CN106769358B - Method for purifying aqueous phase after demulsification of oil adjuvant vaccine - Google Patents
Method for purifying aqueous phase after demulsification of oil adjuvant vaccine Download PDFInfo
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Abstract
The invention provides a method for purifying a water phase after demulsification of an oil adjuvant vaccine, which comprises the following steps: performing gel electrophoresis on an antigen sample subjected to demulsification of the foot-and-mouth disease vaccine; cutting one protein lane for staining to determine the band position of the target protein; then cutting the gel on the unstained gel, and carrying out sample treatment on the gel at the same position as the stained target protein band; and performing ultrasonic extraction on the treated gel block, and performing vacuum freeze drying or low-temperature freeze centrifugal concentration on the obtained supernatant. According to the invention, the antigen after demulsification is separated and purified by electrophoresis, so that the purity of the antigen is improved, the detection repeatability of the purified sample is good, the accuracy of the detection result is improved, the signal-to-noise ratio of the map can be obviously reduced, and the target molecule is highlighted. In addition, the invention specifically adopts a partial dyeing mode in the gel electrophoresis purification step, thereby simplifying the antigen processing step, increasing the antigen recovery rate and reducing the instrument maintenance cost.
Description
Technical Field
The invention relates to the technical field of foot-and-mouth disease vaccine detection, in particular to a method for purifying a water phase after demulsification of an oil adjuvant vaccine.
Background
In the foot-and-mouth disease vaccine detection, because the components in the oil adjuvant are complex, impurities such as a plurality of surfactants, immunopotentiators and the like are dissolved in a water phase, and the existing protein qualitative and quantitative detection method is very easily interfered by the impurities when detecting the protein, so that data fluctuation is caused, the detection process of the oil adjuvant vaccine is influenced by directly using the protein qualitative and quantitative detection method after demulsification, signal covering or interference is caused, the strength of an antigen signal is reduced, even the antigen in the antigen can not be effectively detected, and a method capable of removing the impurities is not available in the industry, so that a purification step is introduced after demulsification to remove various impurities in the water phase after demulsification, the detection is further facilitated by using the protein qualitative and quantitative detection method, and the problem that the impurities in the sample after demulsification reflect a more real sample state is solved.
The mainstream sample purification means in the prior art are various chromatographs, and the amount of samples required in the chromatographic process is large, so that about 30-50% of sample loss (diffusion is more easily caused when the initial sample amount is low) is caused, and the device dependence is high. The antigen concentration in the oil adjuvant vaccine is microgram level, a part of antigen is lost after the demulsification process, and then the antigen is purified by using a chromatography mode, so that the antigen recovery rate is extremely low, the antigen cannot be used for detection, if the demulsification amount of the vaccine is increased, the vaccine is wasted, the service life of chromatography packing is shortened by accelerating impurities in the oil adjuvant, and the economy is poor.
In the existing gel electrophoresis purification technology, the components of the separated complex sample are all biomolecules and congeners thereof, but not other substances with complex properties, wherein the sample state of the processed sample components is simpler compared with that of the water phase after demulsification. The existing protein electrophoresis in-gel enzymolysis technology has more procedures, complex steps and low recovery rate after operation, and the sample which can be recovered after the treatment of a trace sample can not meet the detection requirement.
At present, all domestic manufacturers explore a practical method, which can treat the antigen in the vaccine subjected to demulsification treatment and reduce the influence of impurities in the antigen on the subsequent qualitative and quantitative detection and analysis. Substances such as surfactants and the like in the water phase after demulsification are known as the most difficult-to-treat impurities in the industry, and the water phase after demulsification cannot be directly detected by the prior art in the industry. The components of the sample aimed at in the invention are complex, the concentration of protein in the vaccine is low, the vaccine is not suitable for the traditional purification method, and the water phase after demulsification contains impurities such as surfactant and the like, so that the impurities exist in a free state, the amphiphilic property of the water phase can be combined with the protein, the combined bond of the impurities and the protein can be opened under the action of an electric field force, and the separation and purification can be realized in the gel. Compared with the traditional mode, the method for purifying the protein by the gel electrophoresis greatly reduces the required sample amount, greatly reduces the dependence on operators and instruments and equipment, and can separate protein-bound impurities from the protein.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for purifying a water phase after demulsification of an oil adjuvant vaccine.
The purpose of the invention is realized by the following technical scheme:
the invention provides a method for purifying a water phase after demulsification of an oil adjuvant vaccine, which comprises the following steps:
s1, performing gel electrophoresis on the antigen sample after the foot-and-mouth disease vaccine demulsification;
s2, cutting one protein lane to dye and determine the position of a target protein band;
s3, cutting off the gel which is not dyed and has the same position with the target protein band dyed in the step S2 for sample processing;
s4, performing ultrasonic extraction on the processed gel block, and performing vacuum freeze drying or low-temperature freeze centrifugal concentration on the obtained supernatant.
Preferably, in step S1, the conditions of the gel electrophoresis are: treating with 5% acrylamide concentrated gel and 12% acrylamide separation gel at 210V for 30-40 min. .
Preferably, in step S2, the staining is silver staining or coot staining.
Preferably, in step S3, the sample processing specifically includes the following steps:
a1, passing the gel containing the target protein through ddH2Cleaning with O, shaking to dewater with acetonitrile, adding NH containing dithiothreitol4HCO3Performing first incubation by using an aqueous solution;
a2, shaking and dehydrating the gum block after the first incubation with acetonitrile, and adding NH containing iodoacetamide4HCO3The aqueous solution was incubated for a second time.
Preferably, the first incubation conditions are: incubating at 56 deg.C for 30 min; the second incubation conditions were: incubate for 20min at RT in dark.
Preferably, the temperature of ultrasonic extraction is 0-8 ℃, ultrasonic extraction is carried out for 15min, and the adopted extraction liquid is 80% ACN/5% FA or 80% ACN/0.05% TFA. By adopting ultrasonic extraction, the target components in the glue can be accelerated to enter the solvent, the extraction is promoted, and the extraction time is shortened.
Preferably, the extraction liquid adopted by the ultrasonic extraction is a mixed solution with the mass content of Acetonitrile (ACN) of 80% and the mass content of Formic Acid (FA) of 5%; or a mixed solution containing acetonitrile 80% by mass and trifluoroacetic acid (TFA) 0.05% by mass.
The extract is more preferably a mixed solution containing 80% by mass of acetonitrile and 5% by mass of formic acid, and FA is preferably used for high-precision detection because FA has low masking of a target signal in a mass spectrum. The invention adopts ultrasonic extraction, utilizes the strong cavitation response effect, mechanical vibration, disturbance effect, high acceleration, emulsification, diffusion, crushing, stirring and other multi-stage effects generated by ultrasonic radiation pressure, increases the molecular motion frequency and speed of the substance, and increases the penetrating power of the solvent, thereby accelerating the target component to enter the solvent and promoting the extraction.
The principle of the invention is as follows: according to the invention, by means of gel electrophoresis, impurities such as residual surfactant in a sample can be separated through molecular weight, and interference of the impurities on subsequent detection can be further reduced by specifically cutting a target protein band. Therefore, high-efficiency purification can be realized, compared with the conventional purification means, the required initial sample amount is lower, and the purity of the antigen in the water phase after demulsification can be greatly improved. In addition, the invention specifically adopts a partial dyeing mode in the gel electrophoresis purification step, thereby simplifying the antigen processing step, increasing the antigen recovery rate and reducing the instrument maintenance cost.
Compared with the prior art, the invention has the following beneficial effects:
1) according to the invention, the antigen after demulsification is separated and purified by electrophoresis, so that the purity of the antigen is improved, and the impurity content of the surfactant in the antigen is basically 0, thus the detection repeatability of the purified sample is good, and the accuracy of the detection result is improved.
2) The conventional gel electrophoresis method has the advantages that the conventional gel electrophoresis method can involve dyeing and decoloring steps, protein loss can be caused in each step in subsequent processing steps, the processing time can be prolonged if the decoloring step is carried out, the decoloring agent can cause the loss of protein when being decolored, meanwhile, the residue of the decoloring agent can also affect the subsequent detection process, the detection instrument can be polluted, and the equipment maintenance and use cost are increased. The method of gel electrophoresis is used for antigen purification after demulsification, and only the dyed sample lane is used as a standard in the method, the sample lane needing subsequent treatment is not dyed, the undyed gel is treated, and the decoloring step is directly skipped, so that the problems of the conventional electrophoresis subsequent treatment can be effectively avoided, the sample recovery rate is improved, and the treatment time is shortened.
3) The antigen after electrophoresis is incubated for two times, wherein the first incubation is used for reducing the antigen polypeptide in the collagen and reducing the disulfide bond between cysteine in the antigen into sulfydryl, namely reducing cysteine. The second incubation is to carry out alkylation protection on the reduced cysteine and histidine after the first incubation, so that the antigen is kept in a denatured state for subsequent enzymolysis and detection. After two times of incubation, the enzymolysis efficiency of the antigen can be improved, and the purpose of greatly reducing the initial sample amount required by detection is achieved.
4) When the sample prepared by the purification method is used for mass spectrum detection, the signal-to-noise ratio of the map can be obviously reduced, and the target molecules are highlighted.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a mass spectrometry scan of a sample prepared in example 1;
FIG. 2 is a mass spectrometry scan of an unpurified sample of comparative example 1;
FIG. 3 is a mass spectrometry scan of the sample prepared in comparative example 2.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1
The embodiment provides a method for purifying an aqueous phase after demulsification of an oil adjuvant vaccine, which comprises the following steps:
1. sample treatment: demulsifying a foot-and-mouth disease vaccine, performing ultrafiltration and washing filtration, freeze-drying to obtain a protein antigen sample (the antigen purity is 8%), adding the protein antigen sample into a protein loading buffer solution, heating at 95 ℃ for 2min, performing gel electrophoresis, uniformly loading the sample in all blank lanes (concentrated gel with 5% of acrylamide by mass and separation gel with 12% of acrylamide by mass), performing silver staining on one protein lane at 210V for 30-40min, determining the position of a band of a target protein, and performing sample treatment on the unstained gel and the gel with the same position of the band of the target protein in the sample after silver staining.
2. Cutting the cut rubber blocks into 1-2 mm3The rubber blocks with the sizes are placed in an EP tube, and 400ul of ACN is added to shake and dehydrate for 3 min.
3. Discarding the liquid, drying at room temperature, adding NH dissolved with DTT4HCO3The solution was incubated at 56 ℃ for 30 min. The NH dissolved with DTT4HCO3The concentration of DTT in the solution was 0.01M.
4. Naturally cooling, discarding liquid, adding 400ul ACN, shaking and dehydrating for 3 min.
5. Discarding the liquid, naturally drying, and adding NH dissolved with iodoacetamide4HCO3Appropriate amount of solution, and incubation for 20min at RT in dark. Said NH containing iodoacetamide dissolved therein4HCO3The concentration of iodoacetamide in the solution was 0.1M.
6. Discard the liquid, add 400ul ACN and shake to dehydrate for 3 min.
7. Discarding the liquid, drying at room temperature, adding 12.5ng/ul Trypsin NH4HCO3Solution, RT imbibition.
8. The liquid was discarded and 50mM NH added4HCO3A proper amount of the glue blocks are soaked, and the temperature is 56 DEG CIncubate for 3 h.
9. The solution was removed and placed in a new EP tube, 200ul of a mixed solution of 80% ACN and 5% FA was added, sonication was performed in an ice-water bath for 15min, which was repeated 3 times, and the supernatant was taken and placed in a new EP tube.
10. The extracted aqueous phase was vacuum freeze dried.
11. The lyophilized powder was dissolved in 100. mu.l of 0.1% TFA solution per tube and analyzed by mass spectrometry.
The mass spectrum of the powder prepared in the embodiment is 611AMU which is one of theoretical molecular weights in the sample shown in figure 1, and it can be obtained from the figure that the abundance of impurities in the figure is reduced to an extremely low degree, the target molecule becomes the main peak of the figure, and the purity of the sample in chromatographic detection reaches more than 90%. FIG. 1 is a mass spectrum scanning spectrum of a sample obtained after the treatment according to the procedure described in this example, wherein 611.22AMU is one of the target molecular weights of the sample in this experiment. The figure shows that the abundance of the target molecular weight is high, and the signal-to-noise ratio of the map is low.
Example 2
The embodiment provides a method for purifying an aqueous phase after demulsification of an oil adjuvant vaccine, which comprises the following steps:
1. sample treatment: demulsifying a foot-and-mouth disease vaccine, performing ultrafiltration and washing filtration, freeze-drying to obtain a protein antigen sample (the antigen purity is 8%), adding the protein antigen sample into a protein loading buffer solution, heating at 95 ℃ for 2min, performing gel electrophoresis, uniformly loading the sample in all blank lanes (adopting a concentrated gel with 5% of acrylamide by mass and a separation gel with 12% of acrylamide by mass), performing examination and dyeing on one protein lane at 210V for 30-40min, determining the position of a band of a target protein, and performing sample treatment on the unstained gel and the gel with the same position of the band of the target protein in the examined and dyed sample.
2. Cutting the cut rubber blocks into 1-2 mm3The rubber blocks with the sizes are placed in an EP tube, and 400ul of ACN is added to shake and dehydrate for 3 min.
3. Discarding the liquid, drying at room temperature, adding NH dissolved with DTT4HCO3The solution was incubated at 56 ℃ for 30 min. The NH dissolved with DTT4HCO3Concentration of DTT in solutionIs 0.01M.
4. Naturally cooling, discarding liquid, adding 400ul ACN, shaking and dehydrating for 3 min.
5. Discarding the liquid, naturally drying, and adding NH dissolved with iodoacetamide4HCO3Appropriate amount of solution, and incubation for 20min at RT in dark. Said NH containing iodoacetamide dissolved therein4HCO3The concentration of iodoacetamide in the solution was 0.1M.
6. Discard the liquid, add 400ul ACN and shake to dehydrate for 3 min.
7. Discarding the liquid, drying at room temperature, adding 12.5ng/ul Trypsin NH4HCO3Solution, RT imbibition.
8. The liquid was discarded and 50mM NH added4HCO3The gel block was washed with an appropriate amount and incubated at 56 ℃ for 3 h.
9. The solution was removed and placed in a new EP tube, 200ul of a mixed solution of 80% ACN and 0.05% TFA was added, sonicated in a water bath at 8 ℃ for 15min, repeated 3 times, and the supernatant was removed and placed in a new EP tube.
10. The extracted aqueous phase was vacuum freeze dried.
11. The lyophilized powder was dissolved in 100. mu.l of 0.1% TFA solution per tube and analyzed by mass spectrometry.
The purity of the powder prepared by the embodiment reaches 85%, and the signal-to-noise ratio of the mass spectrum scanning spectrum of the obtained sample is low.
Comparative example 1
Demulsifying the foot-and-mouth disease vaccine, ultrafiltering, washing, filtering, and freeze-drying to obtain protein antigen sample, and directly adding NH4HCO3Digestion was performed with 1ug trypsin and mass spectrometry was performed.
The mass spectrum of the comparative example is shown in figure 2, and can be obtained from the figure, the abundance of impurities in the figure is high, the abundance of target molecules is depressed by the impurities, the signal interference is serious, and the purity of a sample in chromatographic detection is 10%. Fig. 2 is a mass spectrum scanning spectrum of a sample after demulsification and direct enzymolysis, the spectrum is relatively complex, the target molecular weight abundance is low, the interference of impurity signals is serious, and if components such as a surfactant in the sample are not removed, the performance of a subsequent mass spectrum can be greatly influenced, and the maintenance cost in a later period is increased.
Comparative example 2
The embodiment provides a method for purifying a water phase after demulsification of an oil adjuvant vaccine, which specifically comprises the following steps:
1. dyeing a sample: demulsifying the foot-and-mouth disease vaccine, performing ultrafiltration and washing filtration, adding a protein antigen sample (the antigen purity is 8%) obtained after freeze-drying into a protein sample loading buffer solution, heating at 95 ℃ for 2min, performing gel electrophoresis (5% concentrated gel and 12% separation gel), performing 210V, and performing silver staining for 30-40 min.
2. Digestion of stained samples: the gel was placed in a clean bench petri dish and washed with 75% ethanol and then with ultra pure water.
3. Cutting a target protein strip with a surgical blade in a superclean bench into 1-2 mm3The size block was placed in an EP tube.
4. Adding 300ul of destaining solution into an EP tube, uniformly mixing and standing until brown color completely disappears; the decolorizing solution is prepared as follows: 100mmol/L Na2S2O3And 30mmol/L K3Fe(CN)6The solution is mixed according to the volume ratio of 1: 1.
5. Discard the liquid and add 400ul ddH2And O, cleaning the rubber block until the rubber block becomes transparent.
6. Discard the liquid, add 400ul ACN and shake to dehydrate for 3 min.
7. The liquid was discarded, dried at room temperature, and NH was added thereto at a concentration of DTT of 0.01M4HCO3The solution was incubated at 56 ℃ for 30min in appropriate amounts.
8. Naturally cooling, discarding liquid, adding 400ul ACN, shaking and dehydrating for 3 min.
9. Discarding the liquid, naturally drying, adding NH with iodoacetamide concentration of 0.1M4HCO3Appropriate amount of solution, and incubation for 20min at RT in dark.
10. Discard the liquid, add 400ul ACN and shake to dehydrate for 3 min.
11. Discarding the liquid, drying at room temperature, adding 12.5ng/ul Trypsin NH4HCO3Solution, RT imbibition.
12. The liquid was discarded and 50mM NH added4HCO3A proper amount of the glue blocks are soaked, and the temperature is 56 DEG CIncubate for 3 h.
13. The solution was removed and placed in a new EP tube, 200ul of a mixed solution of 80% ACN and 0.05% TFA was added, sonicated in an ice-water bath for 15min, repeated 3 times, and the supernatant was removed and placed in a new EP tube.
14. The extracted aqueous phase was vacuum freeze dried.
15. The lyophilized powder was dissolved in 100. mu.l of 0.1% TFA solution per tube and analyzed by mass spectrometry.
The purity of the powder obtained by the method of the comparative example is about 70%, and fig. 3 is a mass spectrum scanning map of the sample obtained after the treatment by the steps of the embodiment, compared with fig. 1, the signal-to-noise ratio is higher, and the signal intensity is lower than the order of magnitude of fig. 1, so that the antigen content and the purity are greatly lower than those of the sample in fig. 1.
The invention has many applications, and the above description is only a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made without departing from the principles of the invention and these modifications are to be considered within the scope of the invention.
Claims (5)
1. A method for purifying a water phase after demulsification of an oil adjuvant vaccine is characterized by comprising the following steps:
s1, performing gel electrophoresis on the antigen sample after the foot-and-mouth disease vaccine demulsification;
s2, cutting one protein lane to dye and determine the position of a target protein band;
s3, cutting off the gel which is not dyed and has the same position with the target protein band dyed in the step S2 for sample processing;
s4, performing ultrasonic extraction on the processed rubber block, and performing vacuum freeze drying or low-temperature freeze centrifugal concentration on the obtained supernatant to obtain the finished product;
the antigen sample is obtained by carrying out ultrafiltration and washing filtration on an aqueous phase solution obtained after demulsification treatment and then carrying out freeze-drying or concentration;
in step S3, the sample processing specifically includes the following steps:
a1, passing the unstained gel containing the target protein through ddH2Cleaning with O, shaking to dewater with acetonitrile, adding NH containing dithiothreitol4HCO3Performing first incubation by using an aqueous solution;
a2, shaking and dehydrating the gum block after the first incubation with acetonitrile, and adding NH containing iodoacetamide4HCO3Performing secondary incubation by using the aqueous solution;
the first incubation conditions were: incubating at 56 deg.C for 30 min; the second incubation conditions were: incubate for 20min at RT in dark.
2. The method for purifying the aqueous phase after demulsification of the oil-adjuvant vaccine as claimed in claim 1, wherein in step S1, the conditions adopted by the gel electrophoresis are as follows: treating with 5% acrylamide concentrated gel and 12% acrylamide separation gel at 210V for 30-40 min.
3. The method for purifying the aqueous phase after demulsification of the oil-adjuvant vaccine as claimed in claim 1, wherein in step S2, the staining is silver staining or cootie staining.
4. The method for purifying the aqueous phase after demulsification of the oil-adjuvant vaccine as claimed in claim 1, wherein the temperature of ultrasonic extraction is 0-8 ℃, ultrasonic extraction is carried out for 15min, and the ultrasonic extraction is repeated for 3 times.
5. The method for purifying the aqueous phase after demulsification of the oil-adjuvant vaccine as claimed in claim 1, wherein the extraction liquid adopted by the ultrasonic extraction is a mixed solution containing 80% by mass of acetonitrile and 5% by mass of formic acid; or a mixed solution of acetonitrile with the mass content of 80 percent and trifluoroacetic acid with the mass content of 0.05 percent.
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| CN104471395A (en) * | 2012-06-28 | 2015-03-25 | 生物辐射实验室股份有限公司 | Multi-protein quantitation standard |
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