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Alpha fetoprotein specific binding polypeptide and application

A technology for specific binding of alpha-fetoprotein, applied in the field of preparation of polypeptides, or functional fragments, or variants thereof, which can solve the problems of complex preparation process, poor thermal stability and high cost of antibody molecules, and achieve low and stable preparation cost Good sex, good effect of peptide-specific affinity

Active Publication Date: 2020-11-13
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All of the above immunoassay methods require the use of specific antibodies, but due to the characteristics of the antibody molecule itself, such as belonging to a bivalent multi-domain protein, relying on disulfide bond linkage, and complex glycosylation reaction modes, etc., antibody molecules are hot Poor stability, complex and expensive preparation process, etc. Therefore, it is necessary to develop a new type of alpha-fetoprotein affinity element to solve the above problems

Method used

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  • Alpha fetoprotein specific binding polypeptide and application
  • Alpha fetoprotein specific binding polypeptide and application
  • Alpha fetoprotein specific binding polypeptide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Panning and identification of alpha-fetoprotein-specific binding polypeptides.

[0032] 1. Mutant construction and single clone selection

[0033] Aiming at the 9th, 10th, 11th, 13th, 14th, 17th, 18th, 24th, 25th, 27th, 28th, 32nd and 35th amino acid residues of the immunoglobulin binding region polypeptide of staphylococcal protein A, the abbreviation with NNK as the codon is designed and primers, using degenerate primers to obtain the coding gene of the affimer polypeptide library by over-lapPCR. The gene encoding the polypeptide mutant was cloned into a phage display vector, and the recombinant plasmid was electrotransformed into TG1 host cells to obtain a storage capacity of 5×10 7 Affimer polypeptide mutant library. Use the helper phage to display the mutant polypeptides of the affinity body, and then obtain the phage display library of the polypeptide mutants.

[0034] Alpha-fetoprotein extracted from umbilical cord blood (purchased from fitzgerald, Cat. No. 30-1...

Embodiment 2

[0041] Expression and purification of alpha-fetoprotein-specific binding polypeptides.

[0042] From the 13 polypeptide mutants specifically binding alpha-fetoprotein identified in Example 1, the following six were selected as objects for further research: ZAFP-D2, ZAFP-A3, ZAFP-B7, ZAFP-G9, ZAFP-A2, ZAFP-B8.

[0043] The expression host is E.coli BL21 (DE3) (preserved in our laboratory), the expression vector is pET-21b (preserved in our laboratory), and the schematic diagram of the recombinant vector is attached. Figure 4 , wherein ZAFP represents the above six different alpha-fetoprotein-specific binding polypeptides, and C represents cysteine.

[0044] Transform the recombinant expression vector with correct sequencing into E.coli BL21(DE3), pick a single clone into 5mL LB / A medium (1g tryptone, 0.5g yeast extract, 1g sodium chloride to 100mL, autoclave ; then add the final concentration of 100 μg / mL ampicillin), 37 ° C, 180 rpm overnight culture; inoculate the culture ...

Embodiment 3

[0047] Analysis of binding properties of alpha-fetoprotein-specific binding polypeptides.

[0048] In this experiment, the six alpha-fetoprotein-specific binding polypeptides expressed and purified in Example 2 were used as further research objects, and their affinity characteristics with alpha-fetoprotein were detected by ELISA method, and CEA, EGF, BSA, and insulin were selected to detect 6 A polypeptide is used as an irrelevant antigen negative control, and the specific operation is as follows:

[0049] Dilute AFP / CEA / EGF / BSA / insulin with pH 9.6 carbonate buffer to 10 μg / mL, coat 96-well ELISA plate at 4°C overnight; block with 3% skimmed milk powder-PBS, 37°C for 2h; add band ZAFP-D2, ZAFP-A3, ZAFP-B7, ZAFP-G9, ZAFP-A2, and ZAFP-B8 six recombinant proteins with HA tags were incubated at 37°C for 2 h; diluted HA antibody (purchased from Sangon Bioengineering Co., Ltd. company, Cat. No. D191044), incubated at 37°C for 2 h; added diluted HRP-labeled rabbit anti-mouse seconda...

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Abstract

The invention relates to a specific binding polypeptide for alpha-fetoprotein (AFP) and application of the specific binding polypeptide, and belongs to the field of biotechnology. The polypeptide is obtained by mutating 1-20 amino acid residues in a staphylococcus protein A (SPA) immunoglobulin binding region (the polypeptide sequence is shown in SEQ ID: 2-14), and can be specifically bound with alpha fetoprotein. According to the invention, an alpha fetoprotein specific binding polypeptide coding sequence is obtained by three rounds of enrichment and elutriation from a fully synthetic affinity polypeptide molecular library; a prokaryotic expression system is used for preparing a large amount of obtained polypeptide, the polypeptide is specifically combined with alpha fetoprotein, has no obvious affinity with CEA, EGF, BSA, insulin and other proteins, and has the remarkable advantages of low preparation cost, good stability and the like compared with monoclonal antibodies. The polypeptide and the variant thereof are further used as capture elements in enzyme-linked immunosorbent assay and used for quantitative detection of alpha fetoprotein, the detection sensitivity is 8 ng and 2ng respectively, the detection linear ranges are 10-100 ng and 6-100 ng respectively, and the clinical detection requirements can be met.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a polypeptide specifically binding to alpha-fetoprotein (alpha-fetalprotein, AFP), or a variant thereof, or a functional fragment thereof, and the polypeptide, or a variant thereof, or a functional fragment thereof preparation methods and applications. Background technique [0002] Alpha-fetoprotein is a glycoprotein synthesized by rough endoplasmic reticulum ribose granules in stem cells, which consists of a single peptide chain containing 591 amino acid residues (Bergstrand CG, Czar B et al. Demonstration of a new protein fraction in serum from the human fetus. Scandinavian Journal of Clinical & Laboratory Investigation, 1956, 8(2): 174-174). During embryonic development, alpha-fetoprotein is produced by the fetal liver and yolk sac, and the concentration of embryonic alpha-fetoprotein reaches a peak at about 3 mg / mL at the 15th week, and then slowly decreases until it...

Claims

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Application Information

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IPC IPC(8): C07K14/31C07K19/00C07K14/47C07K1/22G01N33/574G01N33/577
CPCC07K14/31C07K14/4715G01N33/57476G01N33/577C07K2319/00G01N2333/471
Inventor 赵青刁爱坡刘娟娟崔东旭
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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