  • Blood group A antigen epitope mimic peptide and its filtration method

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Embodiment Construction

[0024] 1. Main materials and their sources

[0025] 1) Blood group A antigen monoclonal antibody (clone: ​​NaM87-1F6) was purchased from BD Company, USA.

[0026] 2) The phage display random 12-peptide library is from NEB Company of the United States.

[0027] 3) ELISA microplates were purchased from Greiner, Germany.

[0028] 4) Biotin-labeled goat anti-mouse IgG secondary antibody and horseradish peroxidase-labeled avidin were purchased from Beijing Zhongshan.

[0029] 5) All other reagents were of domestic analytical grade.

[0030] 2. Methods and Results

[0031] 1) Screening of phage peptide library

[0032] 0.1M NaHCO 3 (pH8.6) Dilute the antibody NAM87-1F6, and 50 μl coats an ELISA microwell. Soluble in 0.1M NaHCO 3 (pH 8.6) 5 mg / ml BSA in buffer was used as a control to coat one well at the same time. The coated microwells were placed in a humid chamber and incubated at 4°C for 16 hours. 0.1M NaHCO with 5mg / ml BSA 3 (pH8.6) The microwells were washed with a m...

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Abstract

The invention discloses an epitope polypeptide of blood type A antigen and sieving method with amino acid sequence at Glu-Tyr-Trp-Tyr-Cys-Gly-Met-Asn-Arg-Thr-Gly-Cys or Gln-Ile-Trp-Tyr-Glu-Arg-Thr-Leu-Pro-Phe-Thr-Phe, which comprises the following steps: using NaHCO3 buffer to dilute blood type A antigen specific monoclonal antibody and cover ELISA board; adding bacteriophage into pore with monoclonal antibody; using glycine-alcaine buffer to elute the connected bacteriophage under indoor temperature; augumenting the bacteriophage in the host bacteria ER2738 after neutralizing as input in the next step; sieving for three times; cloning the 32nd bacteriophage after the third sieving; identifying to obtain the product; fitting for detecting blood type A antibody and affinity filtering.

Description

technical field [0001] The invention relates to the field of polypeptide biotechnology, in particular to a blood group A antigen epitope mimic polypeptide and a screening method thereof. Background technique [0002] The chemical structure of A blood group antigens belongs to glycoproteins, and their structures mainly involve the genes of two separate loci; the H locus gene and the A locus gene, these genes do not directly produce antigens, but produce glycosyltransferases, glycosyl transfer Enzymes determine the specificity of A blood group antigens. Therefore, glycosyltransferase is the direct product of the gene, while the monosaccharide or oligosaccharide of the blood group epitope is the indirect product of the gene. The antigenic determinant of A blood group is connected to the polysaccharide precursor or the carrier chain of the blood group substance. The precursors of blood group substances are polysaccharide structures. At present, it is believed that the antigen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C40B30/04G01N33/53G01N33/80
Inventor 胡丽华汤兆明李一荣崔天盆
Owner 胡丽华
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