CN112521460A - Chromatography process for purifying recombinant porcine parvovirus VP2 protein - Google Patents
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Abstract
The invention relates to a purification process of a recombinant porcine parvovirus, in particular to a chromatography process for purifying a VP2 protein of the recombinant porcine parvovirus, belonging to the field of biological products for livestock. The chromatographic process for purifying the recombinant porcine parvovirus VP2 protein takes recombinant porcine parvovirus VP2 protein fermentation broth, performs low-temperature high-speed centrifugation, discards precipitates, takes supernate, clarifies and filters the supernate through a filter, adjusts the pH value to be the same as that of a balance solution, samples the supernate into an anion chromatographic column, and elutes the recombinant porcine parvovirus VP2 protein combined with the chromatographic column. The purity of the purified protein reaches 90 percent by SDS-PAGE gel analysis.
Description
Technical Field
The invention relates to a purification process of a recombinant porcine parvovirus, in particular to a chromatography process for purifying a VP2 protein of the recombinant porcine parvovirus, belonging to the field of biological products for livestock.
Background
Porcine Parvovirus (PPV) can cause reproductive disorders in gilts at first birth, leading to stillbirth, mummification, malformed and weak piglets of pigs, etc., which seriously jeopardize the development of the pig industry for a long time. Pigs of all ages, breeds and sexes can be infected with PPV, especially the first-birth sows are most susceptible to infection. The virus can be discharged through the mouth and nose of the sick pig and the excrement, and can continuously infect the whole pig group for a long time. If the sow is infected with the virus in the early gestation period, the embryonic mortality rate can reach 80-100%, and even if the sow survives after gestation infection, the piglet can carry a large amount of virus for a long time.
The porcine parvovirus VP2 protein is a main structural protein of the porcine parvovirus, occupies more than 60% of the surface of a Virus capsid, contains main B cell and T cell epitopes of the Virus, can be self-assembled into viroid Particles (VLPs) by recombinant expression of VP2 protein, can induce an organism to generate neutralizing antibodies, and can be used as a porcine parvovirus genetic engineering vaccine.
At present, most porcine parvovirus vaccines are all whole virus vaccines, and as the average diameter of PPV is between 20 nm and 26 nm, most host cell proteins, nucleic acid fragments, endotoxin and other impurities cannot be removed in the membrane-packed concentration, washing and filtering process, and high-purity vaccines cannot be obtained, so that the side reaction is strong after vaccine injection, and the immunogenicity is reduced.
In order to reduce side reactions of vaccines and enhance immunogenicity, researchers replace traditional viral vaccines with recombinant porcine parvovirus VP2 protein genetic engineering vaccines, most researchers solve the problem of recombinant protein purification by adding His tags, but the His tag protein purification cost is high, and substances such as imidazole and the like need to be removed by desalting after purification, so that the production cost is further increased, and therefore, a low-cost, convenient and rapid porcine parvovirus VP2 protein purification process is urgently needed.
Disclosure of Invention
The invention aims to provide a chromatography process for purifying recombinant porcine parvovirus VP2 protein, and high-purity VP2 protein can be obtained by the chromatography process.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a chromatography process for purifying recombinant porcine parvovirus VP2 protein, which comprises the following steps:
1) taking crude fermentation liquor of porcine parvovirus VP2 protein, centrifuging at low temperature and high speed, discarding the precipitate, and keeping supernatant porcine parvovirus VP2 protein fermentation clear liquor;
2) clarifying and filtering the porcine parvovirus VP2 fermented clear liquid through a filter to obtain a porcine parvovirus VP2 protein sample stock solution;
3) adjusting the pH value of the porcine parvovirus VP2 protein sample stock solution to be the same as the pH value of the equilibrium solution adopted in the step (4) to obtain a porcine parvovirus VP2 protein loading solution;
4) and purifying the porcine parvovirus VP2 protein sample solution by using an anion exchange chromatography column to obtain the high-purity porcine parvovirus VP2 protein with impurities such as foreign proteins, pigments and the like removed.
The chromatographic process for purifying the recombinant porcine parvovirus VP2 protein takes recombinant porcine parvovirus VP2 protein fermentation broth, performs low-temperature high-speed centrifugation, discards precipitates, takes supernate, clarifies and filters the supernate through a filter, adjusts the pH value to be the same as that of a balance solution, samples the supernate into an anion chromatographic column, and elutes the recombinant porcine parvovirus VP2 protein combined with the chromatographic column. The process can obtain recombinant porcine parvovirus VP2 protein with purity as high as 90%.
The traditional anion exchange chromatography mode is that proteins are negatively charged in solution at a pH above their isoelectric point and bind to an anion exchange medium as they flow through the medium. The isoelectric point of the porcine fine VP2 protein is 6, most of the foreign proteins in the fermentation liquid are also acidic proteins, and the protein can be combined with a medium in a solution with the pH value of a buffer solution of more than 6, so that a good separation effect is difficult to achieve. In this case, researchers often neglect the importance of the local charge of the protein, and the invention finds that the porcine fine VP2 protein can be combined with an anion exchange medium even in the environment lower than the isoelectric point of the protein, so that the high-purity porcine fine VP2 protein can be obtained.
Preferably, in the step 1), the low-temperature high-speed centrifugation temperature is 4-8 ℃, the rotation speed is 10000-12000 r/min, and the centrifugation time is 20-30 min.
Preferably, in step 2), the filter is a sterile filter with a pore size of 0.45 μm.
Preferably, in step 3), the pH is adjusted using 0.01-0.1M acetic acid solution.
Preferably, the filler used in the anion exchange chromatography in step 4) is DEAE Purose 6 Fast Flow.
Preferably, the anion exchange chromatography in step 4) uses two solutions of an equilibration solution and an eluent, wherein the equilibration solution is 20-50 mM sodium acetate buffer solution with a pH range of 4.0-6.5, and the eluent is 20-50 mM sodium acetate buffer solution with 1M sodium chloride with a pH range of 4.0-6.5.
Preferably, in step 4), the anion exchange chromatography process is:
washing the ion exchange column with sterile 0.1-1M sodium hydroxide solution for 3 column volumes, and washing the chromatographic column with balancing solution until the effluent end of the chromatographic column detects UV280 and the baseline of the conductance and pH value is stable, wherein the conductance and the pH value are the same as those of the balancing solution;
then pumping the porcine parvovirus VP2 protein sample solution obtained in the step 3) into a chromatographic column for sample loading, continuously eluting the chromatographic column by using a balance solution after the sample loading is finished, eluting the VP2 protein adsorbed on the chromatographic column by using an eluent when the UV280 value is reduced to 0.100AU,
collection was started when the UV280 value started to rise and was above 0.100AU, and was stopped when the UV280 value dropped to 0.100 AU.
Preferably, when the step 4) is carried out by using an anion exchange column for purification, the sample flow rate of the porcine parvovirus VP2 protein is 120-150 cm/h, and the pressure is controlled to be less than 0.3 MPa.
The invention has the beneficial effects that:
1) the invention solves the technical problem of obtaining the high-purity recombinant porcine fine VP2 protein genetic engineering vaccine. The recombinant porcine fine VP2 protein is purified by one-step method through one-step anion exchange chromatography, impurities such as foreign protein and pigment are effectively removed, and a high-purity protein sample is obtained.
2) The method is simple to operate, and compared with affinity chromatography for labeling the recombinant protein, the method omits the steps of subsequent desalting and the like, and saves equipment and labor cost.
3) The recombinant porcine fine VP2 protein purification process can obtain a high-purity porcine fine genetic engineering vaccine, reduces side reactions caused by impurities such as foreign proteins, accounting, endotoxin and the like, and improves the safety and immunogenicity of the vaccine.
Drawings
FIG. 1 is an A280 map of recombinant porcine fine VP2 protein sample solution flowing through DEAE Purose 6 Fast Flow medium chromatographic column;
FIG. 2 is a photograph of SDS-PAGE analysis of a recombinant porcine fine VP2 protein loading solution before purification and flow-through and eluate collected during purification using the process of the present invention, wherein the loading samples of each lane are as follows: lane M, marker, Lane 1, fermentation stock of recombinant porcine fine VP2 protein, Lane 2, purified recombinant porcine fine VP2 protein, Lane 3, flow-through.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1
A chromatography process for purifying recombinant porcine parvovirus VP2 protein comprises the following specific steps:
1 solution preparation
Balance liquid: 20 mM sodium acetate buffer, pH 5.0,
eluent: 20 mM sodium acetate buffer, 1.0M sodium chloride, pH 5.0.
2 preparation of recombinant porcine fine VP2 protein sample solution
Centrifuging 20 mL of the crude recombinant porcine fine VP2 protein fermentation liquid at low temperature and high speed, adjusting the pH value to 5.0 by using 0.01M acetic acid after taking supernatant at the temperature of 4 ℃ and 10000 r/min for 30 min, and filtering by using a 0.45 mu M filter to obtain the recombinant porcine fine VP2 protein loading liquid.
3 purification Using Prepack DEAE Purose 6 Fast Flow 1 mL column
Cleaning of
The column was washed with deionized water at a flow rate of 1 mL/min for 5 column volumes.
Balancing
And (3) balancing 5 column volumes of the chromatographic column by using a balancing solution at the flow rate of 1 mL/min, and zeroing the ultraviolet A280 absorption detection baseline after the balancing is finished.
Sample loading
20 mL of the recombinant porcine parvovirus VP2 protein loading solution was loaded onto the column at a flow rate of 1 mL/min, and the flow-through solution was collected when the UV started to rise.
Leaching with water
After the sample loading is finished, cleaning the chromatographic column by using a balance liquid at the flow rate of 1 mL/min for at least 5 column volumes, and stopping collecting the flow-through liquid after the ultraviolet is stable.
Elution is carried out
And eluting the recombinant porcine fine VP2 protein bound on the chromatographic column by using eluent at the flow rate of 1 mL/min, starting collection when ultraviolet light rises, and stopping collection when the ultraviolet light drops to 0.1 AU.
Cleaning and preserving
Washing the chromatographic column with deionized water at the flow rate of 1 mL/min until the conductivity detection result is less than 0.05 mS/cm, and washing the chromatographic column with 20 v% ethanol at the flow rate of 1 mL/min for 5 column volumes for storage.
A280 map of the recombinant porcine fine VP2 protein loading solution when purified by DEAE Purose 6 Fast Flow medium is shown in FIG. 1.
The purity detection result of the purified recombinant porcine parvovirus VP2 protein is shown in figure 2, the yellow recombinant porcine parvovirus VP2 protein crude fermentation broth is colorless, transparent and clear after purification, most of the foreign proteins flow through, and the VP2 protein is combined with the medium, so that the concentration of the recombinant porcine parvovirus VP2 protein is realized during purification.
The purity results of the recombinant porcine parvovirus VP2 protein fermentation stock solution and the purified recombinant porcine parvovirus VP2 protein sample are detected by SDS-PAGE gel electrophoresis, the results are shown in figure 2, the protein purity is obviously improved, the purity is up to 90% by analysis, and meanwhile, the concentration of the recombinant porcine parvovirus VP2 protein is realized.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (8)
1. A chromatography process for purifying recombinant porcine parvovirus VP2 protein is characterized by comprising the following steps:
1) taking crude fermentation liquor of porcine parvovirus VP2 protein, centrifuging at low temperature and high speed, discarding the precipitate, and keeping supernatant porcine parvovirus VP2 protein fermentation clear liquor;
2) clarifying and filtering the porcine parvovirus VP2 fermented clear liquid through a filter to obtain a porcine parvovirus VP2 protein sample stock solution;
3) adjusting the pH value of the porcine parvovirus VP2 protein sample stock solution to be the same as the pH value of the equilibrium solution adopted in the step (4) to obtain a porcine parvovirus VP2 protein loading solution;
4) and purifying the porcine parvovirus VP2 protein sample solution by using an anion exchange chromatography column to obtain the high-purity porcine parvovirus VP2 protein with impurities such as foreign proteins, pigments and the like removed.
2. The method of claim 1, wherein: in the step 1), the low-temperature high-speed centrifugation temperature is 4-8 ℃, the centrifugation speed is 10000-12000 r/min, and the centrifugation time is 20-30 min.
3. The chromatography process for purifying the recombinant porcine parvovirus VP2 protein as claimed in claim 1, wherein: in step 2), the filter is a sterile filter with a pore size of 0.45 microns.
4. The chromatography process for purifying the recombinant porcine parvovirus VP2 protein as claimed in claim 1, wherein: in step 3), 0.01-0.1M acetic acid solution is used for adjusting the pH value.
5. The chromatography process for purifying the recombinant porcine parvovirus VP2 protein as claimed in claim 1, wherein: the filler used in the anion exchange chromatography in the step 4) is DEAE Purose 6 Fast Flow.
6. The chromatography process for purifying the recombinant porcine parvovirus VP2 protein as claimed in claim 1, wherein: the anion exchange chromatography in step 4) uses two solutions of equilibrium solution and eluent, wherein the equilibrium solution is 20-50 mM sodium acetate buffer solution, the pH range is 4.0-6.5, and the eluent is 20-50 mM sodium acetate buffer solution containing 1M sodium chloride, the pH range is 4.0-6.5.
7. The chromatography process for purifying the recombinant porcine parvovirus VP2 protein as claimed in claim 1, wherein: in the step 4), the anion exchange chromatography process comprises the following steps:
washing the ion exchange column with sterile 0.1-1M sodium hydroxide solution for 3 column volumes, and washing the chromatographic column with balancing solution until the effluent end of the chromatographic column detects UV280 and the baseline of the conductance and pH value is stable, wherein the conductance and the pH value are the same as those of the balancing solution;
then pumping the porcine parvovirus VP2 protein sample solution obtained in the step 3) into a chromatographic column for sample loading, continuously eluting the chromatographic column by using a balance solution after the sample loading is finished, eluting the VP2 protein adsorbed on the chromatographic column by using an eluent when the UV280 value is reduced to 0.100AU,
collection was started when the UV280 value started to rise and was above 0.100AU, and was stopped when the UV280 value dropped to 0.100 AU.
8. The chromatography process for purifying the recombinant porcine parvovirus VP2 protein as claimed in claim 1, wherein: and 4) when the anion exchange column is used for purification, the sample loading flow rate of the porcine parvovirus VP2 protein is 120-150 cm/h, and the pressure is controlled to be less than 0.3 MPa.
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Cited By (2)
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| CN113150126A (en) * | 2021-04-20 | 2021-07-23 | 开江县动物疫病预防控制中心 | Rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof |
| CN113957059A (en) * | 2021-11-26 | 2022-01-21 | 山东滨州沃华生物工程有限公司 | Method for purifying porcine reproductive and respiratory syndrome virus by one-step column chromatography process |
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