Antigen epitope peptide of alpha fetoprotein, nucleic acid, preparation method of nucleic acid, recombinant vector, host cell, hybridoma cell, monoclonal antibody and kit

A technology for recombining host cells and hybridoma cells, which can be applied in the fields including weak immunogenicity and poor specificity, and achieves the effects of strong immunogenicity, low detection limit and good specificity.

Active Publication Date: 2012-01-18
BEIJING LEADMAN BIOCHEM
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] An object of the present invention is to overcome the disadvantages of weak immunogenicity and poor specificity of the existing alpha-fetoprotein epitope peptides, and provide a strong immunogenicity and good specificity antigenic epitope peptides of alpha-fetoprotein

Method used

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  • Antigen epitope peptide of alpha fetoprotein, nucleic acid, preparation method of nucleic acid, recombinant vector, host cell, hybridoma cell, monoclonal antibody and kit
  • Antigen epitope peptide of alpha fetoprotein, nucleic acid, preparation method of nucleic acid, recombinant vector, host cell, hybridoma cell, monoclonal antibody and kit
  • Antigen epitope peptide of alpha fetoprotein, nucleic acid, preparation method of nucleic acid, recombinant vector, host cell, hybridoma cell, monoclonal antibody and kit

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Experimental program
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Effect test

preparation Embodiment 1

[0059] A) Obtaining the nucleotide sequence of the antigenic epitope peptide encoding alpha-fetoprotein

[0060] A-1) RT-PCR in vitro amplification of cDNA genome

[0061] Take 100 mg of human liver cancer cell hepG2 (purchased from ATCC) preserved in liquid nitrogen, and extract total RNA; RT-PCR reaction conditions: reverse transcription at 50°C for 30 minutes, denaturation at 94°C for 2 minutes, 94°C for 45 seconds, and 58°C for 30 seconds , 72°C for 45s, cycled 33 times, and extended at 72°C for 7min. A large number of cDNA fragments of the target gene were amplified by PCR. The reaction conditions were: 94°C for 2min, 94°C for 30s, 61°C for 30s, 72°C for 30s, 33 cycles, and 72°C for 7min.

[0062] A-2) Obtaining the Nucleotide Sequence Encoding Antigen Epitope Peptide

[0063] The following sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd.:

[0064] AFP-1:

[0065] Forward primer 5'-CATGCCATGGGACATTCAGAC-3'

[0066] Reverse primer 5'-CCGCTCGAGGCATTC...

preparation Embodiment 2

[0097] The test kit of the present invention comprises the following components:

[0098] (1) Coating buffer (pH9.60.05M carbonate buffer): NaHCO 3 1.59 g, NaHCO 3 2.93 grams, add distilled water to 1000ml;

[0099] (2) Washing buffer (PH7.4 phosphate buffer): 0.15M is KH 2 PO 4 0.2 g, Na 2 HPO 4 12H 2 O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-200.05% 0.5ml, add distilled water to 1000ml;

[0100] (3) Blocking solution: 0.2-1.0 grams of bovine serum albumin (BSA), 0.5-1.0 grams of casein, and add washing buffer to 100 ml.

[0101] (4) Diluent: Bovine Serum Albumin (BSA) 0.1 g, add washing buffer to 100 ml, or mix 5-10% with washing liquid such as goat serum and rabbit serum for use.

[0102] (5) stop solution (2M H 2 SO 4 ): 178.3ml of distilled water, 21.7ml of concentrated sulfuric acid (98%) was added dropwise.

[0103] (6) Substrate buffer (PH5.0 citric acid phosphate): 0.2MNa 2 HPO 4 (28.4 g / L) 25.7ml, 0.1M citric acid (19.2 g / L) 24.3ml, add distilled water 50ml.

...

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Abstract

The invention discloses antigen epitope peptide of alpha fetoprotein, wherein an amino acid sequence of the antigen epitope peptide is an amino acid sequence (1) shown as SEQ ID NO: 1, an amino acid sequence (2) shown as SEQ ID NO: 3 or an amino acid sequence which is formed by deleting, adding and / or substituting one or more amino acids for the amino acid sequence (1) or (2) with unchanged function of the antigen epitope peptide of the alpha fetoprotein. The invention also discloses a nucleotide sequence for coding the antigen epitope peptide of the alpha fetoprotein and a preparation methodthereof. The nucleotide sequence comprises a recombinant vector of the nucleotide sequence, a recombinant host cell of the recombinant vector, a monoclonal hybridoma cell and a monoclonal antibody and comprises a kit for detecting the alpha fetoprotein of the antigen epitope peptide and / or the monoclonal antibody. The antigen epitope peptide of the alpha fetoprotein has high specificity; and the kit for detecting the alpha fetoprotein prepared by the monoclonal antibody obtained by the antigen epitope peptide has high sensitivity.

Description

technical field [0001] The present invention relates to an antigenic epitope peptide, a nucleotide sequence encoding the antigenic epitope peptide and a method for preparing the nucleotide sequence, a recombinant vector comprising the nucleotide sequence, and a recombinant vector comprising the recombinant vector The recombinant host cell, the monoclonal antibody hybridoma cell and the monoclonal antibody produced by the antigenic epitope peptide, and the protein detection kit including the above-mentioned monoclonal antibody. More specifically, the present invention relates to an antigenic epitope peptide of alpha-fetoprotein, a nucleotide sequence encoding the antigenic epitope peptide of alpha-fetoprotein and a method for preparing the nucleotide sequence, including the nucleoside A recombinant vector of acid sequence, a recombinant host cell comprising the recombinant vector, a monoclonal antibody hybridoma cell and the monoclonal antibody produced thereof against the anti...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K16/18C12N15/12C12N15/10C12N15/63C12N1/21C12N5/20G01N33/577C12R1/91
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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