CN113307857B

CN113307857B - Scaffold proteins derived from epidermal growth factor, lectin and Tat proteins

Info

Publication number: CN113307857B
Application number: CN202110049785.1A
Authority: CN
Inventors: 艾时斌; 杨帆; 刘鑫; 韩晓芳; 陈晶
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-01-14
Filing date: 2021-01-14
Publication date: 2022-11-01
Anticipated expiration: 2041-01-14
Also published as: CN113307857A

Abstract

The present invention relates to recombinant scaffold proteins derived from epidermal growth factor, lectin and Tat proteins. The recombinant scaffold protein designed by the invention has an amino acid recombinant sequence derived from a receptor binding domain of human epidermal growth factor, a collagen-like domain of human fibrin, a glycosyl recognition domain of human mannose-binding lectin and Tat protein of HIV-1 virus. The scaffold protein has the capability of displaying functional proteins, is particularly suitable for constructing libraries in vitro display or related systems, has the characteristics of high affinity and high specificity with targets of glycoprotein binding activity, transmembrane and nuclear translocation activity, and can be widely applied and researched in various fields of biology, medicine, environment, agriculture and food. The invention also relates to various uses of the scaffold, including in therapy, diagnostics, environmental and safety monitoring, synthetic biology and research, and to cells and cell cultures expressing the scaffold proteins.

Description

Scaffold proteins derived from epidermal growth factor, lectin and Tat proteins

Technical Field

The present invention belongs to the field of protein engineering. More specifically, the invention relates to recombinant scaffold proteins derived from epidermal growth factor, lectin and Tat proteins.

Background

For decades, many affinity functional proteins capable of specifically binding to target molecules have been obtained through biotechnology, and are widely used in many fields such as biology, medicine, environment, agriculture, food, and the like. Among these affinity functional proteins, antibodies are typical. Antibodies have long been used extensively in clinical disease therapy, diagnosis and basic research, and have become effective tools for human beings to overcome diseases. However, antibodies as macromolecular proteins have natural limitations, such as extra large molecular weight, low tissue permeability, poor stability, high production cost, large dosage, potential immunogenicity, etc. In order to overcome the problems, people can design or select certain proteins as scaffold proteins (or called mother nuclear proteins), on the basis of ensuring that the main structure of the scaffold proteins is not changed, randomized mutation is carried out on 8-30 amino acid sites in the molecules of the scaffold proteins, so as to construct a protein combinatorial library for screening, and then display technologies such as phage, yeast, cells, ribosome, mRNA, cDNA or SNAP and the like are carried out, so that the affinity functional proteins capable of being specifically combined with target molecules can be obtained.

The existing scaffold proteins, such as the scaffold protein Sac7d derived from Sulfolobus acidocaldarius with the patent application number of 200780050902.4, the scaffold protein derived from the Z domain of staphylococcus aureus protein a with the patent application number of 201180021908.5, the scaffold protein derived from fibronectin type III domain "FN3" with the patent application number of 201180029216.5, the scaffold protein derived from a plant cysteine protease inhibitor with the patent application number of 80022070.5, the scaffold protein derived from a bile triene binding protein and the scaffold protein derived from an ankyrin repeat sequence, and the like, have shown great potential commercial values and gradually become new basic research hotspots and technical innovation points.

Compared with the antibody, the affinity functional protein obtained based on the scaffold protein has the following advantages: (1) the molecular weight is small, the tissue permeability is strong, and the focus part can be reached more quickly; (2) the molecular stability is good; (3) binders with higher affinity for target binding can be obtained; (4) superior to antibodies obtained by immunization in discriminating small differences between related small molecules; (5) the range of action targets is wide; (6) low immunogenicity; (7) the production can be carried out by adopting an engineering means, and the cost is low; (8) can realize the design of double-target or multi-target specific functional protein.

Glycoproteins are proteins containing oligosaccharide chains, which are covalently linked to certain amino acid residues in the peptide chain. Glycoproteins are ubiquitous in animals, plants and microorganisms, and are of a wide variety and wide function. The glycoproteins are mainly provided with the following types: (1) soluble glycoproteins including proteins such as enzymes, hormones, antibodies, complements, growth factors, interferons, lectins and toxins present in mucus secreted by intracellular fluids, body fluids and glands; (2) membrane-bound proteins include enzymes, receptors, lectins, and transporters. Such proteins are often involved in cell recognition and may serve as surface markers or surface antigens for particular cells or cells at particular stages; (3) structural glycoproteins, including, for example, collagen and various non-collagen glycoproteins (fibronectin, laminin, etc.), are insoluble macromolecular glycoproteins in the extracellular matrix. Their functions are not only supporting, linking and buffering as structural components of the extracellular matrix, but also, more importantly, are involved in the recognition, adhesion and migration of cells and regulate their proliferation and differentiation.

Epidermal Growth Factor (EGF) is a polypeptide consisting of 53 amino acids, wherein 20-31 amino acid residues (CMYAALDKYAC) in EGF molecule are Receptor Binding Domains (RBD). Research shows that the binding of the RBD of EGF and an Epidermal Growth Factor Receptor (EGFR) has high specificity, high selectivity and high affinity. As the most important ligand, EGF is involved not only in the proliferation and differentiation of normal cells but also in the formation and progression of tumors. Since EGFR is closely related to malignant transformation, adhesion, metastasis, angiogenesis and the like of tumor cells such as lung cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, bladder cancer, brain glioma, head and neck tumors and the like, EGFR has been intensively studied and clinically applied as a target for a variety of common clinical malignant tumors, and is of great interest.

Ficolin is a C-type lectin protein and is a key molecule in the body's natural immunity. Research proves that ficolin contains collagen-like domain (CLR) and fibrin-like domain (FGD), FGD participates in recognition of carbohydrate ligands on the surface of pathogenic microorganisms by interacting with lipopolysaccharide and mannose-like structures on the surface of pathogenic microorganisms, and CLR has the functions of activating complement agglutinin pathway and opsonophagocytosis and plays an important role in natural immunity.

Mannose-binding lectin (MBL) is a Ca²⁺The structure of the dependent C-type lectin protein is divided into the following parts from the N end to the C end in sequence: signal peptide region, cysteine-rich region, collagen region, coiled-coil region (also called neck region), and carbohydrate-recognition domain (CRD). Wherein CRD is recognition functional region of MBL, and can selectively recognize and combine glycoprotein with mannose, N-acetylmannosamine, N-acetylglucosamine and fucose as glycosyl, and the glycoprotein is distributed on the surface of various pathogenic microorganisms such as bacteria, virus, fungi, parasites, etc. After the microorganisms enter the human body, the CRD of MBL recognizes and binds to surface glycoproteins of pathogenic microorganisms, and then, complement lectin pathway is activated by MBL-associated serine proteases (MASPs), opsonophagocytosis is regulated, and pathogens and infected cells are eliminated. Thus, MBL plays an important role in the immune defense and immune surveillance process of the body. In addition, MBL is involved in the growth, invasion, and metastasis of malignant tumors such as gastric cancer, liver cancer, ovarian cancer, lung cancer, breast cancer, and colon cancer by binding to glycoproteins in tumor cells, or by MASPs and complements. Because MBL plays an important role in inflammatory response, and regulates and restricts a plurality of inflammatory factors (IL-1 beta, IL-12, IL-18, TNF-alpha and the like) to cause the reduction of the sensitivity of tissues to insulin response and the disturbance of glycolipid metabolism, the MBL is closely related to the occurrence and development of diabetes.

The Tat protein is one of important regulatory proteins coded by human immunodeficiency virus type 1 (HIV-1) and has a transcription activation function. The Tat protein consists of 86-101 amino acids (Albini A., et al. HIV-1Tat protein chemistry of chemikiness. Proc. Natl. Acad. Sci. USA 1998 95.

Disclosure of Invention

It is an object of the present invention to provide a non-naturally occurring recombinant scaffold protein derived from epidermal growth factor, lectin and Tat protein. Further, the recombinant scaffold protein is derived from the amino acid sequence of the human epidermal growth factor receptor binding domain, the amino acid sequence of the collagen-like domain of human fibronectin, the amino acid sequence of the glycosyl recognition domain of human mannose-binding lectin and the amino acid sequence of the Tat protein of human immunodeficiency virus type I. The scaffold protein has glycoprotein binding activity, transmembrane and nuclear translocation activity and the ability to display affinity functional proteins. In order to realize the purpose of obtaining the affinity functional protein based on the scaffold protein provided by the invention, on the basis of ensuring that the main structure of the scaffold protein designed by the invention is not changed, 8-30 amino acid sites in the molecule are subjected to random mutation to construct a protein combinatorial library for affinity screening of target molecules, and then the affinity functional protein capable of being specifically combined with the target molecules can be obtained by display technologies such as bacteriophage, yeast, cells, ribosome, mRNA, cDNA or SNAP.

Preferably, the target molecule is one of a peptide, a protein, an oligosaccharide, a single-stranded DNA, a double-stranded DNA, an RNA, a metal ion, or a carbohydrate.

Preferably, the screening process employs phage, yeast, cell, ribosome, mRNA, cDNA, or SNAP display technologies.

A scaffold protein having an amino acid recombination sequence derived from a receptor binding domain of human epidermal growth factor, a collagen-like domain of human fibronectin, a glycosyl recognition domain of human mannose-binding lectin, and a Tat protein of HIV-1 virus.

Preferably, the scaffold protein further comprises at least one heterologous peptide sequence inserted into any region of the scaffold protein.

Preferably, the scaffold protein is a protein having an amino acid sequence shown in SEQ ID NO 1, or a variant having a truncated amino acid residue and a partial amino acid residue substituted in the sequence shown in SEQ ID NO 1, or a variant having a deleted amino acid residue and a partial amino acid residue substituted in the sequence shown in SEQ ID NO 1; or the scaffold protein is a protein of an amino acid sequence shown by SEQ ID NO 2, or a variant of the sequence of SEQ ID NO 2 in which amino acid residues are truncated and a part of the amino acid residues are substituted, or a variant of the sequence of SEQ ID NO 2 in which amino acid residues are deleted and a part of the amino acid residues are substituted; or the scaffold protein is a protein of an amino acid sequence shown by SEQ ID NO 3, or a variant of the sequence of SEQ ID NO 3 in which amino acid residues are truncated and a part of the amino acid residues are substituted, or a variant of the sequence of SEQ ID NO 3 in which amino acid residues are deleted and a part of the amino acid residues are substituted; or the scaffold protein is a protein of an amino acid sequence shown by SEQ ID NO 4, or a variant of the sequence of SEQ ID NO 4 in which amino acid residues are truncated and a part of the amino acid residues are substituted, or a variant of the sequence of SEQ ID NO 4 in which amino acid residues are deleted and a part of the amino acid residues are substituted; or the scaffold protein is a protein of an amino acid sequence shown by SEQ ID NO 5, or a variant of the sequence of SEQ ID NO 5 in which amino acid residues are truncated and a part of the amino acid residues are substituted, or a variant of the sequence of SEQ ID NO 5 in which amino acid residues are deleted and a part of the amino acid residues are substituted; or the scaffold protein is a protein of an amino acid sequence shown by SEQ ID NO 6, or a variant in which an amino acid residue in the sequence of SEQ ID NO 6 is truncated and a part of the amino acid residues is substituted, or a variant in which an amino acid residue in the sequence of SEQ ID NO 6 is deleted and a part of the amino acid residues is substituted.

Preferably, the sequence of said variant has at least 20% identity with the corresponding sequence of SEQ ID NO 1 to SEQ ID NO 6, respectively.

Preferably, the sequence of said variant has at least 50% identity with the corresponding sequence of SEQ ID NO 1 to SEQ ID NO 6, respectively.

Use of a scaffold protein in a medicament for targeted therapy.

Preferably, the scaffold protein of the invention is used as a base, and the following are obtained by a display technology: (1) the application of the affinity functional protein taking glycoprotein on the surface of the virus as a target in anti-coronavirus medicines; (2) the application of the affinity functional protein taking the N-methyl-D-aspartate receptor as a target spot in the medicine for treating diabetes; (3) the application of affinity functional protein taking vascular endothelial growth factor as a target spot in the medicine for treating corneal neovascularization; (4) the application of the affinity functional protein taking tumor necrosis factor-alpha as a target spot in the medicine for treating autoimmune diseases; (5) the application of affinity functional protein with epidermal growth factor receptor-2 as target in preparing medicine for treating malignant tumor; (6) the application of the affinity functional protein with the protein convertase subtilisin 9 as a target point in the preparation of the hypolipidemic drugs.

The invention has the advantages that: compared with the existing scaffold protein derived from single protein, the invention takes the recombinant protein of four polypeptides or proteins (RBD of human EGF, CLR of human Ficolin, CRD of human MBL and Tat protein of HIV-1 virus) as the scaffold protein, derives the nascent affinity function protein which does not exist in the natural world by performing directional and randomized double modification on amino acid residues in the recombinant scaffold protein molecule, can retain the favorable biophysical properties (namely glycoprotein binding activity, transmembrane activity and nuclear translocation activity) of a parent protein (scaffold protein), obtains the nascent function protein with high affinity and high specificity to a specific target by screening, and is used for targeted therapy of diseases.

Drawings

FIG. 1 shows the plasmid map of the coronavirus S protein affinity functional protein Spfitin expression vector pxSpfinin;

FIG. 2 is a SDS-PAGE detection chart of coronavirus S protein affinity functional protein Spfitin; m: protein molecular weight Marker;1: expression products before Ni-NTA purification; 2: protein purified by Ni-NTA;

FIG. 3 is a graph showing the interaction between the S protein of coronavirus and the affinity binding protein Spfitin detected by SPR;

FIG. 4 shows the histopathological diagrams of the protection effect of the Spfitin on the acute lung injury induced by the animal infection virus, and 4A is a normal group; 4B saline control group; 4C is a Spfitin experimental group;

FIG. 5 is a plasmid map of an expression vector pxNmfitin of N-methyl-D-aspartate receptor affinity functional protein Nmfitin;

FIG. 6 is a SDS-PAGE of the N-methyl-D-aspartate receptor affinity binding protein Nmfitin; m: protein molecular weight Marker;1: expression products before Ni-NTA purification; 2: protein purified by Ni-NTA;

FIG. 7 is a flow cytometric assay of the binding activity of Nmfitin to cellular N-methyl-D-aspartate receptor;

FIG. 8, bar graph of the effect of Nmfitin on insulin secretion from islet beta cells;

FIG. 9 is a bar graph of the effect of Nmfitin on blood glucose and blood lipid levels in diabetic model animals; 9A is a histogram of the influence of Nmfitin on blood lipid of a diabetes model animal, and 9B is a histogram of the influence of Nmfitin on blood glucose of the diabetes model animal;

FIG. 10 is the plasmid map of vascular endothelial growth factor affinity binding protein Vefitin expression vector pxVefinin;

FIG. 11 is a SDS-PAGE picture of vascular endothelial growth factor affinity binding protein Vefitin; m: protein molecular weight Marker;1: expression products before Ni-NTA purification; 2: protein purified by Ni-NTA;

FIG. 12 is a graph showing the interaction between vascular endothelial growth factor and affinity binding protein Vefitin detected by SPR;

FIG. 13 is a graph showing that the MTT method detects that Vefitin inhibits vascular endothelial growth factor-induced vascular endothelial cell proliferation;

FIG. 14, graphs of the expression of relevant factors in corneal tissue after Vefitin and dexamethasone treatment; 14A is a expression diagram for detecting VEGF, VEGFR-1 and VEGFR-2 by Western blot; 14B is a histogram of VEGF, VEGFR-1 and VEGFR-2 expression;

FIG. 15, plasmid map of tumor necrosis factor-alpha affinity binding protein Tnfitin expression vector pxTnfitin;

FIG. 16, SDS-PAGE detection of TNF- α affinity binding protein Tnfitin expression. M: protein molecular weight Marker;1: expression products before Ni-NTA purification; 2: protein purified by Ni-NTA;

FIG. 17, SPR detection of the interaction of TNF- α with the affinity binding protein Tnfitin;

FIG. 18 is a graph showing the effect of affinity binding protein Tnfitin on TNF- α induced cytotoxicity;

FIG. 19 is a graph showing the therapeutic effect of Tnfitin on a mouse rheumatoid arthritis model;

FIG. 20 is a plasmid map of an affinity binding protein Hefitin expression vector pxHefitin of epidermal growth factor receptor-2;

FIG. 21 is an SDS-PAGE graph showing the expression of the epidermal growth factor receptor-2 affinity binding protein Hefitin; m: protein molecular weight Marker;1: expression products before Ni-NTA purification; 2: protein purified by Ni-NTA;

FIG. 22, SPR detection of the interaction diagram of epidermal growth factor receptor-2 and affinity binding protein Hefitin;

FIG. 23 is a graph showing the inhibitory effect of Hefitin on tumor cell proliferation;

FIG. 24 is a graph showing the antitumor effect of Hefitin on a tumor-bearing animal model;

FIG. 25 is a graph of the survival analysis of Hefitin on a tumor-bearing animal model;

FIG. 26 is a plasmid map of proprotein convertase subtilisin 9 affinity binding protein Pcfintin expression vector pxPcfintin;

FIG. 27, SDS-PAGE detecting expression of proprotein convertase subtilisin 9 affinity binding protein Pcfitin; m: protein molecular weight Marker;1: expression products before Ni-NTA purification; 2: protein purified by Ni-NTA;

FIG. 28 is a diagram showing the interaction between subtilisin 9 and Pcfitin, an affinity binding protein, before SPR detection;

FIG. 29, effect of Pcfitin on cellular uptake of low density lipoprotein; 29A is a fluorescence image of cells of DMEM culture solution group stained by DiI-LDL; 29B is a fluorescence image of cell nuclei of a DMEM culture solution group stained by Hoechst 33342; 29C is a double-staining fusion picture of DiI-LDL and Hoechst33342 of DMEM culture solution group cells; 29D is a DII-LDL stained fluorescence diagram of anti-PCSK9 control cells; 29E is a fluorescence image of cell nuclei of an anti-PCSK9 control group stained by Hoechst 33342; 29F is a double-staining fusion map of DiI-LDL and Hoechst33342 of anti-PCSK9 control cells; 29G is a fluorescent image of cells of the Pcfitin experimental group stained by DiI-LDL; 29H is a fluorescence image of cell nuclei of a Pcfitin experimental group stained by Hoechst 33342; 29I is a double-staining fusion picture of DiI-LDL and Hoechst33342 of cells in a Pcfitin experimental group;

FIG. 30 shows the effect of Pcfitin on expression of low density lipoprotein receptor in mouse liver tissue; 30A is a diagram of detecting the expression level of hepatic LDL receptors by Western blot, and 30B is a bar chart of the expression level of animal hepatic cell LDL-R.

Detailed Description

The invention is further described with reference to specific examples. It should be noted that, with the development and improvement of display technology, the following examples of screening affinity functional proteins by cDNA display technology are only used to illustrate the present invention, and those skilled in the art can also use phage, yeast, cell, ribosome, mRNA or SNAP display technology to screen affinity functional proteins with high affinity and specificity to other specific targets than the present embodiment based on the scaffold proteins of the present invention. Thus, many modifications may be made without departing from the spirit of the invention, and all changes that come within the scope or range of equivalents thereof are intended to be embraced therein. In the examples of the present invention, reagents and consumables are commercially available products unless otherwise specified.

Example 1: construction of recombinant vectors

The 5' -end noncoding region sequence required for cDNA display was first designed: <xnotran> GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTATTTTTACAACAATTACCAACAACAACAACAAACAACAACAACATTACATTTTACATTCTACAACTACAAGCCACCATGGAATTC ( T7 , 5' - , kozak ), T7 Ω. </xnotran> The 3' -end noncoding region sequence required for cDNA display was then designed: <xnotran> TTTCCCCGCCGCCCCCCGCCCTTGTCCGCCAAGCTTGTGATGATGATGGTGGTGAGACCCTCCGCCTGAGCCTCCACCATCATTTGTCCATCCTTCATC ( 6 × His-tag, ), tolA _ His. </xnotran> The T7 omega and tolA _ His have a 40bp homologous region in the middle and are sent to a gene synthesis company for synthesis. DNA Assembly was performed according to the instructions of the NEBBuilder HiFi DNA Assembly Master Mix kit (NEB), and equimolar amounts of DNA fragments were added, followed by addition of the DNA Assembly mixture (20. Mu.L in total volume), and the mixture was left at 50 ℃ for 1 hour. Connecting the assembled product with a plasmid pMD18-T, standing overnight at 16 ℃, transforming the obtained product into DH5 alpha competent bacteria, extracting positive clone plasmid DNA by using a plasmid extraction kit (TaKaRa) to perform enzyme digestion identification and sequencing, and naming the obtained product as a pT7-tolA recombinant vector after the sequencing is correct.

Example 2: synthesis of puromycin-biotin cross-linked compounds

Take 20nM puromycin fragment (PS): (5' -thio-Modifier C6) -TC- (fluorescein-dT) - (12-carbon polyethylene glycol)₄CC-Puromycin (BEX), was added to 4mM tetrachlorophenol and 50mM phosphate buffer (pH 7.0), mixed well at room temperature, and desalted by NAP-5 desalting column. Another 10nM biotin fragment (BS): CCCGTGCAGCTGTTTCATC- (Biotin-dT) -CGGAAACAGCTGCACCCCCCCGCCCC CCG- (Amino-Modifier C6 dT) -CCT (BEX) and 2. Mu.M of 6- (maleimido) hexanoic acid succinimidyl ester crosslinker were added to 0.2M phosphate buffer (pH 7.0) and mixed well, incubated at 37 ℃ for 30min, and excess crosslinker was removed by ethanol precipitation at 4 ℃. The precipitate was washed twice with 70% pre-cooled ethanol, dissolved in 0.2M phosphate buffer (pH 7.0), PS added and stirred overnight at 4 ℃. Adding 5mM DTT (dithiothreitol), stirring at 37 deg.C for 30min to stop reaction, precipitating with ethanol to remove excessive PS, dissolving the precipitate in phosphate buffer, and purifying by HPLC to obtain PS-BS linked compound.

Example 3: preparation of biotin (biotin) -labeled protein-Streptavidin (SA) magnetic beads

SA magnetic beads (Invitrogen) with solution A (diethyl pyrophosphate treated water, 0.1M Na)OH,0.05M NaCl) and solution B (diethyl pyrophosphate treated water, 0.1M NaCl), then added to 2 Xbinding buffer [20mM Tris-HCl pH 8.0,2mM ethylenediaminetetraacetic acid (EDTA), 2M NaCl,0.2% Triton X-100%]And preserving at-20 ℃ for later use. Respectively taking a proper amount of coronavirus S protein (Spike) S₁Subunit, N-methyl-D-aspartate receptor (NMDAR), vascular Endothelial Growth Factor (VEGF), tumor necrosis factor-alpha (TNF-alpha), epidermal growth factor receptor-2 (HER 2) and proprotein convertase subtilisin 9 (PCSK 9) protein were dissolved in 0.2M phosphate buffer (pH 7.0), sufficient Sulfo-NHS-biotin (Pierce) was added and placed on ice for 2h to react to produce Spike-biotin, NMDAR-biotin, VEGF-biotin, TNF-alpha-biotin, HER2-biotin and PCSK9-biotin compounds, SA magnetic beads were added and reacted at room temperature for 15min, and Spike-biotin-SA magnetic beads, NMDAR-biotin-SA, VEGF-biotin-SA magnetic beads, TNF-alpha-SA magnetic beads, HER 2-biotin-magnetic beads and PCSK9-biotin magnetic beads were separately obtained by affinity binding of biotin SA and stored at-20 ℃.

Example 4: cDNA display coronavirus S protein (Spike) affinity functional protein Spfitin

1. Constructing a cDNA display gene library. The spinous process protein (S protein) of coronavirus is the most important glycoprotein on the surface of the virus, and the virus infects cells through the binding of the S protein to receptors on host cells. The pathogenicity of the virus is closely related to the S protein. Thus, the viral S protein is an important target for anti-coronavirus drugs. The gene encoding amino acid residues at

positions

9, 10, 150, 152, 163, 164, 165, 166, 184, 185, 186, 187, 188, 189 and 190 of the sequence of SEQ ID NO 1 was subjected to randomized mutation using a degenerate oligonucleotide encoding NNS triplets (where N = a, C, T or G, and S = C or G), and the designed DNA fragment was named spfitinib lib, with 40bp homologous regions at both ends to DNA fragments of T7 Ω and tolA _ His, respectively, and was sent to the gene synthesizer for synthesis. Then, using pT7-tolA plasmid as a template, and using T7 omega _ F: ataaacttttagagacttctgggga and T7 Ω _ R: ATGCGCCATCGTAT is an upstream primer and a downstream primer, a T7 Ω DNA fragment (5' -end noncoding region required for cDNA display) was PCR-amplified, and the DNA fragment was PCR-amplified using the upstream primer tolA _ His _ F: GTACCCATAGG and downstream primer tolA _ His _ R: ATACGATGGCGCATTTGTC is subjected to PCR reaction,the tolA _ His DNA fragment (cDNA showing the desired 3' -non-coding region) was amplified. The PCR reaction system is as follows: pfu PCR mix 12.5. Mu.L, template 1. Mu.L, primers 1. Mu.L, ddH were added to 25. Mu.L of the reaction system₂O9.5. Mu.L. The PCR reaction conditions were: pre-denaturation at 94 ℃ for 3min; denaturation at 95 ℃ for 30s, annealing at 45 ℃ for 30s, extension at 72 ℃ for 1min,25 cycles; extension was carried out at 72 ℃ for 1min, agarose gel electrophoresis was carried out, and purified DNA was recovered using a recovery kit (Promega). Assembling T7 omega, spFitinLib and tolA _ His DNA fragments, adding equimolar DNA fragments according to the instructions of NEBBuilder HiFi DNA Assembly Master Mix kit (NEB), adding DNA Assembly mixture, and reacting at 50 ℃ for 1h. Agarose gel electrophoresis, recovery kit (Promega) recovery of purified DNA.

2. In vitro transcription and cross-linking reactions. According to RiboMAX^TMThe recovered DNA was transcribed in vitro to produce mRNA and purified according to Rneasy Mini Kits (Qiagen) kit instructions, product instructions for Large Scale RNA Production System-T7 (Promega). Then, the purified mRNA was complementarily crosslinked with the PS-BS compound by adding the transcription product, a ligation buffer (500 mM Tris-HCl pH 7.5, 100mM MgCl) to the reaction system₂100mM DTT,10mM ATP), PS-BS compound, RNase-free H₂O, T4 Polynucleotide kinase (NEB) and T4 RNA ligase (Takara), and crosslinking reaction at 25 ℃ for 90min. The cross-linked product was purified using the RNeasy MinElute clear Kit (Qiagen).

3. And (3) displaying the cDNA in vitro. According to

T7 Quick for PCR DNA cell-free expression kit (Promega) instructions, the purified mRNA-puromycin ligation product was added to TNT T7 Quick for PCR 40. Mu.L, 1mM methionine 1. Mu.L, 50mM magnesium acetate 1. Mu.L, RNase-free water 50. Mu.L and incubated at 30 ℃ for 1h. Then, KCl and MgCl are added into the reaction system₂Allowing the mixture to reach final concentrations of 800mM and 80mM, respectively, incubating at 37 deg.C for 1h to form mRNA-puromycin-protein fusion, adding SA magnetic beads for use, binding reaction at room temperature for 15min, resuspending in 2 Xbinding buffer, adding reverse transcription buffer (50 mM Tris-HCl pH 8.3, 75mM KCl,3mM M)gCl₂50mM DTT,0.5mM dNTPmix) and SuperScriptIII reverse transcriptase (Invitrogen) at 40 ℃ for 10min to produce mRNA/cDNA-puromycin-protein fusions. With buffer M (10 mM Tris-HCl pH 7.5, 10mM MgCl₂1mM DTT,50mM NaCl) was washed twice, the mRNA/cDNA-puromycin-protein was transferred to 40mL of buffer M, pvuII enzyme (Takara) and BAS were added, reaction was carried out at 37 ℃ for 1H, the mRNA/cDNA-puromycin-protein was separated from the magnetic SA beads, purified by Ni-NTA affinity chromatography using 6 XHis tag in the cDNA display molecule, and RNase T1 (Ambion) and RNase H (Takara) were added for 10min at 37 ℃ to decompose the mRNA to form cDNA-puromycin-protein.

4. And (4) affinity screening. The purified cDNA-puromycin-protein was transferred into a screening buffer (50 mM Tris-HCl pH 7.6,1mM EDTA,500mM NaCl,0.1% Tween-20), spike-biotin-SA magnetic beads were added, gently shaken at room temperature for 1 hour, placed on a magnetic stand for affinity screening, the magnetic beads were washed 5 times with the screening buffer, and cDNA-puromycin-protein which had not bound to the Spike protein was discarded. And using the screened product as a template, using T7 omega _ F and tolA _ His _ R as primers to perform PCR amplification, performing agarose gel electrophoresis, recovering and purifying by a recovery kit (Promega), and using the product as the next cycle, namely transcription-cDNA in-vitro display-screening. A total of 6 rounds of screening were performed.

5. Cloning, expressing and purifying the Spfitin gene. The product recovered by screening is treated by EcoRI/HindIII double enzyme digestion, is connected with pMD-18T plasmid, is kept overnight at 16 ℃, constructs pxSpfinin recombinant plasmid (the structure is shown in figure 1), and is transferred into competence E.coli BL21 (DE 3) for induction expression after verification. The recombinant single colony is inoculated in LB culture medium containing ampicillin and cultured overnight with shaking at 37 ℃. Inoculating to fresh 2YT-A culture medium containing ampicillin, and shake culturing at 37 deg.C to D₆₀₀0.6-1.0, adding isopropyl-beta-D-thiogalactoside (IPTG) for induction, and carrying out shake culture at 37 ℃ for 3-6 h. Centrifuging at 8000r/min for 10min, collecting thallus, suspending in lysis buffer, ultrasonically crushing, centrifuging, collecting supernatant, and purifying with Ni-NTA affinity chromatography. As shown in FIG. 2, the purified product of Ni-NTA showed a single band of about 32kDa in size as determined by SDS-PAGE, indicating that the affinity binding protein Spfitin of Spike was obtained.

Example 5: affinity of Spfitin for Spike binding

The Spike protein S1 subunit was diluted to 10mM with sodium citrate solution (pH 5.5) and each protein was coupled to a CM5 sensor core according to the amino coupling kit instructions. Spfitin was diluted with HBS-EP buffer (GE Healthcare) to 4.0nM, 2.0nM, 1.0nM, 0.5nM and 0.0nM. Spfitin solutions with different concentrations respectively flow through Spike protein S1 subunit coupling chip channels at the flow rate of 30 mu L/min at 25 ℃, the action is carried out on the Spike protein S1 subunit coupling chip channels, and response signals are collected in real time as shown in figure 3. After the concentration of each kind of the Spfitin solution is circulated, 10mM glycine hydrochloride buffer solution (pH 1.5) is injected into the surface of the chip to run for 30s for regeneration, and after the circulation is finished, the binding affinity K of the Spfitin and the Spike is obtained_DAt 2.6nM, it was shown that Spfitin binds to Spike with high affinity.

Example 6: spfitin inhibiting proliferation effect of cells infected by recombinant adenovirus carrying Spike

According to an S protein gene encoded by a coronavirus gene sequence (with the accession number of MN 908947) registered in Genbank, optimizing the S protein gene by adopting a gene optimization method of codon optimization and humanization to obtain an optimized S protein gene sequence, performing segmented synthesis and finishing assembly by a gene delivery company, performing PCR amplification, and cloning the S protein gene into a pDC316-mCMV-EGFP plasmid to obtain a pDC316-mCMV-EGFP-S shuttle plasmid. Extracting plasmids by using a plasmid extraction kit, and performing enzyme digestion identification by using Nhe I and Hind III respectively. Taking human embryo kidney 293Ad⁵⁺Cells at 5X 10⁵Cells/well were seeded in 6-well plates. The cells were cultured at 37 ℃ for 24 hours in a DMEM (Dulbecco's modified eagle medium) medium containing 10% calf serum. Get AdMax^TMThe 4 mug of backbone plasmid (pBHG 1ox _ E1,3 Cre) of adenovirus system and 1 mug of pDC316-mCMV-EGFP-S shuttle plasmid are dissolved in 300 mug of serum-free DMEM culture medium and mixed evenly. Taking Lipofectamine ^TM2000 mu.L of the suspension was dissolved in 300. Mu.L of serum-free DMEM medium and mixed well. Add backbone plasmid (pBH-Glox-E1, 3 Cre) 4. Mu.L, pDC316-mCMV-EGFP-S shuttle plasmid 1. Mu.L, and Lipofectamine to each well^TM10 μ L. The culture was continued for 24h, and the transfection efficiency was determined by observing green fluorescent cells under a fluorescent microscope. After the cells are full of the plate holes, the cells are transferred to 25cm²Continuously culturing in a cell culture bottle, and transferring to 75cm after full growth²The cells were cultured in a cell culture flask and observed for signs of cell toxicity. When the cells show grape-like clusters, namely cytopathic effect (CPE), the cells are gently blown off and collected, PBS is used for resuspension, and the cells are repeatedly frozen and thawed 3 times at-80 ℃ and 37 ℃ to fully release the virus in the cells. Centrifuging at 3000rpm for 5min, collecting virus-containing supernatant, and storing at-80 deg.C.

Get 293Ad⁵⁺The cells were cultured at 1X 10⁴The number of cells/well was put into a 96-well plate, and cultured at 37 ℃ for 24 hours in DMEM medium containing 10% fetal bovine serum. The supernatant containing the virus is added into each well and then divided into groups, the Spfitin experimental group is added with Spfitin to the final concentration of 1 mug/mL, and the blank control group is added with culture medium. 37 deg.C, 5%₂The culture was carried out in an incubator for 48 hours. According to Clontech Adeno-X^TMThe Rapid Titer Kit instruction determines the virus Titer and evaluates the virus proliferation inhibition effect of the Spfitin on in vitro cells. The results showed that the virus titer of the blank control group was 2.71. + -. 1.05X 10¹⁰ifu/mL. The virus titer of the experimental group of Spfitin was 5.44. + -. 1.17X 10⁴if/mL, compared with the virus titer of the blank control group, the virus titer of the Spfitin experimental group is obviously reduced, and the difference is obvious (p)<0.05, t test), which shows that the Spfitin has the effect of inhibiting the proliferation of cells infected by recombinant adenovirus carrying Spike.

Example 7: the Spfitin plays a protective role in acute lung injury induced by animal infection virus

Selecting male Kunming mouse, injecting supernatant containing Spike recombinant adenovirus through tail vein according to 0.1mL/kg body weight to form acute lung injury animal model, setting as lung injury group, and taking tail vein injected animal with PBS of same volume as normal group. The animals in the lung injury model group are randomly divided into a Spfitin experimental group and a normal saline control group, and each group comprises 6 mice. Spfitin is injected into animals in a Spfitin experimental group through tail vein according to the dose of 10mg/kg, 0.2mL of normal saline is injected into animals in a control group through tail vein, pathological tissue examination is carried out on the left lung of a mouse after 72h, 10% tissue homogenate is prepared on the right lung of the mouse, and the levels of inflammatory cytokines of interleukin-6 (IL-6), IL-8, IL-10 and tumor necrosis factor-alpha (TNF-alpha) in lung tissues are detected by ELISA. The pathological examination results of the lung tissues of the mice are shown in fig. 4A, 4B and 4C, the animal lung hemorrhage of the normal saline control group is changed, the lung is changed, and the lung tissue injury is obvious. The levels of IL-6, IL-8, IL-10 and TNF- α in the lung tissue of each group of animals are shown in the following table:

table 1: changes in IL-6, IL-8, IL-10 and TNF-alpha levels in lung tissue of each group of animals

As can be seen from the above table, after the animal lung injury induced by the virus is treated by the Spfitin, inflammatory cytokines (IL-6, IL-8, IL-10 and TNF-alpha) in animal lung tissues are reduced compared with a normal saline group, and the result shows that the Spfitin has a good treatment effect on the mouse lung injury induced by the virus.

Example 8: the cDNA shows the affinity functional protein Nmfitin of the N-methyl-D-aspartate receptor (NMDAR)

Genes encoding amino acid residue expression at

positions

35, 36, 52, 53, 54, 65, 66, 67, 68, 86, 87, 88, 89, 90, 91 and 92 of the sequence of SEQ ID NO 2 were randomly mutated using degenerate oligonucleotides encoding NNS triplets (where N = a, C, T or G, and S = C or G), and the designed DNA fragment was designated nmfitinib lib, with 40bp homology regions at both ends to DNA fragments of T7 Ω and tolA _ His, respectively, and was synthesized by gene synthesis companies. Then, pT7-tolA plasmid is used as a template, assembly of T7 omega, nmFitinLib and tolA _ His DNA fragments is carried out by using NEBBuilder HiFi DNA Assembly Master Mix kit (NEB) according to the method described in example 4, a cDNA display gene library is constructed, in-vitro transcription and cross-linking reaction and cDNA in-vitro display are carried out, NMDAR-biotin-SA magnetic beads are adopted, a target product is subjected to affinity screening from a cDNA-puromycin-protein compound formed by cDNA in-vitro display, transcription-cDNA in-vitro display-screening is carried out for 6 cycles in total, the product is subjected to agarose gel electrophoresis, a recovery kit (Promega) is recovered and purified, and is connected with pMD-18T plasmid, a pxNptin recombinant plasmid (the structure is shown in figure 5) is constructed overnight at 16 ℃, and after verification, the pxNfin recombinant plasmid is transferred into a competence E.coli BL21 (DE 3) for induction expression, and then ultrasonic disruption, centrifugation and Ni-NTA affinity chromatography are carried out. As shown in FIG. 6, after the expression product was purified by Ni-NTA, a single band with a size of about 32kDa was detected by SDS-PAGE, suggesting that the affinity functional protein Nmfitin of NMDAR was obtained.

Example 9: flow cytometry for detecting binding activity of Nmfitin and cell NMDAR

Using RIPA (radio-ionization assay) buffer [1% ethylphenylpolyethylene glycol (NP-40), 1% sodium deoxycholate, 0.1% SDS,150mM NaCl,25mM Tris-HCl, pH 7.4%]After the gastric cancer cell SGC-7901, the liver cancer cell SMMC-772 and the normal human mammary gland cell MFC-10A are cracked, the total cell protein is extracted. Centrifuging at 4 deg.C and 13000r/min for 15min, collecting supernatant, and storing at-20 deg.C. The BCA protein quantification kit detects protein concentration, 100 μ g of each protein is subjected to SDS-PAGE electrophoresis, voltage-stabilized ice bath electrotransfer onto nitrocellulose membrane, 5% skim milk blocking for 1h, primary antibody incubation for 4 ℃ overnight (

NMDAR 2B antibody

1, 100, gapdh antibody 1. The results are shown in FIG. 6, positive expression of NMDAR was found in SGC-7901 and SMMC-772 cells, while negative expression of NMDAR was found in MFC-10A cells. Taking SGC-7901, SMMC-772 and MFC-10A cells for trypsinization, centrifuging, resuspending and counting. At a rate of 1 × 10 per bottle⁷The number of individual cells was put in a cell culture flask at 37 ℃ with CO₂Grouping after culturing for 24h in an incubator. The experimental group was incubated with 5. Mu.M FITC labeled kit for fluorescently labeled Nmfitin cells, and the control group was incubated with FITC cells at 37 ℃ for 1 hour. PBS was washed 2 times and detected using flow cytometry (FACS Calibur). The results show that relative fluorescence intensity was higher in NMDAR-positively expressing SGC-7901 and SMMC-772 cells than in NMDAR-negatively expressing MFC-10A cells after incubation of the cells with fluorescently labeled Nmfitin, as shown in fig. 7, indicating that fluorescently labeled Nmfitin is specific for binding to NMDAR-positively expressing cells.

Example 10: nmfitin for promoting islet cells to secrete insulin

Collecting islet beta cells MIN-6 at a dose of 1 × 10 per bottle⁷The number of each cell was placed in a cell culture flask, and cultured at 37 ℃ for 24 hours in DMEM (Dulbecco's modified eagle medium) medium containing 10% fetal bovine serum, and then divided into an Nmfitin + high-sugar test group, an Nmfitin + low-sugar test group, a buffer + high-sugar control group, and a buffer + low-sugar control group. The cells were discarded, washed 1 time with PBS, and 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer (15mM HEPES,5mM KCl,120mM NaCl,2mM CaCl) was added₂10 μ M Glycine, 24mM NaHCO₃And 1mg/mL fetal bovine serum), nmfitin + high sugar test group added with 10 μ MNmfitin and 16.7mM glucose in HEPES buffer, nmfitin + low sugar test group added with 10 μ MNmfitin and 2mM glucose in HEPES buffer, HEPES + high sugar test group added with 16.7mM glucose in HEPES buffer, HEPES + low sugar control group added with 2mM glucose in HEPES buffer, incubated at 37 ℃ for 2h, supernatant was taken and insulin secreted from cells was detected using insulin detection kit (immunochromatography), and as a result, nmfitin promoted insulin secretion from MIN-6 cells, showing statistical difference compared to HEPES control group, as shown in fig. 8.

Example 11: therapeutic Effect of Nmfitin on diabetes model animals

Female SD rats are injected with 50mg/kg streptozotocin in the abdominal cavity to establish a diabetes animal model, and after high-sugar high-fat feed (100 g white sugar, 17g lard, 250g eggs, a proper amount of salt and vitamins) is fed adaptively for 3 days every day, an Nmfitin experimental group and a normal saline control group are randomly divided into 5 animals in each group. The experimental group is administrated by tail vein injection according to the dose of 10mg/kg, the control group is injected with normal saline once a week and fed with high sugar and high fat every day, the animals are weighed after 3 weeks, blood is taken to detect the blood sugar, triglyceride (TG), high Density Lipoprotein (HDL), low Density Lipoprotein (LDL) and insulin level, the result is shown in figure 9, the weight, the blood sugar, the blood TG and the blood LDL value of the animals of the Nmfitin group are lower than those of the control group, the blood insulin and HDL of the animals of the Nmfitin group are higher than those of the control group, and the Nmfitin group shows statistical difference compared with the normal saline control group (figure 9), which shows that the Nmfitin has repairing effect on insulin cell damage induced by streptozotocin, can improve the blood insulin level of diabetes animals induced by streptozotocin, and has antagonistic effect on blood sugar and blood fat increase.

Example 12: cDNA shows Vascular Endothelial Growth Factor (VEGF) affinity binding protein Vefitin

The gene expressing the amino acid residues at positions 121, 122, 138, 139, 140, 151, 152, 153, 154, 172, 173, 174, 175, 176, 177 and 178 of the sequence encoding SEQ ID NO 3 was subjected to randomized mutation using degenerate oligonucleotides encoding NNS triplets (where N = A, C, T or G, and S = C or G), and the designed DNA fragment was named VeFitinLib, with 40bp homology regions at both ends to DNA fragments of T7. Omega. And tolA _ His, respectively, and was sent to the gene synthesizer for synthesis. Then pT7-tolA plasmid is used as a template, assembling T7 omega, vefitinib and tolA _ His DNA fragments by using NEBBuilder HiFi DNA Assembly Master Mix kit (NEB) according to the method described in example 4, constructing cDNA display gene library, in vitro transcription and cross-linking reaction and cDNA in vitro display, adopting VEGF-biotin-SA magnetic beads, screening out affinity target products from cDNA-puromycin-protein compound formed by cDNA in vitro display, then performing transcription-cDNA in vitro display-screening for 6 cycles, performing agarose gel electrophoresis on the products, recovering and purifying the kit (Promega), connecting with pMD-18T plasmid, overnight at 16 ℃, constructing pxVefin recombinant competent plasmid (structure shown in figure 10), transferring into E.coli BL21 (DE 3) for induction expression, and then performing ultrasonic disruption, centrifugation and Ni-NTA affinity chromatography purification. The expression product Ni-NTA is detected by SDS-PAGE before and after affinity purification. The result shows that as shown in FIG. 11, a distinct band is formed at about 32kDa after Ni-NTA affinity purification, which indicates that VEGF affinity binding protein Vefitin is obtained.

Example 13: affinity of Vefitin for binding to VEGF

VEGF was diluted to 10mM with sodium citrate solution (pH 5.5) and each protein was coupled to a CM5 sensor core according to the amino coupling kit instructions. Vefitin was diluted with HBS-EP buffer (GE Healthcare) to 1.6nM, 0.8nM, 0.4nM, 0.2nM, 0.1nM and 0.0nM. At 25 ℃, vefitin solutions with different concentrations respectively flow through a VEGF coupling chip channel at the flow rate of 30 mu L/min and act with the VEGF coupling chip channel to collect response signals in real time, and the results are shown in a figure12. After the concentration of each Vefitin solution is circulated, 10mM glycine hydrochloride buffer solution (pH 1.5) is injected into the surface of the chip to run for 30s for regeneration, and after the circulation is finished, the binding affinity K of Vefitin and VEGF is obtained_DAt 0.45nM, it was shown that Vefitin binds VEGF with high affinity.

Example 14: effect of Vefitin on vascular endothelial cell proliferation

Culturing human lung cancer cell A549 in DMEM medium containing 10% fetal calf serum until 70% confluence, culturing in serum-free medium for 48 hr, collecting culture supernatant, filtering with 0.22 μm filter membrane, and storing at-80 deg.C. Taking normal human umbilical vein endothelial cells HUVEC, incubating the cells with the spare culture supernatant, and collecting when the cells are converged to 80%, namely the tumor vascular endothelial cells Td-EC.

HUVEC and Td-EC cells were taken at 5X 10 per well³The number of the cells is put into a 96-well culture plate, and the cells are incubated at 37 ℃ until the cells are attached to the wall and then grouped. Vefitin (diluted with serum-free DMEM medium) was added to the cells of the experimental group at different concentration gradients of 5, 10, 15 and 20. Mu.g/mL, and the control group was added with the medium. Each group of cells was plated with three duplicate wells and cultured at 37 ℃ for 48 hours. Before measurement, culture well supernatants were discarded, 10. Mu.L of a newly prepared tetramethyltetrazolium blue (MTT) solution was added to each well at 0.5mg/mL, incubation was continued at 37 ℃ for 48 hours, 150. Mu.L of dimethyl sulfoxide (DMSO) was added to each well supernatant, and after mixing, the A value of the cells was measured at a wavelength of 490nm using a DG3022 type microplate reader, and the cell proliferation inhibition (%) = (1-test well A value/control well A value) × 100% was calculated. The experimental results show that Vefitin has an inhibiting effect on VEGF-induced vascular endothelial cell proliferation, and the Vefitin dose is increased according to the cell proliferation inhibition rate shown in figure 13.

Example 15: therapeutic effect of Vefitin on inhibiting corneal neovascularization of rat

An animal model of rat corneal neovascularization is prepared by adopting an alkali burn method. SD rats were anesthetized by ether inhalation, and a piece of filter paper immersed in 1M NaOH solution was applied to the center of the right cornea of the rat, after which the conjunctival sac was rinsed with normal saline and antibiotic eye solution was dropped to prevent infection, and 1% atropine eye ointment was applied to the eyes to prevent posterior iris adhesion. Animals with corneal alkali burn are divided into Vefitin experimental group and bevacizumab pairControl and saline blank, 6 of them. Each group was given 25 μ L of subconjunctival injection in the right eye of the rat once every other day for 7 times. The conjunctival congestion, corneal edema and corneal neovascularization were observed daily. After 4 weeks (28 days) the length and number of new vessels growing centripetally from the corneoscleral edge were measured and recorded, according to the formula: s = C/12X 3.1416[ r ]²-(r-l)²]And calculating the area of the new blood vessels, wherein C is the number of the peripheral clock points of the cornea accumulated by the new blood vessels, l is the length of the longest blood vessel of the new blood vessels extending into the cornea from the corneal limbus, and r is the corneal radius. The comparison of the growth area of the new blood vessels in the cornea of each group of animals is shown in Table 2.

Table 2: the maximal length and the proliferative area of the cornea neovascularization of each group of animals

As can be seen from the above table, the corneal neovascularization of animals in the Vefitin group had a length and area smaller than those of the dexamethasone control group and the normal saline control group (p < 0.05), indicating that corneal neovascularization of rats can be inhibited after Vefitin is applied.

Furthermore, to further understand whether Vefitin inhibits corneal neovascularization, it was associated with blocking VEGF interaction with vascular endothelial growth factor receptor-1 (VEGFR-1) or vascular endothelial growth factor receptor-2 (VEGFR-2) after targeting VEGF. Experiment each group randomly selected 2 rats, excised corneal tissue and ground to a fine powder with liquid nitrogen, added with pre-cooled RIPA lysate, homogenized, centrifuged at 13000r/min at 4 ℃ for 15min, supernatant was taken, protein concentration was determined with BCA protein quantification kit, 100 μ g of each protein was subjected to SDS-PAGE electrophoresis, transferred to nitrocellulose membrane, blocked with 5% skim milk for 1h, primary anti-incubation overnight at 4 ℃ (VEGF, VEGFR-1 and VEGFR-2 antibody 1. The result is shown in figure 14, vefitin can obviously reduce the expression of VEGF, VEGFR-1 and VEGFR-2 in corneal tissues, and shows that the Vefitin can block the interaction of VEGF and its receptor after being combined with VEGF in a targeted manner, thereby playing a role in inhibiting corneal neovascularization.

Example 16: cDNA display tumor necrosis factor-alpha (TNF-alpha) affinity binding protein Tnfitin

TNF-alpha is a cytokine for promoting inflammatory response, and is related to the occurrence and development of various autoimmune diseases such as rheumatoid arthritis, psoriasis, crohn's disease and the like. Therefore, the drug designed by taking the TNF-alpha as a target point can effectively block the inflammatory reaction process mediated by the TNF-alpha and treat related diseases. The present invention uses degenerate oligonucleotides encoding NNS triplets (where N = a, C, T or G, and S = C or G), randomly mutates the gene expressing the amino acid residues at positions 133, 134, 150, 151, 152, 163, 164, 165, 166, 184, 185, 186, 187, 188, 189 and 190 of the sequence encoding SEQ ID NO 4, and names the designed DNA fragment tnfininlib with 40bp homology regions at both ends to DNA fragments of T7 Ω and tolA _ His, respectively, and sends it to the gene synthesis company for synthesis. Then, pT7-tolA plasmid is used as a template, assembly of T7 omega, tnFitinLib and tolA _ His DNA fragments is carried out by using NEBBuilder HiFi DNA Assembly Master Mix kit (NEB) according to the method described in example 4, a cDNA display gene library is constructed, in vitro transcription and cross-linking reaction and cDNA in vitro display are carried out, TNF-alpha-biotin-SA magnetic beads are adopted, a target product is subjected to affinity screening from a cDNA-puromycin-protein compound formed by cDNA in vitro display, the cDNA-puromycin-protein compound is subjected to transcription-cDNA in vitro display-screening for 6 cycles of circulation, the product is subjected to agarose gel electrophoresis, a recovery kit (Promega) is recovered and purified, and is connected with pMD-18T plasmid, overnight at the temperature of 16 ℃, pxTnfin recombinant plasmid (the structure is shown in figure 15) is constructed, and is transferred into BL21 (DE 3) for induction expression, and is subjected to ultrasonic disruption, purification and Ni-NTA affinity chromatography. The results of SDS-PAGE detection before and after the purification of the product are shown in FIG. 16, and a single band appears at the position of about 32kDa of the product after the purification of Ni-NTA, which indicates that the TNF-alpha affinity binding protein Tnfitin is obtained.

Example 17: affinity of Tnfitin for TNF-alpha binding

TNF-alpha was diluted to 10mM with sodium citrate solution (pH 5.5) and each protein was coupled to a CM5 sensor core according to the amino coupling kit instructions. Tnfitin was diluted with HBS-EP buffer (GE Healthcare) to 500nM, 100nM, 50nM, 10nM and 5nM. Tnfitin solutions with different concentrations were flowed through the channels of the TNF-. Alpha.coupling chip at a flow rate of 30. Mu.L/min at 25 ℃ respectively and acted thereon, and response signals were collected in real time and shown in FIG. 17. After the concentration of each Tnfitin solution is circulated, 10mM glycine hydrochloride buffer solution (pH 1.5) is injected into the surface of the chip to run for 30s for regeneration, and after the circulation is finished, the binding affinity K of the Tnfitin and TNF-alpha is obtained_DAt 6.3nM, it was shown that Tnfitin binds TNF- α with high affinity.

Example 18: tnfitin inhibits TNF-alpha mediated cytotoxic effects

The test takes mouse fibroblast L929 as a research object, and judges whether the combination of Tnfitin and TNF-alpha has an inhibiting effect on TNF-alpha mediated cytotoxic effect. At a rate of 5X 10 per hole³And (2) counting the number of cells, putting the L929 cells into a 96-well culture plate, adding 10ng/mL TNF-alpha, dividing the cell into a Tnfitin experimental group and a TNF-alpha control group, diluting the experimental group according to 10 times of gradient, adding Tnfitin, respectively setting 3 multiple wells, and setting a culture medium blank control group as a natural death control. After culturing at 37 ℃ for 48h, adding 10 mu L of MTT solution into each well, adding DMSO after 4h, and detecting by using a microplate reader to obtain a cell A value according to the formula: t = (1-Tnfitin test well or TNF- α control well a value/blank well a value) calculate cytotoxic effect; and calculating the inhibition rate (%) of cytotoxic effect according to the formula = (1-T)_Tnfitin/T_TNF-α) X 100%. The results are shown in FIG. 18, in which Tnfitin has an inhibitory effect on TNF-. Alpha.induced cytotoxic effects, and is enhanced with increasing dose of Tnfitin.

Example 19: therapeutic effect of Tnfitin on mouse rheumatoid arthritis

Bovine type II Collagen (CII) was dissolved in 0.1M acetic acid at a concentration of 2g/L, stirred overnight at 4 ℃ and then mixed with Freund's complete adjuvant (4 mg/mL) in equal volume and emulsified to prepare CII emulsion. Selecting male Kunming mouse, injecting 0.1mL of C II emulsion into right hind foot sole of mouse to cause inflammation, and injecting 0.1mL again after 3 weeks (21 days) to establish arthritis animal model. Mice successfully modeled were randomly divided into a Tnfitin experimental group (8 mg/kg), a dexamethasone control group (1 mg/kg) and a normal saline control group, 10 mice per group. The administration of each group was started 21 days after the onset of inflammation, and the animals were subjected to intraperitoneal injection 1 time every 2 days to observe the swelling of joints and the like and to score the inflammation of joints (score 0: no swelling; score 1: red swelling of little toe joint; score 2: swelling of toe joint and foot sole; score 3: swelling of paw below ankle joint; score 4: swelling of all paws including ankle joint), and the sum of the inflammation scores of 4 joints per mouse was expressed by Arthritis index (Arthritis index) and scored 1 time every 4 days, and the results were shown in FIG. 19. And after 4 weeks (28 days) of treatment, taking animal blood, measuring the contents of blood RF and C-reactive protein (CRP) by using an ELISA detection kit for arthritis factor (RF) and C-reactive protein (CRP), taking animal joint synovial fluid, and detecting the levels of TNF-alpha and IL-10 in the joint synovial fluid by using an ELISA detection kit for TNF-alpha and interleukin-10 (IL-10), wherein the results are shown in the following table 3:

table 3: biochemical index changes in blood and joint synovial fluid of various groups of animals

As can be seen from the above table, after the treatment of the CII-induced arthritis in the animals by using Tnfitin and dexamethasone, the RF and CRP in the blood of the animals are reduced compared with those in the normal saline group, the TNF-alpha content in the synovial fluid of the joints is reduced, but the IL-10 level in the synovial fluid of the joints is increased. The mice in the normal saline group are observed to have more obvious rear foot swelling, 4 toes gradually swell, the skin of the feet is congested, the toes deform and lameness is shown; as shown in fig. 16, the arthritis index of the saline control group gradually increased with the passage of time, however, the arthritis index of the treatment group was lower than that of the arthritis model control group after treatment with Tnfitin and dexamethasone, clinical symptoms such as toe swelling, hind-foot skin hyperemia, and lameness of the treated mice were significantly reduced, and symptoms of most of the mice were substantially disappeared. The result shows that the Tnfitin has good treatment effect on the mouse rheumatoid arthritis induced by the CII.

Example 20: the cDNA shows the affinity binding protein Hefitin of epidermal growth factor receptor-2 (HER 2)

The genes encoding for the expression of the amino acid residues at

positions

54, 55, 56, 64, 65, 66, 77, 78, 79, 80, 98, 99, 100, 101, 102 and 103 of the sequence SEQ ID NO 5 were subjected to randomized mutation using degenerate oligonucleotides encoding NNS triplets (where N = a, C, T or G, S = C or G) and the designed DNA fragment was named hepitinlib, with 40bp homologous regions at both ends to the DNA fragments of T7 Ω and tolA _ His, respectively, and sent to the gene synthesis company for synthesis. Then, pT7-tolA plasmid is used as a template, assembly of T7 omega, heFitinlib and tolA _ His DNA fragments is carried out by using NEBBuilder HiFi DNA Assembly Master Mix kit (NEB) according to the method described in example 4, after cDNA display gene library, in vitro transcription and cross-linking reaction and cDNA in vitro display are constructed, HER2-biotin-SA magnetic beads are adopted to carry out affinity screening of a target product from a cDNA-puromycin-protein compound formed by cDNA in vitro display, the target product is subjected to 6 cycles of transcription-cDNA in vitro display-screening, the product is subjected to agarose gel electrophoresis, a recovery kit (Promega) is recovered and purified, and is connected with pMD-18T plasmid, overnight at 16 ℃, pxHefintin recombinant sensing plasmid (structure shown in figure 20) is constructed, and is transferred into E.coli BL21 (DE 3) for induction expression, and then ultrasonic disruption, ni-NTA affinity chromatography purification are carried out. The purified product of Ni-NTA is detected by SDS-PAGE, a single band appears, the molecular size is about 32kDa, and HER2 affinity binding protein Hefitin is obtained (as shown in figure 21).

Example 21: affinity of hepitin for binding to HER2

HER2 was diluted to 10mM with sodium citrate solution (pH 5.5) and each protein was coupled to a CM5 sensor core separately according to the amino coupling kit instructions. Hefitin was diluted with HBS-EP buffer (GE Healthcare) to 1.6nM, 0.8nM, 0.4nM, 0.2nM, 0.1nM and 0.0nM. Hefitin solutions of different concentrations were flowed through the HER2 conjugate chip channel at a flow rate of 30. Mu.L/min at 25 ℃ and acted upon, respectively, and response signals were collected in real time, and the results are shown in FIG. 22. After the concentration of each Hefitin solution is circulated, 10mM glycine hydrochloride buffer solution (pH 1.5) is injected into the surface of the chip to run for 30s for regeneration,after the circulation, the binding affinity K of the Hefitin and HER2 is obtained_DAt 0.45nM, indicating that hepitin binds with high affinity to HER 2.

Example 22: hefitin inhibits tumor cell proliferation activity

Human breast cancer cell BT-474 with HER2 positive expression in logarithmic growth phase, human breast cancer cell MDA-MB-453 and human breast cancer cell MFC-7 with HER2 negative expression are taken at the rate of 5 multiplied by 10 per hole³The number of the cells is put into a 96-well culture plate, and the cells are incubated at 37 ℃ until the cells are attached to the wall and then grouped. Hefitin with different concentration gradients of 2 ug/mL, 1 ug/mL, 0.5 ug/mL, 0.25 ug/mL, 0.125 ug/mL, 0.0625 ug/mL, 0.03125 ug/mL, 0.015 ug/mL and 0 ug/mL (diluted with serum-free DMEM medium) was added to cells of Hefitin experimental group, trastuzumab (HER 2 antibody) with the same concentration gradient as that of Hefitin control group was also added to cells of trastuzumab (HER 2 antibody) control group, and blank control group (medium) was set as natural death control. Each group of cells was plated with three duplicate wells and cultured at 37 ℃ for 48 hours. Adding 10 mu L of MTT solution into each well, adding DMSO after 4h, detecting by using a microplate reader to obtain a cell A value, and obtaining a value according to a formula: t = (1-Hefitin experimental wells or trastuzumab control well a value/blank well a value) × 100 cytotoxicity was calculated. The results are shown in fig. 23, where hepitin has inhibitory effect on cell proliferation, and increased with increasing dose of hepitin.

Example 23: antitumor activity of Hefitin

Inoculating the MDA-MB-453 cell suspension to the right side near hind limb subcutaneous surface of an SPF-level SCID mouse of 5-6 weeks old, selecting an animal with good tumor growth and no necrosis after 2 weeks, dislocating and killing cervical vertebrae, taking out solid tumors under an aseptic condition, cutting into small tumor blocks, and transplanting to the right side near hind limb subcutaneous surface of a nude mouse. 2 weeks after tumor transplantation, mice with good tumor growth were randomly assigned to a Hefitin experimental group and a normal saline control group, and 5 mice per group were selected. On the 14 th day after tumor transplantation, hefitin was injected into the tail vein of the animals at a dose of 10mg/kg once a week (7 days), and 0.2mL of physiological saline was injected into the tail vein of the animals in the control group. The major (a) and minor (b) diameters of the tumor body were measured and recorded 2 to 3 times per week before and after administration, according to the formula: v = (a × b)²) Calculating tumor volume (V), then calculating relative tumor volume RTV = V_t/V₀(wherein V₀Tumor volume measured for initial administration, V_tTumor volume for each measurement). Then, the relative tumor proliferation rate T/C (%) = T is calculated_RTV/C_RTV×100％(T_RTV: experimental group relative tumor volume; c_RTV: control group relative tumor volume). As a result, as shown in FIG. 24, MDA-MB-453 is a highly malignant human breast cancer cell line, and it can be seen that the tumor body grown after the transplantation of the tumor by the experimental animal is in various forms such as a round form, an oval form and a strip form (about day 7), the tumor nodules are hard in texture and increase rapidly. Observed after group treatment, the tumor volume at the time of primary treatment (day 7) of the Hefitin group averaged 234. + -. 56mm³After 3 treatments, the tumor volume decreased significantly from day 21. By day 35, the tumor size was 110. + -.48 mm³Tumor size in combination with saline (2288. + -. 120 mm)³) In contrast, the difference was significant (P)<0.05). Because the MDA-MB-53 cell line is a highly malignant cell line, the tumor grows fast. Mice with transplanted tumors in the saline group died about 3 weeks after administration because of no effective treatment. Compared with the normal saline group, the survival time of the animals in the Hefitin group is obviously prolonged (the result is shown in figure 25).

Example 24: the cDNA shows the proprotein convertase subtilisin/kexin 9 (PCSK 9) affinity binding protein Pcfitin

The gene expressing the amino acid residues at positions 140, 141, 142, 150, 151, 152, 163, 164, 165, 166, 184, 185, 186, 187, 188 and 189 of the sequence encoding SEQ ID NO 6 was subjected to randomized mutation using a degenerate oligonucleotide encoding NNS triplets (where N = a, C, T or G, and S = C or G), and the designed DNA fragment was named pcfitinib lib, with 40bp homologous regions at both ends to DNA fragments of T7 Ω and tolA _ His, respectively, and was sent to the gene synthesis company for synthesis. Then, pT7-tolA plasmid is used as a template, assembly of T7 omega, pcFitinLib and tolA _ His DNA fragments is carried out by using NEBBuilder HiFi DNA Assembly Master Mix kit (NEB) according to the method described in example 4, after cDNA display gene library, in vitro transcription and cross-linking reaction and cDNA in vitro display are constructed, PCSK9-biotin-SA magnetic beads are adopted to carry out affinity screening of a target product from a cDNA-puromycin-protein compound formed by cDNA in vitro display, the target product is subjected to transcription-cDNA in vitro display-screening for 6 cycles in total, the product is subjected to agarose gel electrophoresis, a recovery kit (Promega) is recovered and purified, and is connected with pMD-18T plasmid, a pxNmfitin recombinant plasmid (structure 26) is constructed overnight at 16 ℃, the pxNmfitin recombinant plasmid is transferred into E.coli BL21 (DE 3) for induction expression, and then ultrasonic disruption, purification and Ni-NTA affinity chromatography are carried out. The expression product was detected by SDS-PAGE, and the results are shown in FIG. 27. After Ni-NTA purification, a single obvious band appears in the product, the size is about 32kD, and the PCSK9 affinity binding protein Pcfitin is obtained.

Example 25: pcfitin affinity for binding to PCSK9

PCSK9 was diluted to 10mM with sodium citrate solution (pH 5.5) and each protein was coupled to a CM5 sensor core separately according to the amino coupling kit instructions. Pcfitin was diluted with HBS-EP buffer (GE Healthcare) to 1.6nM, 0.8nM, 0.4nM, 0.2nM, 0.1nM and 0.0nM. At 25 ℃, pcfitin solutions with different concentrations respectively flow through the PCSK9 coupled chip channel at a flow rate of 30 μ L/min and act thereon, and response signals are collected in real time, with the results shown in fig. 28. After the concentration of each Pcfitin solution is circulated, 10mM glycine hydrochloride buffer solution (pH 1.5) is injected into the surface of the chip to run for 30s for regeneration, and after the circulation is finished, the binding affinity K of the Pcfitin and the PCSK9 is obtained_D0.45nM, indicating that Pcfitin binds PCSK9 with high affinity.

Example 26: pcfitin promotes cellular uptake of low-density lipoprotein cholesterol (LDL-C) by inhibiting PCSK9

Hepatocyte HepG2 at 1X 10 per vial⁷The number of the cells is put into a culture flask, the cells are cultured for 24 hours by using DMEM culture solution containing 10% fetal bovine serum, the culture solution is discarded, serum-free DMEM culture solution is replaced, and the cells are divided into a Pcfitin experimental group, an anti-PCSK9 (PCSK9 monoclonal antibody) control group and a blank medium control group after 50nM LDL-C and 45nM PCSK9 are added. Supernatants from each group of cells were assayed for LDL-C content using a CX7 biochemical analyzer (Beckman). Placing sterile cover glass in 6-well cell culture plate, adding 1 × 10 per well⁵Culturing individual hepatocyte HepG2 cell at 37 deg.C for 24 hr, removing culture solution, replacing serum-free DMEM culture solution, adding 45nM PCSK9 into cell, and performing Pcfitin experimentGroup, control group and blank control group. Pcfitin was added to the Pcfitin group to a final concentration of 1. Mu.g/mL, anti-PCSK9 was added to the control group to a final concentration of 1. Mu.g/mL, and then 10. Mu.g/mL DiI-LDL (Sigma) was added to each group of cells, which were cultured at 37 ℃ for 4 hours, washed 2 times with PBS containing 0.4% bovine serum albumin, then fixed with 4% paraformaldehyde in PBS for 10min, washed 3 times with PBS, stained for 20min with Hoechst33342, washed 3 times with PBS, and the remaining liquid was aspirated, and an anti-fluorescence quencher was added dropwise and observed with a fluorescence microscope. The observation results of the cells of each group are shown in FIG. 29. After the cells absorb DiI-LDL and Hoechst33342, the DiI dye enables the cells to show red fluorescence color development, the Hoechst33342 dye enables the cell nuclei to show blue fluorescence color development, and the red fluorescence intensity of the Pcfitin and anti-PCSK9 groups is obviously increased compared with that of the blank group, which indicates that the Pcfitin and anti-PCSK9 promote the HepG2 cells to take in the DiI-LDL.

Example 27: pcfitin inhibition of influence of PCSK9 on animal lipid metabolism

The male Kunming mice are divided into a Pcfitin experimental group, an anti-PCSK9 (PCSK 9 monoclonal antibody) control group and a physiological saline blank group, 6 mice in each group are administrated by intravenous injection through the tail of animals according to the dose of 10mg/kg, the mice are fed with high fat feed (common feed +21% fat +0.15% cholesterol) once a week, and the blood and liver of the animals are taken for examination after 2 weeks (14 days). Blood PCSK9 levels were determined as described in the PCSK9 ELISA Kit (MBL) instructions and blood TC (total cholesterol), TG (triglycerides), LDL-C (low density lipoprotein cholesterol) and HDL-C (high density lipoprotein cholesterol) levels were measured using a CX7 biochemical analyzer (Beckman). The results are given in Table 4 below:

table 4: comparison of blood PCSK9, TC, TG, LDL-C and HDL-C levels in various groups of animals

Note: p <0.05 compared to blank group.

As can be seen from the table above, the blood PCSK9 level of animals in the Pcfitin and anti-PCSK9 group is obviously reduced compared with that in the physiological saline group, so that the blood TC, TG and LDL-C levels of the animals in the Pcfitin and anti-PCSK9 group are reduced, the blood HDL-C concentration is increased, and the Pcfitin and anti-PCSK9 influence lipid metabolism by inhibiting PCSK9, thereby achieving the effect of reducing blood fat.

The animal liver adopts Western blot to detect the expression level of the hepatic LDL receptor, namely, the liver is washed by normal saline, then the liver is blotted by filter paper, weighed, 0.5cm multiplied by 0.5cm tissue is taken, after the treatment of RIPA lysate, SDS-PAGE electrophoresis is carried out, membrane is transferred, 5 percent skimmed milk powder TBS-T solution is sealed for 1h, anti-LDL-R primary antibody is added, the overnight standing is carried out at 4 ℃, and a secondary antibody marked by horseradish peroxidase is used for incubation for 1h and development is carried out. As shown in FIGS. 30A and 30B, the expression level of LDL-R in hepatocytes of animals in the Pcfitin and anti-PCSK9 groups was significantly higher than that of the saline group.

SEQUENCE LISTING

<110> Eihe bin

<120> scaffold proteins derived from epidermal growth factor, lectin and Tat protein

<160> 6

<210> 1

<211> 292

<212> PRT

<213> Artificial Sequence

<400> 1

Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Gly Cys Pro Gly

1 5 10 15

Leu Pro Gly Ala Pro Gly Asp Lys Gly Glu Ala Gly Thr Asn Gly Lys

20 25 30

Arg Gly Glu Arg Gly Pro Pro Gly Pro Pro Gly Lys Ala Gly Pro Pro

35 40 45

Gly Pro Asn Gly Ala Pro Gly Glu Pro Ser Pro Asp Gly Asp Ser Ser

50 55 60

Leu Ala Ala Ser Glu Arg Lys Ala Leu Gln Thr Glu Met Ala Arg Ile

65 70 75 80

Lys Lys Trp Leu Thr Phe Ser Leu Gly Lys Gln Val Gly Asn Lys Phe

85 90 95

Phe Leu Thr Asn Gly Glu Ile Met Thr Phe Glu Lys Val Lys Ala Leu

100 105 110

Cys Val Lys Phe Gln Ala Ser Val Ala Thr Pro Arg Asn Ala Ala Glu

115 120 125

Asn Gly Ala Ile Gln Asn Leu Ile Lys Glu Glu Ala Phe Leu Gly Ile

130 135 140

Thr Asp Glu Lys Thr Glu Gly Gln Phe Val Asp Leu Thr Gly Asn Arg

145 150 155 160

Leu Thr Tyr Thr Asn Trp Asn Glu Gly Glu Pro Asn Asn Ala Gly Ser

165 170 175

Asp Glu Asp Cys Val Leu Leu Leu Lys Asn Gly Gln Trp Asn Asp Val

180 185 190

Pro Cys Ser Thr Ser His Leu Ala Val Cys Glu Phe Pro Ile Met Glu

195 200 205

Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro

210 215 220

Lys Thr Ala Ser Asn Asn Cys Tyr Cys Lys Arg Cys Cys Leu His Cys

225 230 235 240

Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys

245 250 255

Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Lys Thr His Gln

260 265 270

Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp Pro Thr

275 280 285

Gly Pro Lys Glu

290

<210> 2

<211> 292

<212> PRT

<213> Artificial Sequence

<400> 2

Gly Cys Pro Gly Leu Pro Gly Ala Pro Gly Pro Lys Gly Glu Ala Gly

1 5 10 15

Thr Asn Gly Lys Arg Gly Glu Arg Gly Pro Pro Gly Pro Pro Gly Lys

20 25 30

Ala Gly Pro Pro Gly Pro Asn Gly Ala Pro Gly Glu Pro Ser Pro Asp

35 40 45

Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg Lys Ala Leu Gln Thr Glu

50 55 60

Met Ala Arg Ile Lys Lys Trp Leu Thr Phe Ser Leu Gly Lys Gln Val

65 70 75 80

Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu Ile Met Thr Phe Glu Lys

85 90 95

Val Lys Ala Leu Cys Val Lys Phe Gln Ala Ser Val Ala Thr Pro Arg

100 105 110

Asn Ala Ala Glu Asn Gly Ala Ile Gln Asn Leu Ile Lys Glu Glu Ala

115 120 125

Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu Gly Gln Phe Val Asp Leu

130 135 140

Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp Asn Glu Gly Glu Pro Asn

145 150 155 160

Asn Ala Gly Ser Asp Glu Asp Cys Val Leu Leu Leu Lys Asn Gly Gln

165 170 175

Trp Asn Asp Val Pro Cys Ser Thr Ser His Leu Ala Val Cys Glu Phe

180 185 190

Pro Ile Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Met Glu

195 200 205

Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro

210 215 220

Lys Thr Ala Ser Asn Asn Cys Tyr Cys Lys Arg Cys Cys Leu His Cys

225 230 235 240

Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys

245 250 255

Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Lys Thr His Gln

260 265 270

Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp Pro Thr

275 280 285

Gly Pro Lys Glu

290

<210> 3

<211> 292

<212> PRT

<213> Artificial Sequence

<400> 3

Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser

1 5 10 15

Gln Pro Lys Thr Ala Ser Asn Asn Cys Tyr Cys Lys Arg Cys Cys Leu

20 25 30

His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly

35 40 45

Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Lys Thr

50 55 60

His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp

65 70 75 80

Pro Thr Gly Pro Lys Glu Gly Cys Pro Gly Leu Pro Gly Ala Pro Gly

85 90 95

Pro Lys Gly Glu Ala Gly Thr Asn Gly Lys Arg Gly Glu Arg Gly Pro

100 105 110

Pro Gly Pro Pro Gly Lys Ala Gly Pro Pro Gly Pro Asn Gly Ala Pro

115 120 125

Gly Glu Pro Ser Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg

130 135 140

Lys Ala Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe

145 150 155 160

Ser Leu Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu

165 170 175

Ile Met Thr Phe Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala

180 185 190

Ser Val Ala Thr Pro Arg Asn Ala Ala Glu Asn Gly Ala Ile Gln Asn

195 200 205

Leu Ile Lys Glu Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu

210 215 220

Gly Gln Phe Val Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp

225 230 235 240

Asn Glu Gly Glu Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu

245 250 255

Leu Leu Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His

260 265 270

Leu Ala Val Cys Glu Phe Pro Ile Cys Met Tyr Ile Glu Ala Leu Asp

275 280 285

Lys Tyr Ala Cys

290

<210> 4

<211> 292

<212> PRT

<213> Artificial Sequence

<400> 4

Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Met Glu Pro Val

1 5 10 15

Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro Lys Thr

20 25 30

Ala Ser Asn Asn Cys Tyr Cys Lys Arg Cys Cys Leu His Cys Gln Val

35 40 45

Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg

50 55 60

Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Lys Thr His Gln Val Ser

65 70 75 80

Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp Pro Thr Gly Pro

85 90 95

Lys Glu Gly Cys Pro Gly Leu Pro Gly Ala Pro Gly Asp Lys Gly Glu

100 105 110

Ala Gly Thr Asn Gly Lys Arg Gly Glu Arg Gly Pro Pro Gly Pro Pro

115 120 125

Gly Lys Ala Gly Pro Pro Gly Pro Asn Gly Ala Pro Gly Glu Pro Ser

130 135 140

Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg Lys Ala Leu Gln

145 150 155 160

Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe Ser Leu Gly Lys

165 170 175

Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu Ile Met Thr Phe

180 185 190

Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala Ser Val Ala Thr

195 200 205

Pro Arg Asn Ala Ala Glu Asn Gly Ala Ile Gln Asn Leu Ile Lys Glu

210 215 220

Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu Gly Gln Phe Val

225 230 235 240

Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp Asn Glu Gly Glu

245 250 255

Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu Leu Leu Lys Asn

260 265 270

Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His Leu Ala Val Cys

275 280 285

Glu Phe Pro Ile

290

<210> 5

<211> 292

<212> PRT

<213> Artificial Sequence

<400> 5

Gly Cys Pro Gly Leu Pro Gly Ala Pro Gly Asp Lys Gly Glu Ala Gly

1 5 10 15

Thr Asn Gly Lys Arg Gly Glu Arg Gly Pro Pro Gly Pro Pro Gly Lys

20 25 30

Ala Gly Pro Pro Gly Pro Asn Gly Ala Pro Gly Glu Pro Cys Met Tyr

35 40 45

Ile Glu Ala Leu Asp Lys Tyr Ala Cys Ser Pro Asp Gly Asp Ser Ser

50 55 60

Leu Ala Ala Ser Glu Arg Lys Ala Leu Gln Thr Glu Met Ala Arg Ile

65 70 75 80

Lys Lys Trp Leu Thr Phe Ser Leu Gly Lys Gln Val Gly Asn Lys Phe

85 90 95

Phe Leu Thr Asn Gly Glu Ile Met Thr Phe Glu Lys Val Lys Ala Leu

100 105 110

Cys Val Lys Phe Gln Ala Ser Val Ala Thr Pro Arg Asn Ala Ala Glu

115 120 125

Asn Gly Ala Ile Gln Asn Leu Ile Lys Glu Glu Ala Phe Leu Gly Ile

130 135 140

Thr Asp Glu Lys Thr Glu Gly Gln Phe Val Asp Leu Thr Gly Asn Arg

145 150 155 160

Leu Thr Tyr Thr Asn Trp Asn Glu Gly Glu Pro Asn Asn Ala Gly Ser

165 170 175

Asp Glu Asp Cys Val Leu Leu Leu Lys Asn Gly Gln Trp Asn Asp Val

180 185 190

Pro Cys Ser Thr Ser His Leu Ala Val Cys Glu Phe Pro Ile Met Glu

195 200 205

Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro

210 215 220

Lys Thr Ala Ser Asn Asn Cys Tyr Cys Lys Arg Cys Cys Leu His Cys

225 230 235 240

Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys

245 250 255

Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Lys Thr His Gln

260 265 270

Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp Pro Thr

275 280 285

Gly Pro Lys Glu

290

<210> 6

<211> 292

<212> PRT

<213> Artificial Sequence

<400> 6

Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser

1 5 10 15

Gln Pro Lys Thr Ala Ser Asn Asn Cys Tyr Cys Lys Arg Cys Cys Leu

20 25 30

His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly

35 40 45

Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Lys Thr

50 55 60

His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp

65 70 75 80

Pro Thr Gly Pro Lys Glu Gly Cys Pro Gly Leu Pro Gly Ala Pro Gly

85 90 95

Asp Lys Gly Glu Ala Gly Thr Asn Gly Lys Arg Gly Glu Arg Gly Pro

100 105 110

Pro Gly Pro Pro Gly Lys Ala Gly Pro Pro Gly Pro Asn Gly Ala Pro

115 120 125

Gly Glu Pro Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Ser

130 135 140

Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg Lys Ala Leu Gln

145 150 155 160

Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe Ser Leu Gly Lys

165 170 175

Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu Ile Met Thr Phe

180 185 190

Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala Ser Val Ala Thr

195 200 205

Pro Arg Asn Ala Ala Glu Asn Gly Ala Ile Gln Asn Leu Ile Lys Glu

210 215 220

Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu Gly Gln Phe Val

225 230 235 240

Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp Asn Glu Gly Glu

245 250 255

Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu Leu Leu Lys Asn

260 265 270

Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His Leu Ala Val Cys

275 280 285

Glu Phe Pro Ile

290

Claims

1. A recombinant scaffold protein derived from epidermal growth factor, lectin and Tat protein, said scaffold protein consisting of an amino acid recombination sequence derived from the receptor binding domain of human epidermal growth factor, the collagen-like domain of human fibronectin, the glycosyl recognition domain of human mannose-binding lectin and Tat protein of HIV-1 virus;

the scaffold protein is SEQ ID NO: 1; or the scaffold protein is SEQ ID NO: 2; or the scaffold protein is SEQ ID NO: 3; or the scaffold protein is SEQ ID NO: 4; or the scaffold protein is SEQ ID NO: 5; or the scaffold protein is SEQ ID NO: 6.

2. Use of the scaffold protein according to claim 1 for the preparation of an anti-coronavirus drug, wherein the scaffold protein is used as a basis to obtain an affinity functional protein targeting coronavirus S protein by a display technique; obtaining the affinity functional protein taking the N-methyl-D-aspartate receptor as a target point, and the application in preparing diabetes drugs; obtaining affinity functional protein taking vascular endothelial growth factor as a target spot, and applying the affinity functional protein in preparing a medicine for treating corneal neovascularization; obtaining affinity functional protein with tumor necrosis factor-alpha as target spot and its application in preparing medicine for autoimmune disease; obtaining affinity functional protein with epidermal growth factor receptor-2 as target spot, and applying in preparing malignant tumor medicine; obtaining the affinity functional protein with the protein invertase subtilisin 9 as the target point, and the application in preparing the hypolipidemic drug.