Purification method suitable for preparing poultry multi-combined vaccine by escherichia coli expression process

Abstract

The invention discloses a purification method suitable for preparing a poultry multi-combined vaccine by an escherichia coli expression process, and belongs to the technical field of bioengineering pharmacy. The invention discloses a purification method suitable for preparing a poultry multi-combined vaccine by an escherichia coli expression process. A molecular adsorption precipitation method, an isoelectric point precipitation method, a salting-out method, a liquid phase separation method and other methods are combined and improved. The novel method for removing endotoxin disclosed by the invention is effective, in addition, part of protein is flocculated after formaldehyde inactivation, and the content of impure protein can be obviously reduced after filtration and removal. After being treated by the method, the content of endotoxin in the final recombinant protein solution does not exceed 500EU / mL, and the purity of the recombinant protein reaches 60% or above.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering pharmaceuticals, and more particularly to a purification method suitable for preparing a multi-unit vaccine for poultry using an Escherichia coli expression process. Background Art

[0002] Escherichia coli is a host bacteria commonly used in the production of recombinant proteins. It has the advantages of clear genetic background, simple culture operation, high transformation and transduction efficiency, fast growth and reproduction, low cost, and can quickly and large-scale production of target proteins. However, the large-scale production of recombinant proteins based on Escherichia coli will produce a large amount of impurities and endotoxins. In the preparation process of the vaccine, if a large amount of impurities and endotoxins cannot be removed, it will cause fever, stress, decreased feeding and other symptoms in the immunized animals, and also affect the absorption of the vaccine. The vaccine injection site is prone to cause inflammatory reactions, which in turn affects the ketone quality of meat products.

[0003] In the process of large-scale factory preparation of recombinant proteins based on the E. coli expression system, the problem of how to remove impurities and endotoxins efficiently, at low cost, and easily for large-scale factory production operations is also a difficult problem that plagues the development of the industry.

[0004] Currently known methods for removing impurities from recombinant proteins mainly include: the first is the ammonium sulfate method, which is based on the different solubility of proteins in ammonium sulfate of different solubility to separate and remove impurities. The advantages are low cost and simple operation, but the disadvantage is that the purity of the separated protein is not high. The second is the chromatography method, including molecular sieve, ion exchange, affinity chromatography, hydrophobic chromatography and other methods, among which molecular sieve chromatography is also called gel filtration chromatography or size exclusion chromatography. Molecular sieve chromatography is a chromatography technique that uses a porous gel with a certain pore size range as a stationary phase to separate the components in a mixture according to their molecular size; the ion exchange method is based on the principle that when the pH of the solution is appropriate, the target protein has a negative (positive) charge, so it can have a strong binding with the anion (cation) ion exchange medium, while the impurities flow through, and the target protein on the column is removed with a high salt buffer; the affinity purification method is based on the principle that an appropriate ligand is immobilized on a chromatographic matrix to synthesize an affinity medium so that it specifically binds to the target protein; the hydrophobic chromatography method is based on the principle that some proteins in the impurities have strong hydrophobicity, but will agglutinate under high salt conditions and cannot be attached to the hydrophobic chromatography column. Therefore, a hydrophobic medium that can bind to the target protein can be selected to remove this part of the protein. The advantage of the chromatography method is that it can effectively remove impurities, the purity of the target protein is high, the efficiency is high, and it is suitable for large-scale production and use. The disadvantage is that in the process of removing impurities, specific equipment and fillers are required, and the cost of use is high. At the same time, it is also necessary to explore suitable purification methods based on the characteristics of the purified target protein.

[0005] There are several methods for removing endotoxins: 1. Liquid phase separation method, the principle is to use Triton X-114 to bind to the lipid part of endotoxins, and extract endotoxins multiple times (2-3 times) through liquid phase separation to effectively remove the latter; this method is simple to operate and has low cost; the disadvantage is that multiple centrifuges are required in large-scale production, and the time is long and the efficiency is low. 2. Ultrafiltration method, according to the difference in molecular weight between the target protein and endotoxin, select an ultrafiltration membrane with a pore size larger than the target protein as the first ultrafiltration, intercept endotoxins, collect the target protein, and then select an ultrafiltration membrane with a pore size smaller than the target protein as the second ultrafiltration, intercept the target protein, and remove endotoxins; this method requires the selection of a suitable ultrafiltration membrane, and at the same time has extremely high requirements for the viscosity of the protein solution. If the viscosity is too high, the protein is easy to adhere to the ultrafiltration membrane, blocking the ultrafiltration membrane, affecting the ultrafiltration effect, and the protein loss is high. At present, this method is less used in the removal of endotoxins from Escherichia coli. 3. Chromatography. Refer to the analysis of "chromatography" in the above "method for removing impure proteins". You can choose a suitable chromatography method to remove endotoxins based on their negative charge and hydrophobic properties.

[0006] Purification of avian embryo-derived antigens is currently mainly based on centrifugation. Through continuous flow centrifugation equipment such as tubular centrifuges or butterfly centrifuges, impurities such as egg yolk and cell membrane in antigens can be removed. The efficiency is relatively high in large-scale production, but this method cannot effectively remove foreign proteins in the allantoic fluid. In addition, some manufacturers process antigens by filtration. Through multi-stage filtration or deep filtration, the purity of antigens can be significantly improved, but this method requires the investment of consumables and the purification cost is relatively high.

[0007] Therefore, providing a purification method suitable for preparing poultry multi-vaccine by E. coli expression process is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0008] In view of this, the present invention provides a purification method suitable for preparing poultry multi-vaccines using Escherichia coli expression technology, which can be used to purify recombinant proteins or chicken embryo-derived antigens; it is a method for purifying Escherichia coli-derived recombinant proteins and avian embryo-derived antigens that is efficient, low-cost, and easy to operate in large-scale factory production.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] A purification method for preparing a multi-combination vaccine for poultry using an Escherichia coli expression process comprises the following steps:

[0011] 1) The E. coli cells collected by centrifugation were re-dissolved in PBS solution at a ratio of 1: (5-10), with the pH value of the PBS solution being 3-4; high-pressure homogenization was performed 1-3 times, and the transmittance OD was measured. 6000.1~0.5;

[0012] 2) Centrifuge the homogenized bacterial solution at 7000-12000 r / min for 20-40 min and collect the supernatant;

[0013] 3) adding ammonium sulfate to the supernatant prepared in step 2) at a ratio of 8 to 12% w / v, shaking well and placing at 2 to 8° C. overnight, then centrifuging at 7000 to 12000 r / min for 20 to 40 min, and collecting the supernatant;

[0014] 4) adding ammonium sulfate to the supernatant prepared in step 3) at a ratio of 13-18% w / v, shaking well and placing at 2-8° C. overnight, then centrifuging at 7000-12000 r / min for 20-40 min to collect the precipitate;

[0015] 5) The target protein precipitate collected after the previous pretreatment was redissolved in a PBS solution with a pH value of 3 to 4 at a ratio of 1: (5 to 10), and after it was dissolved, 0.1 to 0.3% w / v EDTA was added, and after it was completely dissolved, it was placed at 2 to 8°C overnight; then centrifuged at 7000 to 12000 r / min for 20 to 40 minutes, and the supernatant was collected;

[0016] 6) adding 0.1-0.5% w / v chitosan to the supernatant collected in step 5) to completely dissolve it, and leaving it at 2-8°C overnight; then centrifuging at 7000-12000 r / min for 20-40 min, and collecting the supernatant;

[0017] 7) Add 1% to 2% v / v Triton X-114 to the supernatant collected in step 6), shake well, and place at 2 to 8°C overnight; take out the supernatant on the next day, place in a 20 to 30°C water bath for 1 to 2 hours, then set the centrifugation temperature to 24 to 26°C, centrifuge at 7000 to 12000 r / min for 20 to 40 minutes, and collect the supernatant protein solution; the endotoxin of the target protein solution is less than 500 EU / ml;

[0018] 8) Add 0.1-0.3% v / v formaldehyde solution to the supernatant protein solution collected in step 7), the concentration of the formaldehyde solution is 35-40%, shake well and let stand at 2-8° C. for 3-7 days, then filter through 10 μm and 1 μm filters in sequence to collect the filtrate; the purpose of this step is to remove impurities.

[0019] Furthermore, the purification method suitable for preparing poultry multi-vaccine by E. coli expression process is used in purifying E. coli recombinant protein.

[0020] Furthermore, the purification method suitable for preparing poultry multi-vaccine by Escherichia coli expression process is used in removing endotoxin.

[0021] Furthermore, the purification method suitable for preparing poultry multi-vaccine by E. coli expression process is used in purifying antigens in poultry subunit vaccines expressed by E. coli.

[0022] Furthermore, a method for purifying an avian embryo-derived antigen solution comprises the following specific steps:

[0023] (1) If the antigen is mixed with the filtrate obtained in step 8) to prepare a combined vaccine, the antigen can be mixed and stored at 2-8°C overnight, and then the antigen is centrifuged in a tubular centrifuge or a butterfly centrifuge at a speed of 5000-20000 r / min, and the antigen clarity is increased by more than 70%;

[0024] (2) For preparing pure avian embryo-derived vaccine, add 0.01-0.1% w / v EDTA, then add 0.05-0.1% w / v chitosan, stir evenly to dissolve it completely, incubate it at 2-8°C overnight, and then centrifuge the antigen in a tubular centrifuge or butterfly centrifuge at a speed of 5000-20000 r / min. The antigen clarity is increased by more than 70%.

[0025] In the previous study, the inventors of the present invention used the ammonium sulfate method combined with the liquid phase separation method to remove endotoxins, but the endotoxins could only be removed to about 3000EU / ml. It is speculated that the main reason should be that the small molecule endotoxins produced by homogenization are difficult to be removed by centrifugation. Therefore, chitosan is added to make it positively charged in an acidic environment, and the negatively charged endotoxins are adsorbed to form macromolecules, which are formed under the action of EDTA to form nanoparticles, and then removed by high-speed centrifugation; in addition, endotoxins can neutralize the negative charge on the surface under acidic conditions, reduce the rejection reaction, and easily form precipitation; based on the above two considerations, the existing process is optimized.

[0026] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a purification method suitable for preparing poultry multi-unit vaccines by E. coli expression process, creatively combining and improving various methods such as molecular adsorption precipitation method, isoelectric point precipitation method, salting-out method and liquid phase separation method, which have not been reported at home and abroad. The new method for removing endotoxins shown by the present invention is effective. In addition, a part of the protein is flocculated after formaldehyde inactivation, and after being removed by filtration, the content of foreign proteins can be significantly reduced. After being treated by the method of the present invention, the content of endotoxins in the final recombinant protein solution is no more than 500EU / mL, and the purity of the recombinant protein reaches more than 60%. In addition, a method for removing foreign proteins in avian embryo antigen solution was found during the research process, which provides a new method for purifying poultry vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The accompanying drawings are safety test results of the present invention;

[0029] Among them, A: control group; B: experimental group. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] The present invention selects Escherichia coli to express the chicken infectious bursal disease VP2 protein vector (refer to Li Tiantian, Jiang Dawei, Ji Pengchao, et al. Prokaryotic expression of infectious bursal disease virus VP2 protein and evaluation of its immune effect [J]. Henan Agricultural Science, 2020, 49(09): 136-142. DOI: 10.15933 / j.cnki.

[0033] 1004-3268.2020.09.017.), after fermentation, the cells were collected by centrifugation and processed as follows:

[0034] 1) The E. coli cells collected by centrifugation were re-dissolved in PBS solution at a ratio of 1:10, and the pH value of the PBS solution was 3.0; high-pressure homogenization was performed for 3 times, and the transmittance OD was measured. 600 is 0.4.

[0035] 2) Centrifuge the homogenized bacterial solution at 8000 r / min for 20 min and collect the supernatant.

[0036] 3) Add ammonium sulfate to the supernatant prepared in step 2) at a ratio of 8% w / v, shake well and place at 2-8° C. overnight, then centrifuge at 8000 rpm for 20 min to collect the supernatant.

[0037] 4) Add ammonium sulfate to the supernatant prepared in step 3) at a ratio of 13% w / v, shake well and place at 2-8°C overnight, then centrifuge at 8000 rpm for 20 min to collect the precipitate.

[0038] 5) The target protein precipitate collected after the previous pretreatment was re-dissolved in a PBS solution with a pH value of 3.0 at a ratio of 1:10. After it was dissolved, 0.1% w / v EDTA was added and allowed to dissolve completely. The precipitate was placed at 2-8°C overnight. The supernatant was then collected by centrifugation at 8000 r / min for 20 min.

[0039] 6) Add 0.2% w / v chitosan to the supernatant collected in step 5) to completely dissolve it, and place it at 2-8°C overnight; then centrifuge at 8000 r / min for 20 min and collect the supernatant.

[0040] 7) Add 2% v / v TritonX-114 to the supernatant collected in step 6), shake well, and place at 2-8°C overnight; take out the next day, place in a 25°C water bath for 1 hour, then set the centrifugation temperature to 26°C, centrifuge at 12000r / min for 40 minutes, and collect the supernatant protein solution. The endotoxin content of the target protein solution is 50-100EU / ml.

[0041] 8) Add 0.1% v / v formaldehyde solution to the supernatant protein solution collected in step 7), the concentration of the formaldehyde solution is 35-40%, shake well and let stand at 2-8°C for 3 days, then filter through 10μm and 1μm filters in turn to collect VP2 protein filtrate. The target protein content is 3.5g / L by BCA method and SDS-PAGE electrophoresis, and the protein purity is 76.8% by SDS-PAGE electrophoresis grayscale analysis.

[0042] Comparative Example 1

[0043] This case is a comparative example of Example 1 and is processed as follows:

[0044] 1) The E. coli cells collected by centrifugation were re-dissolved in PBS solution at a ratio of 1:10, and the pH value of the PBS solution was 7.0; high-pressure homogenization was performed for 3 times, and the transmittance OD was measured. 600 is 0.3.

[0045] 2) Add ammonium sulfate to the solution after high pressure homogenization in step 1), the addition ratio of ammonium sulfate to the supernatant is 8% w / v, shake well and place at 2-8° C. overnight, then centrifuge at 8000 r / min for 20 min, and collect the supernatant.

[0046] 3) Add ammonium sulfate to the supernatant prepared in step 2) at a ratio of 13% w / v, shake well and place at 2-8° C. overnight, then centrifuge at 8000 rpm for 20 min to collect the precipitate.

[0047] 4) The target protein precipitate collected after the pretreatment was re-dissolved in a PBS solution with a pH value of 7.0 at a ratio of 1:10. After it was dissolved, 2% v / v Triton X-114 was added, shaken well, and placed at 2-8°C overnight. The next day, it was taken out, placed in a 25°C water bath for 1 hour, and then the centrifugation temperature was set to 26°C, and centrifuged at 12000r / min for 40 minutes to collect the supernatant protein solution. This treatment was repeated 3 times, and the endotoxin content of the target protein solution was 3500-4000EU / ml.

[0048] 5) Add 0.1% v / v formaldehyde solution to the protein solution collected in step 4), the concentration of the formaldehyde solution is 35-40%, shake well and let stand at 2-8°C for 3 days. The content of the target protein detected by BCA method and SDS-PAGE electrophoresis is 3.3g / L, and the protein purity detected by SDS-PAGE electrophoresis grayscale analysis is 30.5%.

[0049] Example 2

[0050] The present invention selects Escherichia coli to express the chicken infectious bursal disease VP2 protein vector (refer to Li Tiantian, Jiang Dawei, Ji Pengchao, et al. Prokaryotic expression of infectious bursal disease virus VP2 protein and evaluation of its immune effect [J]. Henan Agricultural Science, 2020, 49(09): 136-142. DOI: 10.15933 / j.cnki.

[0051] 1004-3268.2020.09.017.), after fermentation, the cells were collected by centrifugation and processed as follows:

[0052] 1) The E. coli cells collected by centrifugation were re-dissolved in PBS solution at a ratio of 1:5, and the pH value of the PBS solution was 4.0; high-pressure homogenization was performed once, and the transmittance OD was measured. 600 is 0.4.

[0053] 2) Centrifuge the homogenized bacterial solution at 12000r / min for 40min and collect the supernatant.

[0054] 3) Add ammonium sulfate to the supernatant prepared in step 2) at a ratio of 12% w / v, shake well and place at 2-8° C. overnight, then centrifuge at 12000 rpm for 40 min to collect the supernatant.

[0055] 4) Add ammonium sulfate to the supernatant prepared in step 3) at a ratio of 18% w / v, shake well and place at 2-8°C overnight, then centrifuge at 12000 r / min for 40 min to collect the precipitate.

[0056] 5) The target protein precipitate collected after the previous pretreatment was re-dissolved in a PBS solution with a pH value of 4.0 at a ratio of 1:5. After it was dissolved, 0.3% w / v EDTA was added and allowed to dissolve completely. The mixture was placed at 2-8°C overnight. The mixture was then centrifuged at 12000 r / min for 40 min and the supernatant was collected.

[0057] 6) Add 0.5% w / v chitosan to the supernatant collected in step 5) to completely dissolve it, and place it at 2-8°C overnight; then centrifuge at 12000 r / min for 40 min and collect the supernatant.

[0058] 7) Add 1% v / v TritonX-114 to the supernatant collected in step 6), shake well, and place at 2-8°C overnight; take out the next day, place in a 25°C water bath for 1 hour, then set the centrifugation temperature to 24°C, centrifuge at 7000r / min for 40 minutes, and collect the supernatant protein solution. The endotoxin content of the target protein solution is 50-100EU / ml.

[0059] 8) Add 0.3% v / v formaldehyde solution to the supernatant protein solution collected in step 7), the concentration of the formaldehyde solution is 35-40%, shake well and let stand at 2-8°C for 7 days, then filter through 10μm and 1μm filters in turn to collect the filtrate. The BCA method and SDS-PAGE electrophoresis were used to detect the target protein content of 3.1g / L, and the protein purity was 78.5% by SDS-PAGE electrophoresis grayscale analysis.

[0060] Example 3

[0061] The present invention selects Escherichia coli to express the chicken infectious bursal disease VP2 protein vector (refer to Li Tiantian, Jiang Dawei, Ji Pengchao, et al. Prokaryotic expression of infectious bursal disease virus VP2 protein and evaluation of its immune effect [J]. Henan Agricultural Science, 2020, 49(09): 136-142. DOI: 10.15933 / j.cnki.

[0062] 1004-3268.2020.09.017.), after fermentation, the cells were collected by centrifugation and processed as follows:

[0063] 1) The E. coli cells collected by centrifugation were re-dissolved in PBS solution at a ratio of 1:8, and the pH value of the PBS solution was 3.5; high-pressure homogenization was performed twice, and the transmittance OD was measured. 600 is 0.4.

[0064] 2) Centrifuge the homogenized bacterial solution at 10,000 r / min for 30 min and collect the supernatant.

[0065] 3) Add ammonium sulfate to the supernatant prepared in step 2) at a ratio of 10% w / v, shake well and place at 2-8°C overnight, then centrifuge at 10,000 rpm for 30 min to collect the supernatant.

[0066] 4) Add ammonium sulfate to the supernatant prepared in step 3) at a ratio of 15% w / v, shake well and place at 2-8°C overnight, then centrifuge at 10,000 rpm for 30 min to collect the precipitate.

[0067] 5) The target protein precipitate collected after the previous pretreatment was re-dissolved in a PBS solution with a pH value of 3.5 at a ratio of 1:8. After it was dissolved, 0.2% w / v EDTA was added and allowed to dissolve completely. The mixture was placed at 2-8°C overnight. The mixture was then centrifuged at 10,000 r / min for 30 min and the supernatant was collected.

[0068] 6) Add 0.3% w / v chitosan to the supernatant collected in step 5) to completely dissolve it, and place it at 2-8°C overnight; then centrifuge at 10000 r / min for 30 min and collect the supernatant.

[0069] 7) Add 1.5% v / v TritonX-114 to the supernatant collected in step 6), shake well, and place at 2-8°C overnight; take out the next day, place in a 25°C water bath for 1 hour, then set the centrifugation temperature to 25°C, centrifuge at 10,000 r / min for 30 minutes, and collect the supernatant protein solution. The endotoxin content of the target protein solution is 50-100 EU / ml.

[0070] 8) Add 0.2% v / v formaldehyde solution to the supernatant protein solution collected in step 7), the concentration of the formaldehyde solution is 35-40%, shake well and let stand at 2-8°C for 5 days, then filter through 10μm and 1μm filters in turn to collect the filtrate. The BCA method and SDS-PAGE electrophoresis were used to detect the target protein content of 2.9g / L, and the protein purity was 77.5% by SDS-PAGE electrophoresis grayscale analysis.

[0071] Example 4

[0072] The present invention selects Escherichia coli to express avian adenovirus fiber-2 protein vector (refer to Du Dongying, Huo Huanyan, Tian Hui, et al. Comparison of immune effects of group I avian adenovirus (type 4) fiber-2 protein subunit vaccine and whole virus inactivated vaccine [J]. Chinese Journal of Zoonotic Infectious Diseases, 2023, 31(03): 67-73. DOI: 10.19958 / j.cnki.cn31-2031 / s.20230324.002.), after fermentation and culture, the bacteria are collected by centrifugation and processed as follows:

[0073] 1) The E. coli cells collected by centrifugation were re-dissolved in PBS solution at a ratio of 1:10, and the pH value of the PBS solution was 3.1; high-pressure homogenization was performed for 3 times, and the transmittance OD was measured. 600 is 0.3.

[0074] 2) Centrifuge the homogenized bacterial solution at 12000r / min for 20min and collect the supernatant.

[0075] 3) Add ammonium sulfate to the supernatant prepared in step 2) at a ratio of 8% w / v, shake well and place at 2-8° C. overnight, then centrifuge at 8000 rpm for 40 min to collect the supernatant.

[0076] 4) Add ammonium sulfate to the supernatant prepared in step 3) at a ratio of 13% w / v, shake well and place at 2-8°C overnight, then centrifuge at 8000 rpm for 40 min to collect the precipitate.

[0077] 5) The target protein precipitate collected after the previous pretreatment was re-dissolved in a PBS solution with a pH value of 3.1 at a ratio of 1:10. After it was dissolved, 0.2% w / v EDTA was added and allowed to dissolve completely. The mixture was placed at 2-8°C overnight. The mixture was then centrifuged at 8000 r / min for 30 min and the supernatant was collected.

[0078] 6) Add 0.1% w / v chitosan to the supernatant collected in step 5) to completely dissolve it, and place it at 2-8°C overnight. Then centrifuge at 8000 r / min for 40 minutes and collect the supernatant.

[0079] 7) Add 2% v / v TritonX-114 to the supernatant collected in step 6), shake well, and place at 2-8°C overnight. Take out the next day, place in a 25°C water bath for 1 hour, then set the centrifuge temperature to 25°C, centrifuge at 12000r / min for 20 minutes, and collect the supernatant protein solution. The endotoxin of the target protein solution is 100-200EU / ml.

[0080] 8) Add 0.1% v / v formaldehyde solution to the supernatant protein solution collected in step 7), the concentration of the formaldehyde solution is 35-40%, shake well and let stand at 2-8°C for 3 days, then filter through 10μm and 1μm filters in turn to collect the filtrate. The BCA method and SDS-PAGE electrophoresis were used to detect the target protein content of 2.6g / L, and the protein purity was 63% by SDS-PAGE electrophoresis grayscale analysis.

[0081] Comparative Example 2

[0082] This case is a comparative example of Example 3 and is processed as follows:

[0083] 1) The E. coli cells collected by centrifugation were re-dissolved in PBS solution at a ratio of 1:10, and the pH value of the PBS solution was 7.0; high-pressure homogenization was performed for 3 times, and the transmittance OD was measured. 600 is 0.4.

[0084] 2) Add ammonium sulfate to the solution homogenized under high pressure in step 1) at a ratio of 8% w / v in the supernatant, shake well and place at 2-8° C. overnight, then centrifuge at 8000 rpm for 40 min to collect the supernatant.

[0085] 3) Add ammonium sulfate to the supernatant prepared in step 2) at a ratio of 13% w / v, shake well and place at 2-8° C. overnight, then centrifuge at 8000 rpm for 40 min to collect the precipitate.

[0086] 4) The target protein precipitate collected after the pretreatment was re-dissolved in a PBS solution with a pH value of 7.0 at a ratio of 1:10. After it was dissolved, 2% v / v Triton X-114 was added and placed at 2-8°C overnight. The next day, it was taken out, placed in a 25°C water bath for 1 hour, and then the centrifugation temperature was set to 25°C, and centrifuged at 12000r / min for 20 minutes to collect the supernatant protein solution. This treatment was repeated 3 times, and the endotoxin content of the target protein solution was 3000-3500EU / ml.

[0087] 5) Add 0.1% v / v formaldehyde solution to the supernatant protein solution collected in step 4), the concentration of the formaldehyde solution is 35-40%, shake well and let stand at 2-8°C for 3 days. The target protein content detected by BCA method and SDS-PAGE electrophoresis is 2.1g / L, and the protein purity detected by SDS-PAGE electrophoresis grayscale analysis is 28.5%.

[0088] Example 5

[0089] The present invention selects Escherichia coli to express avian adenovirus fiber-2 protein vector (refer to Du Dongying, Huo Huanyan, Tian Hui, et al. Comparison of immune effects of group I avian adenovirus (type 4) fiber-2 protein subunit vaccine and whole virus inactivated vaccine [J]. Chinese Journal of Zoonotic Infectious Diseases, 2023, 31(03): 67-73. DOI: 10.19958 / j.cnki.cn31-2031 / s.20230324.002.), after fermentation and culture, the bacteria are collected by centrifugation and processed as follows:

[0090] 1) The E. coli cells collected by centrifugation were re-dissolved in PBS solution at a ratio of 1:5, and the pH value of the PBS solution was 4.0; high-pressure homogenization was performed once, and the transmittance OD was measured. 600 is 0.4.

[0091] 2) Centrifuge the homogenized bacterial solution at 7000 r / min for 40 min and collect the supernatant.

[0092] 3) Add ammonium sulfate to the supernatant prepared in step 2) at a ratio of 12% w / v, shake well and place at 2-8° C. overnight, then centrifuge at 12000 rpm for 20 min to collect the supernatant.

[0093] 4) Add ammonium sulfate to the supernatant prepared in step 3) at a ratio of 18% w / v, shake well and place at 2-8°C overnight, then centrifuge at 12000 r / min for 20 min to collect the precipitate.

[0094] 5) The target protein precipitate collected after the previous pretreatment was re-dissolved in a PBS solution with a pH value of 4.0 at a ratio of 1:10. After it was dissolved, 0.3% w / v EDTA was added and allowed to dissolve completely. The mixture was placed at 2-8°C overnight. The mixture was then centrifuged at 12000 r / min for 40 min and the supernatant was collected.

[0095] 6) Add 0.5% w / v chitosan to the supernatant collected in step 5) to completely dissolve it, and place it at 2-8°C overnight. Then centrifuge at 12000 r / min for 20 min and collect the supernatant.

[0096] 7) Add 1% v / v TritonX-114 to the supernatant collected in step 6), shake well, and place at 2-8°C overnight. Take out the next day, place in a 30°C water bath for 1 hour, then set the centrifugation temperature to 24°C, centrifuge at 7000r / min for 40 minutes, and collect the supernatant protein solution. The endotoxin of the target protein solution is 100-200EU / ml.

[0097] 8) Add 0.3% v / v formaldehyde solution to the supernatant protein solution collected in step 7), the concentration of the formaldehyde solution is 35-40%, shake well and let stand at 2-8°C for 7 days, then filter through 10μm and 1μm filters in turn to collect the filtrate. The BCA method and SDS-PAGE electrophoresis were used to detect the target protein content of 2.2g / L, and the protein purity was 67% by SDS-PAGE electrophoresis grayscale analysis.

[0098] Example 6

[0099] The present invention selects to use chicken Newcastle disease virus liquid cultured with avian embryos, inactivates it with formaldehyde, and then purifies it, and the method is as follows:

[0100] Take the chicken Newcastle disease virus inactivated solution and add 0.1% w / v EDTA and 0.1% w / v chitosan. After dissolving, store at 2-8℃ overnight, then centrifuge at 8000r / min for 30min, and take the supernatant to detect OD 600 The OD of the untreated Newcastle disease virus inactivated solution was 0.215. 600 It is 0.876, and the clarity is increased by 75%.

[0101] Example 7

[0102] The present invention selects H9 subtype avian influenza virus liquid cultured with avian embryos, inactivates it with formaldehyde, and then purifies it in the following manner:

[0103] Take the H9 subtype avian influenza virus inactivated solution and add 0.1% w / v EDTA and 0.05% w / v chitosan. After dissolving, store at 2-8°C overnight, then centrifuge at 8000r / min for 30min, and take the supernatant to detect OD 600 The OD of the untreated H9 subtype virus inactivated solution was 0.245. 600 The value is 0.835, and the clarity is increased by 71%.

[0104] Example 8

[0105] The inactivated Newcastle disease virus solution, the inactivated H9 subtype avian influenza virus solution and the filtrate of the infectious bursal disease virus VP2 protein collected after the treatment in Example 1 were mixed in a ratio of 2:2:1; the mixture was centrifuged at 2-8°C overnight, and the supernatant was collected and the absorbance value (OD 600 ) was 0.264, and the absorbance before centrifugation (OD 600 ) is 1.232, and the clarity is improved by 79%.

[0106] Example 9

[0107] Take 96 parts of the supernatant treated by centrifugation after mixing the inactivated Newcastle disease virus liquid, the H9 subtype avian influenza virus inactivated liquid, and the infectious bursal disease virus VP2 protein filtrate treated in Example 8, add 4 parts of sterilized Tween-80, shake thoroughly, and use it as the water phase after the Tween-80 is completely dissolved; take 94 parts of white oil for injection, add 2% aluminum stearate, heat and dissolve, add 6 parts of SIBEN-80, mix, and sterilize under high pressure as the oil phase; take the water phase and the oil phase and mix and emulsify them in a ratio of 1:2 to prepare an oil emulsion vaccine (hereinafter referred to as "New Flow Method") as the experimental group; at the same time, select the untreated mixed solution as the control group, prepare the oil emulsion vaccine according to the same water-oil ratio, and immunize SPF chickens respectively for safety and efficacy tests. And compare the vaccine absorption. The results are as follows:

[0108] 1) Safety test: 20 SPF chickens aged 21 days were selected and randomly divided into two groups, with 10 chickens in each group. The experimental group and the control group were immunized with the new flu vaccine respectively. Each chicken was injected intramuscularly with 1 ml. An autopsy was performed 14 days after immunization to observe the vaccine absorption.

[0109] Results Figure 1 : No systemic or local adverse reactions occurred in both groups. There were obvious differences in the muscle injection sites during autopsy. All 5 injection sites in the control group had a small amount of inflammatory reaction, while only 1 injection site in the experimental group had a small amount of inflammatory reaction.

[0110] 2) Effectiveness test:

[0111] (1) Newcastle disease

[0112] 25 21-day-old SPF chickens were used, 10 in each experimental group and vaccine control group were immunized, 20 μl of vaccine was injected subcutaneously, and the other 5 were used as blank controls. 21 days after immunization, blood was collected from each chicken, serum was separated, and HI antibody titer was determined according to the appendix of the current "Chinese Veterinary Pharmacopoeia". The results are shown in Table 1, showing that there was no significant difference in antibody levels.

[0113] Table 1

[0114]

[0115] (2) H9 subtype avian influenza

[0116] 25 21-day-old SPF chickens were used, 10 in each experimental group and vaccine control group were immunized, 0.3 ml of vaccine was injected subcutaneously, and the other 5 were used as blank controls. 21 days after immunization, blood was collected from each chicken, serum was separated, and HI antibody titer was determined according to the appendix of the current "Chinese Veterinary Pharmacopoeia". The results are shown in Table 2, showing that there was no significant difference in antibody levels.

[0117] Table 2

[0118]

[0119] (3) Infectious bursal disease

[0120] 25 21-day-old SPF chickens were used, 10 in each experimental group and vaccine control group were immunized, 0.3 ml of vaccine was injected subcutaneously, and the other 5 were used as blank controls. 21 days after immunization, blood was collected from each chicken, serum was separated, and agar immunodiffusion test was performed. The results are shown in Table 3, showing that there was no significant difference in antibody levels.

[0121] Table 3

[0122]

[0123] Conclusion: Through safety experiment comparison, the experimental group can obviously reduce the inflammatory response of the vaccine, while the control group has high endotoxin and foreign protein, and there is an inflammatory response after immunization, which will clinically affect the quality of meat ketones. The present invention indirectly solves the clinical poor absorption and inflammatory response of the vaccine by solving the foreign protein and endotoxin residues in the vaccine, thereby ensuring the safety and quality of meat products.

[0124] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A purification method suitable for preparing poultry multi-combination vaccines using E. coli expression technology, characterized in that: The following steps are involved: 1) The E. coli cells collected by centrifugation were re-dissolved in PBS solution at a ratio of 1: (5-10), with the pH value of the PBS solution being 3-4; high-pressure homogenization was performed 1-3 times, and the transmittance OD was measured. 600 0.1~0.5; 2) Centrifuge the homogenized bacterial solution at 7000-12000 r / min for 20-40 min and collect the supernatant; 3) adding ammonium sulfate to the supernatant prepared in step 2) at a ratio of 8 to 12% w / v, shaking well and placing at 2 to 8° C. overnight, then centrifuging at 7000 to 12000 r / min for 20 to 40 min, and collecting the supernatant; 4) adding ammonium sulfate to the supernatant prepared in step 3) at a ratio of 13-18% w / v, shaking well and placing at 2-8° C. overnight, then centrifuging at 7000-12000 r / min for 20-40 min to collect the precipitate; 5) The target protein precipitate collected after the previous pretreatment was redissolved in a PBS solution with a pH value of 3 to 4 at a ratio of 1: (5 to 10), and after it was dissolved, 0.1 to 0.3% w / v EDTA was added, and after it was completely dissolved, it was placed at 2 to 8°C overnight; then centrifuged at 7000 to 12000 r / min for 20 to 40 minutes, and the supernatant was collected; 6) adding 0.1-0.5% w / v chitosan to the supernatant collected in step 5) to completely dissolve it, and leaving it at 2-8°C overnight; then centrifuging at 7000-12000 r / min for 20-40 min, and collecting the supernatant; 7) Add 1% to 2% v / v Triton X-114 to the supernatant collected in step 6), shake well, and place at 2 to 8°C overnight; take out the supernatant the next day, place in a 20 to 30°C water bath for 1 to 2 hours, then set the centrifugation temperature to 24 to 26°C, centrifuge at 7000 to 12000 r / min for 20 to 40 minutes, and collect the supernatant protein solution; The endotoxin content of the target protein solution is less than 500EU / ml; 8) Add 0.1-0.3% v / v formaldehyde solution to the supernatant protein solution collected in step 7), the concentration of the formaldehyde solution is 35-40%, shake well and let stand at 2-8° C. for 3-7 days, then filter through 10 μm and 1 μm filters in sequence to collect the filtrate.

2. Use of the purification method according to claim 1, which is suitable for preparing poultry multi-vaccine by E. coli expression process, in purifying E. coli recombinant proteins.

3. Use of the purification method for preparing poultry multi-vaccine by E. coli expression process as described in claim 1 in removing endotoxin.

4. Use of the purification method for preparing poultry multi-unit vaccines by E. coli expression process as described in claim 1 in purifying antigens in poultry subunit vaccines expressed by E. coli.

5. A method for purifying an avian embryo-derived antigen solution, characterized in that: The specific steps are as follows: (1) If the antigen is mixed with the filtrate obtained in step 8) to prepare a combined vaccine, the antigen can be mixed and stored at 2-8°C overnight, and then the antigen is centrifuged in a tubular centrifuge or a butterfly centrifuge at a speed of 5000-20000 r / min, and the antigen clarity is increased by more than 70%; (2) For preparing pure avian embryo-derived vaccine, add 0.01-0.1% w / v EDTA, then add 0.05-0.1% w / v chitosan, stir evenly to dissolve it completely, incubate it at 2-8°C overnight, and then centrifuge the antigen in a tubular centrifuge or butterfly centrifuge at a speed of 5000-20000 r / min. The antigen clarity is increased by more than 70%.

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