CN110981946A

CN110981946A - Solution for large-scale production of foot-and-mouth disease virus-like particle antigen and purification and assembly method

Info

Publication number: CN110981946A
Application number: CN201911397713.5A
Authority: CN
Inventors: 李峰; 于作; 邱文英; 李妍; 谢建勇; 吴越; 龚文波; 方鹏飞; 严成; 何承勇; 庄万敏; 朱遥; 王俊; 朱旗; 严欢; 沈皓月; 李捷; 余谦
Original assignee: Huapai Bioengineering Group Co ltd
Current assignee: Huapai Bioengineering Group Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-04-10

Abstract

The invention discloses a solution for large-scale production of foot-and-mouth disease virus-like particle antigen and a purification and assembly method, which belong to the technical field of biological medicine. The method provided by the invention has the advantages of simple process, convenient operation and high production efficiency, the purification yield of the foot-and-mouth disease virus-like particles reaches more than 90%, the in vitro assembly rate reaches 67%, and the method is particularly suitable for industrial production of the foot-and-mouth disease virus-like particle antigen expressed by escherichia coli.

Description

Solution for large-scale production of foot-and-mouth disease virus-like particle antigen and purification and assembly method

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a solution for large-scale production of foot-and-mouth disease virus-like particle antigens and a purification and assembly method.

Background

Foot-and-Mouth Disease is an acute, highly contagious and rapidly long-distance transmissible animal Disease mainly caused by Foot-and-Mouth Disease Virus (FMDV), is a virulent infectious Disease which has the greatest threat to animal husbandry development in the world at present, is listed as an infectious Disease which needs to be reported by the world animal health Organization (OIE), and is determined as a Disease species for first-stage immune purification prevention and control in the national animal epidemic Disease prevention and control long-term development plan. At present, the foot-and-mouth disease is mainly immune controlled by using the whole virus inactivated vaccine at home and abroad, but the epidemic prevention effect is not satisfactory. The vaccine can not completely meet the requirement of thorough purification in the target of preventing and controlling animal epidemic diseases due to the defects of higher production cost, possible toxic substance dispersing biological safety problem caused by incomplete inactivation in the vaccine production process, long period of screening and matching vaccine strains and the like.

Virus-like particles (virus-like particles VLPs) are a genetic engineering subunit form which is newly appeared in recent years, and the advantages of the virus-like particles include no genetic material required by virus replication and excellent biological safety; can simulate the natural structure of the virus to ensure that the conformation dependent epitope is correctly presented; can be combined with cell surface receptors to enter cells like natural virus particles, thereby inducing stronger immune response; on the basis of not influencing the structure of VLPs, certain amino acid sequences can be inserted or deleted according to needs and artificially modified, so that the pleiotropic characteristic of simultaneous immunization of various viruses is realized, and the like. Thus, VLPs are considered to be the best vaccine candidate currently able to replace whole virus vaccines as vaccines.

In the prior art, the preparation method of the foot-and-mouth disease virus-like particles generally adopts a laboratory method for preparing the foot-and-mouth disease virus-like particles by a prokaryotic expression system, and comprises the steps of recombinant expression vector construction, protein expression and purification and in-vitro assembly. However, the method is not suitable for the requirement of large-scale production due to high production cost and low production efficiency.

Disclosure of Invention

The invention aims to: aiming at the defects in the prior art, the method provides a process method for purifying and in-vitro assembling the foot-and-mouth disease virus-like particle antigen which is suitable for large-scale production and has low cost, and the foot-and-mouth disease virus-like particles with high purity and high assembly rate are prepared by purifying the recombinant protein fermentation liquor of the foot-and-mouth disease virus expressed by escherichia coli.

The technical scheme adopted by the invention is as follows:

a solution for the large-scale production of foot-and-mouth disease virus-like particle antigens comprises the following components:

buffer a solution: 15-20mM sodium dihydrogen phosphate, 2-10mM disodium hydrogen phosphate, 100-; dissolving the components in purified water, adjusting pH to 7.5-8.0, and filtering with 0.22 μm filter;

buffer B solution: 15-20mM sodium dihydrogen phosphate, 2-10mmM mM disodium hydrogen phosphate, 100-500mM sodium chloride, 50-100mM imidazole; dissolving the components in purified water, adjusting pH to 7.5-8.0, and filtering with 0.22 μm filter;

buffer C solution: 15-20mM sodium dihydrogen phosphate, 2-10mmM disodium hydrogen phosphate, 100-500mM sodium chloride, 300-500mM imidazole; dissolving the components in purified water, adjusting pH to 7.5-8.5, and filtering with 0.22 μm filter;

buffer D solution: 10-50mM Tris-HCl, 200-500mM sodium chloride, 1-5mM calcium chloride, 3-10% glycerol; the components were dissolved in purified water, the pH was adjusted to 7.5-8.5, and filtration was performed using a 0.22 μm filter.

The Buffer A, the Buffer B and the Buffer C solutions are purified solutions, the ratio of the components is determined through experiments, and the finally obtained formula can maximize the purification yield of the foot-and-mouth disease virus-like particles to more than 90%;

the Buffer D solution is an in-vitro assembly solution, the proportion of each component is determined through tests, the formula is particularly suitable for assembling foot-and-mouth disease virus-like particles by membrane dialysis, the protein assembly rate is high (67%) and the properties are stable.

The solution for the large-scale production of the foot-and-mouth disease virus-like particle antigen is applied to the production of the virus particle antigen and the preparation of the virus-like particle vaccine.

The purification and assembly method for the foot-and-mouth disease virus-like particle antigen large-scale production by adopting the solution comprises the following steps:

s1, carrying out continuous flow centrifugation on recombinant protein fermentation liquor of foot-and-mouth disease virus expressed by escherichia coli to collect thalli, carrying out heavy suspension on the thalli by using Buffer A solution, homogenizing and crushing the thalli, filtering, and collecting filtered clear liquid;

s2, carrying out column balance on the clear liquid obtained in the step S1 by using a Buffer A solution, then loading, washing the column by using a Buffer B solution, finally eluting by using a Buffer C solution, and collecting target protein;

and S3, adding small ubiquitin-like modified protease into the target protein obtained in the step S2, performing enzyme digestion, performing circulating dialysis by using a buffer D solution, and collecting a concentrated solution at a backflow end to obtain the protein.

Further, continuous flow centrifugation was performed at 9000r/min in the step of S1; collecting the thallus, then suspending the thallus by using Buffer A, homogenizing and crushing the thallus by using a high-pressure homogenizer at 800-1100bar until the bacteria liquid is uniform, and then filtering by using a 100-300K membrane package.

Further, in the step S2, 5-10 column volumes are balanced by Buffer A for the Ni-NTA agarose resin column, and the balanced flow rate is 200-; the sample loading flow rate is 200-1000 cm/h; after the sample loading is finished, washing 5-10 column volumes by using Buffer B, wherein the washing flow rate is 200-1000 cm/h; the elution flow rate was 200-1000 cm/h. The liquid flow in the pipe is downward from the top of the resin column.

Further, the mass ratio of the target protein to the small ubiquitin-like modified protease in the step S3 is 80-100: 1.

Further, S3 is carried out by shaking enzyme digestion for 6-8h at 2-8 ℃.

Further, in the step S3, circulating dialysis is carried out for 45-50 hours at 2-8 ℃ by using an 8-20k membrane pack.

Further, the inlet speed in the circulating dialysis is 20-30L/h, and the permeation speed is 100-200 mL/h.

The foot-and-mouth disease virus-like particle antigen obtained by the purification and assembly method.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the invention provides a purification and assembly process method capable of producing foot-and-mouth disease virus-like particle antigen in large scale and a corresponding solution formula, which purifies and prepares high-purity foot-and-mouth disease virus-like particles with high assembly rate from foot-and-mouth disease virus protein fermentation liquor expressed by escherichia coli;

2. in the invention, the purification process comprises 4 links of balancing, loading, washing and eluting, compared with a laboratory method adopted in conventional production, the method has the advantages of simple operation, large handling capacity and short purification time, the conventional laboratory purification method generally adopts few Ni-NTA fillers (within 10 ml), and the handling capacity of feed liquid is at most dozens of milliliters; the purification method adopts 10-30L of Ni-NTA filler, the treatment capacity of the feed liquid can reach 100-200L, and the purification can be completed in one day, thereby laying a foundation for the subsequent assembly of the virus-like particles and the preparation of the virus-like particle vaccine;

3. in the invention, the assembly process comprises 2 links of enzyme digestion and assembly, wherein the membrane package is adopted for dialysis assembly, compared with a laboratory method adopted in conventional production, the treatment capacity is large, the time consumption is short, the conventional laboratory assembly method is generally carried out by adopting a dialysis bag, a large amount of assembly liquid needs to be slowly dialyzed at 4 ℃ overnight, the capacity of the dialysis bag is limited, and the protein treatment capacity is limited; the assembly method adopts membrane-packed dialysis assembly, the protein treatment capacity is 20-30L/h, the assembly can be completed within several hours, and the method is very suitable for the industrial production of the foot-and-mouth disease virus-like particle antigen;

4. the method of the invention has the advantages that the purification yield of the foot-and-mouth disease virus-like particles reaches more than 90 percent, the in vitro assembly rate reaches 67 percent, and the method is particularly suitable for the industrial production of the foot-and-mouth disease virus-like particle antigen expressed by escherichia coli;

5. the invention can be used for producing foot-and-mouth disease virus-like particle antigens expressed by escherichia coli in a large scale and can also be used for producing virus-like particle antigens expressed by the escherichia coli and other viruses.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph showing the results of protein purification according to the present invention;

FIG. 2 is a diagram showing the result of the enzyme digestion according to the present invention;

FIG. 3 is an electron micrograph of an in vitro assembled virus-like particle of type O;

FIG. 4 is a graph showing the particle size measurement of virus-like particles;

figure 5 is a sucrose density gradient centrifuge plot.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The purification and assembly method for the large-scale production of the foot-and-mouth disease virus-like particle antigen provided by the preferred embodiment of the invention comprises the following specific steps:

(1) preparing a solution: respectively preparing Buffer A, Buffer B, Buffer C and Buffer D solutions;

buffer a solution: contains 18mM sodium dihydrogen phosphate, 5mM disodium hydrogen phosphate, 300mM sodium chloride and 2% Trition-X100.

Buffer B solution: containing 18mM sodium dihydrogen phosphate, 5mmM mM disodium hydrogen phosphate, 300mM sodium chloride, 80mM imidazole.

Buffer C solution: containing 18mM sodium dihydrogen phosphate, 5mmM disodium hydrogen phosphate, 300mM sodium chloride, 400mM imidazole.

Buffer D solution: containing 30mM Tris-HCl, 300mM sodium chloride, 2mM calcium chloride, 6% glycerol.

(2) And (3) treating fermentation liquor: carrying out continuous flow centrifugation on foot-and-mouth disease virus recombinant protein fermentation liquor expressed by escherichia coli at 9000r/min to collect thalli, carrying out heavy suspension on the thalli by using Buffer A, and carrying out homogenization and crushing on the thalli for 2 times by using a high-pressure homogenizer at 1000bar until the bacterial liquid is uniform. Filtering, and collecting the filtrate.

(3) Affinity chromatography

1) Column balancing: the Ni-NTA agarose resin column was first equilibrated with Buffer A for 8 column volumes at an equilibrium flow rate of 800cm/h, with the liquid in the line flowing downward from the top of the resin column.

2) Loading: and (3) loading the treated fermentation liquor to a resin column, wherein the loading flow rate is 800cm/h, and the liquid in the pipeline flows downwards from the top of the resin column.

3) Washing: after the loading was complete, 8 column volumes were washed with Buffer B at a flow rate of 800cm/h, with the liquid flow being downward from the top of the resin column.

4) And (3) elution: eluting the target protein by using Buffer C, collecting an elution peak, wherein the elution flow rate is 800cm/h, and the liquid flow direction is downward from the top of the resin column.

(4) Digestion and assembly

1) Enzyme digestion: adding small ubiquitin-like modified protease into the collected target protein solution according to the mass ratio of the target protein solution to the small ubiquitin-like modified protease of 90:1, and carrying out enzyme digestion for 7 hours at 5 ℃ in a shaking table.

2) Assembling: and (3) carrying out circulating dialysis for 48 hours, wherein the dialysate is Buffer D solution, the inlet speed is set to be 25L/h, the permeation speed is set to be 150mL/h, and the whole process is at 5 ℃, and collecting concentrated solution at the backflow end to obtain the final product.

Example 2

buffer a solution: contains 15mM sodium dihydrogen phosphate, 2mM disodium hydrogen phosphate, 100mM sodium chloride and 1% Trition-X100.

Buffer B solution: containing 15mM sodium dihydrogen phosphate, 2mmM disodium hydrogen phosphate, 100mM sodium chloride, 50mM imidazole.

Buffer C solution: containing 15mM sodium dihydrogen phosphate, 2mmM disodium hydrogen phosphate, 100mM sodium chloride, 300mM imidazole.

Buffer D solution: containing 10mM Tris-HCl, 200mM sodium chloride, 1mM calcium chloride, 3% glycerol.

(2) And (3) treating fermentation liquor: carrying out continuous flow centrifugation on foot-and-mouth disease virus recombinant protein fermentation liquor expressed by escherichia coli at 9000r/min to collect thalli, carrying out heavy suspension on the thalli by using Buffer A, and carrying out homogenizing and crushing on the thalli for 2 times by using a high-pressure homogenizer at 800bar until the bacterial liquid is uniform. Filtering, and collecting the filtrate.

(3) Affinity chromatography

1) Column balancing: the Ni-NTA agarose resin column was first equilibrated with Buffer A for 5 column volumes at an equilibrium flow rate of 200cm/h, with the liquid in the line flowing downward from the top of the resin column.

2) Loading: and (3) loading the treated fermentation liquor to a resin column, wherein the loading flow rate is 200cm/h, and the liquid in the pipeline flows downwards from the top of the resin column.

3) Washing: after completion of the loading, the column was washed with Buffer B for 5 column volumes at a flow rate of 200cm/h and a liquid flow direction downward from the top of the resin column.

4) And (3) elution: eluting the target protein by using Buffer C, collecting an elution peak, wherein the elution flow rate is 200cm/h, and the liquid flow direction is downward from the top of the resin column.

(4) Digestion and assembly

1) Enzyme digestion: adding small ubiquitin-like modified protease into the collected target protein solution according to the mass ratio of the target protein solution to the small ubiquitin-like modified protease of 80:1, and carrying out enzyme digestion for 6 hours at 2 ℃ in a shaking table.

2) Assembling: and (4) carrying out circulating dialysis for 45 hours, wherein the dialysate is Buffer D solution, the inlet speed is set to be 20L/h, the permeation speed is set to be 100mL/h, and the whole process is at the temperature of 2 ℃, and collecting concentrated solution at the backflow end to obtain the final product.

Example 3

buffer a solution: contains 20mM sodium dihydrogen phosphate, 10mM disodium hydrogen phosphate, 500mM sodium chloride and 5% Trition-X100.

Buffer B solution: containing 20M sodium dihydrogen phosphate, 10mmM disodium hydrogen phosphate, 500mM sodium chloride, 100mM imidazole.

Buffer C solution: containing 20mM sodium dihydrogen phosphate, 10mM 10mmM disodium hydrogen phosphate, 500mM sodium chloride, 500mM imidazole.

Buffer D solution: containing 50mM Tris-HCl, 500mM sodium chloride, 5mM calcium chloride, 10% glycerol.

(2) And (3) treating fermentation liquor: carrying out continuous flow centrifugation on foot-and-mouth disease virus recombinant protein fermentation liquor expressed by escherichia coli at 9000r/min to collect thalli, carrying out heavy suspension on the thalli by using Buffer A, and carrying out homogenization and crushing on the thalli for 2 times by using a high-pressure homogenizer at 1100bar until the bacterial liquid is uniform. Filtering, and collecting the filtrate.

(3) Affinity chromatography

1) Column balancing: the Ni-NTA agarose resin column was first equilibrated with Buffer A for 10 column volumes at an equilibrium flow rate of 1000cm/h, with the liquid in the line flowing downward from the top of the resin column.

2) Loading: and (3) loading the treated fermentation liquor into the resin column, wherein the loading flow rate is 1000cm/h, and the liquid in the pipeline flows downwards from the top of the resin column.

3) Washing: after completion of the loading, 10 column volumes were washed with Buffer B at a flow rate of 1000cm/h and a liquid flow direction was downward from the top of the resin column.

4) And (3) elution: eluting the target protein by using Buffer C, collecting an elution peak, wherein the elution flow rate is 1000cm/h, and the liquid flow direction is downward from the top of the resin column.

(4) Digestion and assembly

1) Enzyme digestion: adding small ubiquitin-like modified protease into the collected target protein solution according to the mass ratio of the target protein solution to the small ubiquitin-like modified protease of 100:1, and carrying out enzyme digestion for 8 hours at 8 ℃ in a shaking table.

2) Assembling: and (3) carrying out circulating dialysis for 50 hours, wherein the dialysate is Buffer D solution, the inlet speed is set to be 30L/h, the permeation speed is set to be 200mL/h, and the whole process is at 8 ℃, and collecting concentrated solution at the backflow end to obtain the final product.

Experimental example 1

The purification effect of the target protein and the enzyme digestion effect of the target protein in the example 1 are detected by SDS-PAGE electrophoresis, and the results are shown in figure 1 and figure 2, which shows that the target protein with the expected size (smtVP 0: 45kDa, smtVP 3: 36kDa, smtVP 1: 35kDa) is obtained, and the purity reaches 94%; after the enzyme cleavage, the target protein with the expected size (VP 0: 33kDa, VP 3: 24kDa, VP 1: 23kDa) is generated.

Experimental example 2

Mu.l of the virus-like particle-containing liquid prepared in example 1 was applied to a 200 mesh copper net, adsorbed at room temperature for 2 to 3min, the copper net was blotted with filter paper, stained with 3% phosphotungstic acid, and the virus-like particles assembled in vitro were observed by transmission electron microscopy, and as a result, particles similar in morphology and size to foot-and-mouth disease virus particles were observed, as shown in FIG. 3.

Experimental example 3

The particle size of the virus-like particles obtained in example 1 was measured by Dynamic Light Scattering (DLS), and the particle size of the assembly was 20 to 30nm as shown in FIG. 4.

Experimental example 4

The efficiency of assembly of the virus-like particles prepared in example 1 was examined by density gradient centrifugation.

Firstly, adding enzyme-digested protein liquid to the upper layer of a centrifugal tube containing 20-45% (w/v) sucrose density gradient, centrifuging for 3 hours at 35000r/min, collecting in parts, measuring the light absorption value at 280nm by using an ultraviolet spectrophotometer, drawing a curve, and calculating the percentage of the assembled part corresponding to the whole peak at 280nm in the whole sample.

As a result, as shown in fig. 5, the assembled virus-like particles and unassembled proteins were separated by sucrose density gradient centrifugation, and in the fractional harvest OD280nm curve, the sample corresponding to the peak was detected by DLS as a portion assembled into virus-like particles, and the protein content in this portion of the sample was 67% of the total sample.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. The solution for the scale production of the foot-and-mouth disease virus-like particle antigen is characterized by comprising the following components:

buffer a solution: 15-20mM sodium dihydrogen phosphate, 2-10mM disodium hydrogen phosphate, 100-;

buffer B solution: 15-20mM sodium dihydrogen phosphate, 2-10mmM disodium hydrogen phosphate, 100-500mM sodium chloride and 50-100mM imidazole;

buffer C solution: 15-20mM sodium dihydrogen phosphate, 2-10mmM disodium hydrogen phosphate, 100-500mM sodium chloride and 300-500mM imidazole;

buffer D solution: 10-50mM Tris-HCl, 200-500mM sodium chloride, 1-5mM calcium chloride and 3-10% glycerol.

2. The use of the solution for the large-scale production of foot-and-mouth disease virus-like particle antigens as claimed in claim 1 in the production of virus particle antigens and the preparation of virus-like particle vaccines.

3. The method for purifying and assembling the foot-and-mouth disease virus-like particle antigen using the solution of claim 1, comprising the steps of:

and S3, adding small ubiquitin-like modified protease into the target protein obtained in the step S2, performing enzyme digestion, performing circulating dialysis by using Buffer D solution, and collecting concentrated solution at a backflow end to obtain the protein.

4. The purification and assembly process according to claim 3, wherein the step of S1 is continuous flow centrifugation at 9000 r/min.

5. The purification and assembly method as claimed in claim 3, wherein the Ni-NTA agarose resin column in the S2 step is equilibrated with Buffer A for 5-10 column volumes at an equilibrium flow rate of 200-1000 cm/h; the sample loading flow rate is 200-1000 cm/h; after the sample loading is finished, washing 5-10 column volumes by using Buffer B, wherein the washing flow rate is 200-1000 cm/h; the elution flow rate was 200-1000 cm/h.

6. The method for purification and assembly according to claim 3, wherein the mass ratio of the target protein to the small ubiquitin-like modified protease in the step S3 is 80-100: 1.

7. The purification and assembly method according to claim 3, wherein the step S3 is performed by shaking for 6-8h at 2-8 ℃.

8. The purification and assembly method according to claim 3, wherein the S3 step is performed by circulating dialysis with 8-20k membrane at 2-8 deg.C for 45-50 hr.

9. The purification and assembly method as claimed in claim 8, wherein the inlet speed in the circulating dialysis is 20-30L/h, and the permeation speed is 100-200 mL/h.

10. A foot-and-mouth disease virus-like particle antigen obtained by the purification and assembly method according to any one of claims 3 to 9.