Recombinant protein of recombinant human Fc and P-selectin, detection product and preparation method and application thereof

Abstract

The invention discloses a recombinant protein of recombinant human Fc and P-selectin, a detection product as well as a preparation method and application of the detection product, and relates to the technical field of tumor cell capture. The recombinant protein of the recombinant human Fc and P-selectin comprises a C-type lectin, an EGF-like domain and a human Fc segment from an N terminal to a C terminal. The recombinant protein is combined with the target cells only through the interaction structural domain of the P-selectin, so that non-specific adhesion is reduced, and the purity of the target cells and the capture efficiency of the target cells are improved.

Description

Technical Field

[0001] This invention relates to the field of tumor cell capture technology, and more specifically, to a recombinant protein of recombinant human Fc and P-selectin, a detection product, a preparation method thereof, and its application. Background Technology

[0002] Microfluidics has important applications in the capture of circulating tumor cells (CTCs), but traditional methods face two major challenges: background interference caused by nonspecific cell adhesion and insufficient specific capture efficiency.

[0003] Non-specific cell adhesion is one of the main challenges in high-throughput target cell separation using microfluidic technology, leading to reduced separation efficiency and limiting high-throughput processing capabilities. Studies have shown that robust and efficient protein coating processes are key to achieving effective target cell capture. Furthermore, previous research has demonstrated the existence of non-domain-specific interactions between proteins (Johnson ME et al., 2011; Krishnan R et al., 2022). Therefore, in immunoaffinity assays using intact protein molecule coatings, background non-specific adhesion may occur, affecting target cell purity and capture efficiency.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant protein of recombinant human Fc and P-selectin, a detection product, a preparation method thereof, and an application thereof to reduce non-specific adhesion and improve the purity and capture efficiency of target cells.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a recombinant protein of recombinant human Fc and P-selectin, which comprises, from the N-terminus to the C-terminus, a C-type lectin, an EGF-like domain, and a human Fc segment, wherein the EGF-like domain and the human Fc segment may or may not have a linker peptide, wherein the amino acid sequences of the C-type lectin and the EGF-like domain are as shown in SEQ ID NO: 1-2, respectively, and the human Fc segment is selected from human IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc.

[0007] Secondly, the present invention also provides a detection product, a separation product, or an enrichment product, comprising: a solid support, the aforementioned recombinant protein, and an affinity protein, wherein the affinity protein binds to the Fc region of the recombinant protein.

[0008] Thirdly, the present invention also provides a detection product, separation product or enrichment product, comprising: a solid support, and having an affinity protein layer on the solid support for binding the Fc region of the recombinant protein described above, and having a recombinant protein layer on the surface of the affinity protein layer, the recombinant protein layer comprising the recombinant protein.

[0009] Fourthly, the present invention also provides a method for preparing a detection product, a separation product, or an enrichment product, comprising the following steps: A solution of recombinant protein is coated onto a solid support coated with an affinity protein layer to form a recombinant protein layer. The recombinant protein solution refers to a solution obtained by dissolving the recombinant protein in a coating solution. The coating solution is a buffer solution containing 1 mM CaCl2 and 1 mM MgCl2, with a pH of 7.4 ± 0.1.

[0010] Fifthly, the present invention also provides the use of recombinant proteins, detection products, separation products, or enrichment products in any of the following: (1) Prepare a kit for detecting circulating tumor cells and / or peritoneal metastatic cells in the subjects; (2) Prepare a kit for isolating or enriching circulating tumor cells and / or peritoneal metastatic cells of the subject; (3) Screening for anti-metastatic drugs; (4) Isolate or enrich circulating tumor cells and / or peritoneal metastatic cells of the subject.

[0011] The present invention has the following beneficial effects: This invention recombines a truncated 158-amino acid P-selectin with a human Fc fragment to obtain a recombinant protein that includes a C-type lectin domain and an EGF-like domain of P-selectin. The lectin domain can specifically bind to glycosylation modifications on the cell surface under flow shear force, and the EGF-like domain can also promote the strong adhesion of P-selectin to cells expressing P-selectin ligands through synergistic effects with other molecules.

[0012] Therefore, the recombinant protein provided by this invention has the function of specifically binding to P-selectin ligands. When coated on a solid support, it can effectively capture cells expressing P-selectin ligands. By truncating P-selectin, the capture process can be ensured to be "domain-specific interaction", that is, binding to target cells only through the interaction domain of P-selectin, reducing non-specific adhesion and improving the purity and capture efficiency of target cells.

[0013] Furthermore, compared to conventional methods of achieving covalent fixation of biomolecules through chemical modification of glass surfaces, this invention first coats an affinity protein onto a solid-phase support, and then achieves directional binding between the affinity protein and the recombinant protein through the interaction between the affinity protein and the Fc fragment. Therefore, the detection, separation, or enrichment products provided by this invention have the advantages of rapid, simple, and efficient preparation methods, and the affinity protein and recombinant protein exhibit ideal directionality.

[0014] When coating recombinant proteins, choosing a buffer containing CaCl2 and MgCl2 can effectively improve the capture effect on metastatic cells, increasing both the capture rate and the number of metastatic cells captured. CaCl2... 2 ⁺ is an essential ion for maintaining the conformation and binding activity of P-selectin, and its concentration (1 mM) ensures the stability of the lectin domain; Mg 2+ Capture efficiency may be further optimized by regulating the conformation of cell surface glycoproteins or enhancing the binding affinity of P-selectin to carbohydrates. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a graph showing the results of Western blot analysis. Figure 2 Image of SDS-PAGE results; Figure 3 The image shows the immunofluorescence results of different Protein A coating concentrations in Experiment Example 1. Figure 4 The image shows the immunofluorescence results of different coating concentrations of recombinant protein in Experiment Example 2. Figure 5 Figure 1 shows the experimental results of the effect of different coating solutions on the number of metastatic cells captured by the microfluidic chip. Figure 6 Figure 1 shows the adhesion rate test results of three recombinant proteins from different sources on a microfluidic chip wafer. Figure 7 The graph shows the adhesion test results of commercially available recombinant protein and recombinant protein 02a from Example 1 on a microfluidic chip. Detailed Implementation

[0017] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] In a first aspect, the present invention provides a recombinant protein of recombinant human Fc and P-selectin, comprising from the N-terminus to the C-terminus: a C-type lectin, an EGF-like domain, and a human Fc segment, wherein the EGF-like domain and the human Fc segment may or may not have a linker peptide, wherein the amino acid sequences of the C-type lectin and the EGF-like domain are as shown in SEQ ID NO: 1-2, respectively, and the human Fc segment is selected from human IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc.

[0020] This recombinant protein possesses the ability to capture cancer cells through carbohydrate interactions between P-selectin and its ligands. In this invention, a truncated 158-amino acid recombinant P-selectin was recombined with a human Fc fragment. The resulting recombinant protein includes a C-type lectin domain and an EGF-like domain of P-selectin. The lectin domain specifically binds to glycosylation modifications on the cell surface under flow shear forces, while the EGF-like domain can also promote the strong adhesion of P-selectin to cells expressing P-selectin ligands through synergistic interactions with other molecules.

[0021] Therefore, the recombinant protein provided by this invention has the function of specifically binding to P-selectin ligands. When coated on a solid support, it can effectively capture cells expressing P-selectin ligands. By truncating P-selectin, the capture process can be ensured to be "domain-specific interaction", that is, binding to target cells only through the interaction domain of P-selectin, reducing non-specific adhesion and improving the purity and capture efficiency of target cells.

[0022] SEQ ID NO: 1: WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTWTWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALC.

[0023] SEQ ID NO: 2: YTASCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRE.

[0024] In a preferred embodiment of the present invention, the amino acid sequences of IgG1Fc, IgG2Fc, IgG3Fc or IgG4Fc are shown in SEQ ID NO: 3-6, respectively.

[0025] SEQ ID NO: 3: PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0026] SEQ ID NO: 4: ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0027] SEQ ID NO: 5: ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK.

[0028] SEQ ID NO: 6: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0029] In a preferred embodiment of the present invention, the linker peptide is selected from flexible linker peptides or rigid linker peptides.

[0030] In a preferred embodiment of the present invention, the linker peptide is selected from (GGGGS). x (Gly)8, (Gly)6, (EAAAK) n ,A(EAAAK)4ALEA(EAAAK)4A,PAPAP,AEAAAKEAAAKA,(Ala-Pro) m (10 – 34 aa), VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG or LE, x=1~4, n=1~3.

[0031] VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRRR, and GFLG are all cleavable linker peptides. Using cleavable linker peptides helps release captured metastatic cancer cells in the chip.

[0032] Secondly, the present invention also provides a detection product, a separation product, or an enrichment product, comprising: a solid support, the aforementioned recombinant protein, and an affinity protein, wherein the affinity protein binds to the Fc region of the recombinant protein.

[0033] In one embodiment, those skilled in the art can coat affinity proteins onto a solid support and then coat recombinant proteins onto the affinity proteins to obtain corresponding detection products, separation products, or enrichment products.

[0034] The detection principle of the above-mentioned detection products is as follows: Tumor cell samples are introduced into the sample inlet of a solid-phase carrier, which is coated with affinity proteins and recombinant proteins. For example, when the solid-phase carrier is a microfluidic chip, affinity proteins and recombinant proteins are coated at the bottom of the channel, so that the sample flows under the corresponding wall shear stress. When peritoneal metastatic cells are captured by P-selectin under the flow conditions (peritoneal metastatic cells and non-metastatic cells have different binding abilities to P-selectin under flow conditions), the peritoneal metastatic cells are separated from other cells in the sample.

[0035] Selectins are calcium-dependent carbohydrate-binding molecules, with Fc-P-selectin being a "key recognition molecule" for capturing metastatic cancer cells. Its function is to recognize and bind to specific carbohydrate ligands (such as sialylated Lewis X antigen, sLeX; or sialylated Lewis A antigen, sLeA) on the surface of metastatic cancer cells through its "ligand-binding domain," thereby achieving specific capture of target cells.

[0036] In a preferred embodiment of the present invention, the detection product, separation product, or enrichment product further includes a coating solution, which is a buffer solution containing 1-1.2 mM CaCl2 and 1-1.2 mM MgCl2, with a pH of 7.4 ± 0.1.

[0037] When coating recombinant proteins, choosing a buffer containing CaCl2 and MgCl2 can effectively improve the capture effect on metastatic cells, increasing both the capture rate and the number of metastatic cells captured. CaCl2... 2 ⁺ is an essential ion for maintaining the conformation and binding activity of P-selectin, and its concentration (1 mM) ensures the stability of the lectin domain; Mg 2+Capture efficiency may be further optimized by regulating the conformation of cell surface glycoproteins or enhancing the binding affinity of P-selectin to carbohydrates.

[0038] In a preferred embodiment of the present invention, the buffer solution is selected from either a 1×TBS solution or a 1×PBS solution.

[0039] This invention minimizes nonspecific cell adhesion by using PBS solution or Tris-based buffer. The special use of the Tris-based buffer is to provide an amine group in the initiation solution, which helps prevent nonspecific adhesion by competing with or masking amine groups on the substrate or cell surface.

[0040] TBS stands for tris(hydroxymethyl)aminomethane, and its main components are Tris, NaCl, and KCl.

[0041] The inventors unexpectedly discovered that PBS was more effective than TBS in minimizing nonspecific cell adhesion.

[0042] In a preferred embodiment of the present invention, the affinity protein is selected from Protein A or Protein G. Protein A is preferred; Protein A is a bacterial protein that specifically binds to the Fc region of the recombinant protein without affecting the C-type lectin-like domain and EGF-like domain of the recombinant protein.

[0043] The affinity protein also carries a label; the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0044] The aforementioned markers refer to substances possessing properties that can be directly observed with the naked eye or detected by instruments, such as luminescence, color development, and radioactivity. These properties enable qualitative or quantitative detection of the corresponding target analytes. In practical applications, those skilled in the art can select appropriate markers based on detection conditions or actual needs. Regardless of the marker used, it falls within the scope of protection of this invention.

[0045] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5...). .5, Cy3, etc. or similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0046] In optional embodiments, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0047] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and18 F.

[0048] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0049] In optional embodiments, nanoparticle-based markers include, but are not limited to, nanoparticles and colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0050] In a preferred embodiment of the present invention, the solid support is selected from a chip or an adsorption column.

[0051] The products to be detected, separated, or enriched are selected from reagent kits, microfluidic chips, or detectors.

[0052] A chip can also be called a suspension array or a liquid array. It consists of a carrier and proteins (affinity protein layers) bound to the surface of the carrier.

[0053] The aforementioned carrier can be made of various materials and in various forms, such as preferably a container with a flat bottom. A more typical preferred example is multi-well plates, microplates, microfluidic-based devices (e.g., microfluidic chips), petri dish-like containers, etc., which are widely used in biochemical assays, and are not limited thereto.

[0054] The preferred microfluidic chip is a straight-channel microfluidic chip, which uses a straight channel rather than a complex channel to avoid clogging.

[0055] The microfluidic chip is selected from T-type chip, flow focusing chip or coaxial flow chip PDMS chip or metal droplet generator or PMMA microfluidic chip.

[0056] Furthermore, the above-mentioned kit may also include at least one of the following: buffer solution, detection reagent, diluent (such as PBS or TBS solution), and washing solution, and is not limited thereto.

[0057] Thirdly, the present invention also provides a detection product, a separation product, or an enrichment product, comprising: a solid support having an affinity protein layer on the solid support for binding the Fc region of the recombinant protein described above, and a recombinant protein layer on the surface of the affinity protein layer, the recombinant protein layer comprising the recombinant protein.

[0058] In one embodiment, the affinity protein is disposed on a solid support in the form of an affinity protein layer or coating, the affinity protein layer or coating containing saturated affinity protein, the saturation density of the affinity protein ensuring uniform fixation of the recombinant protein.

[0059] Unlike the aforementioned methods that require the preparation of products for detection, separation, or enrichment, in this embodiment, the affinity protein layer and recombinant protein layer are pre-coated on a solid-phase support. This allows the finished product to be used directly for the detection, separation, or enrichment of target cells.

[0060] In one embodiment, the density of affinity proteins in the affinity protein layer is 0.375~0.625 ug / cm³. 2 The density range of recombinant proteins in the recombinant protein layer is 0.025 ug / cm³. 2 Up to 0.625 ug / cm 2 .

[0061] Within the aforementioned density range, it exhibits superior cell capture performance.

[0062] In a preferred embodiment of the present invention, the affinity protein is selected from Protein A or Protein G. Protein A is selected as the affinity protein because it exhibits superior capture effect on metastatic cells.

[0063] The affinity protein may or may not carry a label; the label is selected from at least one of fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0064] In a preferred embodiment of the present invention, the solid support is selected from a chip or an adsorption column.

[0065] In a preferred embodiment of the present invention, the detection product, separation product, or enrichment product is selected from reagent kits, microfluidic chips, or detectors.

[0066] A chip can also be called a suspension array or a liquid array. It consists of a carrier and proteins (affinity protein layers) bound to the surface of the carrier.

[0067] The aforementioned carrier can be made of various materials and in various forms, such as preferably a container with a flat bottom. A more typical preferred example is multi-well plates, microplates, microfluidic-based devices (e.g., microfluidic chips), petri dish-like containers, etc., which are widely used in biochemical assays, and are not limited thereto.

[0068] The preferred microfluidic chip is a straight-channel microfluidic chip, which uses a straight channel rather than a complex channel to avoid clogging.

[0069] The microfluidic chip is selected from T-type chip, flow focusing chip or coaxial flow chip PDMS chip or metal droplet generator or PMMA microfluidic chip.

[0070] Furthermore, the above-mentioned kit may also include at least one of the following: buffer solution, detection reagent, diluent (such as PBS or TBS solution), and washing solution, and is not limited thereto.

[0071] Fourthly, the present invention also provides a method for preparing a detection product, a separation product, or an enrichment product, comprising the following steps: A solution of recombinant protein is coated onto a solid support coated with an affinity protein layer to form a recombinant protein layer. The recombinant protein solution refers to a solution obtained by dissolving the recombinant protein in a coating solution. The coating solution is a buffer solution containing 1 mM CaCl2 and 1 mM MgCl2, with a pH of 7.4 ± 0.1.

[0072] In a preferred embodiment of the present invention, the final concentration of the recombinant protein in the coating solution is 5-25 μg / mL. At this coating concentration, superior immunofluorescence intensity is achieved, resulting in better detection and capture performance.

[0073] For example, the final concentration of recombinant protein in the solution is 25 μg / mL, 26 μg / mL, 27 μg / mL, 28 μg / mL, 29 μg / mL, 30 μg / mL, 31 μg / mL, 32 μg / mL, 33 μg / mL, 34 μg / mL, or 35 μg / mL.

[0074] In a preferred embodiment of the present invention, after coating the recombinant protein solution, the process further includes blocking, which is performed using 1%-5% BSA.

[0075] In a preferred embodiment of the present invention, the method for coating an affinity protein layer onto a solid support includes: coating the solid support with an affinity protein at a final concentration of 20-30 μg / mL, incubating, and removing unbound affinity protein. At the above coating concentration, superior immunofluorescence intensity is achieved, resulting in superior detection and capture effects.

[0076] The final coating concentration of the affinity protein is, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 μg / mL.

[0077] Fifthly, the present invention also provides the use of recombinant proteins, detection products, separation products, or enrichment products in any of the following: (1) Prepare a kit for detecting circulating tumor cells and / or peritoneal metastatic cells in the subjects; (2) Prepare a kit for isolating or enriching circulating tumor cells and / or peritoneal metastatic cells of the subject; (3) Screening for anti-metastatic drugs; (4) Isolate or enrich circulating tumor cells and / or peritoneal metastatic cells of the subject.

[0078] The subject refers to a person.

[0079] Peritoneal metastatic cells are selected from cells of at least one of the following cancer metastases: Ovarian cancer, stomach cancer, colorectal cancer, pancreatic cancer, appendix cancer, primary peritoneal cancer, liver cancer, bile duct cancer, uterine cancer, etc.

[0080] The above-mentioned use (3) for screening anti-metastatic drugs includes, but is not limited to, the following methods: (a) Contacting metastatic cells with a test reagent; (b) Treating metastatic cells to specifically bind to P-selectin; (c) Detecting metastatic cells that have been in contact with the test reagent but do not specifically bind to P-selectin, wherein the metastatic cells that do not bind to P-selectin indicate that these cells have been in contact with a test reagent with anti-metastatic properties.

[0081] The inventors performed molecular docking on the extracellular domain of P-selectin and found that P-selectin binds to ligands in any of the following ways, and the binding probability of sites (1)-(3) to ligands decreases sequentially.

[0082] Recombinant proteins bind ligands through at least one of the following mechanisms: (1) The ligand is bound to the 82-89aa amino acid (one loop) and the 99-107 amino acid (one loop) of the recombinant protein; that is, the 123-130 (one loop) and 140-148 (one loop) amino acids of the P-selectin before truncation.

[0083] (2) The ligand binds through amino acids at positions 26-32aa (a small loop), 62-71 (a loop), 116-119 and 135-138 (a small loop) of the recombinant protein; that is, corresponding to amino acids at positions 67-73 (a small loop), 104-112 (a loop), 157-160 and 176-179 of the P-selectin before truncation.

[0084] (3) The ligand binds through amino acids 119-133 (one loop) of the recombinant protein. That is, it corresponds to amino acids 160-174 of the P-selectin before truncation.

[0085] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0086] Example 1 This embodiment provides a recombinant protein of recombinant human Fc and P-selectin, the amino acid sequence of which is shown in SEQ ID NO: 7.

[0087] WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTWTWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTASCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVREGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0088] This amino acid sequence was synthesized by Abclonal / Yurogen. The expression vector for 02a was pcDNA3.4-signal 02a (C-hFc tag), and the expression vector for 02b was pcDNA3.4-signal 02b (C-hFc tag). The signal peptide was naturally removed during protein purification. The host cells were all Expi293F. The C-terminus is Fc-tagged.

[0089] The preparation method of recombinant protein is as follows: (1) Prepare expression plasmids and achieve efficient transient transfection; (2) Protein expression was expanded in 293F cells, with each expression strain having a 30 mL culture system; (3) Use systems such as Gator to quickly assess expression levels; (4) Protein purification was performed using a one-step affinity chromatography method; (5) Quality testing: a. Protein purity is analyzed by reducing SDS-PAGE; b. The final protein concentration is determined, which should be better than 0.5 mg / mL (if the stability of the protein at high concentrations is permissible); c. The final protein buffer system is 1X PBS.

[0090] The results of the Western blot analysis were referenced. Figure 1 As shown, Figure 1 Lane 1 contains pCDNA3.4-signal 02a-SELP (42-199aa)-C-terminal hFC tag Expi293F cell culture supernatant, with 5 μL added to each well; Lane 2 contains pCDNA3.4-signal 02b-SELP (42-199aa)-C-terminal hFC tag Expi293F cell culture supernatant, with 5 μL added to each well. Lane 3 is the positive control (hFC tag protein, 0.05 mg / mL), with 5 μL added to each well. The theoretical molecular weight is 44 KD; the apparent molecular weight is 60 KD.

[0091] In Western blotting experiments: Blocking buffer: 3% skim milk powder, incubated at room temperature for 1 hour. Primary antibody: Donkey AntiHuman IgG (H+L)-HRP, diluted 1:5000. Exposure time: 60 seconds.

[0092] Figure 1 The results showed that the target recombinant protein was successfully prepared. Secretory expression of the SELP(42-199aa)-C-hFC tag recombinant protein was successfully achieved in the Expi293F expression system.

[0093] Further SDS-PAGE analysis was performed, and the experimental results were referenced. Figure 2 As shown, Figure 2 Lane 1 contains the pCDNA3.4-signal 02a-SELP(42-199aa)-C-hFC tag, in a non-reduced state, 3.5 μL per well; Lane 2 contains the pCDNA3.4-signal 02b-SELP(42-199aa)-C-hFC tag, in a non-reduced state, 3.5 μL per well; Lane 3 contains the protein molecular weight standard (Marker); Lane 4 contains BSA (0.4 mg / mL), 7 μL per well; Lane 5 contains the pCDNA3.4-signal 02a-SELP(42-199aa)-C-hFC tag, in a reduced state, 3.5 μL per well; Lane 6 contains the pCDNA3.4-signal 02b-SELP(42-199aa)-C-hFC tag, in a reduced state, 3.5 μL per well. Theoretical molecular weight: 44kDa; Apparent molecular weight: 60kDa; Electrophoretic gel type: 4-20% gradient gel.

[0094] Experimental results show that, due to glycosylation modification, the molecular weight of the target protein on SDS-PAGE is higher than the theoretical value.

[0095] After one step of Protein A affinity purification, 1.36 mg of SELP(42-199aa)-C-hFC(signal 02a) recombinant protein with a concentration of 0.8 mg / mL and a purity of 90% was obtained.

[0096] Similarly, after one-step Protein A affinity purification, 1.36 mg of SELP(42-199aa)-C-hFC(signal 02b) recombinant protein with a concentration of 0.8 mg / mL and a purity of 90% was obtained.

[0097] The pCDNA3.4-signal 02b-SELP(42-199aa)-C-hFC tag system was selected for subsequent large-scale expression.

[0098] Example 2 This embodiment provides a microfluidic chip in which the channels are coated with affinity protein (Protein A) and recombinant proteins of recombinant human Fc and P-selectin provided in Example 1.

[0099] The specific preparation method is as follows: 1. Reagents The experiment used 1x PBS.

[0100] 3% bovine serum albumin (BSA) is used for blocking.

[0101] BSA dissolved in 1x PBS (filtered through a 0.22 μM filter); 20 μg / mL Protein A (ProA); ProA dissolved in 1x PBS; PBS solution of recombinant protein at 25 μg / mL: Recombinant human Fc and P-selectin recombinant proteins were dissolved in 1x PBS solution containing 1 mM calcium chloride (CaCl2) and 1 mM magnesium chloride (MgCl2).

[0102] 2. The chip coating process is as follows: 2.1 Cleaning the chip Use a P200 pipette to clean the chip channels twice with 200 μL of 75% ethanol each time. (1) Slowly inject into each channel to remove excess ethanol; (2) Take care to avoid introducing air bubbles into the flow channel; (3) After washing the chip with 75% ethanol, rinse the flow channel twice with PBS, 200 μL each time.

[0103] 2.2 Protein A coating (1) Remove the PBS from the flow channel and slowly inject 100 μL of Protein A solution into each flow channel to avoid the generation of air bubbles; (2) This process operates one flow channel at a time to avoid the formation of bubbles after the flow channel dries; (3) Place in a bacterial culture dish and incubate at room temperature (24℃) for 1 hour; (4) Without aspirating Pro A, slowly wash each flow channel with sterile PBS, 1 mL each time, to remove unbound Protein A. The final Protein A coating density is 0.5 ug / cm³. 2 .

[0104] 2.3 Coating of recombinant proteins (1) Remove the PBS from the flow channel and slowly add PBS solution of 25 μg / mL recombinant protein, 60 μL / flow channel, to avoid the generation of bubbles; (2) This process operates one flow channel at a time to avoid the formation of bubbles after the flow channel dries; (3) Incubate at room temperature (24℃) for 2 hours in a closed environment; or incubate overnight at 4℃ under humidified conditions; (4) Without aspirating the recombinant protein solution, slowly wash each flow channel with sterile PBS, 1 mL each time, to remove unbound recombinant protein solution.

[0105] 2.4 BSA coating (1) Slowly add 200 μL of 3% BSA to each channel using a P200 pipette; (2) Incubate in a closed environment at room temperature (24℃) for 15 minutes; (3) Do not aspirate BSA, but slowly wash each flow channel once with 1 mL PBS.

[0106] Thus, a microfluidic chip coated with Protein A and recombinant protein was prepared.

[0107] The specific experimental methods for cell capture and fluorescence experiments in Examples 1-5 below are as follows: 1.1 Reagents required The experiment used 1x PBS; 3% Bovine serum albumin (BSA); 5 mM EDTA; RPMI 1640 medium (5% fetal bovine serum FBS, 1% P / S); Buffer (PBS (0.1% Ca)) 2+ 0.1%Mg 2+ ) or TBS (0.2% Ca 2+ 0.1%Mg 2+ )).

[0108] 1.2 Cell Treatment (1) Preheat PBS, and incubate PBS (0.1% Ca2+ 0.1% Mg2+), 1640 (5% FBS + 1% PS) and 5 mM EDTA in a 37°C water bath for 15 min; (2) Remove the cells (HeyA8 high-transfer cells) from the incubator, and discard the culture medium by aspirating it with a 5mL pipette. Wash once with 2mL PBS. (3) Add 2 mL of 5 mM EDTA and quickly transfer to a 37°C incubator and time for 5 min; (4) Gently pipette and collect the cells, then transfer them to a 15 ml centrifuge tube containing 2 mL of 1640 (5% FBS) to terminate digestion; (5) Centrifuge (1000 rpm, 5 min), add appropriate amount of buffer (0.1% Ca2+). 2 +0.1%Mg 2+ Resuspend the cells and agitate them thoroughly. (If necessary, live cell tracer dyes can be used to label the cells for subsequent cell quantification analysis.)

[0109] (6) Count cells using a hemocytometer, prepare 1 mL of cell suspension (1×10⁵ - 2.5×10⁵ cells / mL) for subsequent experiments, and retest the cell concentration after preparation to ensure a cell density of 1×10⁵. 5 -2.5×10 5 per mL. 1.3 Chip Fabrication Preparation 1.3.1 Reagent Preparation (1) Washing buffer: (PBS (0.1% Ca2+ 0.1% Mg2+) or TBS (0.2% Ca2+ 0.1% Mg2+)); (2) Trypan blue; (3) 1640 RPMI (5%FBS 1%PS) 3mL; (4) 3 mL of 75% ethanol; 1.3.2 Chip Fabrication Preparation (1) Connect the Longer pump device, draw 75% ethanol into the connector with the needle of the syringe, let it stand for a while, and then discharge the 75% ethanol; (2) Rinse the tubing and needle twice with cleaning buffer to remove any 75% ethanol residue; (3) Draw 100 μL of air from the syringe tip and fill the syringe with clean cleaning buffer to the 300 μL mark; (4) In the perfusion mode of 3500μL / h, turn on the pump and wait for the liquid to overflow. Then insert the metal connector into the chip to rinse the chip. Use a pipette to remove the overflowing PBS in time. When there is 100μL left in the syringe, insert the memory. When liquid overflows from the memory, you can stop the pump. The excess liquid in the memory can be removed and discarded with a pipette.

[0110] 1.3.3 Loading Cells Mix the cell suspension thoroughly by pipetting, add 100 μL of the cell suspension to the memory, adjust the extraction mode to 1500 μL / h, and turn on Pump to load the cells; 1.3.4 Elution of non-adhesive cells Once the cells have been loaded and the liquid level in the storage container is near the bottom, add 100 μL of washing buffer to rinse away any non-adhering cells. Repeat this process three times to ensure that all non-adhering cells have been eluted. 1.4 Collection of non-adhesive cells (1) If cells are labeled with live cell tracer dyes, full-length imaging scans can be performed using a high-resolution optical microscope, followed by analysis software to quantify the cells. (2) Discard any residual liquid in the memory to prevent abnormal leakage. (2) Disconnect the metal connector and collect the non-adherent cells into a 15mL centrifuge tube for counting and later use.

[0111] 1.5 Elution of Adhesive Cells (1) After leaving a small amount of air in the syringe, draw 100 μL of 1640 RPMI (5% FBS 1% PS); (2) Reconnect the connector to the chip; (3) Add 100 μL of 1640 RPMI (5% FBS 1% PS) to the memory; (4) Set the pump to 3500 μL / h extraction mode and turn on the pump. Wait until the liquid level in the storage tank is half full. (5) Pause the Pump switch, push and pull the syringe plunger, and flush with a rapid flow to detach the adherent cells from the chip; 1.6 Collection of Adhesive Cells (1) Add 100 μl of 1640 RPMI (5% FBS) to elute the adhering cells (twice in total), adjust the pump to the extraction mode, set the flow rate to 3500 μl / h, and collect the adhering cells; after all the liquid in the chip has been collected, disconnect the metal connector at the chip end.

[0112] (2) Transfer the adherent cells from the syringe and tubing to centrifuge tubes for later use (insert the metal connector into the bottom of the 15mL centrifuge tube to avoid cell loss); 1.7 Cell Count (1) Centrifuge non-adhesive / adhesive cells at 400g for 5 min, discard the supernatant and mix thoroughly (retain 30-50 μL for counting), and calculate the adhesion rate; (2) Take 10 μL and mix with trypan blue 1:1 and stain for 3 min.

[0113] Adhesion rate calculation formula Adhesion rate (%) = (Number of adherent cells according to analysis software / Adhesive + Non-adhesive) × 100%.

[0114] Experimental Example 1 To improve the detection efficiency of microfluidic chips, the coating concentration of Protein A was screened in this experimental example.

[0115] In this experimental example, Protein A was labeled with Alexafluor488, and Alexafluor488-Protein A was used for gradient dilution coating and immunofluorescence staining.

[0116] Specifically, following step 2.2 of Example 2, Protein A was coated into the flow channels of the microfluidic chip at concentrations of 0, 5, 10, 15, 20, 25, and 30 μg / mL, respectively. Other steps were the same as in Example 2.

[0117] Figure 3 The results show the fluorescence signal and intensity quantification for each tested Protein A concentration, and the optimal concentration range of 15-25 μg / mL Protein A was selected as achieving stable coating. The results also show that the fluorescence signal tends to plateau above 20 μg / mL Protein A, indicating that coating has reached saturation; therefore, a Protein A concentration of 20 μg / mL was chosen as optimal.

[0118] Experimental Example 2 Immunofluorescence staining was performed on the serially diluted coating material (recombinant protein) in the microfluidic chip prepared in Example 2 using Alexafluor488-anti-P selectin antibody.

[0119] Figure 4 The results showed that 5-25 μg / mL of Pselectin was the optimal concentration range for achieving stable coating. Figure 4 The lower right figure shows the results of the microfluidic chip adhesion experiment, demonstrating that 15-30 μg / mL of Pselectin can capture highly metastatic cancer cells. The cell capture efficiency of recombinant whole protein Pselectin / human Fc is optimal at a concentration of 25 μg / mL; therefore, 25 μg / mL was selected as the optimal concentration.

[0120] Experimental Example 3 This experiment tested the effect of different coating solutions on the number of metastatic cells captured by a microfluidic chip.

[0121] Results reference Figure 5 As shown, using 1x PBS containing 1mM calcium chloride (CaCl2) and 1mM magnesium chloride (MgCl2) to decoat recombinant proteins can effectively increase the number of metastatic cells captured by microfluidic chips. Figure 5 The x-axis represents the total number of cells loaded with the sample, and the y-axis represents the percentage of captured cells out of the total number of cells.

[0122] Experiment Example 4 The adhesion rate of 10 μg / mL Pselectin (P-selectin interaction domain / Fc) from three different sources (pcDNA3.4-signal 02a, pcDNA3.4-signal 02b, and the recombinant protein sequence of Novoprotein SEQ ID NO: 8 in Example 1) to the microfluidic chip was compared. The Pselectin coating was measured using fluorescent labeling, and the results were compared with those obtained from [previous data]. Figure 6 As shown, although the Novoprotein (i.e., the novo group) has high adhesion, its fluorescence signal value is unstable. Therefore, the 02a P-selectin interaction domain / Fc recombinant protein was selected.

[0123] SEQ ID NO: 8: WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTWTWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTASC QDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVREASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. Among these, amino acids 159-488 represent human IgG1.

[0124] Experimental Example 5 Adhesion rate was tested by fabricating microfluidic chips using commercially available recombinant whole protein / human Fc compared to the P-selectin interaction domain / Fc recombinant protein prepared in Example 1.

[0125] Figure 7 The results showed that the 02a P-selectin interaction domain / fc was superior in capturing highly metastatic cancer cells, and it could effectively capture highly metastatic cancer cells at concentrations of 5ug / ml-25ug / ml. Figure 7 The right image is a representative picture of the adhesion test, showing the different abilities of the 02a P-selectin interaction domain / fc in capturing highly metastatic cancer cells.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant protein of human Fc and P-selectin, characterized in that, comprises, from N-terminus to C-terminus: a C-type lectin, an EGF-like domain, and a human Fc segment, with or without a connecting peptide between the EGF-like domain and the human Fc segment, wherein the amino acid sequences of the C-type lectin and the EGF-like domain are respectively shown as SEQ ID NOs: 1-2, and the human Fc segment is selected from a human IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc; Preferably, the amino acid sequences of the IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc are respectively shown as SEQ ID NOs: 3-6. Preferably, the connecting peptide is selected from a flexible connecting peptide or a rigid connecting peptide. Preferably, the connecting peptide is selected from (GGGGS) x , (Gly)8, (Gly)6, (EAAAK) n , A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro) m (10 - 34 aa), VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, E DVVCCSMSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG or LE, x = 1-4, n = 1-3.

2. A detection product, isolation product or enrichment product, characterized in that, comprises: a solid phase carrier, the recombinant protein of claim 1, and an affinity protein that binds to the Fc region of the recombinant protein.

3. The detection, isolation or enrichment product of claim 2, wherein, The detection product, separation product, or enrichment product further comprises a coating solution, wherein the coating solution is a buffer solution containing 1-1.2 mM CaCl2 and 1-1.2 mM MgCl2, and the pH of the coating solution is 7.4±0.

1. Preferably, the buffer solution is selected from a 1×TBS solution or a 1×PBS solution.

4. The detection, isolation or enrichment product according to claim 2, characterized in that, The affinity protein is selected from Protein A or Protein G. Preferably, the affinity protein further carries a label; the label is selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label. Preferably, the solid phase carrier is selected from a chip or an adsorption column. Preferably, the detection product, separation product, or enrichment product is selected from a kit, a microfluidic chip, or a detector.

5. A detection product, isolation product or enrichment product, characterized in that, comprises: a solid phase carrier, and the solid phase carrier has an affinity protein layer for binding to the Fc region of the recombinant protein of claim 1, and the surface of the affinity protein layer has a recombinant protein layer comprising the recombinant protein.

6. The detection, isolation or enrichment product according to claim 5, characterized in that, The affinity protein in the affinity protein layer is selected from Protein A or Protein G. Preferably, the affinity protein carries or does not carry a label; the label is selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label. Preferably, the density of the affinity protein in the affinity protein layer is in the range of 0.375 ~ 0.625 ug / cm 2 Preferably, the density of the recombinant protein in the recombinant protein layer is in the range of 0.025 ug / cm 2 to 0.625 ug / cm 2 ; Preferably, the solid phase carrier is selected from a chip or an adsorption column. Preferably, the detection product, separation product, or enrichment product is selected from a kit, a microfluidic chip, or a detector.

7. A method of producing a detection product, isolation product or enrichment product according to any one of claims 5 to 6, wherein, comprises the following steps: coating a solution of the recombinant protein on a solid phase carrier coated with an affinity protein layer to form a recombinant protein layer, and the solution of the recombinant protein refers to a solution obtained by dissolving the recombinant protein in a coating solution; the coating solution is a buffer solution containing 1 mM CaCl2 and 1 mM MgCl2, and the pH of the coating solution is 7.4±0.

1.

8. The production method according to claim 7, characterized by, When coating the solution of the recombinant protein, the final concentration of the recombinant protein in the solution of the recombinant protein is 5-25 μg / mL.

9. The production method according to claim 8, characterized by, After coating the solution of the recombinant protein, blocking is further included, and the blocking is performed with 1%-5% BSA; Preferably, the method for coating the affinity protein layer on the solid phase carrier comprises: coating the affinity protein with a final concentration of 20-30 μg / mL on the solid phase carrier, incubation, and removing the unbound affinity protein.

10. The recombinant protein of claim 1, the detection product, separation product or enrichment product of any one of claims 2-4, or the detection product, separation product or enrichment product of any one of claims 5-6 for use in any one of: (1) preparing a kit for detecting circulating tumor cells and / or peritoneal metastasis cells in a subject; (2) preparing a kit for separating or enriching circulating tumor cells and / or peritoneal metastasis cells in a subject; (3) screening anti-metastasis drugs; (4) separating or enriching circulating tumor cells and / or peritoneal metastasis cells in a subject; Preferably, the recombinant protein binds to the ligand by at least one of the following: (1) by the amino acids at positions 82-89 and 99-107 of the recombinant protein; (2) by the amino acids at positions 26-32, 62-71, 116-119 and 135-138 of the recombinant protein; (3) by the amino acids at positions 119-133 of the recombinant protein.

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