CN113046343A - Preparation and application method of lysozyme affinity filler - Google Patents
Preparation and application method of lysozyme affinity filler Download PDFInfo
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Abstract
The invention discloses a preparation and application method of lysozyme affinity filler, which is characterized in that the lysozyme is fixed on a solid phase carrier by bonding with hydroxyl, carboxyl, amino or epoxy on the surface of the solid phase carrier to obtain the lysozyme affinity filler, the lysozyme antibody obtained by other separation methods such as lysozyme antibody fusion protein and microorganism metabolites is separated by using the lysozyme affinity filler, the separation efficiency is high, and the purity of the obtained lysozyme antibody is up to more than 95%.
Description
Technical Field
The invention belongs to the technical field of biotechnology pharmaceutical engineering, and particularly relates to a preparation and application method of lysozyme affinity filler.
Background
Lysozyme, also known as muramidase or N-acetylmuramyl glycan hydrolase, ruptures bacteria primarily by breaking the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, breaking down the cell wall insoluble mucopolysaccharide into soluble glycopeptides, causing the contents of the ruptured cell wall to escape. Lysozyme can be directly combined with viruses with negative electricity to form double salts with DNA, RNA and proteins of a radiation-free group, so that the viruses are inactivated. Therefore, the lysozyme has the functions of antibiosis, antiphlogosis, antivirus, and the like.
Lysozyme is an important component of the endogenous immune system of various animal tissues, and the normal content of lysozyme is different from that of different tissues; changes in lysozyme content in a variety of tissues are often associated with certain diseases. For example, an abnormal concentration of lysozyme in the urine of blood means a disease in the blood or kidney; lysozyme deficiency in neonates may lead to bronchogenic lung dysplasia; lysozyme deficiency in the recipe may increase the incidence of dysentery. Therefore, the detection of lysozyme in vivo plays a very important role in the diagnosis of many diseases.
At present, domestic lysozyme detection mainly adopts a micrococcus flavus turbidimetry method, a flat plate diffusion method, an electrochemiluminescence method and the like, and the methods generally have the defects of poor specificity, repeatability or accuracy and the like. With the wide application of immunological techniques, recombinant antibodies are also more adopted in the field of detection; compared with the traditional polyclonal antibody, the recombinant antibody has unique advantages, can be expressed in a prokaryotic expression system, has extremely short period, large production capacity and low cost, and has important significance for large-scale detection and production of increasing daily life.
At present, the common separation methods for lysozyme antibodies include crystallization, precipitation, ion exchange, affinity chromatography, etc., wherein the affinity chromatography technology has the advantages of high yield, high purity, capability of keeping natural state of biological macromolecules, etc., the conventional recombinant antibodies can be separated by adopting affinity fillers such as protein A, protein G, etc., for example, patent CN109336968A discloses a method for purifying antibodies by protein A affinity chromatography, and the following protein A affinity chromatography columns are recycled to purify target antibodies: the filler is MabSelect LX, Amsphere A3 or Praesto Jetted A50, the column height is 5-15 cm, and the linear flow rate is 200-500 cm/h; before eluting a target antibody by using an eluent, a buffer solution with the pH value of 5-6 and the conductivity of 20-100 mS/cm is used for washing a column bed, and the patent selects a filler with high load, good mass transfer capacity and high running flow speed, so that the medium cost is reduced, and the purification efficiency is improved; however, this method is mainly used for purification by binding to an antibody Fc fragment, and cannot be used for separation and purification of an antibody such as a single-chain antibody or a non-Fc fragment such as a nanobody.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method and an application method of lysozyme affinity filler, which are characterized in that lysozyme is bonded on a solid phase carrier to prepare the filler of a high-affinity lysozyme antibody, the lysozyme antibody obtained by other ways of separating lysozyme antibody fusion protein, microbial metabolites and the like is separated by using the affinity lysozyme filler, the separation efficiency is high, and the purity of the obtained lysozyme antibody is up to more than 95%.
The technical scheme provided by the invention is as follows:
the preparation method of the lysozyme affinity filler comprises the steps of bonding the lysozyme with hydroxyl, carboxyl, amino or epoxy on the surface of a solid phase carrier, and fixing the lysozyme on the solid phase carrier to obtain the lysozyme affinity filler.
Preferably, the solid phase carrier is a polymer microsphere, agarose gel, dextran, cellulose, polycarbonate, digestive fiber, silica or magnetic microsphere, etc.
Preferably, the specific preparation steps of the lysozyme affinity filler are as follows;
(1) and (3) activating the filler: adjusting the pH value of the solid phase carrier solution to 13-14, adding an activating agent and a reducing agent for reaction, and cleaning with deionized water after the reaction is finished;
(2) ligand coupling: adding lysozyme and a coupling buffer solution without free amino into the system reacted in the step (1), adjusting the pH value to be 8-10, reacting, and cleaning with deionized water after the reaction is finished;
(3) and (3) blocking reaction: and (3) adding a sealing agent into the system reacted in the step (2), adjusting the pH value of the system to be 8-9, reacting, and cleaning with deionized water after the reaction is finished to obtain the lysozyme affinity filler.
More preferably, in the step (1), the activating agent is epoxy chloropropane, the reducing agent is a sodium borohydride solution, the reaction temperature is 20-40 ℃, and the reaction time is 2-6 hours; in the step (2), the coupling buffer solution without free amino is carbonate or phosphate, the reaction temperature is 20-50 ℃, and the reaction time is 8-24 hours; in the step (3), the reaction temperature is 25-40 ℃, the reaction time is 1-2 h, and the sealing agent is ethanolamine.
Preferably, the specific preparation steps of the lysozyme affinity filler are as follows;
(1) the activated solid phase carrier is cleaned by HCl solution and then is cleaned by coupling liquid;
(2) dissolving lysozyme and adding the lysozyme into the solid phase carrier cleaned in the step (1) for reaction;
(3) and (3) washing the filler obtained in the step (2) by deionized water, adding a sealing liquid for reaction, removing the sealing liquid after the reaction is finished, and washing to obtain the lysozyme antibody affinity filler.
More preferably, in step (1), the coupling solution is 0.2M NaHCO at pH83And 0.5M NaCl; in the step (2), the volume ratio of the solid phase carrier to the lysozyme is 1: (1-2) oscillating and reacting at 25-30 ℃ for 2-4 h or reacting at 4-6 ℃ overnight.
More preferably, in the step (3), the reaction condition is oscillation reaction at 25-30 ℃ for 1-2 h; the cleaning method after removing the sealing liquid comprises the following steps: the filler is washed by deionized water with 2-3 times of column volume, then the mixed solution of 0.1M acetic acid-sodium acetate and 0.5M NaCl with the pH value of 4 is washed for 1-2 times, then the mixed solution of 0.1M Tris-HC with the pH value of 8 and 0.5M NaCl is washed for 1-2 times, and finally the deionized water is used for repeatedly washing for 2-3 times.
The invention also discloses an application method of the lysozyme affinity filler, which comprises the following steps:
(1) balancing lysozyme affinity filler by using a balancing liquid;
(2) adding the lysozyme antibody crude product solution into the lysozyme affinity filler after being balanced in the step (1);
(3) eluting with 0.1M, pH 3-4 glycine to obtain the purified lysozyme antibody pure product.
Preferably, the equilibrium solution is phosphate buffer solution with pH of 7-8, and the elution speed is 1-2 ml/min.
Preferably, the lysozyme antibody is recombinantly expressed in a host cell.
More preferably, the lysozyme antibody is recombinantly expressed in the host cell by: Psmart-P is taken as a vector, and EcoRI and Hind are arranged at the upstream and the downstream of the vector
The two enzyme cutting sites are inserted with lysozyme antibody sequence (GACGTCCAGCTCGTCGAATCCGGCGGCGGCTCCGTCCAGGCCGGCGGCTCCCTCCGCCTCTCCTGCGCCGCCTCCGGCTCCACCGACTCCATCGAATACATGACCTGGTTCCGCCAGGCCCCCGGCAAGGCCCGCGAAGGCGTCGCCGCCCTCTACACCCACACCGGCAACACCTACTACACCGACTCCGTCAAGGGCCGCTTCACCATCTCCCAGGACAAGGCCAAGAACATGGCCTACCTCCGCATGGACTCCGTCAAGTCCGAAGACACCGCCATCTACACCTGCGGCGCCACCCGCAAGTACGTCCCCGTCCGCTTCGCCCTCGACCAGTCCTCCTACGACTACTGGGGCCAGGGCACCCAGGTCACCGTCTCCTCC), recombinant plasmid is constructed and transformed into prokaryotic cells for fusion protein expression; preferably, a flag tag (DYKDDDDK) can be inserted at the C-terminus of the lysozyme antibody sequence as a screening tag.
More preferably, the lysozyme antibody is recombinantly expressed in the host cell by: taking pSmart-E as a vector, inserting a kozak sequence (GCCACCAUGG) at the upstream of a signal peptide in the vector, inserting a lysozyme antibody sequence (GACGTGCAGCTGGTGGAGAGCGGCGGCGGCAGCGTGCAGGCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCAGCACCGACAGCATCGAGTACATGACCTGGTTCCGCCAGGCCCCCGGCAAGGCCCGCGAGGGCGTGGCCGCCCTGTACACCCACACCGGCAACACCTACTACACCGACAGCGTGAAGGGCCGCTTCACCATCAGCCAGGACAAGGCCAAGAACATGGCCTACCTGCGCATGGACAGCGTGAAGAGCGAGGACACCGCCATCTACACCTGCGGCGCCACCCGCAAGTACGTGCCCGTGCGCTTCGCCCTGGACCAGAGCAGCTACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC) at the downstream of the signal peptide in the vector, constructing a recombinant plasmid, and transforming the recombinant plasmid into eukaryotic cells for expression of fusion protein; preferably, the C-terminus of the lysozyme antibody sequence may be inserted with 6 × his (hhhhhhhh) or flag (dykdddddddk) as a screening tag.
Compared with the prior art, the invention has the following technical advantages:
(1) the recombinant plasmid designed by the invention can increase the solubility of the fusion protein, reduce the probability of the fusion protein forming inclusion bodies and improve the expression quantity of the fusion protein; flag (DYKDDDDK) or His label is used as a double label to provide a basis for the affinity chromatography separation of the subsequent fusion protein.
(2) Compared with the separation medium used for ion exchange chromatography in the prior art, the lysozyme affinity filler prepared by the invention has higher specificity separation efficiency and simple operation, not only can separate fusion protein expressed by recombinant plasmids, but also can directly purify lysozyme antibody obtained by other approaches such as microbial metabolites, cell lysate and the like without excessive subsequent optimization operation, compared with the conventional affinity chromatography filler, the used ligand has low cost and stronger specificity combination, the sample loading amount is large when the lysozyme antibody or antibody fusion protein is purified, the separation purity reaches more than 95 percent, and the lysozyme affinity filler can be produced in industrial scale.
Drawings
FIG. 1 is a schematic diagram of the recombinant plasmid obtained in example 1
FIG. 2 is the polyacrylamide gel electrophoresis detection chart of prokaryotic expression target protein in example 1
FIG. 3 is a polyacrylamide gel electrophoresis test of the target protein after purification in example 1
FIG. 4 is a schematic diagram of the recombinant plasmid obtained in example 2
FIG. 5 is the polyacrylamide gel electrophoresis detection chart of the eukaryotic expression target protein in example 2
FIG. 6 is a photograph showing polyacrylamide gel electrophoresis detection of the target protein after purification in example 2.
Detailed Description
The invention will now be further described with reference to the accompanying drawings and specific embodiments.
Example 1
It should be noted that the solid phase carrier NHS-Activated Beads 4FF after activation in this example is produced by Changzhou Tiandi and Biotech Co.
The invention discloses a preparation method of lysozyme affinity filler (through coupling of carboxyl on the surface of microspheres), which comprises the following steps:
(1) NHS-Activated Beads 4FF was washed three times with 1mM HCl solution by suction filtration and 0.2M NaHCO at pH83Washing with a mixed solution of 0.5M NaCl;
(2) adding lysozyme solution with the same volume into the washed NHS-Activated Beads 4FF, carrying out oscillation reaction for 4 hours at 28 ℃, collecting coupling filler after the reaction is finished, washing the coupling filler with ionized water, adding 1M ethanolamine with 2 times of column volume, and carrying out oscillation reaction for 1 hour at 28 ℃;
(3) and (3) taking out the reaction system in the step (2), draining ethanolamine, cleaning the filler for 1 time by using deionized water with 3 times of column volume, cleaning for 2 times by using a mixed solution of 0.1M acetic acid-sodium acetate and 0.5M NaCl with the pH value of 4, cleaning for 2 times by using a mixed solution of 0.1M Tris-HC and 0.5M NaCl with the pH value of 8, and repeatedly washing for 2-3 times by using deionized water to obtain the lysozyme affinity filler.
The invention also discloses an application method of the lysozyme affinity filler, which comprises the following steps:
preparation of fusion protein
(1) As shown in figure 1, Psmart-P is used as a vector to construct a recombinant plasmid, wherein the Psmart-P is a modified vector based on a standard vector pET28a, is a complete expression vector and has a promoter, an enhancer, a terminator, a replication initiation site, antibiotic resistance and the like, a lysozyme antibody sequence (GACGTCCAGCTCGTCGAATCCGGCGGCGGCTCCGTCCAGGCCGGCGGCTCCCTCCGCCTCTCCTGCGCCGCCTCCGGCTCCACCGACTCCATCGAATACATGACCTGGTTCCGCCAGGCCCCCGGCAAGGCCCGCGAAGGCGTCGCCGCCCTCTACACCCACACCGGCAACACCTACTACACCGACTCCGTCAAGGGCCGCTTCACCATCTCCCAGGACAAGGCCAAGAACATGGCCTACCTCCGCATGGACTCCGTCAAGTCCGAAGACACCGCCATCTACACCTGCGGCGCCACCCGCAAGTACGTCCCCGTCCGCTTCGCCCTCGACCAGTCCTCCTACGACTACTGGGGCCAGGGCACCCAGGTCACCGTCTCCTCC) is inserted into two enzyme cutting sites of EcoRI and HindIII at the upstream and the downstream of the vector to obtain the recombinant plasmid, the lysozyme antibody sequence can be used as a tag sequence at the N end of a fusion protein, and a flag tag (DYKDDDDK) is inserted into the C end of the sequence to be used as a screening tag;
(2) transferring the recombinant plasmid into an escherichia coli competent cell, culturing for 4h at 37 ℃, and adding IPTG (isopropyl-beta-thiogalactoside) for induction expression.
Detecting the expressed fusion protein by polyacrylamide gel electrophoresis, as shown in FIG. 2, wherein I is the electrophoresis image of the fusion protein without the target sequence, and II is the electrophoresis image of the fusion protein in this example; III is Mark electrophoresis diagram, the designed molecular weight of the target fusion protein in this example is 30KD, while the electrophoresis diagram in II has a distinct electrophoresis band at the position close to 30KD, and the electrophoresis diagram in I has no electrophoresis band at the corresponding position, which shows that the target sequence in this example is successfully inserted into the target site on the carrier, and the expression quantity of the target fusion protein in this example is calculated to be more than 1 g/L.
II, separation of fusion protein
(1) Using phosphate buffer solution with pH7.4 to balance the lysozyme affinity filler;
(2) ultrasonically crushing the escherichia coli cells after the expression is finished, taking supernatant, and adding the supernatant into the lysozyme affinity filler obtained in the step (1);
(3) eluting with 0.1M, pH3 glycine, and collecting the target fraction to obtain the pure lysozyme antibody fusion protein.
The purified lysozyme antibody fusion protein pure product is detected by polyacrylamide gel electrophoresis, as shown in figure 3, an electrophoretogram of a crude lysozyme antibody fusion protein I, electrophoretograms of non-lysozyme antibody fusion proteins collected in an elution process II and III, an electrophoretogram of lysozyme antibody fusion proteins collected in an IV, positions corresponding to the lysozyme antibody fusion proteins IV in the electrophoretograms II and III, the band color of the lysozyme antibody fusion proteins is quite light, and the bands of other substances except the lysozyme antibody fusion proteins in the electrophoresis graph IV are fewer and not obvious, the purity of the purified lysozyme antibody fusion proteins is higher than 95 percent through calculation, and the purified loading capacity of the filler prepared in the embodiment is higher than 20mg/ml through protein concentration determination.
Example 2
The invention discloses a preparation method of lysozyme affinity filler (coupled by microsphere surface hydroxyl), which comprises the following steps:
(1) and (3) activating the filler: adding 90ml of 1M NaOH into 100ml of agarose gel solution, adjusting the pH value to 14, adding 15ml of epichlorohydrin and 200mg of sodium borohydride, reacting for 2 hours at 40 ℃, cleaning with deionized water after the reaction is finished, and collecting the filler;
(2) ligand coupling: adding 100ml of 10mg/ml lysozyme and then adding 0.1M Na into the system after the reaction in the step (1)2CO3Adjusting the reaction systemReacting for 24 hours at 25 ℃ after the pH value is 9, cleaning with deionized water after the reaction is finished, and collecting the filler;
(3) and (3) blocking reaction: and (3) adding 100ml of 1M ethanolamine into the system reacted in the step (2), adjusting the pH of the reaction system to 9, reacting for 1h at 40 ℃, and cleaning with deionized water after the reaction is finished to obtain the lysozyme affinity filler.
The invention also discloses an application method of the lysozyme affinity filler, which comprises the following steps:
preparation of fusion protein
(1) As shown in FIG. 4, pSmart-E was used as a vector to construct a recombinant plasmid, which comprises a promoter, an enhancer, a terminator, a replication initiation site, an antibiotic resistance site, etc., and a constitutive promoter (cmv) and a signal peptide were used to increase the protein expression level; inserting a kozak sequence (GCCACCAUGG) at the upstream of a signal peptide in the vector, and inserting a lysozyme antibody sequence (GACGTGCAGCTGGTGGAGAGCGGCGGCGGCAGCGTGCAGGCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCAGCACCGACAGCATCGAGTACATGACCTGGTTCCGCCAGGCCCCCGGCAAGGCCCGCGAGGGCGTGGCCGCCCTGTACACCCACACCGGCAACACCTACTACACCGACAGCGTGAAGGGCCGCTTCACCATCAGCCAGGACAAGGCCAAGAACATGGCCTACCTGCGCATGGACAGCGTGAAGAGCGAGGACACCGCCATCTACACCTGCGGCGCCACCCGCAAGTACGTGCCCGTGCGCTTCGCCCTGGACCAGAGCAGCTACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC) at the downstream; obtaining recombinant plasmid, wherein the lysozyme antibody sequence can be used as a tag sequence of the N end of the fusion protein, and a flag tag (DYKDDDDK) is inserted into the C end of the sequence and used as a screening tag;
(2) transferring the recombinant plasmid into Chinese hamster ovary cells (CHO cells), culturing at 37 deg.C under 6% carbon dioxide at 125 rpm to 0.5 × 106Cells/ml, during the culture, using Lipofectamin2000 and plasmid transfection, three days later collecting the cultured cell supernatant.
Detecting the expressed fusion protein by polyacrylamide gel electrophoresis, as shown in fig. 5, i is Mark electrophoresis, ii is the electrophoresis of the fusion protein without the target sequence, iii is the electrophoresis of the fusion protein in this embodiment, the designed molecular weight of the target fusion protein in this embodiment is 35KD, the electrophoresis of iii has an obvious electrophoresis band at the position of 30-40 KD, and the electrophoresis of ii has no obvious electrophoresis band at the corresponding position, which indicates that the target sequence in this embodiment is successfully inserted into the target site on the vector, and the expression of the target protein in this embodiment is greater than 50mg/L by calculation.
II, separation of fusion protein
(1) Using phosphate buffer solution with pH8 to balance lysozyme affinity filler;
(2) carrying out ultrasonic disruption on hamster ovary cells after expression, taking supernatant, and adding the supernatant to lysozyme affinity packing in the step (1);
(3) eluting with 0.1M, pH4 glycine, and collecting the target fraction to obtain the expressed fusion protein.
Adopting polyacrylamide gel electrophoresis to detect the purified lysozyme antibody fusion protein, as shown in figure 6, wherein I is a Mark electrophoresis pattern, II is an electrophoresis pattern of a fusion protein crude product, III is an electrophoresis pattern of the collected lysozyme antibody fusion protein, and in the electrophoresis pattern of III, except an obvious lysozyme antibody fusion protein band at a position of 30-40 KD, the bands of other substances are fewer and not obvious, and the purity of the purified lysozyme antibody fusion protein is more than 95 percent through calculation.
Claims (10)
1. A preparation method of lysozyme affinity filler is characterized in that: the lysozyme is fixed on the solid phase carrier by bonding with hydroxyl, carboxyl, amino or epoxy on the surface of the solid phase carrier to obtain the lysozyme affinity filler.
2. The method for preparing lysozyme affinity filler according to claim 1, wherein: the specific preparation steps of the lysozyme affinity filler are as follows;
(1) and (3) activating the filler: adjusting the pH value of the solid phase carrier solution to 13-14, adding an activating agent and a reducing agent for reaction, and cleaning with deionized water after the reaction is finished;
(2) ligand coupling: adding lysozyme and a coupling buffer solution without free amino into the system reacted in the step (1), adjusting the pH value to be 8-10, reacting, and cleaning with deionized water after the reaction is finished;
(3) and (3) blocking reaction: and (3) adding a sealing agent into the system reacted in the step (2), adjusting the pH value of the system to be 8-9, reacting, and cleaning with deionized water after the reaction is finished to obtain the lysozyme affinity filler.
3. The method for preparing lysozyme affinity filler according to claim 2, wherein: in the step (1), the activating agent is epoxy chloropropane, the reducing agent is a sodium borohydride solution, the reaction temperature is 20-40 ℃, and the reaction time is 2-6 h; in the step (2), the coupling buffer solution without free amino is carbonate or phosphate, the reaction temperature is 20-50 ℃, and the reaction time is 8-24 hours; in the step (3), the reaction temperature is 25-40 ℃, the reaction time is 1-2 h, and the sealing agent is ethanolamine.
4. The method for preparing lysozyme affinity filler according to claim 1, wherein: the specific preparation steps of the lysozyme affinity filler are as follows;
(1) the activated solid phase carrier is cleaned by HCl solution and then is cleaned by coupling liquid;
(2) dissolving lysozyme and adding the lysozyme into the solid phase carrier cleaned in the step (1) for reaction;
(3) and (3) washing the filler obtained in the step (2) by deionized water, adding a sealing liquid for reaction, removing the sealing liquid after the reaction is finished, and washing to obtain the lysozyme antibody affinity filler.
5. A method of making a lysozyme affinity filler according to claim 4, wherein: in the step (1), the coupling solution is 0.2M NaHCO with the pH value of 83And 0.5M NaCl; in the step (2), the volume ratio of the solid phase carrier to the lysozyme is 1: (1-2) oscillating and reacting at 25-30 ℃ for 2-4 h or reacting at 4-6 ℃ overnight.
6. A method of making a lysozyme affinity filler according to claim 4, wherein: in the step (3), the reaction condition is that the oscillation reaction is carried out for 1-2 h at the temperature of 25-30 ℃; the cleaning method after removing the sealing liquid comprises the following steps: the filler is washed by deionized water with 2-3 times of column volume, then the mixed solution of 0.1M acetic acid-sodium acetate and 0.5M NaCl with the pH value of 4 is washed for 1-2 times, then the mixed solution of 0.1M Tris-HC with the pH value of 8 and 0.5M NaCl is washed for 1-2 times, and finally the deionized water is used for repeatedly washing for 2-3 times.
7. A method of using lysozyme affinity filler according to claim 1, characterized by comprising the steps of:
(1) balancing lysozyme affinity filler by using a balancing liquid;
(2) adding the lysozyme antibody crude product solution into the lysozyme affinity filler after being balanced in the step (1);
(3) eluting with 0.1M, pH 3-4 glycine to obtain the purified lysozyme antibody pure product.
8. The method of using lysozyme affinity filler according to claim 7, wherein: the lysozyme antibody can be obtained by recombinant expression in host cells.
9. The method of using lysozyme affinity filler according to claim 8, wherein: the recombinant expression method of the lysozyme antibody in the host cell comprises the following steps: Psmart-P is taken as a vector, and EcoRI and Hind are arranged at the upstream and the downstream of the vector
The two enzyme cutting sites of (2) are inserted into a lysozyme antibody sequence (GACGTCCAGCTCGTCGAATCCGGCGGCGGCTCCGTCCAGGCCGGCGGCTCCCTCCGCCTCTCCTGCGCCGCCTCCGGCTCCACCGACTCCATCGAATACATGACCTGGTTCCGCCAGGCCCCCGGCAAGGCCCGCGAAGGCGTCGCCGCCCTCTACACCCACACCGGCAACACCTACTACACCGACTCCGTCAAGGGCCGCTTCACCATCTCCCAGGACAAGGCCAAGAACATGGCCTACCTCCGCATGGACTCCGTCAAGTCCGAAGACACCGCCATCTACACCTGCGGCGCCACCCGCAAGTACGTCCCCGTCCGCTTCGCCCTCGACCAGTCCTCCTACGACTACTGGGGCCAGGGCACCCAGGTCACCGTCTCCTCC), a recombinant plasmid is constructed and transformed to the original sitePerforming fusion protein expression in the nuclear cells; preferably, a flag tag (DYKDDDDK) can be inserted at the C-terminus of the lysozyme antibody sequence as a screening tag.
10. The method of using lysozyme affinity filler according to claim 8, wherein: the recombinant expression method of the lysozyme antibody in the host cell comprises the following steps: taking pSmart-E as a vector, inserting a kozak sequence (GCCACCAUGG) at the upstream of a signal peptide in the vector, inserting a lysozyme antibody sequence (GACGTGCAGCTGGTGGAGAGCGGCGGCGGCAGCGTGCAGGCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCAGCACCGACAGCATCGAGTACATGACCTGGTTCCGCCAGGCCCCCGGCAAGGCCCGCGAGGGCGTGGCCGCCCTGTACACCCACACCGGCAACACCTACTACACCGACAGCGTGAAGGGCCGCTTCACCATCAGCCAGGACAAGGCCAAGAACATGGCCTACCTGCGCATGGACAGCGTGAAGAGCGAGGACACCGCCATCTACACCTGCGGCGCCACCCGCAAGTACGTGCCCGTGCGCTTCGCCCTGGACCAGAGCAGCTACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC) at the downstream of the signal peptide in the vector, constructing a recombinant plasmid, and transforming the recombinant plasmid into eukaryotic cells for expression of fusion protein; preferably, the C-terminus of the lysozyme antibody sequence may be inserted with 6 × his (hhhhhhhh) or flag (dykdddddddk) as a screening tag.
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