CN103145798B

CN103145798B - Polypeptide of specific binding rare earth nanoparticle and screening method thereof

Info

Publication number: CN103145798B
Application number: CN201210260132.9A
Authority: CN
Inventors: 温龙平; 张云娇
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2012-06-07
Filing date: 2012-07-25
Publication date: 2014-09-10
Anticipated expiration: 2032-07-25
Also published as: CN103145798A

Abstract

The invention provides a polypeptide of a specific binding of rare earth nanoparticle and a screening method thereof. The polypeptide can be combined on surface of the nanoparticle to form a coating layer, thereby improving suspension ability of rare earth nanoparticles in water, preventing aggregation between particles, reducing interaction between particles, and reducing nonspecific adhesion ability of rare earth nanoparticles with cell surface, cell culture plate surface and other medium surfaces.

Description

Polypeptide specifically combined with rare earth nanoparticles and screening method thereof

Technical Field

The invention belongs to the field of nano biology, and particularly relates to a polypeptide specifically bound with rare earth nanoparticles and a screening method thereof.

Background

The up-conversion luminescent nanoparticles (UCN) based on rare earth are used as a new generation of bioluminescent markers, and compared with down-conversion luminescent markers such as organic fluorescent dyes and quantum dots, the up-conversion luminescent nanoparticles have the advantages of good photostability, high chemical stability, narrow absorption and emission bands, long luminescent life, small potential biotoxicity and the like. The upconversion luminous marker adopts near-infrared continuous laser as an excitation source, has the remarkable advantages of deeper light penetration depth, no biological background fluorescence interference, almost no damage to biological tissues and the like, and becomes an ideal marker for biological imaging. In addition, the rare earth up-conversion nano material is rare earth doped fluoride nano particles, has lower phonon energy, can reduce non-radiative transition and improve luminous intensity, is separated from a plurality of matrixes such as oxides, sulfides, phosphides and the like, and is widely applied to the fields of analysis and detection, disease treatment and the like in recent years.

However, rare earth upconversion nanoparticles as inorganic nanoparticles have presented a number of problems prior to their use in vivo applications and clinical trials. For example, it is easily agglomerated and insoluble in water, and is difficult to apply to the in vivo environment; when the compound is applied to in vivo biological imaging and in vivo photodynamic therapy, the compound can be non-specifically adhered to a plurality of tissues, cells and biomolecules to influence the action effect; enter cells or animals, which can cause severe autophagy and even cell death.

Currently, the surface modification method for inorganic nanoparticles mainly uses silica gel (CN 101434748), fatty acid (CN 1400167), polyethylene glycol (CN 101038290), chitosan (CN 101411893), protein, polypeptide, etc. to improve the surface characteristics of nanoparticles by various covalent and non-covalent means. Among these methods, of note is the surface modification by encapsulation with a coating of a specific binding peptide, which has uneconomical versatility, biocompatibility, simplicity and extendibility. At present, specific binding polypeptides mainly reported at home and abroad are mainly aimed at binding tissues, organs, cells and proteins in vivo and in vitro. For example, polypeptides that specifically bind to normal or cancerous tissue organs (CN 101531706, CN101827583A, CN1563078, CN102060909A, CN 101891803A); polypeptides that specifically bind to normal or cancerous cells (CN 101918433A, CN1709905, CN1763082, CN101033251, CN 1900108); and polypeptides that specifically bind to proteins in vivo (CN 102060913A, CN1823087, CN1262688, CN102105487A, CN101146822, CN1721432, CN1687128, CN101225108, CN101113164, CN 101481418). However, there are few reports of specific binding polypeptides for inorganic nanoparticles, the only ones being binding polypeptides for titanium, silver, silicon materials (CN 1829734) and high affinity binding peptides for metallic nickel (CN 1911956).

Disclosure of Invention

The invention provides a method for screening out polypeptide with the capacity of specifically binding rare earth nanoparticles by using a phage display library by using a phage display technology. The method of the invention comprises the following steps: (a) mixing and incubating a phage display polypeptide library and rare earth nanoparticles; (b) recovering the phage bound to the rare earth nanoparticles; (c) the amplified and recovered phage is used for the next round of combination screening of the rare earth nanoparticles; (d) repeating the steps (a) to (c) at least once; (e) and (4) picking a monoclonal from the recovered phage, and sequencing to obtain the encoded display polypeptide. In the examples, the polypeptide identified using the methods of the invention has a nucleotide sequence encoding display polypeptide CTARSPWIC (RE-0, SEQ ID NO: 1). The phage carrying the displayed polypeptide shows the ability to specifically bind to rare earth nanoparticles with high affinity.

The invention also provides the chemical synthesis of high affinity polypeptide and analogues thereof, which specifically bind to rare earth nanoparticles, and the analogues comprise amino acid sequences identified by phage display technology screening. Specific polypeptides, including amino acid sequences comprising phage-displayed peptides, and analogs thereof, have been shown to be capable of highly specific binding to rare earth nanomaterials. These rare earth nanomaterials include rare earth oxides, rare earth sulfides, rare earth oxysulfides, up-conversion luminescent nanomaterials based on rare earth doping, and all compounds containing rare earth elements. Any production techniques for such polypeptides or proteins known to those skilled in the art, including but not limited to expression of the peptides or proteins by standard molecular biology techniques such as recombinant techniques, isolation of the peptides or proteins from natural sources, or chemical synthesis of such peptides or proteins, are included within the scope of the present invention.

In a preferred embodiment, a polypeptide having sequence ACTARSPWICG (RE-1, SEQ ID NO: 2) was synthesized, where RE-1 includes the sequence of RE-0, the RE-0 sequence plus the two terminal amino acids A and G, derived from the coat protein of bacteriophage M13. In other embodiments, polypeptide analogs of RE-1 are also synthesized. These polypeptide analogs include RE-3 (ACWPATRISCG, SEQ ID NO: 4), which is formed by leaving each of the 2 amino acids at both ends of RE-1 unchanged and disrupting the sequence of the middle seven amino acids; RE-2X (ACTARSPWICGGGACTARSPWICG, SEQ ID NO: 13), which is composed of two RE-1 s. The high affinity ability of these polypeptides and their analogs to specifically bind to rare earth nanomaterials has been demonstrated.

In other embodiments, polypeptide analogs of RE-1 are also synthesized. These polypeptide analogs include RE-2 (TARSPWI, SEQ ID NO: 3), which is composed of the middle seven amino acids remaining after removal of 2 amino acids from both ends of RE-1; RE-4 (AATARSPWICG, SEQ ID NO: 5), which is a single amino acid substitution of RE-1 (C → A); RE-5 (ACAARSPWICG, SEQ ID NO: 6), which is a single amino acid substitution of RE-1 (T → A); RE-6 (ACTAASPWICG, SEQ ID NO: 7), which is a single amino acid substitution of RE-1 (R → A); RE-7 (ACTARAPWICG, SEQ ID NO: 8), which is a single amino acid substitution of RE-1 (S → A); RE-8 (ACTARSAWICG, SEQ ID NO: 9), which is a single amino acid substitution of RE-1 (P → A); RE-9 (ACTARSPAICG, SEQ ID NO: 10), which is a single amino acid substitution of RE-1 (W → A); RE-10 (ACTARSPWACG, SEQ ID NO: 11), which is a single amino acid substitution of RE-1 (I → A); RE-11 (ACTARSPWIAG, SEQ ID NO: 12), which is a single amino acid substitution of RE-1 (C → A). These polypeptides and analogs thereof have demonstrated the ability to bind rare earth nanomaterials with general affinity.

In other embodiments, polypeptide analogs of RE-1 are also synthesized. These polypeptide analogs include RE-Ag (ACTARSPWICGGGNPSSLFRYLPSD, SEQ ID NO: 14), which is composed of RE-1 and metallic silver specific binding peptide (NPSSLFRYLPSD), which has been shown to bind specifically with high affinity to both rare earth nanoparticles and metallic silver. RE-Ti (RKLPDAGGGACTARSPWICG, SEQ ID NO: 15), which is composed of RE-1 and metallic titanium specific binding peptide (RKLPDA), has been demonstrated to bind specifically with high affinity to both rare earth nanoparticles and metallic titanium. RE-AT (CLSYYPSYCGGACTARSPWICG, SEQ ID NO: 16), which is composed of RE-1 and apoptotic cell targeting peptide (CLSYYPSYC), has been shown to not only bind specifically with high affinity to rare earth nanoparticles, but also target rare earth nanoparticles to the apoptotic cell surface. RE-PH (SMSIARLGGACTARSPWICG, SEQ ID NO: 17), which is composed of RE-1 and prostate tissue targeting peptide (SMSIARL), has been shown to not only bind specifically with high affinity to rare earth nanoparticles, but also target rare earth nanoparticles to prostate tissue. RE-BH (CLEVSRKNCGGACTARSPWICG, SEQ ID NO: 18), which consists of RE-1 and brain tissue targeting peptide (CLEVSRKNC), has been shown to not only bind specifically with high affinity to rare earth nanoparticles, but also target rare earth nanoparticles to brain tissue. RE-LH (CGFECVRQCPERCGGACTARSPWICG, SEQ ID NO: 19), which consists of RE-1 and lung tissue targeting peptide (CGFECVRQCPERC), has been shown to not only bind specifically with high affinity to rare earth nanoparticles, but also target rare earth nanoparticles to lung tissue. RE-DH (ACTARSPWICGGPKKKRKVC, SEQ ID NO: 20), which is composed of RE-1 and a nucleic acid localization Peptide (PKKKRKV), has been shown to not only bind specifically with high affinity to rare earth nanoparticles, but also target rare earth nanoparticles to the nucleus. RE-SMAC (ACTARSPWICGGGAVPIAQK, SEQ ID NO: 21), which is composed of RE-1 and the pro-apoptotic polypeptide SMAC (AVPIAQK), has been shown to not only bind specifically with high affinity to rare earth nanoparticles, but also target rare earth nanoparticles to apoptotic cells. RE-1-RGD (CRGDCGGACTARSPWICG, SEQ ID NO: 22), which is composed of RE-1 and tumor cell targeting peptide RGD, has been shown to not only bind specifically with high affinity to rare earth nanoparticles, but also target rare earth nanoparticles to tumor cells.

The invention also provides isolated nucleotides, homologs and analogs that encode, or hybridize under stringent conditions to, a nucleotide sequence encoding, for example, polypeptide RE-0 (SEQ ID NO: 1), RE-1 (SEQ ID NO: 2), RE-2X (SEQ ID NO: 13), RE-3 (SEQ ID NO: 4) or a portion thereof, or a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 13. SEQ ID NO: 4 or the amino acid sequences shown in the parts of the sequences have the high-affinity ability of specifically binding the rare earth nanoparticles. Further, the invention also provides nucleotides, homologs and analogs including those encoding SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 13. SEQ ID NO: 4, a portion thereof, or a nucleotide sequence that is the complement thereof.

The invention also provides a method for evaluating the suspension capacity of the nano material, which comprises the following steps: (a) placing the nanometer material suspension in an ultraviolet detection pool; (b) the ultraviolet characteristic absorption wavelength of the nano material is used for carrying out real-time observation and scanning on the nano material in the sample cell to obtain a time dynamics curve; (c) and (4) evaluating the suspension capacity of the nano material according to a time kinetic curve.

The invention also provides a method for evaluating the sedimentation rate and the diffusion capacity of the nano material and the interaction with cells, which comprises the following steps: (a) culturing cells on a cover glass, respectively placing the cells with the front side facing upwards or the cells with the front side facing downwards in a cell culture plate, and placing the cover glass on a small glass body; (b) pouring the nano material culture medium into a cell culture plate, so that the cells are fully immersed in the culture medium, and the position of the cell cover glass is just positioned at the middle height of the liquid; (c) observing the bound nanomaterial on the cell surface with a fluorescence microscope equipped with an infrared laser; (d) and evaluating the sedimentation rate and the diffusion capacity of the nano material and the interaction with cells according to the fluorescence observation result.

The invention also provides a device for evaluating the sedimentation rate and the diffusion capacity of the nano material and the interaction with cells, which comprises a cell culture plate, a cover glass and a small glass body, wherein the small glass body is positioned in the cell culture plate, and the cover glass is erected on the small glass body.

The invention provides a polypeptide capable of specifically binding rare earth nanoparticles with high affinity, which can be bound on the surfaces of the nanoparticles to form a coating layer, so that the suspension performance of the rare earth nanoparticles in water is improved, the aggregation among the particles is prevented, the interaction among the particles is reduced, and the nonspecific adhesion capacity of the rare earth nanoparticles to the surfaces of cells, the surfaces of cell culture plates and other media is also reduced.

Drawings

FIG. 1 shows the specificity with Nd₂O₃Number of phage bound results.

FIG. 2 is a graph showing the confirmation of REOB-1 phage and Nd₂O₃PCR results plot of binding capacity.

FIG. 3 shows the different concentrations of RE-1 short peptide vs. REOB-1 phage and Nd₂O₃Results of inhibition of binding ability.

FIG. 4 is a graph showing the binding ability of RE-1 short peptide with different inorganic nano-materials.

FIG. 5 is a graph showing the results of the characterization of UCN binding to RE-1. Wherein, FIG. 5A is a transmission electron micrograph of the combination of UCN and RE-1 short peptide; FIG. 5B is a transmission electron micrograph of UCN bound to REOB-1 phage; FIG. 5C is a scanning electron micrograph of the binding of UCN to RE-1 short peptide.

Fig. 6 is a graph showing the results of particle size analysis of the upconversion luminescent nanomaterials. Wherein, fig. 6A is a particle size distribution of UCN; FIG. 6B is a particle size distribution of UCP; FIG. 6C shows the particle size distribution of UCN-S.

FIG. 7 is a graph of the binding of the upconversion luminescent nanomaterials to RE-1. Wherein FIG. 7A (UCN) and FIG. 7B (UCP) are combined time gradient curves; FIG. 7C (UCN), FIG. 7D (UCP) and FIG. 7E (UCN-S) are combined concentration gradient curves.

FIG. 8 is a graph of the effect of the environment on the binding of the upconversion luminescent nanomaterial to RE-1. Wherein FIG. 8A (UCN) and FIG. 8B (UCP) are the effect of different pH values; FIG. 8C (UCN) and FIG. 8D (UCP) are the effect of different salt ion concentrations; FIG. 8E (UCN) and FIG. 8F (UCP) are the effects of different temperatures.

FIG. 9 shows the dissociation of RE-1 bound to the upconversion luminescent nanomaterial over time. Wherein, fig. 9A shows the dissociation result of UCN; fig. 9B shows the dissociation result of UCP.

FIG. 10 is a graph showing the results of time-kinetic curves of RE-1 improving the suspension ability of the upconversion luminescent nanomaterial. Wherein, fig. 10A is a time kinetic curve of UCN; fig. 10B is a time kinetic curve of UCP.

FIG. 11 is a graph showing the results of the detection of nonspecific adhesion ability of RE-1 to reduce the upconversion luminescent nanomaterial. Wherein, fig. 11A and 11B are detection results of UCNs; FIGS. 11C and 11D show the results of UCN-S detection.

FIG. 12 is a graph showing the results of the detection of RE-1 reducing the diffusion ability of the upconversion luminescent nanomaterial in a solution. Wherein, fig. 12A and 12B are schematic diagrams of the experimental apparatus; fig. 12C, 12D, and 12E show the detection results of UCNs; fig. 12F, 12G, and 12H show the detection results of UCP.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Examples1. Screening specificity and Nd by phage display technology₂O₃Conjugated short peptides

For screening specificity and Nd₂O₃The library of bound peptides, provided by New England Biolabs (Mass, MA), is a library of heptapeptides containing disulfide bonds (Ph.D. -C7C). The random fragments in this library are flanked by cysteine residues that can be oxidized during phage assembly to form disulfide bonds, thus forming a cyclic peptide that interacts with the target. This library contained over two billion clones. The random peptide in the library was at the amino terminus of the small coat protein pIII, so five copies were expressed per phage particle. The position of the phage expression random sequence in the ph.d. -C7C library was preceded by alanine-cysteine. A short linker sequence (glycine-serine) is contained between the random peptide and the pIII protein. (Ph.D.^TM-C7C phage peptide display kit, http:// www.neb.com/nebecom/products E8120. asp). 2. mu.l of 10¹⁰Phage and 1ml Nd₂O₃(1 mg/ml, available from Whitzel chemical, Guangzhou.) and incubated at 37 ℃ for 2h in a shaker, after centrifugation at 12000rpm for 10min, the pellet was washed 10 times with TBST (containing 2% Tween-20, pH 7.5) to bind Nd₂O₃The phage were recovered and then mixed with 0.5ml of E.coli ER2378 (purchased from New England Biolabs, Mass., MA), incubated for 0.5h and amplified for 6h in 20ml of LB medium (tryptone 0.2 g; yeast extract 0.1 g; sodium chloride 0.2 g). The amplified phage were resuspended in phosphate buffer (150 mM NaCl, 50mM Tris-HCl, pH 7.5) and used for a second round of selection. Approximately 100 phages were recovered in the first round, and the third round of amplified phages were mixed with Nd₂O₃The binding efficiency of (A) was improved by two orders of magnitude over the phage library (FIG. 1).

The third round of recovered phage was plated on LB plates containing X-gal (5-bromo-4-chloro-3-indolyl-. beta. -D-galactolactose) and IPTG (isoproyl-. beta. -D-thiogalactoside), and 15 blue plaques were randomly picked and sequenced using an automatic DNA sequencer (ABI 3730). 12 of these (designated REOB-1) contained identical inserts, and the encoded peptide sequence was CTARSPWIC (RE-0, SEQ ID NO: 1).

Example 2Identification of REOB-1 phage and Nd₂O₃Bonding force of (2)

The titer was 10⁵The REOB-1 phage and the titer of 10⁸AP-1 phage (random phage randomly selected from phage library, used as a control, encoding a peptide sequence of CNATLPHQC, SEQ ID NO: 23) of (1) was mixed with 100. mu.l of Nd₂O₃(1 mg/ml) was incubated at 37 ℃ for 2h, centrifuged at 12000rpm for 10min, the pellet was washed 10 times with TBST (containing 2% Tween-20, pH 7.5), and phage bound in the pellet were eluted with 1ml of 0.2M glycine-hydrochloric acid (pH 2.2) containing 1mg/ml BSA and plated on LB plates. Randomly pick 50 blue plaques, and each plaque is subjected to a double PCR reaction simultaneously. One was performed with a primer specific to REOB-1 phage (5'-CGAGGTCGCCTTGGATTTGC-3') and one was performed with a primer specific to AP-1 phage (5'-GACCAGTCGCATCCGCAGCA-3'). The additional primers required for the PCR reaction consist of sequences on the vector, shared for both reactions. The PCR product was subjected to 0.8% agarose gel electrophoresis, and the result was photographed by a gel imaging system (Tanon 1600, Tanon, China). The results show that, since the titers of the added REOB-1 phage and AP-1 phage differ by a factor of 1000, the results of PCR REOB-1: AP-1= 12: 1, thus REOB-1 phage and Nd₂O₃The binding force was 12000 times higher than for the AP-1 phage.

Examples3. Chemical synthesis of specific binding peptides and analogs thereof

The polypeptides in this application were synthesized by Shanghai Jier Biochemical company using standard FMOC solid phase synthesis in an automated polypeptide synthesizer (CS 536-1381, CS Bio Co., Menlo Park, Calif.) and purified to greater than 95% purity by HPLC and molecular weights were determined using a mass spectrometer (ThermoFisher Scientific, Thermo, USA).

The peptide of sequence ACTARSPWICG (RE-1, SEQ ID NO: 2) was synthesized as follows. The terminal amino acids alanine and glycine were derived from the M13 surface protein. RE-1 was synthesized using standard FMOC solid phase synthesis, generally by the manual synthesis procedure as follows: approximately 0.2mM Fmoc-Gly-Wang resin was added to a manual reaction tube (Peptide International) and DMF was added to swell it for 2 h. The Fmoc protecting group was then removed by addition of 20% piperidine/DMF for 2min and the deprotection step was repeated once and ice reacted for 20 min. A positive Kaiser test indicated free amino groups on the resin. Amino acid residues were added to the resin according to the following cycle method: a three-fold excess of Fmoc protected amino acids, a three-fold excess of HOBT and a three-fold excess of DIPEA/DMF were added to the reaction tube according to the sequence of RE-1. In N₂The coupling reaction was carried out for 2h in stream and the reaction tubes were evacuated with a vacuum pump. The N-terminally protected peptide was washed with DMF (5X 1 min) to remove excess reactants, and then the Fmoc protecting group was removed with 20% piperidine/DMF as before. The reaction tube was washed with DMF to remove the piperidine and repeated steps were performed to couple the next amino acid in the sequence. Synthesis of N with side chain protection^aAfter Fmoc-Ala-Cys-Thr-Ala-Arg-Ser-Pro-Trp-Ile-Cys-Gly-resin conjugate, the peptide resin conjugate was washed with DMF (4X 1 min) and dried in vacuo. The amino-terminal Fmoc protecting group was removed with 20% piperidine/DMF. Deprotection of the side chains and cleavage of the peptide resin bond were reacted with 10ml of lysis buffer (95% trifluoroacetic acid, 2.5% water and 2.5% triisopropylsilane) for 3h with shaking at 500 rpm. The reaction was transferred to a pre-weighed Erlenmeyer flask and precipitated with ice dry ether. The crude product was dried in vacuo for 48 h. 70-100mg of the crude product was purified by reverse phase high performance liquid chromatography (RP-HPLC) and lyophilized to a powder. The purity of the peptide was determined by analytical RP-HPLC and its molecular weight was determined by mass spectrometry.

Control peptide ACNATLPHQCG (AP-1, SEQ ID NO: 24) and polypeptide analogs and derivatives were synthesized using the same method described above. AP-1 is an 11 peptide with identical terminal amino acids and contains irrelevant internal sequences. FITC was coupled to the amino terminus of RE-1 and AP-1 via Acp.

Examples4. Identification of polypeptide analogs and Nd₂O₃Bonding force of (2)

100. mu.g of Nd₂O₃Mixing with polypeptide such as RE-1, AP-1, RE-2, RE-3, RE-4, RE-5, RE-6, RE-7, RE-8, RE-9, RE-10, etc., respectively, and adding the mixture to a mixer with titer of 10⁸The REOB-1 phage was incubated for 2 hours, and Nd was detected₂O₃Amount of bound REOB-1 phage. IC (integrated circuit)₅₀The values reflect the relative activity of various polypeptides to compete for inhibition of REOB-1 phage binding to neodymia nanometers. The results are shown in Table 1, with polypeptide RE-1 and its analogs and Nd₂O₃With varying degrees of binding.

TABLE 1 Polypeptides RE-1 and analogs thereof with Nd₂O₃Bonding force of (2)

Examples5. RE-1 short peptide inhibits REOB-1 and Nd₂O₃In combination with

Mixing RE-1 short peptide (0. mu.g/ml, 1. mu.g/ml, 10. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 500. mu.g/ml) with different concentration gradient and the content of the mixture with the titer of 10⁸100. mu.l Nd of REOB-1 phage₂O₃(1 mg/ml) was incubated at 37 ℃ for 2h, the pellet was washed 10 times, and bound Nd was recovered₂O₃The bacteriophage of (1). Meanwhile, 5000. mu.g/ml of AP-1 short peptide was used as a control in the same manner. The results show that when RE-1 and REOB-1 are combined with Nd₂O₃In combination, RE-1 couples REOB-1 with Nd as the dose increases₂O₃Has inhibitory effect on binding ability of (A). When it exceeds 10. mu.g/ml, RE-1 almost completely inhibits REOB-1 and Nd₂O₃Binding capacity (FIG. 3). The RE-1 short peptide is proved to have competitive inhibition effect on REOB-1 phage, REOB-1 and Nd₂O₃Due to the specific interaction of the peptide it displays with the nanomaterial surface.

Examples6. Testing the binding capacity of RE-1 short peptide and various nano materials

Mu.g of FITC-RE-1 polypeptide is respectively incubated with 100 mu.l of a plurality of different inorganic nano-materials (1 mg/ml) for 1h at room temperature, then the mixture is centrifuged at 12000rpm for 10min, and after 3 times of precipitate washing, FITC (emission wavelength is 535 nm) is detected by a fluorescence spectrophotometer (RF-5301 PC, Shimadzu, Japan), and a standard curve of the relationship between FITC-RE-1 fluorescence and concentration is used as a control to obtain the binding capacity of the different materials and the FITC-RE-1 polypeptide. The results show that RE-1 has stronger binding capacity with rare earth metal oxide and rare earth element doped up-conversion luminescent nano-material, wherein Y₂O₃，CeO₂，Yb₂O₃，Nd₂O₃The binding ability of UCN (upconversion luminescent nanomaterials), UCN-S (small particles of UCN) and UCP (large particles of UCN) is strongest. And RE-1 with non-rare earth materials, e.g. diamond, TiO₂，SiO₂，SiO₃And nanosilver were almost unbound (fig. 4).

Examples7. Method for synthesizing UCN (NaYF 4: Yb, Er)

Spherical NaYF 4: the 18% Yb, 2% Er nanoparticles were synthesized as follows. Yttrium chloride (0.1562 g), ytterbium chloride (0.0503 g), erbium chloride (0.0055 g) were mixed with 3ml of oleic acid and 17ml of octadecene in a 50ml flask, heated to 160 ℃, and then cooled to room temperature. Sodium hydroxide (0.01 g) and ammonium fluoride (0.148 g) were dissolved in 10ml of methanol and added dropwise to the flask, and stirred for 30min to ensure sufficient reaction of the chloride. The solution was slowly heated to evaporate the methanol, degassed at 100 ℃ for 10min, then heated to 300 ℃ under argon and maintained for 1 h. The solution is naturally cooled and the nano-crystals are precipitated. After precipitation with ethanol and three washes with ethanol/water (1: 1 by volume), the surface was treated with 1M hydrochloric acid for 5h at room temperature to remove oleic acid and washed 10 times with water and cyclohexane. The characteristic absorption peak of oleic acid at 230nm was detected to ensure its complete removal. UCN and UCN-S were synthesized by adding varying amounts of oleic acid (UCN: 3ml oleic acid, UCN-S: 6ml oleic acid). The synthesized UCN was stored in water for use.

Examples8. Observation of binding form of RE-1 short peptide to UCN

The binding pattern of RE-1 short peptide and REOB-1 phage to UCN was observed using a transmission electron microscope. The complex of RE-1 and UCN bound together was washed away with excess, unbound polypeptides and phage, and the bound product was stained with uranium acetate (ph 6.7) and placed on a carbon film-coated copper mesh. The images were observed using a transmission electron microscope TEM (JEM-2100F, JEOL, Japan) at 200 kv (fig. 5A and 5B).

The binding form of RE-1 short peptide to UCN was observed by scanning electron microscope. The complex of RE-1 and UCN bound together was washed away for excess unbound polypeptide, the bound product was placed on a copper plate, and the image was observed using a 15 kV scanning electron microscope SEM (JEOL JSM-6700F, JEOL, Japan) (FIG. 5C).

The results show that REOB-1 phage can be tightly bound to UCN surface with high affinity, and RE-1 short peptide can form stable peptide layer on UCN surface.

Examples9. Identification of up-conversion luminescent nano material (UCN, UCP, UCN-S) in nano scale

The synthesized UCN and UCN-S and UCP (Phosphor Technology, UK) subjected to ultrasonic disintegration sedimentation were dissolved in water, the solution was placed in a square polystyrene cell (DTS 0012, Malvern, UK), and the particle size distribution of the nanoparticles was measured using a Malvern Nano laser particle sizer (Nano ZS90, Malvern, UK) with a 633nm helium/neon laser. The results show that the size scale for UCN is about 92nm (fig. 6A), for UCP is about 500nm (fig. 6B), and for UCN-S is about 20nm (fig. 6C).

Examples10. Testing the binding capacity of RE-1 short peptide and up-conversion luminescent nano material (UCN, UCP, UCN-S)

Mu.g of FITC-RE-1 was incubated with 100. mu.g of UCN for various periods of time (2 min, 10min, 30min, 120min, 720 min), and the amount of FITC-RE-1 fluorescent peptide bound to UCN was then detected using a spectrofluorometer. The results show that RE-1 binds to UCN very rapidly, 50% already at 2min, and almost saturation of binding is achieved at 10min (FIG. 7A).

Mu.g of FITC-RE-1 was incubated with 100. mu.g of UCP for various periods of time (2 min, 10min, 30min, 120min, 720 min), and the amount of FITC-RE-1 fluorescent peptide bound to UCP was then detected using a spectrofluorometer. The results show that the binding of RE-1 to UCP also has a time-kinetic distribution, and at 2min, 50% is already bound, and 10min almost reaches the binding saturation (FIG. 7B).

After incubating 100. mu.g of UCN with different concentrations (5. mu.g, 10. mu.g, 25. mu.g, 50. mu.g, 100. mu.g) of FITC-RE-1 for 1h, excess unbound peptides were washed off, and the amount of FITC-RE-1 fluorescent peptide bound to UCN was then detected using a spectrofluorometer. The results showed that 100. mu.g of UCN was able to bind 47. mu.g of FITC-RE-1 (FIG. 7C).

After incubating 100. mu.g of UCP with different concentrations (1. mu.g, 5. mu.g, 10. mu.g, 20. mu.g) of FITC-RE-1 for 1h, excess unbound peptides were washed off, and the amount of FITC-RE-1 fluorescent peptide bound to UCP was then detected using a spectrofluorometer. The results showed that 20. mu.g of UCP was able to bind 9.2. mu.g of FITC-RE-1 (FIG. 7D).

After incubating 100. mu.g of UCN-S with different concentrations (50. mu.g, 100. mu.g, 200. mu.g, 300. mu.g, 400. mu.g) of FITC-RE-1 for 1h, excess unbound peptides were washed away, and the amount of FITC-RE-1 fluorescent peptide bound to UCN-S was then detected using a spectrofluorometer. The results showed that 400. mu.g of UCN-S was able to bind 301. mu.g of FITC-RE-1 (FIG. 7E).

Examples11. Influence of solution environment on binding capacity of RE-1 short peptide and up-conversion luminescent nano material (UCN, UCP)

After incubating 50. mu.g of FITC-RE-1 with 100. mu.g of UCN dissolved at different pH values (3, 5, 7, 10) for 1h, the excess unbound peptides were washed off and the amount of FITC-RE-1 fluorescent peptide bound to UCN was measured by a spectrofluorometer. The results show that the binding of FITC-RE-1 to UCN is not sensitive to pH changes in the solution environment (FIG. 8A).

After incubating 10. mu.g of FITC-RE-1 with 100. mu.g of UCP dissolved at different pH values (3, 5, 7, 10) for 1h, the excess unbound peptides were washed off and the amount of FITC-RE-1 fluorescent peptide bound to UCP was measured using a spectrofluorometer. The results show that the binding of FITC-RE-1 to UCP is not sensitive to pH changes in the solution environment (FIG. 8B).

After incubating 50. mu.g of FITC-RE-1 with 100. mu.g of UCN dissolved in solutions of different salt ion concentrations (0M, 0.15M, 0.3M, 0.5M sodium chloride) for 1h, excess unbound peptides were washed off, and the amount of FITC-RE-1 fluorescent peptide bound to UCN was then detected using a spectrofluorometer. The results show that the binding of FITC-RE-1 to UCN is not sensitive to changes in the salt ion concentration in the solution environment (FIG. 8C).

After incubating 10. mu.g of FITC-RE-1 with 100. mu.g of UCP dissolved in solutions of different salt ion concentrations (0M, 0.15M, 0.3M, 0.5M sodium chloride) for 1h, excess unbound peptides were washed off, and the amount of FITC-RE-1 fluorescent peptide bound to UCP was then detected using a spectrofluorometer. The results show that binding of FITC-RE-1 to UCP is not sensitive to changes in the salt ion concentration in the solution environment (FIG. 8D).

50 mu g of FITC-RE-1 and 100 mu g of UCN dissolved in water are incubated for 1h in environments with different temperatures (25 ℃, 37 ℃, 55 ℃ and 80 ℃), redundant unbound peptides are washed away, and then the amount of FITC-RE-1 fluorescent peptide bound to UCN is detected by a fluorescence spectrophotometer. The results showed that binding of FITC-RE-1 to UCN was not sensitive to changes in ambient temperature (FIG. 8E).

After incubating 10. mu.g of FITC-RE-1 and 100. mu.g of UCP dissolved in water in different temperature environments (25 ℃, 37 ℃, 55 ℃ and 80 ℃) for 1h, washing off the surplus unbound peptides, and then detecting the amount of FITC-RE-1 fluorescent peptide bound to UCP by using a fluorescence spectrophotometer. The results showed that binding of FITC-RE-1 to UCP was not sensitive to changes in ambient temperature (FIG. 8F).

In summary, RE-1 and the up-conversion luminescent nano-materials (UCN, UCP) have extremely strong binding capacity, and the change of the surrounding environment can not destroy the binding force between the peptide and the nano-materials.

Examples12. Testing the dissociation condition of RE-1 short peptide and up-conversion luminescent nano material (UCN, UCP)

Mu.g of FITC-RE-1 was well bound to 500. mu.g of UCN, excess unbound polypeptides were washed off, the mixture was then divided evenly into five equal portions, gently shaken on a shaker at 37 ℃ and the amount of dissociated FITC-RE-1 in the supernatant was measured with a spectrofluorometer at different time points (0 h, 2h, 12h, 24h, 48 h), respectively. The results showed that RE-1 was stably associated with UCN, and only dissociated after 48h to less than 0.7% (FIG. 9A).

The amount of FITC-RE-1 dissociated in the supernatant was determined by mixing 50. mu.g of FITC-RE-1 with 500. mu.g of UCP according to the above procedure. The results showed that RE-1 was also stably associated with UCP, and only dissociated after 48h to less than 1.5% (FIG. 9B).

Examples13. RE-1 reduces the sedimentation velocity of the up-conversion luminescent nano material (UCN, UCP)

3mg of UCN was combined with 1.5mg of RE-1 and its analogues RE-2, RE-3, RE-5 and PEG, respectively, according to the binding method, excess unbound RE-1 was washed off, the precipitate was resuspended in 3ml of water, and after 1min of sonication, placed in an ultraviolet detection cell (X72053, Alpha Laboratories Ltd, UK), and then continuously observed for 120min at an absorption wavelength of 500nm with an ultraviolet spectrophotometer (DU-640, Beckman, USA), to obtain a time kinetic curve. The results showed that UCN without encapsulated RE-1 short peptide precipitated to the bottom of the pool very quickly, while UCN encapsulated RE-1 short peptide remained suspended after resting for 120min, indicating that RE-1 could effectively reduce the sedimentation rate of UCN (FIG. 10A).

A time kinetic curve was obtained by operating 3mg of UCP with 500. mu.g of RE-1 and its analogues RE-2, RE-3, RE-4, RE-5, RE-6, RE-7, RE-8, RE-9, RE-10 and AP-1, respectively, according to the above procedure. The results showed that UCP without encapsulated RE-1 short peptide precipitated to the bottom of the pool very quickly, while UCP encapsulated RE-1 short peptide remained suspended after resting for 120min, indicating that RE-1 could effectively reduce the sedimentation rate of UCP (FIG. 10B).

Examples14. RE-1 reduces the nonspecific adhesion capability of the up-conversion luminescent nano-material (UCN, UCN-S)

Live Hela cells (from shanghai biochemical institute of China academy of sciences), fixed cells and cell culture plates cultured in 96 wells were treated with unencapsulated and RE-1-encapsulated UCN and UCN-S, respectively, and after 2h of treatment, washed 3 times with PBS, and then observed for UCN nanoparticles bound to live Hela cells, fixed cells and cell culture plates with a fluorescence microscope (Olympus IX71, Olympus, Japan) equipped with a 980nm infrared laser (MDL-980 nm 1W, Changchun New Industries Optoelectronics tech. The results showed that non-specific adhesion of RE-1-coated UCN group to Hela live cells, fixed cells and cell culture plates was reduced by about 10-fold compared to non-RE-1-coated UCN group (fig. 11A and 11B); non-specific adhesion of RE-1-coated UCN-S groups to Hela live cells, fixed cells, and cell culture plates was reduced by about 100-fold compared to RE-1-uncoated UCN-S groups (FIGS. 11C and 11D).

Examples15. RE-1 reduces the diffusion capability of up-conversion luminescent nano-materials (UCN, UCP) in solution

Hela cells are cultured on a cover glass (length and width are 22 mm), the cells at the front side are respectively placed in a 6-well plate, the cover glass is put on a sterilized small glass body (length and width are 2 mm), then 4ml of DMEM culture medium (Gibco, USA) containing the up-conversion luminescent nano materials (UCN, UCP) which are not wrapped and wrapped with RE-1 is slowly poured into a six-well plate, so that the cells are fully immersed in the culture medium, and the position of the cell cover glass is just positioned at the middle height of the liquid. And placed in a cell culture chamber at 37 ℃ for 2h, followed by washing 3 times with PBS, and observing the UCNs bound on the cell surface with a fluorescence microscope (Olympus IX71, Olympus, Japan) equipped with a 980nm infrared laser (MDL-980 nm 1W, Changchun New Industries optics Tech.Co., Ltd., China). The results show that for the experimental group with the cell front facing upwards, the upconversion luminescent nanomaterial group without coating RE-1 has a large amount of non-specific adhesion particles, while the upconversion luminescent nanomaterial group coating RE-1 has a great reduction in the amount of cell surface adhesion upconversion luminescent nanoparticles; for the experiment group with cells facing downwards, the non-specific adhesion particles of the upconversion luminescent nanomaterial group without coating RE-1 are reduced slightly compared with the group with cells facing upwards, but the upconversion luminescent nanoparticles adhered on the surface of the cells are much more than those of the upconversion luminescent nanomaterial group coating RE-1. It is demonstrated that RE-1 not only reduces the sedimentation rate of UCNs and UCPs but also reduces the diffusion ability of the upconversion luminescent nanomaterials, whereby RE-1 achieves a reduction of the nonspecific adhesion ability of the upconversion luminescent nanomaterials (fig. 12A and 12B are schematic diagrams of experimental devices, fig. 12C, 12D and 12E are results of UCNs, and fig. 12F, 12G and 12H are results of UCPs).

Claims

1. A polypeptide that specifically binds to a rare earth nanoparticle, wherein the polypeptide is:

(a) as shown in SEQ ID NO: 1;

(b) polypeptide sequences derived from (a) which are subjected to addition, deletion, order change or substitution of one or more amino acids in the polypeptide sequences defined in (a) and are capable of specifically binding to rare earth nanoparticles;

wherein,

the polypeptide sequence which is added with one or more amino acids in the polypeptide sequence defined by (a) and is derived from (a) and can be specifically combined with the rare earth nanoparticles is shown as SEQ ID NO: 2; the polypeptide sequence which is obtained by deleting one or more amino acids in the polypeptide sequence defined by the step (a) and can be specifically combined with the rare earth nanoparticles and is derived from the step (a) is SEQ ID NO: 3; the polypeptide sequence which is subjected to sequence conversion in the polypeptide sequence defined by the step (a) and is derived from the step (a) and can be specifically combined with the rare earth nanoparticles is SEQ ID NO: 4;

the polypeptide sequence which is substituted by one or more amino acids in the polypeptide sequence defined by the (a) and can be specifically combined with the rare earth nanoparticles and is derived from the (a) is SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12.

2. A nucleotide sequence comprising a nucleotide sequence encoding the polypeptide sequence of claim 1.