CN114950371A - Nano antibody coupling filler and preparation method and application thereof - Google Patents
Nano antibody coupling filler and preparation method and application thereof Download PDFInfo
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- CN114950371A CN114950371A CN202111544867.XA CN202111544867A CN114950371A CN 114950371 A CN114950371 A CN 114950371A CN 202111544867 A CN202111544867 A CN 202111544867A CN 114950371 A CN114950371 A CN 114950371A
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- filler
- nanobody
- coupling
- nano antibody
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28047—Gels
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Dispersion Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of protein separation, and particularly relates to a nano antibody coupling filler, and a preparation method and application thereof. Aiming at the problems of no acid and alkali resistance and poor purification effect of the existing antibody coupling filler; the invention provides a preparation method of a nano antibody coupling filler, which has the defects of harsh use environment, short service life and the like. The nano antibody coupling filler prepared by the invention has good application in gene engineering protein purification and natural protein grabbing, and has stronger specificity compared with the common affinity chromatography filler; compared with common antibody affinity filler, the antibody affinity filler has stronger stress resistance, acid resistance, high temperature resistance and stable structure, and the stability and durability of the performance also enable the antibody affinity filler to be better cleaned and stored.
Description
Technical Field
The invention belongs to the technical field of protein separation, and particularly relates to a nano antibody coupling filler, and a preparation method and application thereof.
Background
The antibody coupling technology is mainly applied to antibody coupling drugs, and the antibody is combined with a cytotoxic small molecule drug to obtain the antibody coupling drug. The first antibody-coupled drug was approved by the FDA in the united states for the treatment of hodgkin lymphoma in 2011. The antibody coupling drug combines the targeting property of the antibody and the killing property of the chemotherapeutic drug, has more remarkable curative effect in tumor treatment, and is concerned by people. Meanwhile, the antibody coupling drug is combined with an antibody guidance system, and a fixed-point killing target is realized, so that the treatment window of the small-molecule drug is widened, and the side effect of the drug is reduced.
However, antibody coupling techniques are limited in their utility in other applications, and have been studied less extensively, particularly in the field of protein isolation and protein purification. Because the antibody has strict requirements on storage temperature, conditions and the like, the antibody is easily degraded and destroyed by the influence of external environment, thereby losing activity. In the field of protein separation and purification, target protein often needs to be captured in a complex environment, and repeated cleaning is often needed for a protein separation and purification system. In the process, the antibody is easily affected by enzymes or acid and alkali, and the pressure and the temperature are also changed, so that the antibody can be seriously damaged, and the antibody can be out of function.
At present, monoclonal antibodies or polyclonal antibodies are coupled with fillers for purification, but the coupled fillers can be prepared by a laboratory or specially customized by a company entrusted with producing the purified fillers, and are not mass-produced market products. Moreover, the conditions for preparing protein by purifying and using antibody coupling filler are very harsh, and the common monoclonal antibody or polyclonal antibody coupling filler used at present has many defects for purifying protein: the acid and alkali resistance is not realized, so that the adsorbed impurities cannot be completely eluted and thoroughly cleaned; the temperature, the pH value and the like can also cause the binding force of the antibody to be greatly reduced and even to be directly inactivated, so that the service life is greatly influenced, and the antibody can be used for 1-2 times.
The nano antibody is a genetically engineered antibody which only consists of a heavy chain antibody variable region, is also called as a VHH antibody, has strong stress resistance compared with the traditional antibody, and still has high activity under the condition of strong acid. At present, no research on protein purification by adopting a nano antibody coupled filler exists, and the invention provides a problem to be solved if the nano antibody coupled filler can be applied to the field of protein purification and how to improve the effect of protein purification by using the nano antibody coupled filler.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the existing antibody coupling filler has the defects of no acid and alkali resistance and poor purification effect; severe using environment, short service life and the like.
The technical scheme for solving the technical problems comprises the following steps: the preparation method of the nano-antibody coupling filler comprises the following steps:
a. filler washing
Placing the filler in a gravity column hollow column, after the acetone preservation solution is drained, washing the filler for 30-40 min by using 5-10 column volumes of pre-cooled HCl with the concentration of 1mM, wherein the washing temperature is 0-4 ℃;
b. coupling
Diluting the washed filler into a solution, mixing the solution with the nano antibody solution, and uniformly mixing the solution at room temperature for 2-3 h;
c. sealing of
After draining and coupling supernatant, adding a sealing liquid, and sealing for 2-4 h at room temperature;
d. preservation of
And c, washing the nano antibody coupling filler obtained in the step c with water, and storing the nano antibody coupling filler in 1-1.5% benzyl alcohol solution or 10-20% ethanol at 0-4 ℃.
Wherein, in the preparation method of the nano-antibody coupling filler, the filler in the step a is gel.
Preferably, in the method for preparing the nanobody-coupled filler, the filler in step a is agarose gel.
More preferably, in the preparation method of the nanobody-coupled filler, the filler in the step a is CNBr-activated prestrain 4FF preactivated sepharose.
In the preparation method of the nano-antibody coupling filler, the concentration of the nano-antibody solution in the step b is 0.5-15 mg/ml.
In the preparation method of the nano-antibody coupling filler, the filler in the step b is diluted by 1mM HCl, and 0.5-1 mL of the filler is added per 1mL of the filler.
In the preparation method of the nano-antibody coupling filler, the volume ratio of the filler in the step b to the nano-antibody solution is 1: 1-1.2.
In the preparation method of the nano-antibody coupling filler, the confining liquid in the step c is 0.1-0.15M Tris-HCl, and the pH value is 8.3-8.5.
Further, in the preparation method of the nanobody-coupled filler, the addition amount of the blocking solution in the step c is as follows: adding 5-10 ml of confining liquid into every 0.5-1 ml of substances left after draining and coupling supernatant.
Wherein, in the preparation method of the nano antibody coupling filler, the washing in the step c adopts I-grade water for washing.
The invention also provides the nano antibody coupling filler directly prepared by the method.
The invention also provides application of the nano-antibody coupled filler in protein separation and purification or natural protein enrichment and purification.
The invention has the beneficial effects that:
the invention provides a nano-antibody coupling filler, which is prepared by adopting natural nano-antibody or other screened nano-antibodies with special functions. Compared with other protein purification and separation methods, the method comprises ion exchange chromatography, molecular sieve, Ni ion affinity chromatography and the like, has antibody target recognition capability and stronger grasping specificity, so that the target protein with extremely high purity can be obtained in the protein separation and purification application, and the separation and purification effect is more excellent. Meanwhile, the nano antibody coupling filler has the target recognition capability, and can specifically recognize the target antigen without manually adding a label, so the nano antibody coupling filler can be applied to the grabbing of natural protein, directly enriches the natural protein to obtain the natural protein with a single component, and has extremely high application value in the research field of the nano antibody coupling filler. The preparation method of the nano antibody coupling filler is simple, and the nano antibody coupling filler has a good effect on protein purification and has a good application value.
Drawings
FIG. 1 shows Nanobody buffer displacement of anti-glycosyltransferase A subunits;
FIG. 2 shows nanobody coupling against glycosyltransferase A subunits;
FIG. 3 shows the effect of nanobody-coupled filler protein purification;
FIG. 4 shows the purification effect of another nanobody-coupled filler protein.
Detailed Description
The invention provides a preparation method of a nano antibody coupling filler, which comprises the following steps:
a. filler washing
Placing the filler in a gravity column hollow column, after the acetone storage solution is drained, washing the filler for 30-40 min by using 5-10 column volumes of precooled HCl with the concentration of 1mM, wherein the washing temperature is 0-4 ℃;
washing on the one hand removes residual acetone and on the other has the effect of activating the filler. According to the invention, the filler is activated firstly, and the activated filler can be coupled and combined with the intervened antibody better, so that the loading capacity is improved.
b. Coupling
Diluting the washed filler into a solution, mixing the solution with the nano antibody solution, and uniformly mixing the solution at room temperature for 2-3 hours;
c. sealing of
After draining and coupling supernatant, adding a sealing liquid, and sealing for 2-4 h at room temperature;
d. preservation of
And c, washing the nano antibody coupling filler obtained in the step c with water, and storing the nano antibody coupling filler in 1-1.5% benzyl alcohol solution or 10-20% ethanol at 0-4 ℃.
Wherein, in the preparation method of the nano-antibody coupling filler, the filler in the step a is gel.
Preferably, in the method for preparing the nanobody-coupled filler, the filler in step a is agarose gel.
More preferably, in the preparation method of the nanobody-coupled filler, the filler in the step a is CNBr-activated prestrain 4FF preactivated sepharose. The filler of the invention can adopt CNBr-Activated Bestarose 4FF preactivated agarose gel, and other agarose gel with similar functions, such as Epoxy-Activated Beads 4FF, NHS Activated-BerPHarose FF and the like, can also achieve the same effect, and is within the protection scope of the invention.
In the preparation method of the nano-antibody coupling filler, the concentration of the nano-antibody solution in the step b is 0.5-15 mg/ml. The method is finally determined through repeated tests, and only when the concentration of the nano antibody solution is 0.5-15 mg/ml, the binding position on the filler is just and fully combined and covered, so that the non-specific impurity combination is reduced, and the capture specificity of the coupling filler in application is improved.
In the preparation method of the nano-antibody coupling filler, the filler in the step b is diluted by 1mM HCl, and 0.5-1 mL of the filler is added per 1mL of the filler.
In the preparation method of the nano-antibody coupling filler, the volume ratio of the filler in the step b to the nano-antibody solution is 1: 1-1.2. At this volume ratio, the mixed solution has a relatively good turbidity, and both the filler and the antibody can be sufficiently dispersed, and have a relatively good contact area with each other, so that the coupling efficiency can be improved.
In the preparation method of the nano-antibody coupling filler, the confining liquid in the step c is 0.1-0.15M Tris-HCl, and the pH value is 8.3-8.5.
Further, in the preparation method of the nanobody-coupled filler, the addition amount of the blocking solution in the step c is as follows: adding 5-10 ml of confining liquid into every 0.5-1 ml of substances left after draining and coupling supernatant.
Wherein, in the preparation method of the nano antibody coupling filler, the washing in the step c adopts I-grade water for washing.
The invention also provides the nano antibody coupling filler directly prepared by the method.
The invention prepares a brand new nano antibody coupling filler, which is a brand new product. At present, the cost of the antibody coupling filler is extremely high, and the conditions for using the antibody coupling filler after the high-cost coupling filler is manufactured are very harsh, so the application range is very small, the commercial value is not high, and the antibody coupling filler is not effectively utilized. The preparation method of the nano antibody coupling filler prepared by the invention is very simple, can be completed by only a few simple steps, greatly saves the cost, can tolerate reagents with various concentrations, has a wider pH range and has a better use effect.
And the nano antibody coupling filler also has good commercial value and practicability, when the nano antibody coupling filler is used, more extreme environments and reagents can be used for improving the coupling efficiency without worrying about the damage of the material by the substances, the fault tolerance rate is higher, more choices can be provided for process production, and the cost has larger optimization space.
The invention also provides application of the nano-antibody coupled filler in protein separation and purification or natural protein enrichment and purification.
The nano antibody coupling filler has the targeting recognition capability of the antibody, has stronger grasping specificity, and can obtain target protein with extremely high purity in the application of protein separation and purification. Due to the target recognition, the method can also be applied to the grabbing of natural protein, so that the natural protein is directly enriched, and the obtained natural protein with a single component has extremely high application value in the research field. Meanwhile, due to the excellent stress resistance, the use cost is greatly reduced, and the method has good commercial value.
The following examples are intended to illustrate specific embodiments of the present invention but are not intended to limit the scope of the invention to the examples.
The glycosyltransferase A subunit nano antibody used in example 2 is a self-made product, the preparation method is disclosed in patent 202110077974.X, and the sequence of the prepared glycosyltransferase A subunit nano antibody is also disclosed in patent 202110077974. X. CNBr-activated Bestarose 4FF preactivated Sepharose was purchased from Bogelong Biotechnology Ltd; HCl and acetic acid were purchased from Kyoko Chemicals, Inc.; NaHCO 2 3 Tris, NaCl, glycine, Na 2 HPO 4 、NaH 2 PO 4 And the like from Biotechnology engineering (Shanghai) Inc. The other reagents are all common commercial products.
Example 1 preservation solution for substituted Nanobody
The invention firstly replaces the nano antibody preservation solution, so that the buffer solution is more suitable for the coupling condition, and the better coupling effect is achieved.
The specific operation steps are as follows:
(1) cleaning: the protein purification apparatus (Bio-Rad, NGC QUEST 100PLUS) was started up, and each channel was washed, with a column pressure limit of 0.4 MPa.
(2) Balancing: washing with I-grade water, and washing the filler at 6ml/min until the baseline is stable; buffer A (0.1M NaHCO) 3 0.5M NaCl, pH 8.3) solution 6ml/min until the baseline is stable;
(3) loading: preserving 6ml of the nano antibody solution at the speed of 6 ml/min;
(4) and (3) re-balancing: after the sample loading is finished, the balance chromatographic column is re-balanced by Buffer A liquid, 6ml/min is washed until the baseline is completely stable, and the nano antibody solution is collected.
(5) Repeating the steps (3) and (4), and loading 3ml of sample each time until the replacement is completed;
(6) cleaning: changing the grade I water and washing until the baseline is stable;
(7) and (3) storage: washing with 20% ethanol water solution until baseline is stable, taking off chromatography column, and storing at 4 deg.C, the results are shown in FIG. 1.
EXAMPLE 2 Nanobody-coupled Sepharose
The specific operation steps are as follows:
(1) gel washing: taking 1mL CNBr-activated Bestarose 4FF preactivated agarose gel in a gravity column empty column, washing with precooled Buffer B (1mM HCl) at 0-4 ℃ for 30min after acetone preservation liquid is drained;
(2) coupling: diluting the washed filler with Buffer B (about 0.5mL of Buffer B is added into 1mL of filler), mixing the diluted filler with the nano antibody solution in the same volume as that of the nano antibody solution in the example 1, and uniformly mixing the mixture at room temperature for 2 hours;
(3) and (3) sealing: after draining and coupling the supernatant, adding a sealing solution (0.1M Tris-HCl, PH 8.3), and sealing at room temperature for 4 h;
(4) and (3) storage: the filler was washed with grade I water and stored in 1% benzyl alcohol solution at 4 ℃.
The nano antibody coupling filler prepared by the embodiment is shown in fig. 2, wherein the first lane is a nano antibody sample, and the second lane is a sample after the nano antibody flows through the filler, so that almost all the nano antibody is coupled with the filler, and the coupling effect is good.
Example 3 verification of separation and purification effects of combined filler protein after coupling
The experimental sample is protein mixed liquor containing a large amount of impurity protein interference. The specific operation steps are as follows:
(1) cleaning: starting up the machine, cleaning each passage, and setting the column pressure limit to 0.3 MPa;
(2) balancing: washing with I-grade water, and washing the filler at 2ml/min until the baseline is stable; changing BufferC (100mM glycine-HCL solution, PH 2.5)2ml/min and washing until the baseline is stable; changing Buffer D (10mM PB, 135mM NaCl, pH 7.4) and washing at 2ml/min until the baseline is stable;
(3) loading 5mL of sample on the loading ring at 0.5mL/min, and collecting a flow-through sample;
(4) and (3) re-balancing: after the sample loading is finished, the Buffer D is used for re-balancing the chromatographic column, and the sample is washed until the base line is stable at 2 mL/min;
(5) and (3) elution: eluting with Buffer E (10mM PB, 135mM NaCl, pH 6.0), 2mL/min, and collecting the elution peak;
(6) cleaning: flushing with Buffer C until the baseline is stable, replacing I-grade water for flushing, and cleaning the filler at 2ml/min until the baseline is stable;
(7) and (3) storage: washing with 1% benzyl alcohol-PBS solution until the baseline is stable, and storing at 4 deg.C.
As shown in FIG. 3, the first lane shows the protein mixture sample containing impurities without being adsorbed by the packing, the second lane shows the sample flowing through after being adsorbed by the coupling packing, and the third lane shows the target protein under elution.
Example 4 verification of separation and purification effects of other coupled combined filler proteins
The experimental filler is another nano-antibody coupling filler, the amino acid sequence of the nano-antibody is shown in SEQ ID NO.1, and the experimental sample is protein mixed liquor containing a large amount of impurity protein interference.
1 nanometer antibody amino acid sequence of SEQ ID NO
MAQVQLQESGGGLVQAGGSLRLSCAASFRKISDFVMGWFRQAPGKEREFVAAGERFIWRANYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADVERVSSIDEFLYDYWGQGTQVTVSSAAAYPYDVPDYGSHHHHHH。
The specific operation steps are as follows:
(1) cleaning: starting up the machine, cleaning each passage, and setting the column pressure limit to 0.3 MPa;
(2) balancing: washing with I-grade water, and washing the filler at 2ml/min until the baseline is stable; changing Buffer C (100mM glycine-HCl solution, pH 2.5)2ml/min and washing until the baseline is stable; buffer D (10mM PB, 135mM NaCl, pH 7.4)2ml/min rinse until baseline plateaus;
(3) loading 5mL of sample on the loading ring at 0.5mL/min, and collecting a flow-through sample;
(4) and (3) re-balancing: after the sample loading is finished, the Buffer D is used for re-balancing the chromatographic column, and the sample is washed until the base line is stable at 2 mL/min;
(5) and (3) elution: eluting with Buffer E (10mM PB, 135mM NaCl, pH 6.0) at 2mL/min, and collecting the elution peak;
(6) cleaning: flushing with Buffer C until the baseline is stable, replacing I-grade water for flushing, and cleaning the filler at 2ml/min until the baseline is stable;
(7) and (3) storage: washing with 1% benzyl alcohol-PBS solution until the baseline is stable, and storing at 4 deg.C.
The result is shown in fig. 4, the first lane is a protein mixture sample containing impurities, the second lane is a sample flowing through after being adsorbed by a coupling filler, and the fourth lane is target protein eluted, so that after the nanobody is replaced, the corresponding target protein can still be captured by the filler of the invention, and the capture is very specific, and the target protein is very pure.
In both example 3 and example 4, the filler is washed by using a reagent with strong acidity, so that the nano antibody coupling filler has strong tolerance; in addition, the nano antibody coupling filler can be washed, stored in ethanol and then used subsequently, can be used repeatedly, and has longer service life.
Sequence listing
<110> Sichuan university Hospital in western China
<120> nano antibody coupling filler and preparation method and application thereof
<141> 2021-12-17
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 145
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala
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Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Phe Arg Lys Ile Ser
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Asp Phe Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Gly Glu Arg Phe Ile Trp Arg Ala Asn Tyr Ala Asp
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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
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Tyr Cys Ala Ala Asp Val Glu Arg Val Ser Ser Ile Asp Glu Phe Leu
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Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His
130 135 140
His
145
Claims (10)
1. The preparation method of the nano antibody coupling filler is characterized by comprising the following steps:
a. filler washing
Placing the filler in a gravity column hollow column, after the acetone preservation solution is drained, washing the filler for 30-40 min by using 5-10 column volumes of pre-cooled HCl with the concentration of 1mM, wherein the washing temperature is 0-4 ℃;
b. coupling
Diluting the washed filler into a solution, mixing the solution with the nano antibody solution, and uniformly mixing the solution at room temperature for 2-3 h;
c. sealing of
After draining and coupling the supernatant, adding a sealing liquid, and sealing for 2-4 h at room temperature;
d. preservation of
And c, washing the nano antibody coupling filler obtained in the step c with water, and storing the nano antibody coupling filler in 1-1.5% benzyl alcohol solution or 10-20% ethanol at 0-4 ℃.
2. The method for preparing nanobody-coupled filler according to claim 1, wherein: the filler in the step a is agarose gel.
3. The method for preparing nanobody-coupled filler according to claim 2, wherein: the filler in the step a is CNBr-activated Bestarose 4FF preactivated agarose gel.
4. The method of preparing nanobody-coupled filler according to claim 1, wherein: and c, the concentration of the nano antibody solution in the step b is 0.5-15 mg/ml.
5. The method of preparing nanobody-coupled filler according to claim 1, wherein: and c, diluting the filler in the step b by using 1mM HCl, and adding 0.5-1 mL of the filler per 1mL of the filler.
6. The method of preparing nanobody-coupled filler according to claim 1, wherein: the volume ratio of the filler to the nano antibody solution in the step b is 1: 1-1.2.
7. The method of preparing nanobody-coupled filler according to claim 1, wherein: and c, the confining liquid in the step c is 0.1-0.15M Tris-HCl, and the pH value is 8.3-8.5.
8. The method of preparing nanobody-coupled filler according to claim 1, wherein: the addition amount of the confining liquid in the step c is as follows: 5-10 ml of confining liquid is added into every 0.5-1 ml of substance left after the drainage coupling supernatant.
9. A nanobody-coupled filler directly prepared by the method of any one of claims 1 to 8.
10. Use of the nanobody-coupled filler of claim 9 for protein isolation purification or enrichment purification of native proteins.
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Application publication date: 20220830 |