CN116047055A - Method for large-scale directional marking of alkaline phosphatase - Google Patents

Abstract

The invention relates to the technical field of biological fixed-point labeling, in particular to a method for large-scale directional labeling of alkaline phosphatase, which comprises the following steps: the method comprises the steps of designing subclones of the CIAP protein fused by the latch-Taq, purifying the CIAP protein fused by the latch-Taq, designing subclones of the C-Myc antibody fused by the Spy-Taq, purifying the C-Myc antibody fused by the Spy-Taq, performing directional coupling on the CIAP and the C-Myc antibody, and performing directional coupling on the CIAP to the C end of the recombinant protein.

Description

Method for large-scale directional marking of alkaline phosphatase

Technical Field

The invention relates to the technical field of biological fixed-point labeling, in particular to a method for large-scale directional labeling of alkaline phosphatase.

Background

The enzyme labeling method is also called an immunoenzymatic technique, and is a biological technique of coupling an antigen, an antibody and an enzyme based on an immune reaction. Plays a critical role in related immune experiments, such as enzyme-linked immunosorbent assay, immunoblotting assay, enzyme immunohistochemical staining and the like. The results of the enzyme labeling technology are easy to observe, and can be detected by related equipment, and can be qualitative and quantitative. Commonly used enzyme-labeled techniques are horseradish peroxidase (HRP) and alkaline phosphatase (CIAP). Alkaline Phosphatase (CIAP) is widely distributed in animal liver, bones, intestines, kidneys, placenta and other tissues, and bovine small intestine alkaline phosphatase (CIAP) is selected as a research object, and the enzyme activity of the CIAP is relatively high and is widely used in enzyme labeling experiments.

At present, two methods for marking alkaline phosphatase are mainly available, one is glutaraldehyde crosslinking marking method, and glutaraldehyde is used as a difunctional crosslinking agent, and can be combined with amino groups on enzyme molecules and protein molecules respectively, so that the method is divided into a one-step method and a two-step method. The protein, enzyme and glutaraldehyde are mixed simultaneously in a one-step process. The method is simple to operate, the enzyme-labeled proteins such as AP, HRP and the like can be used for the method, but the method has great defects, the active product of the enzyme label is very few, the enzyme and the protein are easy to inactivate probably due to the obstruction of a space structure, and the protein is easy to crosslink; the two-step method is to react excessive glutaraldehyde with enzyme to make the amino groups on the enzyme molecule combine with aldehyde groups on glutaraldehyde molecule only, so that the combination of enzyme and enzyme does not occur, and then add protein after removing excessive glutaraldehyde to form enzyme-glutaraldehyde-protein conjugate. Its advantages are uniform label, no self-polymerization, high sensitivity, low productivity and low labeling efficiency. The other method is to use sodium periodate oxidation method, which is to produce a plurality of aldehyde groups by sodium periodate oxidase itself, then the aldehyde groups react with the protein to be marked to obtain the conjugate, and the method is characterized in that the marking effect is greatly improved, but the operation is more complicated, which is a challenge for production personnel.

Disclosure of Invention

In order to overcome the shortcomings of the prior art, the present invention provides a method for large scale directed labeling of alkaline phosphatase.

The technical scheme adopted for solving the technical problems is as follows: a method for large scale directional labeling of alkaline phosphatase comprising the steps of:

s1, designing subcloning of the Catcher-Taq fused CIAP protein, designing and constructing subcloning plasmids, producing the Catcher-Taq fused CIAP protein in a eukaryotic expression system, performing codon optimization on a target gene after software analysis, and constructing the target gene on a eukaryotic expression vector;

s2, purifying the latch-Taq fused CIAP protein, wherein the latch-Taq fused CIAP protein is subjected to affinity purification through a high-tolerance Ni filler, sampling and balancing, then washing impurities with 2-5mM low-concentration imidazole washing buffer solution, eluting with 250-500mM high-concentration imidazole eluting buffer solution, and finally dialyzing the mixed solution to a mixed solution of 20-50mM Tris-HCl, 10-50mM MgCl2 and 10-50 mu M ZnCl2, wherein the pH value of the mixed solution is 6.5-8.5;

s3, subcloning the Spy-Taq fused c-Myc antibody, designing and constructing subcloning plasmids, producing the Spy-Taq fused c-Myc antibody in a eukaryotic expression system, performing codon optimization on a target gene after software analysis, and constructing the target gene on a eukaryotic expression vector;

s4, purifying a c-Myc antibody fused with Spy-Taq, wherein the Spy-Taq fusion Protein is a recombinant expressed antibody and is provided with Fc-Taq, purifying through Protein A filler, balancing after loading, eluting with 0.1M glycine, and finally neutralizing with 1-2M Tris-HCl, wherein the pH of the Tris-HCl is 8;

s5, performing directional coupling on the CIAP and the C-Myc antibody, and performing directional coupling on the CIAP to the C end of the C-Myc antibody at the reaction temperature of 18-25 ℃ for 1-3hr.

Specifically, spy-Taq fusion protein and Spy-latch fusion protein are respectively expressed in a eukaryotic expression system, so that the CIAP is coupled with a recombinant antibody or antigen, the coupling method is to directionally couple the CIAP to the C end of the recombinant protein, the coupling method is simple and rapid, no complex operation is performed, the structure of the protein is not damaged, and the requirement on operators is low, so that the method is suitable for large-scale production, and the problems of low marking rate and complex marking method of the existing CIAP marking protein method are solved.

According to another embodiment of the present invention, in S1 and S2, the eukaryotic expression vector is a pcdna3.1 transient vector or a stable expression vector with a resistance gene.

According to another embodiment of the present invention, further comprising, in S2, the wash buffer is 3mM imidazole and the elution buffer is 250mM imidazole.

According to another embodiment of the present invention, in S2, the mixed solution is 20mM Tris-HCl, 10mM MgCl2 and 10uM ZnCl2, and the pH is 8.

According to another embodiment of the present invention, further comprising, in S4, the glycine is at ph3.0.

According to another embodiment of the present invention, further comprising, in S4, the Tris-HCl is 2M.

According to another embodiment of the present invention, further comprising, in S5, the reaction temperature is 20 ℃.

According to another embodiment of the present invention, further comprising, in S5, the reaction time is 2hr.

According to another embodiment of the present invention, further comprising, in S5, a ratio of the coupling molar concentration of the latch fusion protein to the SPY fusion protein is 1:1 to 1:4.

The beneficial effects of the invention are as follows:

1. the alkaline phosphatase is widely distributed in animal liver, bones, intestines, kidneys, placenta and other tissues, bovine small intestine alkaline phosphatase (CIAP) is selected as a research object, recombinant CIAP expression is carried out in eukaryotic cells, and the activity is consistent with that of natural alkaline phosphatase without obvious difference;

2. the recombinant expression system is flexible, and a required label or a required sequence can be added through a molecular biology means, so that the recombinant expression CIAP, the recombinant expression Spytaq and the recombinant expression SpyCatcher can be applied to other related fields;

3. the stability of the Spytaq and Spycatcher systems is high, covalent bonds can be stably formed under extreme conditions such as low pH, so that the system can realize high-efficiency directional coupling under some special environments;

4. the Spytaq and Spycatcher systems have mild reaction conditions, no complex operation, low requirements on operators and no damage to the structure of the protein, so that the method is suitable for large-scale production and industrialization.

Drawings

The invention will be further described with reference to the drawings and examples.

FIG. 1 is a diagram of SDS-PAGE of non-reducing (Lane 1) and reducing (Lane 2) antibodies to c-Myc;

FIG. 2 is an SDS-PAGE electrophoresis of Cather-CIAP fusion protein reduction (Lane 1) and non-reduction (Lane 2);

FIG. 3 is an SDS-PAGE electrophoresis of the Catcher-CIAP and c-Myc-Spy antibodies before mixing (Lane 2) and after mixing (Lane 1) c-Myc-Spy antibodies;

FIG. 4 is a graph showing the activity of ELISA for detection of different c-Myc antibody coupling methods.

Detailed Description

A method for large scale directional labeling of alkaline phosphatase comprising the steps of:

s1, designing subcloning of the Catcher-Taq fused CIAP protein, designing and constructing subcloning plasmids, producing the Catcher-Taq fused CIAP protein in a eukaryotic expression system, performing codon optimization on a target gene after software analysis, and constructing the target gene on a eukaryotic expression vector;

s2, purifying the latch-Taq fused CIAP protein, wherein the latch-Taq fused CIAP protein is subjected to affinity purification through a high-tolerance Ni filler, sampling and balancing, then washing impurities with 2-5mM low-concentration imidazole washing buffer solution, eluting with 250-500mM high-concentration imidazole eluting buffer solution, and finally dialyzing the mixed solution to a mixed solution of 20-50mM Tris-HCl, 10-50mM MgCl2 and 10-50 mu M ZnCl2, wherein the pH value of the mixed solution is 6.5-8.5;

s3, subcloning the Spy-Taq fused c-Myc antibody, designing and constructing subcloning plasmids, producing the Spy-Taq fused c-Myc antibody in a eukaryotic expression system, performing codon optimization on a target gene after software analysis, and constructing the target gene on a eukaryotic expression vector;

s4, purifying a c-Myc antibody fused with Spy-Taq, wherein the Spy-Taq fusion Protein is a recombinant expressed antibody and is provided with Fc-Taq, purifying through Protein A filler, balancing after loading, eluting with 0.1M glycine, and finally neutralizing with 1-2M Tris-HCl, wherein the pH of the Tris-HCl is 8;

s5, performing directional coupling on the CIAP and the C-Myc antibody, and performing directional coupling on the CIAP to the C end of the C-Myc antibody at the reaction temperature of 18-25 ℃ for 1-3hr.

Specifically, spy-Taq fusion protein and Spy-latch fusion protein are respectively expressed in a eukaryotic expression system, so that the CIAP is coupled with a recombinant antibody or antigen, the coupling method is to directionally couple the CIAP to the C end of the recombinant protein, the coupling method is simple and rapid, no complex operation is performed, the structure of the protein is not damaged, and the requirement on operators is low, so that the method is suitable for large-scale production, and the problems of low marking rate and complex marking method of the existing CIAP marking protein method are solved.

In S1 and S2, the eukaryotic expression vector is a pCDNA3.1 transient vector or a stable expression vector with a resistance gene.

Preferably, in S2, the wash buffer is 3mM imidazole and the elution buffer is 250mM imidazole.

Preferably, in S2, the mixture is 20mM Tris-HCl, 10mM MgCl2 and 10uM ZnCl2, and the pH is 8.

Preferably, in S4, the glycine is at ph3.0.

Preferably, in S4, the Tris-HCl is 2M.

Preferably, in S5, the reaction temperature is 20 ℃.

Preferably, in S5, the reaction time is 2hr.

Preferably, in S5, the coupling molar concentration ratio of the latch fusion protein to the SPY fusion protein is 1:1-1:4.

Specifically, the operation is as follows:

1. subcloning design and total Gene Synthesis

1.1 Total gene synthesis

The total gene synthesis contains the CIAP sequence of Catcher-Taq, the sequence is directly synthesized on a eukaryotic expression vector, bamHI and XhoI enzyme cutting sites are contained on the vector, an 8 XHis tag is reserved at the C end, and the Ni column purification can be carried out subsequently.

1.2 construction of eukaryotic expression plasmid for c-Myc-Spy antibody

The primers C-Myc-spin-F and C-Myc-spin-R are designed for PCR, a spin tag sequence is constructed to the C end of an antibody IgG-Fc by a PCR method, and the fused C-Myc-spin monoclonal antibody eukaryotic expression plasmid is obtained, wherein the PCR is specifically set to be 95 ℃ 30sec,95 ℃ 10sec,55 ℃ 30sec,72 ℃ 90sec,72 ℃ 5min and 4 ℃ for storage. Wherein, the primer sequence of the c-Myc-spy-F is as follows:

gctctgggtgcccggctccaccggagaggtgcacctggtggagag；

the primer sequences for c-Myc-spy-R are:

TCAGCCGGAGCCCCGGAAAGGCCCACATTGTGATGGTGGACGCCTACAGCCCTACAAGC.

1.3 Construction of Catcher-CIAP eukaryotic expression plasmid

Designing primers including a Catcher-CIAP-F and a Catcher-CIAP-R, constructing a Catcher-tag sequence to the N end of an antibody CIAP by a PCR method to obtain a Catcher-CIAP eukaryotic expression plasmid, wherein the PCR is specifically set to be at 95 ℃ 30sec,95 ℃ 10sec,55 ℃ 30sec,72 ℃ 90sec,72 ℃ 5min and 4 ℃ for storage. Wherein, the primer sequence of the latch-CIAP-F is as follows:

GCTCACAGGGAGCAGATCGGAGGAGGATCCCTGATTCCAGTGGAGGAGGA；

the primer sequences for the Catcher-CIAP-R are:

AGGAGAGGCGGCGAGGTGGGCGGCGTCGGGAATAGATGTGGCTGTTGTTG.

2. expression and purification of fusion proteins

2.1 Expression and purification of polypeptide sequences of fusion antibodies

Carrying out plasmid transfection by adopting a PEI max transfection reagent of polyscience, respectively adding 300 mu L of PEI max and 100 mu g of heavy and light chain plasmid fused with the c-Myc-Spy antibody into 10mL of optimem, reversing the materials up and down, mixing the materials uniformly, and standing the materials at room temperature for 5min; then slowly adding PEI max mixed solution into mixed solution containing c-Myc-Spy antibody plasmid, standing at room temperature for 10min, slowly adding into cells, adding corresponding additives after 20h, centrifuging after protein expression for 5 days, and taking culture medium supernatant for purification after cell centrifugation, wherein the specific purification method is as follows:

the c-Myc-Spy antibody is purified by using a rProtein At gravity column, and the culture supernatant after centrifugation is purified, for example, 200mL culture supernatant is purified by using 2mL rProtein At. 2mL of rProtein At filler is filled in a gravity column, the gravity column adopts a specification of 12mL, then a balance solution with a volume of 20 times of column is used for balanced flushing, a pH=7.4 0.01M PBS buffer solution is used as the balance solution, 200mL of culture medium supernatant is added for slow loading after balanced flushing, the loading flow rate is controlled At 2mL/min, the mixture is continuously washed with a volume of 20 times of 0.01M PBS after loading, finally the mixture is eluted with 0.1M glycine (pH=3.0) and collected by a separate tube, 2mL of each tube is collected, 100 mu.L of 2M Tris-HCl buffer solution (pH=8.0) is added in each collection tube before eluting, and purified protein is subjected to SDS-PAGE electrophoresis to detect the purity of c-Myc-Spy antibody and dialyzed into 0.01M PBS (pH=7.4) after 12 hours. Specific electrophoresis conditions are as follows:

loading 3 mu g c-Myc-Spy antibody, electrophoresis with 4-20% SDS-PAGE gradient gel by MOPS buffer, setting parameters as 160V electrophoresis for 40min, and finally coomassie brilliant blue staining to obtain protein size and purity.

As shown in the non-reducing (Lane 1) and reducing (Lane 2) SDS-PAGE electrophoresis of the c-Myc antibody in FIG. 1, the experimental result shows that after c-Myc-Spy tag, the c-Myc-Spy tag is about 150-160kDa in a non-reducing state (Lane 1), and the c-Myc-Spy tag has two heavy chains and light chains of 50kDa and 25kDa in a reducing state (Lane 2), the size and the natural antibody are identical, the band is single, and the purity is more than or equal to 95%.

2.2 Expression and purification of the Catcher-CIAP fusion protein

Carrying out plasmid transfection by adopting a PEI max transfection reagent of polyscience, respectively adding 300 mu L of PEI max and 100 mu g of heavy and light chain plasmid fused with the c-Myc-Spy antibody into 10mL of optimem, reversing the materials up and down, mixing the materials uniformly, and standing the materials at room temperature for 5min; then slowly adding PEI max mixed solution into mixed solution containing c-Myc-Spy antibody plasmid, standing at room temperature for 10min, slowly adding into cells, adding corresponding additives after 20h, centrifuging after protein expression for 5 days, and taking culture medium supernatant for purification after cell centrifugation, wherein the specific purification method is as follows:

the latch-CIAP fusion protein is purified by adopting a gravity column, and the culture medium supernatant after centrifugation is purified, for example, 200mL of culture medium supernatant is purified by adopting 2mL Ni Smart Beads 6FF filler. Filling 2mL Ni Smart Beads 6FF filler into a gravity column, performing balanced flushing on the gravity column by using a 12mL size, performing balanced flushing on the gravity column by using a 20-time column volume balancing solution, performing balanced flushing on the balancing solution by using a 0.01M PBS buffer solution with pH of 7.4, adding 200mL of culture medium supernatant for slow loading, controlling the flow rate of loading at 2mL/min, continuing to perform impurity flushing on the gravity column by using a 20-time column volume 0.01M PBS after loading, eluting by using a 250mM imidazole elution buffer solution (containing 0.3M NaCl and pH of 8.0) and collecting by separating tubes, collecting 2mL of the gravity column per tube, performing SDS-PAGE on purified protein to detect the purity of the Catcher-CIAP fusion protein, and dialyzing into the 0.01M PBS (pH of 7.4) after 12 hours. Specific electrophoresis conditions are as follows:

3 mug of the latch-CIAP fusion protein is loaded, 4-20% SDS-PAGE gradient gel is subjected to electrophoresis by using MOPS buffer, parameters are set to 160V for 40min, and finally coomassie brilliant blue staining is performed to carry out protein size and purity.

As shown in the experimental results of the Cather-CIAP fusion protein reduction (Lane 1) and non-reduction (Lane 2) SDS-PAGE electrophoresis of FIG. 2, the Catcher-CIAP protein can generate a certain oligomerization phenomenon in a non-reduction state (Lane 2), and the band of the Catcher-CIAP fusion protein in the reduction state (Lane 1) is single, and the purity is more than or equal to 90%.

Directional coupling of the latch-CIAP fusion protein to the c-Myc-Spy antibody

10. Mu.g of the latch-CIAP fusion protein and 10. Mu. g c-Myc-Spy antibody are added into a 100. Mu.L system, a reaction buffer solution is 0.01M PBS buffer solution with pH=7.4, the mixed solution is reacted in a mixer for 60min at the temperature of 25 ℃, and then the coupling efficiency is detected by means of SDS-PAGE electrophoresis under the following conditions:

10. Mu.L of the coupled samples were loaded, 4-20% SDS-PAGE gel was electrophoresed in MOPS buffer at 160V for 40min, and finally stained with Coomassie blue. As shown in the SDS-PAGE electrophoresis of the Catcher-CIAP and c-Myc-Spy antibodies before mixing (Lane 2) and after mixing (Lane 1) as shown in FIG. 3, the Catcher-CIAP fusion protein and c-Myc-Spy antibodies migrate to the upper side after mixing (Lane 1 band is larger, in the gel well), indicating that the Catcher-CIAP fusion protein and c-Myc-Spy antibodies are coupled together to change the size of the antibodies, the labeling efficiency is not less than 90%, and is far higher than that of the traditional sodium periodate method (up to 70% labeling efficiency).

ELISA activity test is carried out on coupled CIAP, 50ng of antigen containing c-Myc tag is coated, coating is carried out for 12 hours at 4 ℃, then PBST is used for washing 5 times, coupled c-Myc-CIAP antibody is added, incubation is carried out for 1 hour at 37 ℃, PNPP color development is carried out after PBST is used for washing 5 times, and the steps are as follows:

1. the PNPP chromogenic substrate is placed at room temperature for more than 20 minutes to reach the room temperature; secondly, lightly mixing; 3. adding 100 mu l of pNPP into each hole of the ELISA plate, and gently vibrating the ELISA plate to thoroughly mix the ELISA plate; 4. incubating the ELISA plate for 15-30 minutes at room temperature or longer until the color development is deep enough; 5. adding 2N NaOH into each hole to terminate the reaction, and lightly vibrating the ELISA plate to thoroughly mix the ELISA plate; sixth, absorbance is detected, wavelength 405 and nm.

In a control experiment, an AP (sigma) protein is marked on a c-Myc antibody by adopting a sodium periodate oxidation method, and activity measurement is carried out by adopting an ELISA method, wherein the experimental result is shown in an activity chart of the ELISA detection different c-Myc antibody coupling methods of FIG. 4, the result in the chart shows that the activity (EC 50=0.06 mug/mL) of the antibody is not influenced by using the Spy-Catcher for antibody coupling, and the activity is higher than that of the conventional sodium periodate marking method (EC 50=0.15 mug/mL), so that the structure of the CIAP and the c-Myc antibody is not damaged by coupling the antibody with the CIAP by adopting the method disclosed by the invention, and the coupling method is very simple, so that the method is suitable for large-scale production, and the problem that the conventional marked CIAP method is not suitable for large-scale industrial preparation is solved.

Wherein:

amino acid sequence of CIAP

SEQ ID NO.1

LIPVEEEDPAFWNCQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYKTIGVSAAARYNQCNTTSGNEVTSVMNRAKKAGKAVGVVTTSRVQHASPAGAYAHTVNRNWYSDADLPADAQMNGCQDIATQLVYNMDIDVILGGGRMYMFPEGTPDPEYPYDVNQTGVRKDKRNLVQEWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLQEMTEVALRVLSRNPRGFYLFVEGGRIDHGHHEGKAYMALTDTVMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPSKALDSKSYTSILYGNGPGYALGGGSRPDVNDSTSEDPSYQQQAAVPLASETHGGEDVAVFARGPQAHLVHGVQEETFVAHIMAFAGCVEPYTDCNLPAPTTATSIPDAAHLAASP

Amino acid sequence of Catcher-Taq

SEQ ID NO.2

DSATHIKFSKRELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETVATAITFTVNEQGGKATK

Amino acid sequence of Spy-Taq

SEQ ID NO.3

AHIVDAYSPVM

Binding of Spy tag to Catcher tag is the formation of an isopeptide bond. The spontaneous formation of isopeptidic bonds was found at the earliest in the assembly of the capsid protein of the HK97 phage, and later researchers found that SpyTag and SpyCatcher peptide fragments carry Asp residues and Lys residues, respectively, which form isopeptidic bonds characterized by stable binding, short peptide chains and rapid reactions, from streptococcus pyogenes fibronectin CnaB 2.

The invention obtains the latch-CIAP fusion protein and the C-Myc-Spy antibody by molecular cloning and recombinant expression, and the coupling mode is to form an amide bond spontaneously so as to directionally mark the CIAP to the C end of the C-Myc antibody, so that the stability of the amide bond is very high, and the CIAP fusion protein and the C-Myc-Spy antibody exist stably under the extreme pH condition. Compared with the traditional method, such as glutaraldehyde crosslinking labeling method, the enzyme labeled active product is few, the enzyme and the protein are easy to be deactivated, and the protein itself is easy to crosslink; the sodium periodate oxidation method is complex in operation and is a challenge for production personnel, the latch-Spy system and the eukaryotic expression system are perfectly combined, so that the CIAP and the c-Myc antibody are directionally coupled, the coupling method is simple and rapid, the condition is mild, no complex operation is caused, the structure of the antibody is not damaged by the coupling method, the problem that the conventional marked HRP method is not suitable for large-scale industrialized preparation is solved, and most importantly, the marking efficiency is more than or equal to 90 percent and is far higher than that of the conventional marking method.

The invention is a way of enabling the CIAP to be covalently bound to the antigen or antibody without any chemical modification, has simple operation, controllable process and risk and reduces the uncertainty of the reaction to the greatest extent. The invention utilizes the Catcher-Spy system to combine CIAP with the antibody, the activity of the antibody (EC 50=0.06 mug/mL) is not affected, and the activity of HRP is higher than that of the traditional sodium periodate method (EC 50=0.15 mug/mL), which indicates that the coupling mode of the invention can not damage the structure of the antibody, thus being suitable for large-scale production and solving the problem that the traditional CIAP labeling method is not suitable for large-scale industrialized preparation.

The recombinant expression system has strong flexibility, and can reasonably modify the target gene according to own ideas, so that all proteins expressed by the recombinant expression system can be applied to other related fields.

The above description is illustrative of the invention and is not to be construed as limiting, and it will be understood by those skilled in the art that many modifications, changes or equivalents may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (9)

1. A method for large scale directional labeling of alkaline phosphatase comprising the steps of:

s1, designing subcloning of the Catcher-Taq fused CIAP protein, designing and constructing subcloning plasmids, producing the Catcher-Taq fused CIAP protein in a eukaryotic expression system, performing codon optimization on a target gene after software analysis, and constructing the target gene on a eukaryotic expression vector;

s2, purifying the latch-Taq fused CIAP protein, wherein the latch-Taq fused CIAP protein is subjected to affinity purification through a high-tolerance Ni filler, sampling and balancing, then washing impurities with 2-5mM low-concentration imidazole washing buffer solution, eluting with 250-500mM high-concentration imidazole eluting buffer solution, and finally dialyzing the mixed solution to a mixed solution of 20-50mM Tris-HCl, 10-50mM MgCl2 and 10-50 mu M ZnCl2, wherein the pH value of the mixed solution is 6.5-8.5;

s3, subcloning the Spy-Taq fused c-Myc antibody, designing and constructing subcloning plasmids, producing the Spy-Taq fused c-Myc antibody in a eukaryotic expression system, performing codon optimization on a target gene after software analysis, and constructing the target gene on a eukaryotic expression vector;

s4, purifying a c-Myc antibody fused with Spy-Taq, wherein the Spy-Taq fusion Protein is a recombinant expressed antibody and is provided with Fc-Taq, purifying through Protein A filler, balancing after loading, eluting with 0.1M glycine, and finally neutralizing with 1-2M Tris-HCl, wherein the pH of the Tris-HCl is 8;

s5, performing directional coupling on the CIAP and the C-Myc antibody, and performing directional coupling on the CIAP to the C end of the C-Myc antibody at the reaction temperature of 18-25 ℃ for 1-3hr.

2. The method for large-scale targeted labeling of alkaline phosphatase according to claim 1, wherein in S1 and S2, the eukaryotic expression vector is pcdna3.1 transient vector or stable expression vector with resistance gene.

3. The method for large scale targeted labelling of alkaline phosphatase according to claim 1, wherein in S2 the wash buffer is 3mM imidazole and the elution buffer is 250mM imidazole.

4. The method for large scale targeted labelling of alkaline phosphatase according to claim 1, wherein in S2 the mixture is 20mM Tris-HCl, 10mM MgCl2 and 10um ZnCl2 and the pH is 8.

5. The method for large scale targeted labelling of alkaline phosphatase according to claim 1, wherein in S4, the glycine is at ph3.0.

6. The method for large scale targeted labelling of alkaline phosphatase according to claim 1, wherein in S4 the Tris-HCl is 2M.

7. The method for large scale directional labeling of alkaline phosphatase according to claim 1, wherein in S5 the reaction temperature is 20 ℃.

8. The method for large scale directional labeling of alkaline phosphatase according to claim 1, wherein in S5 the reaction time is 2hr.

9. The method for large scale targeted labelling of alkaline phosphatase according to claim 1, wherein the ratio of coupling molar concentration of Catcher fusion protein to SPY fusion protein in S5 is 1:1-1:4.

Images