CN111925425A

CN111925425A - Alpha-fetoprotein specific binding polypeptide and application thereof

Info

Publication number: CN111925425A
Application number: CN202010798418.7A
Authority: CN
Inventors: 赵青; 刁爱坡; 刘娟娟; 崔东旭
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2020-11-13
Anticipated expiration: 2040-08-12
Also published as: CN111925425B

Abstract

The invention relates to an alpha-fetoprotein (AFP) specific binding polypeptide and application thereof, belonging to the technical field of biology. Wherein the polypeptide is obtained by mutating 1-20 amino acid residues in an immunoglobulin binding region of Staphylococcal Protein A (SPA) (the polypeptide sequence is shown in SEQ ID: 2-14), and can specifically bind to the alpha-fetoprotein. The invention obtains the alpha fetoprotein specificity binding polypeptide coding sequence from the total synthesis affinity body polypeptide molecule library through three rounds of enrichment panning, and utilizes a prokaryotic expression system to carry out mass preparation on the obtained polypeptide, the polypeptide is specifically bound with the alpha fetoprotein, has no obvious affinity with proteins such as CEA, EGF, BSA, insulin and the like, and has the obvious advantages of low preparation cost, good stability and the like compared with a monoclonal antibody. The invention further applies the polypeptide and the variant thereof as a capture element in enzyme-linked immunoassay for quantitative detection of alpha fetoprotein, the detection sensitivity is respectively 8ng and 2ng, the detection linear range is respectively 10 ng to 100ng and 6 ng to 100ng, and the clinical detection requirement can be met.

Description

Alpha-fetoprotein specific binding polypeptide and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a polypeptide specifically binding alpha-fetoprotein (AFP), or a variant or a functional fragment thereof, and a preparation method and application of the polypeptide, or the variant or the functional fragment thereof.

Background

Alpha-fetoprotein is a glycoprotein synthesized by rough endoplasmic reticulum ribosomes in stem cells and consists of a single peptide chain of 591 amino acid residues (Bergstrand CG, Czar B et al. Demonstromation of a new protein fraction in serum from the human sources. Scandinavian Journal of Clinical & Laboratory Investigation, 1956, 8 (2): 174). During embryonic development, fetoprotein is produced by the fetal liver and yolk sac, and the concentration of fetal alpha fetoprotein peaks at week 15 and is approximately 3mg/mL and then slowly decreases until it reaches 20 μ g/mL before birth, whereas the normal adult alpha fetoprotein concentration is less than 25 ng/mL. A great deal of research proves that the alpha-fetoprotein is an important serological marker of primary liver cancer (PHC) and is applied to clinic as a sensitive index after PHC diagnosis and treatment effect evaluation (Kamiyama T, Takahashi M et al. AFP mRNA detected in bone marrow by real-time quantitative RT-PCR analysis precursors and recurrent after clinical diagnosis and treatment for liver cancer of animals of Surgery, 2006, 244 (3): 451). In addition, alpha fetoprotein has important significance for monitoring the development and activity of testicular tumors, and is a sensitive index in the diagnosis and treatment effect evaluation of testicular non-seminoma. The alpha-fetoprotein also has higher guiding significance for early detection of gastric cancer, selection of operation modes and prognosis judgment (Song Yu Hua, Wang shan, Hu Anla: research progress of AFP positive gastric cancer. J. Zhong Hua Mao prevent and treat, 2006, 13 (7): 552-555).

At present, the domestic detection of alpha-fetoprotein mainly adopts the following method: colloidal gold immunochromatographic assay (GICA) (ZHao Y, Zhang G et al. development of a colloidal gold immunological gold immunochromatographic strip for the detection of the polypeptide detection of the floroxacin reactivity. journal of Agricuifinal & Food Chemistry, 2008, 56 (24): 12138. sup. 12142), enzyme immunoassay (ELSIA) (HuangCaiyun, Hansuzui, ZJinghua, Wangxinghui, Lishilong, ZhouXiujin: ELISA assay for the detection of an alpha-fetoprotein heteroplasma for the diagnosis of liver cancer. research and study of marker immunoassay and clinics, 2012, 19 (3): 147. sup. 149), Radioimmunoassay (RIA), chemiluminescence immunoassay (CLISA) (Huangyanping: electrochemiluminescence immunoassay (ECLIA): detect the clinical diagnostic value of alpha-fetoprotein (AFP) on primary liver cancer. 22, 1127), among others, the national chemiluminescence immunoassay (1127) is widely popularized in the market. The above immunoassay methods all require the use of specific antibodies, but due to the characteristics of antibody molecules, such as bivalent multi-domain proteins, disulfide bond-dependent bonds, complex glycosylation patterns, etc., the antibody molecules have poor thermal stability, complex preparation process and high cost, and therefore, the development of new alpha-fetoprotein affinity devices is needed to solve the above problems.

Affibodies (affibodies) are a novel class of affinity ligands, derived from the B-segment of the immunoglobulin binding domain of staphylococcal protein A (Nilsson B, Moks T et al. A synthetic IgG-binding domain based on a protein Engineering, 1987, 1 (2): 107-113), and are single-stranded structures consisting of 58 amino acid residues, which contain 3 alpha-helices. The receptor binding site of the affibody contains 13 amino acid residues, and these mutants formed by Karin Nord replacing the codons of these 13 amino acids with degenerate codons, respectively, constitute an affibody molecule library (Nord K, Nilsson J et al. A combinatorial library of an alpha-bacterial receptor domain. protein Engineering, 1995, 8 (6): 601-608), whereby different affibodies produced recombinantly can have specific binding activity and high affinity for a number of target protein molecules, including insulin, fibrin, Transferrin (TRF), tumor necrosis factor alpha (TNF-alpha), etc., respectively. The affibody polypeptide is similar to antibody in function, and has the advantages of small relative molecular weight, high stability, high affinity, high specificity, flexible application, easy preparation, etc. and may be used widely in biological science. Therefore, if an affibody polypeptide that specifically binds to alpha-fetoprotein is developed, the polypeptide can be used as a high-efficiency affinity ligand for alpha-fetoprotein affinity purification, alpha-fetoprotein quantitative or qualitative detection, and the like.

Disclosure of Invention

The invention is based on a phage display technology platform, utilizes a total synthesis affinity body polypeptide molecular library, selects a series of alpha fetoprotein specific binding polypeptides through three rounds of enrichment panning, and obtains a polypeptide sequence of SEQ ID No: 2-14. The prokaryotic colibacillus expression system is utilized to prepare alpha fetoprotein specific binding polypeptide, and ELISA binding experiment is utilized to verify the binding capacity and binding specificity of the elutriated affinity body polypeptide and the alpha fetoprotein. The results show that the 13 obtained affibody polypeptides can be specifically combined with alpha fetoprotein, and can be used for the affinity purification of the alpha fetoprotein and the quantitative or qualitative detection of the alpha fetoprotein.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the polypeptide sequence specifically bound by the alpha-fetoprotein is obtained by mutating 1-20 amino acid residues in a Staphylococcal Protein A (SPA) immunoglobulin binding region, and is shown as SEQ ID No: 2-14, has high affinity activity for alpha-fetoprotein.

The alpha fetoprotein specific binding polypeptide sequence comprises the polypeptide, or the variant or the functional fragment thereof, and any adjustment and modification of the polypeptide sequence by taking the polypeptide, or the variant or the functional fragment thereof as a core; the modified material includes, but is not limited to, specific proteins, enzymes, fluorescent proteins, fluorescent luminescent groups, biotin, radiation-related groups, and nanomaterials.

The invention also provides a preparation method of the alpha fetoprotein specific binding polypeptide, or the variant or the functional fragment thereof. One skilled in the art may substitute, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to the sequences of the invention to obtain variants of the sequences of the polypeptides or functional fragments thereof, without substantially affecting the affinity properties of alpha-fetoprotein. All of which are considered to be included within the scope of the present invention.

The alpha-fetoprotein specific binding polypeptide sequence is used for identification of alpha-fetoprotein, including but not limited to enzyme-linked immunosorbent assay (ELISA) detection.

The application of the polypeptide in the rapid detection of alpha-fetoprotein and the affinity purification of alpha-fetoprotein.

The application of the polypeptide in qualitative or quantitative detection of alpha-fetoprotein.

The invention has the advantages and positive effects that:

1. the invention is based on phage display technology, elutriates alpha fetoprotein specific binding polypeptide from a total synthesis affinity body polypeptide molecular library through three rounds of enrichment, has good specific affinity of the polypeptide, and has the remarkable advantages of low preparation cost, good stability and the like compared with a monoclonal antibody.

2. The alpha fetoprotein specific binding polypeptide and the polymer thereof obtained by the invention can be used as a capture element in enzyme-linked immunosorbent assay, are used for quantitative detection of alpha fetoprotein, have good detection limit and can meet the clinical detection requirement.

Drawings

FIG. 1 is a graph showing the fold enrichment for each round of phage display screening for polypeptides that specifically bind alpha-fetoprotein.

FIG. 2 shows the ELISA results of positive phage clones screened, and BSA is the negative control.

FIG. 3 is an alignment of the amino acid sequences of selected polypeptides that specifically bind to alpha-fetoprotein, with the red portion being 13 mutation sites.

FIG. 4 is a schematic diagram of 6 polypeptide fusion expression vectors that specifically bind to alpha-fetoprotein.

FIG. 5 shows the results of the optimization of the purification of polypeptides that specifically bind to alpha-fetoprotein.

FIG. 6 shows the results of purification of 6 polypeptides that specifically bind to alpha-fetoprotein.

FIG. 7 shows the results of the in vitro affinity and specificity of 6 polypeptides specifically binding to alpha-fetoprotein.

FIG. 8 is a schematic diagram of an alpha-fetoprotein specific binding polypeptide dimer, trimer, tetramer fusion protein expression vector.

FIG. 9 shows the results of purification of alpha-fetoprotein specific binding polypeptides, dimer fusion proteins.

FIG. 10 shows the affinity and specificity of the alpha-fetoprotein specific binding polypeptide, the dimer fusion protein, and the alpha-fetoprotein.

FIG. 11 shows the thermal stability of alpha-fetoprotein specific binding polypeptides, dimer fusion proteins.

FIG. 12 shows capture of alpha-fetoprotein in ELISA experiments by monomers that bind specifically to polypeptides.

FIG. 13 shows the capture of alpha-fetoprotein in an ELISA assay by the dimer of an alpha-fetoprotein specific binding polypeptide.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention in any way. The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials, reagent materials and the like used are all commercially available products unless otherwise specified.

Example 1

And (3) elutriating and identifying the alpha fetoprotein specific binding polypeptide.

1. Mutant construction and monoclonal selection

Degenerate primers with NNK as codon were designed for amino acid residues at

positions

9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32 and 35 of immunoglobulin binding region polypeptides of staphylococcal protein A, and the genes encoding the library of affinity polypeptides were obtained by over-lap PCR using the degenerate primers. Cloning the polypeptide mutant encoding gene to a phage display vector, and electrically transforming the recombinant plasmid to TG1 host cells to obtain a library with the volume of 5 × 10⁷A library of the affibody polypeptide mutants of (1). And displaying the polypeptide mutant of the affinity body by using the helper phage so as to obtain a polypeptide mutant phage display library.

The alpha-fetoprotein (purchased from fitzgerald, with the product number of 30-1370) extracted from umbilical cord blood is taken as a target, an affibody phage display library is added, and after three rounds of enrichment panning, the third round of captured phage is 10 times enriched compared with the first round, and the enrichment times of the enriched phage in each round are shown in figure 1.

2. Phage ELISA for detecting binding capacity of polypeptide and alpha fetoprotein

After three rounds of panning, 70 monoclonals are selected from the second round of production plates and the third round of production plates, each monoclone is inoculated into 1mL of 2 XYTAG culture medium (1.6g of tryptone, 1g of yeast extract and 0.5g of sodium chloride with constant volume of 100mL, autoclaved, added with 2% glucose with final concentration and 100 mu g/mL of ampicillin) and cultured at 37 ℃ and 180rpm overnight; inoculating the culture into 5mL 2 XYTAG medium at 1: 50, culturing at 37 deg.C and 180rpm to logarithmic phase; adding helper phage M13KO7 (purchased from New England Biolabs, Cat.) at 1: 20, standing at 37 deg.C for 30min for infection, and shake-culturing at 37 deg.C and 180rpm for 30 min; taking 1mL of bacteriophage culture, 7000rpm, centrifuging for 10min, and discarding the supernatant; the cells were resuspended in 5mL of 2 XYTAK medium (1.6g of tryptone, 1g of yeast extract, 0.5g of sodium chloride to 100mL, autoclaved, and then ampicillin and kanamycin to a final concentration of 100. mu.g/mL) and incubated overnight at 30 ℃ and 185 rpm. Centrifuging the culture at 10000rpm and 4 ℃ for 10min, and collecting supernatant to obtain the phage monoclonal displaying the polypeptide mutant.

The alpha-fetoprotein and BSA (negative control) were diluted to 5. mu.g/mL with a carbonate buffer at pH 9.6, coated on a 96-well microplate (purchased from Nunc, cat #) and incubated overnight at 4 ℃; blocking with 3% skimmed milk powder-PBS, and standing at 37 deg.C for 2 hr; adding the collected phage monoclonal supernatant, and incubating for 2h at room temperature; adding HRP-labeled anti-M13 antibody (purchased from Cassia Proteus, cat No. 11973-MM05T-H) diluted 1: 2000, and incubating at room temperature for 2H; adding TMB substrate for developing for 10 min; 2M H was added₂SO₄The reaction was stopped and OD450 read in a microplate reader. In the ELISA procedure, plates were washed 4 times with PBST (PBS + 0.5% Tween-20) at each step. For ELISA results, the clones with positive/negative values > 2.1 were selected as positive clones, and a total of 25 positive clones were selected, and the specific data results of each positive clone are shown in FIG. 2.

3. DNA sequencing

DNA sequencing is carried out on the positive phage clones, 12 clones in 25 clones have the same sequencing result and are named as ZAFP-D2, the sequencing results of other clones are different and are respectively named as ZAFP-C8, ZAFP-G5, ZAFP-H4, ZAFP-H24, ZAFP-A3, ZAFP-B8, ZAFP-H7, ZAFP-C9, ZAFP-G9, ZAFP-A9, ZAFP-A2 and ZAFP-B7, amino acid sequences can be obtained according to the DNA sequencing result and a codon table, and the amino acid sequences of 13 polypeptides specifically binding to the alpha-fetoprotein are shown in figure 3.

Example 2

And (3) expressing and purifying the alpha fetoprotein specific binding polypeptide.

From the 13 polypeptide mutants specifically binding to alpha-fetoprotein identified in example 1, the following six were selected as subjects for further study: ZAFP-D2, ZAFP-A3, ZAFP-B7, ZAFP-G9, ZAFP-A2, ZAFP-B8.

Coli BL21(DE3) (laboratory preservation), pET-21b (laboratory preservation) as expression vector, and FIG. 4 as schematic drawing of recombinant vector, wherein ZAFP represents the specific binding polypeptide of the above 6 different alpha-fetoproteins, and C represents cysteine.

E.coli BL21(DE3) was transformed with the correctly sequenced recombinant expression vector, and the monoclonal was picked up into 5mL LB/A medium (1g tryptone, 0.5g yeast extract, 1g sodium chloride to 100mL, autoclaved, then added with final concentration 100. mu.g/mL ampicillin) and cultured overnight at 37 ℃ at 180 rpm; the culture was inoculated into 200mL of LB/A medium at 1: 50, cultured at 37 ℃ and 180rpm until logarithmic phase, and induced overnight at 20 ℃ and 180rpm with the addition of 0.1mM IPTG at the final concentration. Centrifuging the culture at 4 ℃ and 4400rpm to collect thalli; with 20mL of precooled PBS (8g NaCl, Na)₂HPO₄·12H₂O，0.2g KCl，0.24gNaH₂PO₄·2H₂O), resuspending the thalli, carrying out ultrasonication (for over 3s and stopping for 10s) for 20min, then centrifuging at 12000rpm for 15min, and collecting the supernatant.

Purification of recombinant polypeptide a Ni-TED column (from Biotechnology engineering Co., Ltd., product No. C600803) was selected. First, the Ni-TED column was equilibrated with 10 column volumes of phosphate buffer; filtering the collected protein broken supernatant through a 0.45-micron filter membrane, then loading the sample, performing flow-through for 4 times, and reserving flow-through liquid for SDS-PAGE analysis; the column was washed with 20mM imidazole in phosphate buffer (20 column volumes) to remove contaminating proteins; phosphate buffers containing 50mM, 100mM, 250mM, and 400mM imidazole were prepared, and the target protein was eluted with 2 column volumes, respectively. SDS-PAGE analysis is carried out on protein samples collected by imidazole with different concentrations, and the result is shown in figure 5; according to the SDS-PAGE result, the high purity protein is selected, and dialyzed by phosphate buffer solution, and the dialyzed protein is analyzed by SDS-PAGE, and the results are shown in figure 6, and the six kinds of affinity body polypeptides are successfully purified.

Example 3

Analysis of binding characteristics of alpha-fetoprotein specific binding polypeptides.

In this experiment, six polypeptides specifically binding to alpha-fetoprotein expressed and purified in example 2 were used as further subjects, and their affinity to alpha-fetoprotein was tested by ELISA method, while 6 polypeptides for detecting CEA, EGF, BSA and insulin were selected as negative controls for unrelated antigens, and the following operations were performed:

diluting AFP/CEA/EGF/BSA/insulin to 10 μ g/mL with carbonate buffer of pH 9.6, coating 96-well enzyme label plate, and standing overnight at 4 deg.C; blocking with 3% skimmed milk powder-PBS, and standing at 37 deg.C for 2 hr; adding six recombinant proteins of ZAFP-D2, ZAFP-A3, ZAFP-B7, ZAFP-G9, ZAFP-A2 and ZAFP-B8 with HA tags, and incubating for 2h at 37 ℃; adding diluted HA antibody (purchased from Biotechnology engineering Co., Ltd., product No. D191044), and incubating at 37 deg.C for 2 h; adding diluted HRP-labeled rabbit anti-mouse secondary antibody (purchased from Biotechnology engineering Co., Ltd., product number D110098), and incubating at 37 deg.C for 45 min; adding TMB substrate for developing for 10 min; the reaction was stopped by the addition of 2M H2SO4 and OD450 was read in a microplate reader. In the ELISA procedure, plates were washed 4 times with PBST (PBS + 0.5% Tween-20) at each step. The results are shown in FIG. 7. It can be seen that ZAFP-D2 has the best specific binding capacity.

Example 4

The preparation and affinity characteristic analysis of the alpha-fetoprotein specific binding polypeptide polymer fusion protein.

In this experiment, the polypeptide ZAFP-D2 of example 3, which has high affinity for alpha-fetoprotein and good specificity, was used asSubject of further study, preparation of dimer of ZAFP-D2 (ZAFP-D2)₂Trimer (ZAFP-D2)₃Tetramer (ZAFP-D2)4 was used as a polypeptide for capturing alpha-fetoprotein in ELISA experiments.

Coli BL21 (stored in laboratory), pET-21B (stored in laboratory), dimeric recombinant vector shown in figure gA, trimeric recombinant vector shown in figure 8B, tetrameric recombinant vector shown in figure 8C, wherein C is cysteine, G is G₄SG₄S represents linker GGGGSGGGGS. Protein expression purification procedures reference example 2, in which ZAFP-D2 and its dimer (ZAFP-D2)₂See FIG. 9, ZAFP-D2 and (ZAFP-D2)₂The purification effect is good, the yield is high, and 18.9mg of purified ZAFP-D2 protein and purified dimer (ZAFP-D2) can be respectively obtained from 1L fermentation liquor₂Protein 43.9 mg.

ZAFP-D2 and its dimer (ZAFP-D2)₂Analysis of affinity and specificity with AFP, methods see example 3, FIG. 10, ZAFP-D2 and its dimer (ZAFP-D2)₂The OD450 values measured after AFP incorporation were much higher than in the negative control group.

Example 5

And (3) performing stability experiment of the alpha-fetoprotein specifically binding polypeptide.

In this experiment, the polypeptides ZAFP-D2 and (ZAFP-D2) of example 4 were used₂For further study of the subjects, verification of ZAFP-D2, (ZAFP-D2)₂And the affinity of the alpha-fetoprotein monoclonal antibody to alpha-fetoprotein after heat treatment at different temperatures. The specific method comprises the following steps:

diluting AFP to 10 μ g/mL with carbonate buffer with pH 9.6, coating 96-well enzyme-linked immunosorbent assay plate, and standing overnight at 4 ℃; blocking with 3% skimmed milk powder-PBS, and standing at 37 deg.C for 2 hr; adding ZAFP-D2, (ZAFP-D2)2 and alpha fetoprotein monoclonal antibody treated at 37 deg.C, 50 deg.C, 65 deg.C and 80 deg.C for 10 min; adding diluted HA antibody (purchased from Biotechnology engineering Co., Ltd., product No. D191044), and incubating at 37 deg.C for 2 h; adding diluted HRP-labeled rabbit anti-mouse secondary antibody (purchased from Biotechnology engineering Co., Ltd., product number D110098), and incubating at 37 deg.C for 45 min; adding TMB substrate for developing for 10 min; 2M H was added₂SO₄The reaction was stopped and OD450 read in a microplate reader. In the ELISA procedure, plates were washed 4 times with PBST (PBS + 0.5% Tween-20) at each step. The results are shown in FIG. 11. As can be seen from FIG. 11, the heat stability of the alpha-fetoprotein specific binding polypeptide is significantly better than that of the common monoclonal antibody.

Example 6

The alpha-fetoprotein is captured in ELISA experiment by using the alpha-fetoprotein specific binding polypeptide monomer or polymer.

In this experiment, the polypeptides ZAFP-D2 and (ZAFP-D2) of example 4 were used₂For further study subjects were used to capture alpha-fetoprotein in ELISA experiments. The specific method comprises the following steps:

diluting the polypeptide monomer and polymer to 10 mug/mL by carbonate buffer with pH 9.6, coating 96-hole enzyme label plate, and staying overnight at 4 ℃; blocking with 3% skimmed milk powder-PBS, and standing at 37 deg.C for 2 hr; adding 2 times of gradient diluted alpha-fetoprotein solution with initial concentration of 625ng/mL, incubating at 37 ℃ for 30min, and washing the plate for 6 times by PBST; adding the alpha-fetoprotein polyclonal antibody, and incubating for 2h at 37 ℃; adding a goat anti-rabbit secondary antibody marked by HRP, and incubating for 45min at 37 ℃; adding TMB substrate for developing for 10 min; 2M H was added₂SO₄The reaction was stopped and OD450 read in a microplate reader. In the ELISA procedure, each step was performed 4 times with PBST (PBS + 0.5% Tween-20) unless otherwise specified. The results are shown in FIGS. 12 and 13, respectively, and the peptide A protein specifically binds to the polypeptide ZAFP-D2, the sensitivity is 8ng/mL, and the linear range is 10-100 ng. Alpha fetoprotein specific binding polypeptide dimer (ZAFP-D2)₂The sensitivity is 2ng/mL, and the linear range is 6-100 ng.

Claims

1. A polypeptide specifically binding alpha-fetoprotein (AFP), or a variant or functional fragment thereof, wherein the polypeptide specifically binding AFP is obtained by mutating 1-20 amino acid residues from a Staphylococcal Protein A (SPA) immunoglobulin binding region, and has 1 or more than 1 alpha-fetoprotein binding domain.

2. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide has 1-25 amino acid mutation sites compared with the amino acid sequence of the SPA immunoglobulin binding region (shown as SEQ ID NO: 1).

3. The polypeptide of claim 2, wherein the amino acid sequence of the polypeptide has 1 and more than 1 amino acid mutation at positions 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32 and 35.

4. The polypeptide of claims 1 to 3, which has an amino acid sequence as set forth in SEQ ID NO: 2-13 or a mutant thereof.

5. A fusion protein of a polypeptide according to any one of claims 1 to 4 with another polypeptide, which fusion protein specifically binds to alpha-fetoprotein, comprising:

a. a polypeptide according to claims 1 to 4, or a variant thereof, or a functional fragment thereof;

b. other amino acid residues or polypeptide chains at the amino-or carboxy-terminus of the alpha-fetoprotein specific binding polypeptide, or variant thereof, or functional fragment thereof;

preferably, such other amino acid residues or polypeptide chains include, but are not limited to, one or more cysteines, protein tags (e.g., histidine tags, myc tags, Flag tags, HA tags, etc.), and other functional proteins.

6. The fusion protein of claim 5, wherein the other polypeptide chains at the amino-or carboxy-terminus of the alpha-fetoprotein specific binding polypeptide, or variant thereof, or functional fragment thereof, consists of 1 or more polypeptides according to claims 1 to 4, or variant thereof, or functional fragment thereof, that specifically bind to alpha-fetoprotein.

7. The fusion protein of claim 6, wherein the other polypeptide chain at the amino-terminus or the carboxy-terminus of the alpha-fetoprotein specific binding polypeptide, or variant thereof, or functional fragment thereof, is an enzyme protein having catalytic function or a protein having a function associated with fluorescence emission.

8. A label for covalent or non-covalent attachment of the polypeptide or fusion protein of claims 1 to 7 to another group;

preferably, the label includes, but is not limited to, a fluorescent luminescent group, biotin, a radiation-related group, a nanomaterial, and the like.

9. A method for affinity purification of alpha-fetoprotein using a polypeptide of claims 1 to 8, or a variant or functional fragment thereof, or a fusion protein.

10. Performing alpha-fetoprotein detection using a polypeptide of claims 1 to 8, or a variant or functional fragment thereof, or a fusion protein, or a phage displaying a polypeptide of claims 1 to 7, or a variant or functional fragment thereof, or a fusion protein;

preferably, the alpha-fetoprotein test sample comprises a blood sample, a tissue sample, or the like.