A preparation method of a self-pre-dyed fluorescent protein marker

Abstract

The application relates to the technical field of bioengineering, in particular to a preparation method of a pre-dyed fluorescent protein Marker, which comprises the following steps: preparing a subunit mixture: 1 mg of luminescent protein precipitate is placed in a centrifuge tube, high-speed centrifugation is carried out at 4 DEG C for 5 min, supernatant and precipitate are obtained, the supernatant is completely sucked away by using a pipette gun, and only the precipitate part is reserved. The fluorescent protein Marker is a naturally extracted fluorescent protein, the molecular weight of the fluorescent protein is 20-300 kDa, the fluorescent protein is covalently coupled with protein A and protein G which can be combined with an antibody IgG and an antibody Fc region protein or peptide which can be combined with a secondary antibody, due to the color of the fluorescent protein, the purpose that the protein is pre-dyed protein and fluorescent protein is achieved, transfer film staining or X-ray film imaging is not needed, electrophoresis is simpler, due to the very strong fluorescent signal, even if a trace amount of protein, electrophoresis detection can be realized, and an ideal electrophoresis map is obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a preparation method of a self-pre-stained fluorescent protein Marker. BACKGROUND

[0002] Protein marker is a standard of protein molecular weight size, which is widely used in protein electrophoresis. It is a mixture of proteins or polypeptides with different molecular weight sizes.

[0003] The existing protein Marker development has experienced four development stages of ordinary Marker, pre-stained Marker, exposure Marker and fluorescent protein Marker. Among them, ordinary protein Marker has no dye molecules or marker molecules, and the molecular weight size is exactly the size of the original protein. In the early stage, the protein size can be accurately judged. However, the ordinary Marker cannot be seen during the electrophoresis process, and can only be seen after being dyed together with the target protein, so it cannot play a reference role in the experimental process.

[0004] Pre-stained Marker is a protein (or polypeptide) in ordinary protein Marker, which is covalently coupled with a blue or other color dye after chemical modification. The appearance of pre-stained protein Marker brings great convenience to Western Blot. Researchers can determine the approximate position of the target band according to the size of the blue (or other color) protein Marker on the membrane after membrane transfer, so as to cut and process the membrane.

[0005] Exposure Marker is a mixture of luminescent protein Marker and pre-stained protein Marker with different proportions, which can specifically bind to primary antibody or secondary antibody, so that the final Marker band will appear in the developed picture together with the target protein, avoiding deviation when comparing the picture and the membrane. The preparation process becomes complicated. The exposure pre-stained protein Marker is a fusion protein covalently coupled with active dye remazol, so as to realize the purpose of the same protein being luminescent protein and pre-stained protein. It can combine IgG derived from human, rat, mouse, rabbit and other species, or anti-rabbit and mouse secondary antibody, and indicate the approximate position of the protein during the electrophoresis process and after membrane transfer. Moreover, it can indicate the position of the protein on X film. SUMMARY

[0006] The application aims to provide a preparation method of a self-pre-dyed fluorescent protein Marker, which expands the application field of the fluorescent protein, realizes that the protein is both pre-dyed protein and fluorescent protein, does not need membrane dyeing or X-ray imaging, makes electrophoresis simpler, has very strong fluorescent signal, realizes electrophoresis detection even for trace protein, and has the advantage of obtaining ideal electrophoresis diagram.

[0007] To achieve the above object, the application provides the following technical scheme: a preparation method of a self-pre-dyed fluorescent protein Marker, which comprises the following steps.

[0008] Step one, preparation of subunit mixture:

[0009] 1.1, 1 milligram of luminescent protein precipitate is placed in a centrifuge tube, high-speed centrifugation is carried out at 4 DEG C for 5 minutes, supernatant and precipitate are obtained, the supernatant is completely sucked away by a pipette gun, and only the precipitate is reserved;

[0010] 1.2, 200 microliters of buffer solution are added to the precipitate to fully dissolve, then after high-speed centrifugation at 4 DEG C for 10 minutes, the precipitate is removed, and supernatant solution is obtained;

[0011] 1.3, the supernatant solution is desalted by a fast desalting column or a dialysis bag, dithiothreitol (DTT) is added to the luminescent protein solution after desalting, a mixture with a final concentration of 20-50 micromole / liter is obtained, then after normal temperature reaction for 90 minutes, a subunit mixture is obtained;

[0012] Step two, preparation of derivatized fusion protein:

[0013] 2.1, 1 milligram of fusion protein is dissolved in 200 microliters of 0.1M PBS (PH 7.2-7.4) buffer solution to prepare a 5 milligram / milliliter solution;

[0014] 2.2, a bifunctional reagent is dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a 50 milligram / milliliter mother liquor, 20 microliters of SMCC are added to each milligram of fusion protein, the aluminum foil is sealed, and then the reaction is carried out at room temperature for 60 minutes, so that the amino group on the fusion protein molecule reacts with succinamide to generate derivatized fusion protein;

[0015] 2.3, the buffer solution is exchanged to pre-equilibrate the gel column, the derivatized fusion protein is passed through the column, the fusion protein peak is collected, and the concentration is adjusted;

[0016] Step three, cross-linking preparation:

[0017] 3.1 The subunit mixture is added dropwise to the derivatized fusion protein, mixed gently with a homogenizer, and covalently cross-linked by rotating the aluminum foil at room temperature for 120 minutes (or overnight at 4°C) to obtain a self-pre-stained fluorescent protein marker product.

[0018] Preferably, in 1.1 of step one, the light-emitting protein includes one of phycoerythrin, phycocyanin, allophycocyanin, green fluorescent protein, yellow fluorescent protein, and red fluorescent protein.

[0019] Preferably, in 1.3 of step one, the subunit mixture can also be used with a desalting column or dialysis bag to remove excess DTT in the mixed solution.

[0020] Preferably, in 2.2 of step two, the bifunctional reagent includes succinimidyl-4-(N- methylmaleimide)cyclohexane-1-carboxylate (SMCC).

[0021] Preferably, in 3.1 of step three, after gentle mixing with a homogenizer, covalent cross-linking can also be achieved by sealing the aluminum foil and placing it at 4°C overnight to obtain a self-pre-stained fluorescent protein marker product.

[0022] Preferably, step two includes thiolation of the fusion protein.

[0023] Preparation: 1 milligram of protein A GL fusion protein is dissolved in 200 microliters of 0.1M PBS (pH 7.2-7.4) buffer solution to prepare a 5 milligram / milliliter solution to obtain a pure fusion protein product, which includes the following steps:

[0024] A. 3-(2-pyridyl disulfide) propionic acid N-hydroxysuccinimidyl ester (SPDP) is dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a 50 milligram / milliliter stock solution;

[0025] B. The pure fusion protein product is taken and the concentration is adjusted to 5 milligrams / milliliter or more. SPDP solution is added according to 1 milligram of fusion protein, mixed gently, and stirred at room temperature for 0.5-1.5 hours. Excess SPDP is removed using a centrifugal desalting column, and the collected solution is SPDP-fusion protein;

[0026] C. Tris(2-carboxyethyl)phosphine (TCEP) is dissolved in 0.1M PBS (pH 7.4, 0.1M NaCl) to prepare a 2 milligram / milliliter solution. According to 20 microliters of TCEP solution per milligram of SPDP-fusion protein, gently mix and react at room temperature for 10-15 minutes;

[0027] D. Separate the thiolated product on a PD-10 column and adjust the concentration to 3 mg / ml.

[0028] Preferably, the purity of the fusion protein is greater than 90%.

[0029] Preferably, the SPDP solution comprises no less than 15 μl.

[0030] Preferably, step three 3.1 further comprises adding the activated fusion protein drop by drop into the thiolated fusion protein, mixing gently with a mixer while adding, sealing with aluminum foil and rotating coupling for 120 minutes at room temperature (or overnight at 4°C) to achieve covalent cross-linking of the thiol and the maleimide group to obtain a self-pre-stained fluorescent protein Marker product.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] The fluorescent protein Marker of the present application is a naturally extracted fluorescent protein with a molecular weight of 20-300 kDa, which is covalently coupled with protein A capable of binding antibody IgG, protein G capable of binding secondary antibody, and antibody Fc region protein or peptide capable of being bound by secondary antibody. Since the fluorescent protein has a color, the purpose of protein being pre-stained protein and fluorescent protein is achieved, without the need for membrane transfer staining or X-ray imaging, so that electrophoresis is simpler. Since the fluorescent signal is very strong, even a small amount of protein can achieve electrophoretic detection, and an ideal electropherogram is obtained. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0034] Embodiment one: covalent coupling of fluorescent phycoerythrin subunit and protein A to prepare a self-pre-stained fluorescent protein Marker

[0035] It should be noted that phycoerythrin is isolated and purified from red algae, can emit strong fluorescence, and has a molecular structure of carrier protein connected by thioether bond and open-chain linear extension of tetrapyrrole compound. The molecular weight is about 240,000 daltons, and the subunit composition of the protein is (alpha-beta) 6 gamma. Each alpha-subunit and beta-subunit is about 20,000 daltons, and each gamma subunit is about 30,000 daltons.

[0036] Step one, preparation of a mixture of phycoerythrin alpha, beta and gamma subunits:

[0037] 1.1, 1 mg of phycoerythrin precipitate was placed in a centrifuge tube, and centrifuged at high speed for 5 minutes at 4°C. The supernatant was carefully removed with a pipette, and only the precipitate was reserved.

[0038] 1.2, The precipitate was dissolved in 200 μl of buffer, and centrifuged at high speed for 10 minutes at 4°C. The supernatant was obtained by removing the precipitate.

[0039] 1.3, The orange-red supernatant was desalted using a fast desalting column or a dialysis bag. Then, dithiothreitol (DTT) was added to the desalted phycoerythrin to obtain a mixture with a final concentration of 20-50 μmol / L. The mixture was reacted at room temperature for 90 minutes. The solution was a mixture of phycoerythrin α, β, and γ subunits. The excess DTT was removed using a desalting column or a dialysis bag, and the solution was reserved.

[0040] Step 2, Preparation of derivatized Staphylococcus aureus immunoglobulin G binding protein A (protein A):

[0041] 2.1, 1 mg of protein A was dissolved in 200 μl of 0.1 M PBS (pH 7.2-7.4) buffer solution to prepare a 5 mg / ml solution.

[0042] 2.2, Bifunctional reagent succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a 50 mg / ml stock solution. 20 μl of SMCC was added to each mg of protein A. The aluminum foil was sealed and rotated at room temperature for 60 minutes to react the amino group on the protein A molecule with succinamide to generate derivatized protein A protein.

[0043] 2.3, The gel column was equilibrated with a buffer solution, and the derivatized protein A protein was passed through the column. The protein A protein peak was collected, and the concentration was adjusted to 5 mg / ml.

[0044] Step 3, Preparation of a self-pre-dyed fluorescent protein Marker:

[0045] 3.1, The mixture of phycoerythrin α, β, and γ subunits was added dropwise to the succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) derivatized protein A protein, and mixed gently with a homogenizer. The aluminum foil was sealed and rotated at room temperature for 120 minutes (or overnight at 4°C) to covalently cross-link the thiol group on the phycoerythrin α, β, and γ subunit molecules with the maleimide group, thereby obtaining a self-pre-dyed fluorescent protein Marker product.

[0046] Example 2: Preparation of covalently coupled phycocyanin subunits and protein AG to produce pre-stained fluorescent protein marker

[0047] It should be noted that phycocyanin is isolated and purified from Spirulina, and can emit strong fluorescence. Its molecular structure is a carrier protein connected by a sulfide bond and an open-chain linear extension of a tetrapyrrole compound. Its molecular weight is 260,000 daltons, and its subunit composition is (alpha-beta)6, with each alpha-subunit and beta-subunit being about 20,000 daltons.

[0048] Step 1: Preparation of a mixture of thiolated phycocyanin alpha and beta subunits

[0049] 1.1. Place 1 mg of phycocyanin precipitate in a centrifuge tube and centrifuge at high speed at 4°C for 5 minutes. Carefully aspirate the supernatant with a pipette, leaving only the precipitate.

[0050] 1.2. Dissolve the precipitate in 200 μl of buffer, and centrifuge at high speed at 4°C for 10 minutes. Remove the precipitate to obtain the supernatant solution.

[0051] 1.3. Remove the salt and protective agent from the blue supernatant solution using a fast desalting column or a dialysis bag. Then add dithiothreitol (DTT) to the desalted phycocyanin solution to make the final concentration of the mixture 20-50 μmol / L. React at room temperature for 90 minutes to decompose the large phycocyanin molecules into a mixture of phycocyanin alpha and beta subunits. Remove the excess DTT from the mixture solution using a desalting column or a dialysis bag, and reserve for use.

[0052] Step 2: Preparation of derivatized fusion protein AG (protein AG)

[0053] 2.1. Dissolve 1 mg of protein AG fusion protein in 200 μl of 0.1 M PBS (pH 7.2-7.4) buffer solution to prepare a 5 mg / ml solution.

[0054] 2.2. Dissolve the bifunctional reagent succinimidyl-4-(N-maleimidomethyl cyclohexane-1-carboxylate) (SMCC) in anhydrous dimethyl sulfoxide (DMSO) to prepare a 50 mg / ml stock solution. Add 20 μl of SMCC per mg of protein AG, seal with aluminum foil, and rotate at room temperature for 60 minutes to react the amino groups on the protein AG molecules with the succinamide to produce derivatized protein AG protein.

[0055] 2.3, Exchange buffer pre-equilibrate the gel column, pass the derivatized protein AG protein through the column, collect the protein AG protein peak.

[0056] Step three, preparation of the cross-linking from pre-stained fluorescent protein Marker:

[0057] 3.1, dropwise add the mixture of phycocyanin alpha, beta subunit to the protein AG protein derivatized with succinimidyl-4-(N-methylmaleimide)cyclohexane-1-carboxylate (SMCC), mix gently with a homogenizer while adding, seal with aluminum foil and rotate coupling at room temperature for 120 minutes (or overnight at 4°C), so that the thiol group on the phycocyanin alpha, beta subunit molecule and the maleimide group are covalently cross-linked to obtain the pre-stained fluorescent protein Marker product.

[0058] Example three: preparation of covalently coupled fluorescent allophycocyanin and protein AGL from pre-stained fluorescent protein Marker

[0059] It should be noted that allophycocyanin is isolated and purified from spirulina, which can emit strong fluorescence, and its molecular structure is a carrier protein connected by a thioether bond and an open chain linear extension of a tetrapyrrole compound. The molecular weight is 104,000 daltons, and the protein subunit composition is (alpha-beta)3, with each alpha-subunit and beta-subunit about 17,000 daltons.

[0060] Step two, preparation of derivatized fluorescent allophycocyanin protein molecules:

[0061] 2.1, dissolve 1 mg of protein AGL fusion protein in 200 μl of 0.1 M PBS (pH 7.2-7.4) buffer solution to prepare a 5 mg / ml solution.

[0062] 2.2, dissolve succinimidyl-4-(N-methylmaleimide)cyclohexane-1-carboxylate (SMCC) in anhydrous dimethyl sulfoxide (DMSO) to prepare a 5 mg / ml stock solution. Add 9 μl of SMCC per mg of allophycocyanin, seal with aluminum foil and stir the reaction mixture at room temperature for 30 minutes or stir the reaction at 4°C for 2 hours, so that the amino group on the allophycocyanin molecule reacts with succinamide to form derivatized allophycocyanin.

[0063] 2.3, exchange buffer pre-equilibrate the gel column, pass the derivatized protein AG protein through the column, collect the protein AG protein peak, and adjust the concentration to 5 mg / ml.

[0064] Thiolation of fusion protein AGL (protein AGL):

[0065] Preparation: 1 mg of protein AGL fusion protein was dissolved in 200 μl of 0.1 M PBS (pH 7.2-7.4) buffer solution to prepare a 5 mg / ml solution to obtain a pure fusion protein, which includes the following steps:

[0066] A. 3-(2-pyridyl disulfide) propionic acid N-hydroxy succinimidyl ester (SPDP) was dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a 50 mg / ml stock solution.

[0067] B. Take the pure protein AGL (purity > 90%), adjust the concentration to more than 5 mg / ml, add 15 μl of SPDP solution per mg of protein AGL, mix gently, and stir at room temperature for 0.5-1.5 hours. Remove excess SPDP with a centrifugal desalting column, and collect the SPDP-protein AGL solution.

[0068] C. Tris(2-carboxyethyl)phosphine (TCEP) was dissolved in 0.1 M PBS (pH 7.4, 0.1 M NaCl) to prepare a 2 mg / ml solution. Add 20 μl of TCEP solution per mg of SPDP-protein AGL, mix gently, and react at room temperature for 10-15 minutes.

[0069] D. Separate the thiolated protein AGL on a PD-10 column and adjust the concentration to 3 mg / ml.

[0070] Step three, preparation of self-pre-dyed fluorescent protein Marker from cross-linking:

[0071] 3.1. Add the activated allophycocyanin mixture dropwise to the thiolated protein AGL protein, mix gently with a mixer while adding, seal with aluminum foil, and rotate the coupling at room temperature for 120 minutes (or overnight at 4°C) to covalently cross-link the thiol group with the maleimide group to obtain the self-pre-dyed fluorescent protein Marker product.

[0072] Example four: covalent coupling preparation of self-pre-dyed fluorescent protein Marker of fusion protein of fluorescent protein: green fluorescent protein (GFP) (or yellow fluorescent protein (YFP), or red fluorescent protein (RFP), etc.) and protein G-antibody Fc region;

[0073] It should be noted that green fluorescent protein (Green fluorescent protein, abbreviated as GFP) is a protein composed of about 238 amino acids, which can be excited from blue light to ultraviolet light to emit green fluorescence.

[0074] Thiolation of green fluorescent protein (GFP)

[0075] Preparation: 1 mg of green fluorescent protein (GFP) was dissolved in 200 μl of 0.1 M PBS (pH 7.2-7.4) buffer solution to prepare a 5 mg / ml solution, and the fusion protein was purified, including the following steps:

[0076] A) 3-(2-pyridyl disulfide) propionic acid N-hydroxy succinimidyl ester (SPDP) was dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a 50 mg / ml stock solution.

[0077] B) 20 μl of SPDP solution was added per mg of green fluorescent protein (GFP), and the mixture was gently mixed and stirred at room temperature for 0.5-1.5 hours. Excess SPDP was removed by a centrifugal desalting column, and the collected solution was SPDP-GFP.

[0078] C) Tris(2-carboxyethyl)phosphine (TCEP) was dissolved in 0.1 M PBS (pH 7.4, 0.1 M NaCl) to prepare a 2 mg / ml solution. 20 μl of TCEP solution was added per mg of SPDP-GFP, and the mixture was gently mixed and reacted at room temperature for 10-15 minutes.

[0079] D) The thiolated GFP was separated by PD-10 column and adjusted to a concentration of 5 mg / ml.

[0080] Step two, preparation of derivatized fusion protein protein G-Fc:

[0081] 2.1, 1 mg of fusion protein was dissolved in 200 μl of 0.1 M PBS (pH 7.2-7.4) buffer solution to prepare a 5 mg / ml solution.

[0082] 2.2, Bifunctional reagent succinimidyl-4-(N-maleimide) cyclohexane-1-carboxylate (SMCC) was dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a 50 mg / ml stock solution. 20 μl of SMCC was added per mg of fusion protein, and the mixture was rotated at room temperature for 60 minutes after being sealed with aluminum foil to react the amino group on the fusion protein molecule with succinamide to generate derivatized fusion protein.

[0083] 2.3, The gel column was equilibrated with the exchange buffer, and the derivatized fusion protein was passed through the column. The fusion protein peak was collected and adjusted to a concentration of 5 mg / ml.

[0084] Step three, preparation of cross-linking from pre-stained fluorescent protein Marker:

[0085] 3.1, Add the activated fusion protein drop by drop into the thiolated green fluorescent protein (GFP) protein, mix gently with a homogenizer while adding, seal with aluminum foil and rotate coupling at room temperature for 120 minutes (or overnight at 4°C), so that the thiol and maleimide group realize covalent crosslinking to obtain self-pre-dyed fluorescent protein Marker products.

[0086] It should be noted that the relational terms herein such as first and second and the like are used solely to distinguish one entity or action from another, without necessarily requiring or implying any such actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0087] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, alternatives, and variations can be made in the embodiments without departing from the spirit and scope of the present application as defined by the appended claims and their equivalents.

Claims

1. A method for preparing a self-pre-stained fluorescent protein Marker, the method comprising the following steps: Step 1, preparing a subunit mixture: 1.1, 1 milligram of luminescent protein precipitate is placed in a centrifuge tube, high speed centrifugation at 4℃ for 5 minutes, to obtain supernatant and precipitate, the supernatant is completely absorbed by a pipette, only the precipitate is reserved; 1.2, 200 microliters of buffer is added to the precipitate to dissolve completely, then centrifugation at 4℃ for 10 minutes, remove the precipitate, to obtain the supernatant solution; 1.3, the supernatant solution is desalted by a fast desalting column or dialysis bag, and the desalted luminescent protein solution is added with dithiothreitol DTT to obtain a mixture with a final concentration of 20-50 micromole / liter, then reaction at room temperature for 90 minutes, to obtain the subunit mixture; Step two, preparation of derivatized fusion protein: 2.1, 1 milligram of fusion protein is dissolved in 200 microliters of 0.1M PBS pH 7.2-7.4 buffer solution, to prepare a 5 milligram / milliliter solution; 2.2, the bifunctional reagent is dissolved in anhydrous dimethyl sulfoxide DMSO to prepare a 50 milligram / milliliter stock solution, 20 microliters of succinimidyl-4-n-maleimidomethyl cyclohexane-1-carboxylate SMCC is added for every milligram of fusion protein, the aluminum foil is sealed and rotated at room temperature for 60 minutes, so that the amino group on the fusion protein molecule reacts with succinamide to generate derivatized fusion protein; 2.3, the buffer is exchanged and the gel column is pre-equilibrated, the derivatized fusion protein is passed through the column, the fusion protein peak is collected, and the concentration is adjusted; The preparation of derivatized fusion protein also includes the thiolation of fusion protein: Preparation: 1 milligram of protein A GL fusion protein is dissolved in 200 microliters of 0.1M PBS pH 7.2-7.4 buffer solution, to prepare a 5 milligram / milliliter solution, to obtain a pure fusion protein, which specifically includes the following steps: A, 3-2-pyridine dithioglycollic acid N-hydroxy succinimidyl ester SPDP is dissolved in anhydrous dimethyl sulfoxide DMSO to prepare a 50 milligram / milliliter stock solution; B, take the pure fusion protein, the concentration is adjusted to more than 5 milligrams / milliliter, according to the addition of SPDP solution for every milligram of fusion protein, gently mix, stir at room temperature for 0.5-1.5 hours, remove the excess SPDP with a centrifugal desalting column, and collect the SPDP-fusion protein solution; C, 3-2-carboxyethyl phosphine TCEP is dissolved in 0.1M PBS pH 7.4, 0.1M NaCl, to prepare a 2 milligram / milliliter solution, 20 microliters of TCEP solution is added for every milligram of SPDP-fusion protein, gently mix, and react at room temperature for 10-15 minutes; D, separate the thiolated fusion protein on a PD-10 column, and adjust the concentration to 3 mg / ml, Step three, cross-linking preparation: 3.1, the subunit mixture is added dropwise to the derivatized fusion protein, and gently mixed with a mixer while adding, the aluminum foil is sealed and rotated for coupling at room temperature for 120 minutes or overnight at 4℃, to realize covalent cross-linking and obtain a self-prepared fluorescent protein marker preparation; The step three 3.1 further comprises adding the activated fusion protein drop by drop into the thiolated fusion protein, mixing gently with a homogenizer, sealing with aluminum foil and rotating coupling for 120 minutes at room temperature or overnight at 4℃ to achieve covalent cross-linking of the thiol and the maleimide group to obtain the self-pre-stained fluorescent protein Marker product.

2. The method for preparing a self-prestained fluorescent protein marker according to claim 1, characterized in that: In the step one 1.1, the luminescent protein comprises one of phycoerythrin, phycocyanin, allophycocyanin, green fluorescent protein, yellow fluorescent protein and red fluorescent protein.

3. The method for preparing a self-prestained fluorescent protein marker according to claim 1, characterized in that: In the step one 1.3, the subunit mixture is used after removing the excess DTT in the mixed solution by using a desalting column or a dialysis bag.

4. The method for preparing a self-prestained fluorescent protein marker according to claim 1, characterized in that: In the step three 3.1, after mixing gently with a homogenizer, sealing with aluminum foil and rotating coupling overnight at 4℃, covalent cross-linking is achieved to obtain the self-pre-stained fluorescent protein Marker product.

5. The method for preparing a self-prestained fluorescent protein marker according to claim 1, characterized in that: The purity of the fusion protein pure product is greater than 90%.

6. The method for preparing a self-prestained fluorescent protein marker according to claim 1, characterized in that: The SPDP solution comprises no less than 15 μl. The SPDP solution comprises no less than 15 μl.