CN113061166A

CN113061166A - Large-scale production method for separating and purifying virus VP2 protein

Info

Publication number: CN113061166A
Application number: CN202110381614.9A
Authority: CN
Inventors: 王青竹; 鞠妍; 高宏雷; 刘景利; 王晓龙; 张峣; 孙振峰; 朱庆虎; 王子敬; 牛卉颖
Original assignee: Harbin Weike Biotechnology Development Co
Current assignee: Harbin Weike Biotechnology Development Co
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2021-07-02
Anticipated expiration: 2041-04-09
Also published as: CN113061166B

Abstract

A large-scale production method for separating and purifying virus VP2 protein, belonging to the technical field of biological products. In order to solve the problem that the production of chicken infectious bursal disease vaccine by using Escherichia coli to express infectious bursal disease VP2 protein can generate endotoxin, the invention provides a large-scale production method for separating and purifying virus VP2 protein. The production method of the invention can produce high-concentration bursa of Fabricius VP2 protein, can meet the GMP production requirement of new veterinary drugs, and has the characteristic of low production cost.

Description

Large-scale production method for separating and purifying virus VP2 protein

Technical Field

The invention belongs to the technical field of biological products, and particularly relates to a large-scale production method for separating and purifying virus VP2 protein.

Background

Infectious Bursal Disease (IBD) in chickens is an acute, highly Infectious, immunosuppressive Disease caused by Infectious Bursal Disease Virus (IBDV). The IBDV major structural protein VP2 contains serotype-specific conformation-dependent epitopes capable of inducing the production of neutralizing antibodies, which can passively protect the host from IBDV infection, is the major protective antigen of IBDV, has specificity, and thus becomes the major protein for preparing genetic engineering subunit vaccine for bursal disease. In the prior art, Escherichia coli capable of expressing VP2 gene of infectious bursal disease virus is often used for producing VP2 protein, and further producing chicken infectious bursal disease vaccine. The method for obtaining the bursa of Fabricius VP2 protein needs to crush the Escherichia coli firstly, and the Escherichia coli is crushed to generate a large amount of endotoxin which is a component in the cell wall of gram-negative bacteria and is called lipopolysaccharide inevitably. Lipopolysaccharides are toxic to the host. Endotoxin is released only when bacteria die and lyse or bacterial cells are artificially destroyed, and is called endotoxin. Endotoxin is located in the outermost layer of the cell wall, covering the mucin of the cell wall. Endotoxins of various bacteria have weak and almost identical toxic effects, and cause fever, microcirculation disturbance, endotoxin shock, disseminated intravascular coagulation, and the like. Thus, if endotoxin is not reduced to a certain extent, (severe) side effects may be caused to the vaccinated animals.

In order to control the endotoxin produced by the disruption of E.coli to a certain extent, it is necessary to separate and purify the bursa of Fabricius VP2 protein. The most common and effective way to remove endotoxin is chromatography, but this method requires special filler, which is expensive and exceeds the production cost of poultry vaccine. There is also a relatively inexpensive extraction method, but this method requires repeated centrifugation, and some of the procedures in centrifugation may not meet the GMP specifications of new versions. Because the poultry vaccine is low in market price and needs to control the production cost, the quality of the vaccine must be guaranteed, the protein is purified at high concentration, and the cost in the purification process needs to be controlled, so that the difficulty of subsequent separation and purification is increased. Therefore, a large-scale production method for separating and purifying the bursa of Fabricius VP2 protein, which can purify the bursa of Fabricius VP2 protein at high concentration and reduce the production cost, is urgently needed.

Disclosure of Invention

The invention provides a large-scale production method for separating and purifying virus VP2 protein, which meets the requirements of production cost and GMP (good manufacturing practice) specifications and comprises the following specific steps:

step one, carrying out heavy suspension, crushing by a high-pressure homogenizer and centrifugation on escherichia coli thalli expressing VP2 gene of bursal disease virus to obtain bacterial liquid;

step two, putting the bacterial liquid obtained in the step one into a hollow fiber column with an ultrafiltration pore diameter, repeatedly flushing after closing a permeation end, then opening the permeation end, adjusting the flow rate, concentrating the volume in the column to 1/5-1/10 of the original volume, and then closing the permeation end;

step three, adding an isometric PB buffer solution into the column, closing the permeation end, circulating in the column for 15-30min, opening the permeation end, concentrating the volume of the bacterial liquid again to the volume before the buffer solution is added, and then continuously washing and filtering the bacterial liquid by using the PB buffer solution until the volume of waste liquid at the permeation end reaches 4-8 times of the volume of liquid in the tank;

and step four, adding the mixed liquid containing sodium deoxycholate and sodium chloride with the same volume into the bacterial liquid obtained in the step three, adjusting the pH to 7.0-7.5, closing a valve at the outlet end, circulating in the column for 15min-30min, and then continuously washing and filtering the bacterial liquid by using a PB buffer solution containing sodium deoxycholate and sodium chloride until the volume of the waste liquid at the outlet end reaches 5-10 times of the volume of the liquid in the tank, thereby completing the whole process flow.

Further, the lysate in the step one is 0.1mol/L-0.3mol/L PB buffer solution, and the volume ratio of the wet bacteria weight of the escherichia coli to the lysate is 1: 10.

Further, the crushing pressure in the first step is 800bar, and the process is repeated for 3 times.

Further, the centrifugation in the step one is centrifugation by a disc centrifuge, and the rotating speed is 10000 r/min.

And further limiting, the continuous washing and filtering method in the third step is to add the PB buffer solution into the tank at a constant speed, open the valve at the permeation end, adjust the flow rate at the permeation end to be the same as the injection speed of the buffer solution, and control the pressure at the inlet end to be 6-12 psi.

Further limiting, in the mixed solution containing sodium deoxycholate and sodium chloride in the fourth step, the mass fraction of the sodium deoxycholate is 1% -2%, and the concentration of the sodium chloride is 1M.

Further limiting, in the PB buffer solution containing sodium deoxycholate and sodium chloride in the fourth step, the mass fraction of the sodium deoxycholate is 0.5% -1%, and the concentration of the sodium chloride is 0.5M.

And further limiting, the continuous elution method in the fourth step is that PB buffer solution containing sodium deoxycholate and sodium chloride is added into the column at a constant speed, a valve at the outlet end is opened, the flow rate at the outlet end is adjusted to be the same as the injection speed of the buffer solution, and the pressure at the inlet end is controlled to be 6-12 psi.

Advantageous effects

The method is characterized in that a hollow fiber purification system is used for purifying bursa of Fabricius VP2 protein expressed by escherichia coli, and the specific method comprises the steps of crushing the escherichia coli, adding sodium deoxycholate in a certain proportion, adding an organic phosphate buffer solution as a chelating agent, adjusting the pH value to be proper, depolymerizing endotoxin aggregates into monomers, and opening an endotoxin-protein complex. And simultaneously adding NaCl with a certain concentration into the solution, fully and uniformly mixing, removing endotoxin monomers by using the tangential flow filtration function of the hollow fiber column, and then selecting a membrane column with a proper pore diameter to perform multiple steps of protein precipitation, redissolution, purification and the like. Finally, the bursa of Fabricius VP2 protein with high concentration is produced, thereby meeting the production requirement of the vaccine. The production method of the invention has the following advantages:

1. the production process is simplified, the operation is convenient and fast, the working procedures are simple, multiple operations can be completed by one machine, and the requirements of various production indexes are met.

2. The production cost is controlled within the acceptable range of the poultry vaccine.

3. The whole adjusted production flow more meets the GMP production requirements of new veterinary drugs.

Detailed Description

The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.

Example 1

Step one, adding a thallus lysate (0.1mol/L-0.3mol/L PB buffer solution) into escherichia coli thallus expressing VP2 gene of bursal disease virus according to a ratio of 1:10 (wet thallus weight: lysate), carrying out heavy suspension, then crushing the thallus by using a high-pressure homogenizer, repeating for 3 times, centrifuging the crushed thallus by using a disc centrifuge at a rotating speed of 10000r/min, and collecting the thallus after centrifugation to remove thallus fragments.

And step two, concentrating the crushed bacteria liquid by using a hollow fiber column with an ultrafiltration aperture, operating a machine, sealing a penetrating end, repeatedly flushing the bacteria liquid in the hollow fiber column, opening the penetrating end, adjusting to proper flow rate (different membranes and membrane area flow rates), closing the penetrating end after the volume in the column is concentrated to 1/5-1/10 volume of the original volume, and suspending the machine.

And step three, adding an isometric buffer solution (PB) into the column, closing the permeation end, circulating in the column for 15-30min, fully and uniformly mixing the buffer solution and the bacterial solution, opening the permeation end, concentrating the volume of the solution again to the volume before the buffer solution is added, then adding the buffer solution into the tank at a constant speed, opening a valve of the permeation end, adjusting the flow rate of the permeation end to be the same as the injection speed of the buffer solution, continuously washing and filtering the bacterial solution, controlling the pressure of the inlet end to be 6-12 psi, and suspending the machine when the volume of the waste liquid at the permeation end reaches 4-8 times of the volume of the liquid in the tank.

Step four, adding the mixed liquid containing sodium deoxycholate and sodium chloride with the same volume into the bacterial liquid obtained in the step three, adjusting the pH value to 7.0-7.5, closing a permeation end valve, operating a machine, enabling the bacterial liquid to perform in-column circulation, opening a permeation end after circulating for 15-30min, then adding PB buffer liquid containing sodium deoxycholate and sodium chloride into a tank at a constant speed, opening the permeation end valve, adjusting the flow rate of the permeation end to be the same as the injection speed of the buffer liquid, then performing continuous washing and filtering on the bacterial liquid, controlling the pressure of an inlet end to be 6-12 psi, pausing the machine when the volume of waste liquid of the permeation end reaches 5-10 times of the volume of liquid in the tank, collecting the bacterial liquid into a storage tank, cleaning the machine, and finishing the whole process flow.

According to the agar amplification and endotoxin detection, the conclusion can be drawn that agar amplification results are not obviously changed before and after purification, which indicates that the virus loss is within an acceptable range, and the endotoxin result after bacterial liquid crushing is 20000EU-40000EU, which can reach 1000EU-2000EU after purification, the protein content is reduced by about 80%, and the occurrence of side reactions after immunization is greatly reduced. The overall conditions can meet the requirements of the vaccine.

Claims

1. A large-scale production method for separating and purifying virus VP2 protein is characterized by comprising the following steps:

2. The method according to claim 1, wherein the lysate in step one is 0.1mol/L-0.3mol/L PB buffer, and the volume ratio of the wet bacteria weight of the Escherichia coli to the lysate is 1: 10.

3. The method of claim 1, wherein said crushing of step one is performed at a pressure of 800bar, and is repeated 3 times.

4. The method according to claim 1, wherein the centrifugation of step one is centrifugation with a disc centrifuge at a rotation speed of 10000 r/min.

5. The method of claim 1, wherein the continuous washing and filtering in step three is performed by adding PB buffer solution into the tank at a constant speed, opening the valve at the outlet end, adjusting the flow rate at the outlet end to be the same as the injection speed of the buffer solution, and controlling the pressure at the inlet end to be 6-12 psi.

6. The method according to claim 1, wherein in the mixed solution containing sodium deoxycholate and sodium chloride in the fourth step, the mass fraction of sodium deoxycholate is 1% -2%, and the concentration of sodium chloride is 1M.

7. The method according to claim 1, wherein in the PB buffer containing sodium deoxycholate and sodium chloride in the fourth step, the mass fraction of the sodium deoxycholate is 0.5% -1%, and the concentration of the sodium chloride is 0.5M.

8. The method of claim 1, wherein the continuous elution in step four is performed by adding PB buffer solution containing sodium deoxycholate and sodium chloride at a constant speed into the column, opening the valve at the outlet, adjusting the flow rate at the outlet to be the same as the injection speed of the buffer solution, and controlling the pressure at the inlet to be 6-12 psi.