CN111751468A - Method for purifying lysozyme dimer and preparing standard substance thereof - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/16—Injection
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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Abstract
The invention discloses a method for purifying lysozyme dimer and preparing a standard product thereof, wherein a size exclusion chromatography separation and purification is carried out on a lysozyme monomer/polymer mixture obtained after cross-linking by adopting a high performance liquid phase system. The chromatographic column is a hydrophilic silica gel column; the mobile phase is 50mMPB, 0.5M urea, 0.3M ammonium sulfate and 10 percent acetonitrile; driven at a flow rate of 0.35ml/min, the analysis time of the sample was set to 40 minutes; detection was performed using a UV 280nm ultraviolet detector. And collecting the dimer fraction in the mixture, and desalting by using an ultrafiltration tube after freeze drying. And (3) carrying out concentration determination on the obtained lysozyme dimer aqueous solution by using a BCA quantitative kit, and carrying out freeze-drying after subpackaging to obtain a lysozyme dimer standard substance. The method can be completed in 5 hours at room temperature, and has the advantages of simple process steps, simple and feasible operation, high lysozyme dimer yield, low cost and good effect.
Description
Technical Field
The invention relates to a method for purifying and preparing a dimer, in particular to a method for separating, purifying and preparing a lysozyme dimer, which is applied to the technical field of chemical substance separation and purification and test standard preparation.
Background
The lysozyme is a common spherical basic protein in the nature, has a molecular weight MW of about 14.4kDa, and has the effects of antibiosis, antiphlogosis, antivirus and the like. The lysozyme dimer takes egg white lysozyme as a raw material, modifies monomer lysozyme into the lysozyme dimer, and the dimer retains the enzymatic activity of the monomer lysozyme and also has quite new properties and antibacterial activity [ Ibrahim et al 1991,
et al.2009]. In experimental animals, the dimer has been shown to increase nonspecific antigen reactions and immunoglobulin production, stimulate the enzymatic activity of lung macrophages, prevent antibiotic immunosuppression, eliminate cyclophosphamide immunosuppression, and reduce oxygen free radical levels in the blood and secretions from inflamed mammary glands.
The lysozyme dimer is used as a feed additive for replacing antibiotics, and is mainly used for improving the average daily gain of weaned piglets; the villus height of duodenum is obviously increased, the crypt depth of jejunum is reduced, and the ratio of the villus height of duodenum and jejunum to the crypt depth is obviously increased, so that the intestinal development of piglets is improved, and the absorption of nutrient substances is promoted; obviously enhance the phagocytic rate and the phagocytic index of abdominal macrophages, and improve the phagocytic rate of blood neutral cells, thereby improving the nonspecific immunity function of weaned piglets.
For the separation and purification of the lysozyme monomer/multimer mixture obtained after crosslinking, reverse phase high performance liquid chromatography (RP), Ion Exchange Chromatography (IEC), Size Exclusion Chromatography (SEC), gel electrophoresis separation and molecular filtration can be generally employed to eliminate reagent and eluent residues and purify lysozyme dimers. Wherein Size Exclusion Chromatography (SEC), proteins are separated by their size, or more precisely by their hydrodynamic volume. Patent document CN101734671A discloses a method for monitoring the synthesis of silica gel microspheres by size exclusion chromatography to monitor the synthesis progress and batch reproducibility of silica gel microspheres; patent document with publication number CN 105486792a discloses a ginkgo diterpene lactone meglumine injection and a detection method of macromolecular compounds in raw materials for preparing the same, which are used for process quality control and final product quality evaluation; the patent publication No. CN111220720A discloses a method for detecting the purity of trypsin and its zymogen, which is used for process quality control and final product quality evaluation; patent document CN 104219957a discloses a method for refining triglyceride oils and purified triglyceride oils obtained by such a method, so that refined triglyceride oils of high purity can be produced cost-effectively. The prior art has primarily used size exclusion chromatography to monitor chemical synthesis preparations. The separation effect of the separation method of Size Exclusion Chromatography (SEC) is mainly influenced by the selection of a stationary phase and a mobile phase, different condition parameters adopted for purifying target substances are greatly different, and the molecular weight of lysozyme dimer is determined by using the SEC, so that the purity of the lysozyme dimer is effectively controlled, and the method has the advantages of accuracy, intuition, quickness, small sampling amount and the like.
Disclosure of Invention
In order to solve the problems of the prior art, the invention aims to overcome the defects of the prior art and provide a method for purifying lysozyme dimer and preparing a standard substance thereof, wherein the lysozyme dimer is separated and purified and the standard substance thereof is prepared, the whole process can be finished within a short time of 5 hours at room temperature, the process steps are simple, the operation is simple and feasible, and the purification and detection efficiency is high; the consumption of raw materials is low, the yield of lysozyme dimer is high, the lysozyme monomer can be recycled, and the cost is low; the prepared lysozyme dimer has high standard quality and good effect, and meets the purity requirement of an experimental sample and the requirement of the quality standard of a product after preparation and post-treatment.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a method for purifying lysozyme dimer and preparing a standard product thereof comprises the following steps:
a. taking the mixture of lysozyme monomer and polymer obtained after crosslinking, centrifuging by adopting a high-speed centrifuge to remove insoluble impurities, and taking the supernatant and subpackaging into a chromatographic sampling bottle for later use;
b. b, performing size exclusion chromatography separation and purification treatment on the supernatant obtained in the step a by using a high performance liquid phase system to obtain a separated and purified lysozyme dimer;
in the separation and purification process, the adopted chromatographic column is a hydrophilic silica gel column, the flow rate of a mobile phase is set to be 0.35ml/min, the analysis time is set to be 40 minutes, and a UV 280nm ultraviolet detector is adopted for detection; lysozyme monomer, dimer and polymer can all obtain very good baseline separation;
and (b) during separation and purification, collecting the obtained dimer fraction in the supernatant obtained in the step a, performing freeze drying, performing desalination treatment by using an ultrafiltration tube, performing concentration determination on the obtained lysozyme dimer aqueous solution by using a BCA quantitative kit, subpackaging according to 100 micrograms/tube, and performing freeze drying to obtain the lysozyme dimer standard product with the purity of not less than 99%. Preferably, a lysozyme dimer standard with a purity of 99.2% is obtained.
In a preferred embodiment of the present invention, in the step a, the centrifugal treatment is performed for at least 10 minutes by setting a centrifugal force not lower than 15000 g.
As a preferred technical solution of the present invention, in the step b, the parameters of the hydrophilic silica gel column are as follows: 7.8 × 300ID, 5 μm. Is very suitable for the size exclusion chromatographic separation of lysozyme monomer, dimer and polymer protein mixture.
In the preferred embodiment of the present invention, in the step b, the hydrophilic silica gel column is made of hydrophilic silica gel of tokyo co, model number TSKgel G2000 SWXL.
In a preferred embodiment of the present invention, in the step b, the mobile phase is used, wherein the concentration of PB is 50mM, the concentration of urea is 0.5M, the concentration of ammonium sulfate is 0.3M, and the volume concentration of acetonitrile is 10% (v/v.).
In a preferred embodiment of the present invention, in the step b, the pH of the mobile phase is adjusted to 3.
In a preferred embodiment of the present invention, in step b, the chemical reagent used is analytically pure, and the solution is chromatographically pure.
In the step b, an ultrafiltration centrifugal tube with the cut-off molecular weight not more than 14KD is adopted as the ultrafiltration tube. Further preferably, the ultrafiltration tube adopts an ultrafiltration centrifugal tube with the cut-off molecular weight not more than 3 KD. Preferably, the ultrafiltration tube is a Millipore ultrafiltration tube, the model specification is UFC900396, and the cut-off molecular weight is 3 KD. Because the molecular weight of the lysozyme monomer is about 14KD, the ultrafiltration centrifugal tube with the cut-off of 3KD is most suitable.
In the step b, the ultrafiltration centrifugal speed of the ultrafiltration tube is set to be not lower than 8000rpm, the solution is replaced by adding chromatographic pure water for at least 6 times, and the solution is respectively centrifuged for not less than 20 minutes each time.
In a preferred embodiment of the present invention, in the step b, the BCA quantification kit is a chemical reagent kit manufactured by seimer feishel corporation.
As a preferred technical scheme of the invention, in the step b, a MALDI-TOF mass spectrometer and SDS-PAGE gel separation are adopted for detection, and the purity of the lysozyme dimer is obtained.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1. the method adopts a size exclusion chromatography separation method, and is suitable for monitoring the separation and purification of lysozyme monomer, dimer and polymer protein mixture; the lysozyme monomer, dimer and polymer can be well separated from the base line;
2. the lysozyme dimer is separated and purified and the standard product of the lysozyme dimer is prepared, the whole process can be completed within a short time within 5 hours at room temperature, the efficiency is high, the process steps are simple, and the operation is simple and feasible; the consumption of raw materials is low, the yield of lysozyme dimer is high, the lysozyme monomer can be recycled, and the cost is low; the prepared lysozyme dimer has high standard quality and good effect;
3. the method is simple and easy to implement, low in cost and suitable for popularization and application.
Drawings
FIG. 1 is a graph of size exclusion chromatography of monomers, dimers, and multimers of a preferred embodiment of the method of the invention.
FIG. 2 is a MALDI-TOF mass spectrum of lysozyme dimer according to a preferred embodiment of the present invention.
FIG. 3 is an SDS-PAGE gel of monomers, dimers, and multimers of the method of the preferred embodiment of the invention.
Fig. 4 is a BSC standard curve plotted by the method of the preferred embodiment of the present invention.
Detailed Description
The above-described scheme is further illustrated below with reference to specific embodiments, which are detailed below:
in this embodiment, a method for purifying lysozyme dimer and preparing a standard thereof comprises the following steps:
a. taking 100mL of lysozyme monomer and polymer mixture obtained after crosslinking, carrying out centrifugal purification treatment by adopting a high-speed centrifuge, setting the centrifugal force to be 15000g, carrying out centrifugal treatment for 10 minutes to remove insoluble impurities, and taking supernatant liquid to subpackage into a chromatographic sampling bottle for later use;
b. b, performing size exclusion chromatography separation treatment on the supernatant obtained in the step a by using a high performance liquid phase system to obtain a separated and purified lysozyme dimer;
in the separation and purification process, the adopted chromatographic column is a hydrophilic silica gel column, the flow rate of a mobile phase is set to be 0.35mL/min, the analysis time is set to be 40 minutes, and a UV 280nm ultraviolet detector is adopted for detection;
when separation and purification are carried out, the formula of the mobile phase is as follows: the concentration of PB was 50mM, the concentration of urea was 0.5M, the concentration of ammonium sulfate was 0.3M, the volume concentration of acetonitrile was 10% (v/v.), the chemical reagents used were analytically pure, the solution was chromatographically pure, and the method of preparing 1L mobile phase was performed according to table 1 below:
TABLE 1 flow phase equation table of the present example
In the process of separation and purification, a Nanoacity ultrahigh pressure liquid chromatography system is adopted to carry out size exclusion chromatography separation experiment on the lysozyme monomer/polymer mixture; the chromatographic column is hydrophilic silica gel column prepared from Tosoh corporation (TOSOH corporation) with model specification of TSKgel G2000SWXL, 7.8 × 300ID, 5 μm; the method is very suitable for performing size exclusion chromatographic separation on lysozyme monomer, dimer and polymer protein mixture; the sample volume of the automatic sample injector is 20 uL; driven at a flow rate of 0.35mL/min, the analysis time of the sample was set to 40 minutes; detecting by using a UV 280nm ultraviolet detector; lysozyme monomer, dimer and polymer can all obtain very good baseline separation; as shown in FIG. 1, monomers, dimers and multimers were very well separated; manually collecting lysozyme dimer fraction, freeze-drying, and performing salt treatment by using an ultrafiltration centrifugal tube with the cut-off molecular weight of 3KD, wherein the ultrafiltration centrifugal tube is a Millipore ultrafiltration tube, the model specification is UFC900396, the ultrafiltration centrifugal speed is set to 8000rpm, the solution is replaced by adding chromatographic pure water for 6 times, and the solution is respectively centrifuged for 20 minutes; the lysozyme dimer obtained was analyzed by MALDI-TOF mass spectrometry, as shown in FIG. 2; separating purified dimer from monomer, polymer SDS-PAGE gel for detection, as shown in FIG. 3; the purity of lysozyme dimer reaches more than 99 percent;
then, the aqueous solution of the lysozyme dimer obtained was produced by Saimer Feishel CoThe BCA quantification kit of (1) for concentration determination; the concentration gradient of standard curve BSA was 0,0.025,0.05,0.1,0.2,0.3,0.4, 0.5. mu.g/. mu.L, in a volume of 20. mu.L; diluting the samples by 40 times with water, and taking 20 mu L of the diluted samples, wherein each sample is provided with 3 multiple holes; adding a standard curve solution and a sample solution into a 96-well plate, preparing a color developing agent according to the volume ratio of the solution A to the solution B of 50:1, adding 200ML into each well, incubating at the constant temperature of 37 ℃ for about 45min, detecting the absorbance value by using an enzyme-linked immunosorbent assay under the condition of 562nm, calculating the concentration of the sample to be 5.49ug/uL according to the standard curve, and referring to a standard curve of BSC (basic station controller) shown in figure 4: 1.2463x +0.1209, R2＝0.9963。
Finally, subpackaging according to 100 micrograms/tube and then freeze-drying to obtain the lysozyme dimer standard substance with the purity not lower than 99.2 percent.
The method is adopted to separate and purify the lysozyme dimer and prepare the standard product thereof, the whole process can be completed within 5 hours at room temperature, the process steps are simple, and the operation is simple and feasible; the consumption of raw materials is low, the yield of lysozyme dimer is high, the lysozyme monomer can be recycled, and the cost is low; the prepared lysozyme dimer has high standard quality and good effect.
The embodiments of the present invention have been described with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made according to the purpose of the invention, and any changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the technical scheme of the present invention shall be equivalent substitution ways, as long as the technical principle and inventive concept of the method for purifying lysozyme dimer and preparing the standard thereof according to the present invention are not departed from the technical principle and inventive concept of the method for preparing the lysozyme dimer and the standard thereof, which belong to the protection scope of the present invention.
Claims (10)
1. A method for purifying lysozyme dimer and preparing a standard product thereof is characterized by comprising the following steps:
a. taking the mixture of lysozyme monomer and polymer obtained after crosslinking, centrifuging by adopting a high-speed centrifuge to remove insoluble impurities, and taking the supernatant and subpackaging into a chromatographic sampling bottle for later use;
b. performing size exclusion chromatography separation and purification treatment on the supernatant obtained in the step a by using a high performance liquid phase system to obtain a mixture of separated and purified lysozyme dimer;
in the separation and purification process, the adopted chromatographic column is a hydrophilic silica gel column, the flow rate of a mobile phase is set to be 0.35ml/min, the analysis time is set to be 40 minutes, and a UV 280nm ultraviolet detector is adopted for detection;
and (b) during separation and purification, collecting the obtained dimer fraction in the supernatant obtained in the step a, performing freeze drying, performing desalination treatment by using an ultrafiltration tube, performing concentration determination on the obtained lysozyme dimer aqueous solution by using a BCA quantitative kit, subpackaging according to 100 micrograms/tube, and performing freeze drying to obtain the lysozyme dimer standard product with the purity of not less than 99%.
2. The method for purifying lysozyme dimer according to claim 1 and preparing a standard thereof, wherein the method comprises the following steps: in the step a, when the centrifugal treatment is carried out, the centrifugal force is set to be not less than 15000g, and the centrifugal treatment is carried out for at least 10 minutes.
3. The method for purifying lysozyme dimer according to claim 1 and preparing a standard thereof, wherein the method comprises the following steps: in the step b, the parameters of the hydrophilic silica gel column are as follows: 7.8 × 300ID, 5 μm.
4. The method for purifying lysozyme dimer according to claim 1 and preparing a standard thereof, wherein the method comprises the following steps: in step b, a mobile phase was used in which the concentration of PB was 50mM, urea was 0.5M, ammonium sulfate was 0.3M, and acetonitrile was 10% (v/v.).
5. The method for purifying lysozyme dimer according to claim 1 and preparing the standard thereof, wherein the method comprises the following steps: in said step b, the pH of the mobile phase used is adjusted to 3.
6. The method for purifying lysozyme dimer according to claim 1 and preparing a standard thereof, wherein the method comprises the following steps: in step b, the chemical reagent used is analytically pure and the solution is chromatographically pure.
7. The method for purifying lysozyme dimer according to claim 1 and preparing the standard thereof, wherein the method comprises the following steps: in the step b, the ultrafiltration tube adopts an ultrafiltration centrifugal tube with the cut-off molecular weight not more than 14 KD.
8. The method for purifying lysozyme dimer according to claim 7 and preparing the standard thereof, wherein the method comprises the following steps: in the step b, the ultrafiltration tube adopts an ultrafiltration centrifugal tube with the cut-off molecular weight not more than 3 KD.
9. The method for purifying lysozyme dimer according to claim 1 and preparing the standard thereof, wherein the method comprises the following steps: in the step b, setting the ultrafiltration centrifugal speed of the ultrafiltration tube to be not less than 8000rpm, adding chromatographic pure water for at least 6 times for solution replacement, and respectively centrifuging for not less than 20 minutes each time.
10. The method for purifying lysozyme dimer according to claim 1 and preparing the standard thereof, wherein the method comprises the following steps: in step b, the purity of lysozyme dimer was determined by MALDI-TOF mass spectrometry and SDS-PAGE gel separation.
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