CN103937757B - A kind of Latex agglutination test purification process - Google Patents

Abstract

The invention provides a method for purifying porcine Japanese encephalitis virus (JEV). The method comprehensively adopts a series of processes such as microfiltration clarification and purification method, ultrafiltration concentration purification method, repeated washing and filtration method, etc., and maximizes the increase of JEV. High recovery efficiency and reduced purification cost. The porcine encephalitis virus concentrated liquid finished product prepared by the present invention is especially suitable for preparing vaccines, and the vaccine prepared by the porcine Japanese encephalitis virus concentrated liquid finished product prepared by the present invention is compared with the porcine Japanese encephalitis virus vaccine of the prior art. As far as it is concerned, it has the advantages of high purity, high safety, good uniformity, and good immune effect. At the same time, the invention has simple and convenient process, low cost and outstanding large-scale application prospect.

Description

A kind of purification method of porcine Japanese encephalitis virus

technical field

The invention relates to a method for purifying virus, in particular to a method for purifying porcine Japanese encephalitis virus.

Background technique

Japanese encephalitis is a zoonotic disease caused by Japanese Encephalitis Virus (JEV), which belongs to the Flaviviridae family and belongs to the genus Flavivirus. Pigs are considered to be the most important natural breeding animals for this disease, which can cause abortion, stillbirth, mummified fetuses in pregnant sows, orchitis in boars; clinically, it is characterized by high fever, disturbance of consciousness, convulsions, respiratory failure and meningeal irritation . The prevalence of Japanese encephalitis not only caused serious harm to the pig industry, but also seriously threatened human health.

Vaccines are one of the main ways to prevent the disease. At present, the porcine Japanese encephalitis vaccines commercialized in my country are mainly mouse brain inactivated vaccines and attenuated vaccines. Among them, the attenuated vaccine is the main one, and its safety and effectiveness are mainly affected by factors such as virus titer and antigen purity. If commercialized vaccines are produced directly with cell-propagated virus liquid, they will be affected by impurities such as cell breakdown products, medium components, and cell metabolites, which will cause side effects such as allergic reactions and fever reactions after the vaccine is immunized to animals. Therefore, improving virus titer and antigen purity is the basic way to improve vaccine quality.

Hollow fiber membrane filtration technology belongs to the category of Tangential Flow Filtration (TFF), also known as cross-flow filtration (Cross-Flow Filtration, CFF): the feed liquid circulates on the upper surface of the membrane at a certain flow rate, which is smaller than that of the membrane. Substances with a pore size can pass through the membrane to the permeation end, while substances larger than the pore size of the membrane will be retained by the membrane, thereby achieving the concentration of the target substance and the fractional separation of different substances.

Compared with the traditional flat membrane package, the hollow fiber column has a fiber tubular open flow channel structure, and the tubular flow channel structure without screen avoids the irregular and severe turbulent flow of the feed liquid, so it has lower shear force and mild The operation can effectively prevent the shedding of virus surface glycoproteins and protein aggregation, which is conducive to protecting the integrity of the virus, preventing the aggregation of virus particles and helping the penetration and removal of foreign proteins.

At present, there is no report on the hollow fiber clarification, purification and concentration process of porcine Japanese encephalitis virus.

Contents of the invention

The invention aims to solve the technical problems of poor uniformity and poor immune effect of existing porcine Japanese encephalitis virus vaccines, and provides a porcine Japanese encephalitis virus purification method realized by using a microfiltration clarification and purification system and an ultrafiltration concentration purification system.

To achieve the above technical purpose, the present invention adopts the following technical solutions:

A method for purifying porcine Japanese encephalitis virus, the method is realized by a microfiltration clarification purification system and an ultrafiltration concentration purification system, and the process is as follows:

1 system assembly

Under sterile conditions, the microfiltration clarification and purification system and the ultrafiltration concentration purification system are assembled according to the assembly requirements. A sterile 0.45μm or 0.65μm, intact hollow fiber microfiltration membrane can be installed in the microfiltration clarification and purification system, and a sterile 100KD or 300KD, intact hollow fiber ultrafiltration membrane can be installed in the ultrafiltration concentration purification system.

2 System Integrity Detection

The pressure hold method checks the integrity of the system.

3 system processing

3.1 Cleaning and sterilization

Fill the feed tank of the system with 0.5M NaOH solution, soak for 20 minutes, turn on the circulation pump at 300 rpm, and clean and sterilize the system for 30 minutes.

3.2 Water washing and flux detection

After the sterilization, drain the NaOH solution in the system. Fill the feeding tank of the system with sterile ultrapure water, turn on the circulation pump, circulate at 300rpm for 30 minutes, discard the liquid in the system, and wash it repeatedly until the pH in the system is about 7.0.

3.3 PBS processing

After washing with water, discard the last ultrapure water. Fill the feed tank with 0.1M PBS solution, and turn on the circulation pump at 300rpm for 20min.

In the technical solution described above, the NaOH solution used in steps 3.1 and 3.2 plays a cleaning and sterilizing role, and 50%-80% alcohol solution can also be used, or the system can be sterilized by steam sterilization.

A method for purifying porcine Japanese encephalitis virus, the method is realized by a microfiltration clarification purification system and an ultrafiltration concentration purification system, and the method comprises the following steps:

1) Add sterile Tween 20 into porcine Japanese encephalitis virus liquid at a ratio of 0.5% to 10% (v/v), and shake it;

2) Inject the virus liquid after shaking treatment in step 1 into the feeding tank of the microfiltration clarification and purification system, turn on the circulation pump, and after a certain period of circulation, pass through a 0.45 or 0.65 μm hollow fiber microfiltration column for microfiltration, and collect the permeate for further processing. use;

3) Add sterile 0.1M PBS buffer solution into the retentate in the feeding tank at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump to circulate for a certain period of time, collect the permeate, and obtain the first Wash the filtrate for use;

4) Add sterile 0.1M PBS buffer solution to the retentate in the feed tank that has been processed in step 3) at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump for a certain period of time, and collect Permeate liquid, obtain the second washing filtrate stand-by;

5) Add sterile 0.1M PBS buffer solution to the retentate in the feed tank after processing in step 4) at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump for a certain period of time, and collect Permeate liquid, obtain the third washing filtrate stand-by;

6) Mix the above-mentioned permeate, the first washing filtrate, the second washing filtrate, and the third washing filtrate evenly to obtain a mixed solution;

7) Pour the mixed liquid obtained in step 6) into the feed tank of the ultrafiltration concentration purification system, turn on the circulation pump to circulate for a certain period of time, filter through a 100KD or 300KD hollow fiber ultrafiltration column, discard the permeate, and collect the feed The remaining retained liquid in the tank is the primary concentrated virus liquid;

8) Inject 0.1M PBS into the primary concentrated virus liquid in the feeding tank at a ratio of 1:1 (v/v), resuspend, turn on the circulating pump to circulate for a certain period of time, discard the permeate, and collect it in the feeding tank The remaining retained liquid is the secondary concentrated virus liquid;

9) Inject 0.1M PBS into the secondary concentrated virus liquid in the feed tank at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump, circulate for a certain period of time, discard the washing filtrate, and collect the feed tank The remaining intercepted liquid in the medium is the third-level concentrated virus liquid;

10) Inject 0.1M PBS into the three-stage concentrated virus liquid in the feed tank at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump, circulate for a certain period of time, discard the washing filtrate, and collect the feed tank The remaining retentate in the medium is the finished product of virus concentrate.

Store the finished virus concentrate solution prepared above at -20°C.

The above-mentioned purification method of porcine Japanese encephalitis virus adopts a two-step purification method. The first step is to filter the virus liquid through a clarification and purification filter membrane with a larger pore size to remove cell debris in the venom; The filtrate of the first step of the purification filter membrane is diafiltered to achieve the purpose of removing Tween 20, small molecular proteins, small molecular substances of cell metabolites and other impurities in the virus liquid and realizing the purpose of concentrating the virus.

In the method for purifying porcine Japanese encephalitis virus described above, the shaking treatment time in step 1) is preferably 10 minutes; the ratio in step 1) is preferably 5%; the pore size of the microfiltration membrane in step 2) is preferably 0.65 μm; the pore size of the ultrafiltration membrane described in step 7) is preferably 300KD; the operating conditions for turning on the circulation pump for a certain period of time in steps 2, 3, 4, 5, 7, 8, 9, and 10 are all preferably 400rpm Circulate for 30 minutes; the finished virus concentrated solution prepared by this method is used to prepare vaccines; the above-mentioned virus clarification, purification and concentration method, the volume of the permeate in step 2) can be adjusted according to needs, preferably the volume of the permeate reaches the original solution 90%; the concentration factor in step 7) can be adjusted according to actual needs, preferably more than 10 times concentration.

In the above technical solution, the porcine encephalitis virus liquid described in step 1) refers to the prepared virus stock solution, which has not been diluted; in step 1), Tween 20 is added to emulsify, which can effectively prevent virus particles from aggregating into group, thereby improving the subsequent filtration efficiency.

The microfiltration clarification and purification system refers to the FlexStand hollow fiber clarification and purification system manufactured by GE Company; the microfiltration membrane can be selected from GE Company, the model is CFP-6-D-9A, CFP-4-E-9A The Xample microfiltration column membrane; the ultrafiltration membrane can be selected from Xample ultrafiltration column membranes manufactured by GE Company, model UFP-300-C-9A, UFP-100-C-9A.

The detection method of the virus concentrate finished product of above-mentioned preparation is as follows:

The virus titer and protein concentration of the virus samples during the purification and concentration process were tested to analyze the effectiveness of the concentration process. Wherein the detection method of virus titer is:

The virus titer of each sample was detected by observing the cytopathic changes and measuring TCID _50. The criteria for judging the effectiveness of the purification were: no viable virus could be detected in the filtrate from concentrated purification and repeated diafiltration; the virus recovery rate of the concentrated solution was 80% above.

The determination method of protein content is:

The remaining amount of protein in the concentrated virus liquid was measured with a protein concentration determination kit, and the remaining amount of soluble protein should be less than 10% of the soluble protein in the original solution, indicating that the process is effective.

The content of the invention of the present invention has been described in detail above. The purification method of the present invention comprehensively adopts a series of processes such as microfiltration clarification and purification method, ultrafiltration concentration purification method, repeated diafiltration method, etc., which maximizes the recovery efficiency of JEV and reduces the purification cost. The porcine encephalitis virus concentrated liquid finished product prepared by the present invention is especially suitable for preparing vaccines, and the vaccine prepared by the porcine Japanese encephalitis virus concentrated liquid finished product prepared by the present invention is compared with the porcine Japanese encephalitis virus vaccine of the prior art. , has the advantages of high purity, high safety, good uniformity, good immune effect, etc., and at the same time, the invention has simple process and low cost, and has outstanding large-scale application prospects.

detailed description

The instrument model used in the embodiment is as follows:

FlexStand hollow fiber clarification and purification system;

Xample microfiltration column: CFP-6-D-9A (0.65μm), CFP-4-E-9A (0.45μm);

Xample ultrafiltration column: UFP-300-C-9A(300KD), UFP-100-C-9A(100KD);

All the above instruments were purchased from GE Company.

The reagents used in the examples are as follows:

100L porcine Japanese encephalitis virus liquid, 10 ^8.5 TCID ₅₀ /ml, produced by our company;

BCA protein quantification kit was purchased from Kangwei Century.

JEV Antibody Diagnostic Kit, produced by Beijing Institute of Biological Products.

0.5M NaOH100L;

Sterile 0.1M PBS100L;

Sterile high-purity water 100L;

Tween 20, analytically pure;

Sucrose gelatin protectant.

Example 1

1Operation process

1.1 Pre-processing

1.1.1 Assembly of the system

Under sterile conditions, the clarification and purification system and the concentration system are assembled according to the assembly requirements. A sterile 0.65μm, intact hollow fiber microfiltration membrane can be installed in the clarification system, and a sterile hollow fiber ultrafiltration membrane with a pore size of 300KD can be installed in the concentration system.

1.1.2 System Integrity Detection

The pressure hold method checks the integrity of the purification system.

1.1.3 System processing

Cleaning and Sterilization:

Fill the feed tank of the purification system with 0.5M NaOH solution, soak for 20 minutes, turn on the circulation pump at 300 rpm, and clean and sterilize the system for 30 minutes.

Washing and flux detection:

After the sterilization, drain the NaOH solution in the system. Fill the feeding tank of the system with sterile ultrapure water, turn on the circulation pump, circulate at 300rpm for 30 minutes, discard the liquid in the system, and wash it repeatedly until the pH in the system is about 7.0.

PBS treatment:

After washing with water, discard the last ultrapure water. Fill the feed tank with 0.1M PBS solution, and turn on the circulation pump at 300rpm for 20min.

1.2 Clarification and purification of virus

1.2.1 Treatment of virus liquid

Add 5L of sterile Tween 20 into 100L of porcine Japanese encephalitis virus (JEV) virus liquid, and shake for 10 minutes.

1.2.2 Clarification of virus fluid

After the clarification system is finished, the PBS solution in the system is discarded, the vibrating liquid is poured into the feed tank of the clarification and purification system, the circulation pump is turned on, 400rpm circulates for 30min, and the purification is carried out through a 0.65μm hollow fiber microfiltration column For processing, collect 90L of permeate, and retain 10L of retentate in the feed tank.

1.2.3 Diafiltration of retentate

First diafiltration:

Add 10 L of sterile 0.1 M PBS to the retentate in the feeding tank, resuspend, turn on the circulation pump at 400 rpm for 30 min, collect 10 L of the permeate, and record it as wash filtrate 1.

Second diafiltration:

Add 10L of sterile 0.1M PBS to the retentate in the feeding tank, resuspend, turn on the circulation pump at 400rpm for 30min, collect 10L of permeate, and record it as wash filtrate 2.

The third diafiltration:

Add 10L of sterile 0.1M PBS to the retentate in the feeding tank, resuspend, turn on the circulation pump at 400rpm for 30min, collect 10L of the permeate, and record it as wash filtrate 3.

1.2.4 Collection of virus liquid

The permeate collected in the above clarification and purification process and the three washing filtrates were evenly mixed to obtain 120 L of mixed solution.

1.2.5 Primary enrichment

After the treatment of the concentration system is completed, inject the mixed solution into the feed tank of the concentration system, turn on the circulation pump, circulate at 400rpm for 30min, purify through a 300KD hollow fiber ultrafiltration column, discard the permeate, and leave 10L of retained liquid in the feed tank , recorded as the primary concentrated virus liquid.

1.2.6 Diafiltration of concentrate

First diafiltration:

Inject 10L of 0.1M PBS into the primary concentrate in the feed tank, resuspend, turn on the circulation pump, circulate at 400rpm for 30min, discard 10L of the permeate, and leave 10L of retained liquid in the feed tank, which is recorded as the secondary concentrated virus liquid .

Second diafiltration:

Inject 10L of 0.1M PBS into the secondary concentrate in the feeding tank, resuspend, turn on the circulation pump, circulate at 400rpm for 30min, discard 10L of the wash filtrate, and leave 10L of retained liquid in the feeding tank, which is recorded as the third-level concentrated virus liquid.

The third diafiltration:

Inject 10L of 0.1M PBS into the tertiary concentrate in the feeding tank, resuspend, turn on the circulating pump, circulate at 400rpm for 30min, discard 10L of the washing filtrate, and leave 10L of retained liquid in the feeding tank, which is the finished virus concentrate.

2.2.7 Collection of virus liquid

The finished virus concentrated solution obtained after the above treatment was collected and stored at -20°C.

2 Validity testing

The virus titer and protein concentration of the virus samples during the purification and concentration process were tested to analyze the effectiveness of the concentration process.

2.1 Detection of virus titer

The virus titer of each sample was detected _by observing cytopathic changes and measuring TCID50, and the specific steps were as follows:

2.1.1 Plating and culture of cells

Digest well-grown baby hamster kidney passage cells (BHK-21) with 0.05% EDTA-trypsin, resuspend in cell growth medium to 1×10 ⁵ /ml and inoculate in 96-well cell culture plate, 100 μl /well and cultured in a 37°C, 5% CO ₂ cell incubator for 24 hours.

2.1.2 Gradient dilution of virus

Each collected venom sample was sampled and diluted 10 times with 0.1M PBS for 10 gradients (10 ⁻¹ to 10 ⁻¹⁰ ).

2.1.3 Virus inoculation and cultivation

Inoculate the diluted virus solution in each gradient to the detection cells described in step (1), 100 μl/well, 6 replicates for each gradient, and set the cell control without virus solution at the same time, culture the cells at 37°C, 5% CO ₂ The culture was maintained in the box for 5-7 days.

2.1.4 Statistics and calculation of virus titer

The number of wells with cytopathic changes was counted, and the titer of porcine Japanese encephalitis virus (TCID ₅₀ ) was calculated by the Reed-Muench method. The results confirmed that the titer of the concentrated virus solution was significantly higher than that of the virus solution before concentration. The washing filtrate in the concentration stage and the cell control group had no cytopathic changes, and the virus titer reached 10 ^9.0 TCID ₅₀ /ml after concentration, the washing filtrate in the concentration stage had no potency, and the virus recovery rate was close to 100%.

2.2 Detection of soluble protein

Each sample was measured with BCA protein concentration kit, and the residual rate of soluble protein was calculated to be 9.5%.

3 Vaccine preparation

The virus concentrate prepared by the above process is used as a raw material to further prepare a vaccine, and the process steps are as follows:

3.1 Dilute seedlings

The purified and concentrated JEV venom was diluted with sterile 0.1M PBS to make the virus titer reach 10 ^8.5 TCID ₅₀ /ml, and the purified JEV virus liquid (10 ^8.5 TCID ₅₀ /ml) was mixed with the purified JEV Virus liquid (10 ^8.5 TCID ₅₀ /ml) was added with suitable sucrose gelatin protective agent, respectively, and the vaccines were mixed; the vaccines prepared from the pre-purified JEV virus liquid and the purified JEV virus liquid were respectively recorded as: JEV inactivated vaccine 1 and JEV inactivated vaccine2.

3.2 Freeze-drying

Mix the two parts of seedling preparations evenly, and divide them into vials, each with 2.3ml. Use the same freeze-drying program (pre-freeze the product below -30°C for 3h; program temperature control: -18°C for 30min; -8°C for 30min; -8°C for 15h; 0°C for 30min; 0°C for 5h; 10°C for 2h; 25°C 30min; 25°C 4h) to freeze-dry the vaccine.

4 Vaccine Safety Inspection

JEV live vaccine 1 (before purification), JEV live vaccine 2 (after purification) and sterile 0.1M PBS solution prepared by the above process were used as experimental materials; 4-8 days old healthy susceptible suckling pigs (JEV HI antibody negative) 30 SPF mice with 12 heads and 10-12 g were used as experimental animals. The specific operation steps are as follows:

4.1 Animal grouping

12 piglets were randomly divided into 3 groups with 4 piglets in each group.

4.2 Vaccine Immunization

4.2.1 Safety inspection of suckling pigs Intramuscularly inject 2ml of the two vaccines (including 10 piglets) into the experimental suckling pigs, inject 2ml of PBS into the control group, and inject 4 piglets in each group. Observe the 21st.

4.2.1 Mice Safety Test Subcutaneously inject 0.1ml of the two vaccines (including 0.5 doses) into the experimental mice, inject 0.1ml of PBS into the control group, and at the same time inject the right side of the brain with empty pricks, 4 mice in each group, observe 14th.

4.3 Experimental results

On the 21st day after immunization, the piglets in each group had no local or systemic adverse reactions due to vaccination; on the 14th day after immunization, all the mice in each group were alive and healthy.

5 Vaccine efficacy test

5.1 Detection of vaccine virus titer after lyophilization

Dissolve the lyophilized JEV live vaccine 1 and JEV live vaccine 2 in 1ml of PBS respectively and detect the virus titer, the method is the same as "3.1". Results After lyophilization, the titer of JEV live vaccine 1 was 10 ^7.67 TCID ₅₀ /ml, and that of JEV live vaccine 2 was 10 ^8.0 TCID ₅₀ /ml. That is, the lyophilization loss rate of JEV live vaccine 2 (after purification) was less than that of JEV live vaccine 1 (before purification).

5.2 Immunization test of gilts

Using JEV live vaccine 1, JEV live vaccine 2, and sterile 0.1M PBS solution as experimental reagents, use the JEV antibody diagnostic kit to measure the JEV HI antibody; take 12 70-80kg healthy susceptible gilts (negative for JEVHI antibody) as experimental animals. The specific experimental steps are as follows:

5.2.1 Grouping of test animals

The 12 sows were randomly divided into 3 groups, 4 in each group.

5.2.2 Vaccine Immunization

Two kinds of vaccines and 2ml of PBS were injected intramuscularly into the neck of the experimental gilts, 4 heads in each group.

5.2.3 Blood collection and antibody detection

Blood was collected once a week before and after immunization, a total of 6 times, serum was separated, and the JEV antibody diagnostic kit was used for detection.

5.2.4 Experimental results

The experimental results showed that the antibody level of the second group of sows immunized by JEV live vaccine 2 (after purification) was earlier and higher than that of JEV live vaccine 1 (before purification).

To sum up, the finished product of concentrated and purified solution of porcine encephalitis virus purified by the present invention is suitable for preparing porcine encephalitis virus vaccine, and has good safety and remarkable immune efficacy.

Example 2

1) Add sterile Tween 20 into porcine Japanese encephalitis virus (JEV) virus liquid at a ratio of 10% (v/v), and shake for 10 minutes;

2) Inject the virus liquid after shaking treatment in step 1 into the feeding tank of the microfiltration clarification and purification system, turn on the circulation pump, and after a certain period of circulation, pass through a 0.45 μm hollow fiber microfiltration column to microfiltration, and collect the permeate for use;

3) Add sterile 0.1M PBS buffer solution into the retentate in the feeding tank at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump to circulate for a certain period of time, collect the permeate, and obtain the first wash filtrate;

4) Add sterile 0.1M PBS buffer solution to the retentate in the feed tank that has been processed in step 3) at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump for a certain period of time, and collect Permeate liquid, obtain the second washing filtrate stand-by;

5) Add sterile 0.1M PBS buffer solution to the retentate in the feed tank after processing in step 4) at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump for a certain period of time, and collect The permeate was obtained to obtain the third washing filtrate for use.

6) Mix the above-mentioned permeate, the first washing filtrate, the second washing filtrate, and the third washing filtrate evenly to obtain a mixed solution;

7) Pour the mixed liquid obtained in step 6) into the feed tank of the ultrafiltration concentration purification system, turn on the circulation pump to circulate for a certain period of time, filter through a 100KD hollow fiber ultrafiltration column, discard the permeate, and collect it in the feed tank The remaining retentate is the primary concentrated virus solution;

8) Inject 0.1M PBS into the primary concentrated virus liquid in the feeding tank at a ratio of 1:1 (v/v), resuspend, turn on the circulating pump to circulate for a certain period of time, discard the permeate, and collect it in the feeding tank The remaining retained liquid is the secondary concentrated virus liquid;

9) Inject 0.1M PBS into the secondary concentrated virus liquid in the feed tank at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump, circulate for a certain period of time, discard the washing filtrate, and collect the feed tank The remaining intercepted liquid in the medium is the third-level concentrated virus liquid;

10) Inject 0.1M PBS into the three-stage concentrated virus liquid in the feed tank at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump, circulate for a certain period of time, discard the washing filtrate, and collect the feed tank The remaining retentate in the medium is the finished product of virus concentrate.

Example 3

1) Add sterile Tween 20 into porcine Japanese encephalitis virus (JEV) virus liquid at a ratio of 0.5% (v/v), and shake for 10 minutes;

2) Inject the virus liquid after shaking treatment in step 1 into the feeding tank of the microfiltration clarification and purification system, turn on the circulating pump to circulate at 400rpm for 30min, and then microfiltration through a 0.65μm hollow fiber microfiltration column to collect the permeate stand-by;

3) Add sterile 0.1M PBS buffer solution to the retentate in the feeding tank at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump and circulate at 400rpm for 30min, collect the permeate, Obtain the first wash filtrate stand-by;

4) Add sterile 0.1M PBS buffer solution to the retentate in the feed tank that has been processed in step 3) at a ratio of 1:1 (v/v), resuspend, and turn on the circulation pump to circulate at 400rpm After 30min, the permeate was collected to obtain the second washing filtrate for storage;

5) Add sterile 0.1M PBS buffer solution to the retentate in the feed tank that has been treated in step 4) at a ratio of 1:1 (v/v), resuspend, and turn on the circulation pump to circulate at 400rpm After 30 minutes, the permeate was collected to obtain the third washing filtrate for use.

6) Mix the above-mentioned permeate, the first washing filtrate, the second washing filtrate, and the third washing filtrate evenly to obtain a mixed solution;

7) Pour the mixed liquid obtained in step 6) into the feed tank of the ultrafiltration concentration system, turn on the circulating pump to circulate at 400rpm for 30min, carry out ultrafiltration treatment through a 300KD hollow fiber ultrafiltration column, discard the permeate, and collect The remaining retained liquid in the feed tank is the primary concentrated virus liquid;

8) Inject 0.1M PBS into the primary concentrated virus liquid in the feeding tank at a ratio of 1:1 (v/v), resuspend, turn on the circulation pump and circulate at 400rpm for 30min, discard the permeate, and collect The remaining retained liquid in the tank is the secondary concentrated virus liquid;

9) Inject 0.1M PBS into the secondary concentrated virus liquid in the feeding tank at a ratio of 1:1 (v/v), resuspend, turn on the circulating pump and circulate at 400rpm for 30min, discard the washing filtrate, and collect The remaining retained liquid in the material tank is the third-level concentrated virus liquid;

10) Inject 0.1M PBS into the three-stage concentrated virus liquid in the feeding tank at a ratio of 1:1 (v/v), resuspend, turn on the circulating pump and circulate at 400rpm for 30min, discard the washing filtrate, and collect it into The remaining retained liquid in the material tank is the finished product of virus concentrated liquid.

The embodiments of the present invention have been described in detail above, but the content is only a preferred embodiment of the present invention, and is not intended to limit the present invention. All modifications, equivalent replacements and improvements made within the application scope of the present invention shall be included in the protection scope of the present invention.

Claims (1)

1. a method for purifying Japanese encephalitis virus of porcine is characterized in that using microfiltration clarification purification system and ultrafiltration concentration purification system to realize the purification of virus liquid, and may further comprise the steps: 1.1 Pre-processing 1.1.1 Assembly of the system Under aseptic conditions, the clarification and purification system and the concentration system are assembled according to the assembly requirements; a sterile 0.65 μm, intact hollow fiber microfiltration membrane is installed in the clarification system, and a sterile hollow fiber with a pore size of 300KD is installed in the concentration system Ultrafiltration membrane; 1.1.2 System Integrity Detection Pressure maintenance method to test the integrity of the purification system; 1.1.3 System processing Cleaning and Sterilization: Fill the feed tank of the purification system with 0.5M NaOH solution, soak for 20 minutes, turn on the circulation pump at 300 rpm, and clean and sterilize the system for 30 minutes; Washing and flux detection: After the sterilization is completed, drain the NaOH solution in the system; fill the feed tank of the system with sterile ultrapure water, turn on the circulation pump, circulate at 300rpm for 30 minutes, discard the liquid in the system, and wash it repeatedly until the pH in the system is 7.0; PBS treatment: After washing, discard the last ultrapure water; fill the feeding tank with 0.1M PBS solution, and turn on the circulation pump for 300rpm to circulate and rinse for 20min; 1.2 Clarification and purification of virus 1.2.1 Treatment of virus liquid Add 5L of sterile Tween 20 into 100L of porcine Japanese encephalitis virus (JEV) virus liquid, and shake for 10 minutes; 1.2.2 Clarification of virus fluid After the clarification system is finished, the PBS solution in the system is discarded, the vibrating liquid is poured into the feed tank of the clarification and purification system, the circulation pump is turned on, 400rpm circulates for 30min, and the purification is carried out through a 0.65μm hollow fiber microfiltration column For processing, collect 90L of permeate, and retain 10L of retentate in the feed tank; 1.2.3 Diafiltration of retentate First diafiltration: Add 10L of sterile 0.1M PBS to the retentate in the feed tank, resuspend, turn on the circulation pump at 400rpm for 30min, collect 10L of the permeate, and record it as wash filtrate 1; Second diafiltration: Add 10L of sterile 0.1M PBS to the retentate in the feed tank, resuspend, turn on the circulation pump at 400rpm for 30min, collect 10L of permeate, and record it as wash filtrate 2; The third diafiltration: Add 10L of sterile 0.1M PBS to the retentate in the feeding tank, resuspend, turn on the circulation pump at 400rpm for 30min, collect 10L of permeate, and record it as wash filtrate 3; 1.2.4 Collection of virus liquid Mix the permeate collected in the above clarification and purification process and the three washing filtrates evenly to obtain 120L of mixed solution; 1.2.5 Primary enrichment After the treatment of the concentration system is completed, inject the mixed solution into the feed tank of the concentration system, turn on the circulation pump, circulate at 400rpm for 30min, purify through a 300KD hollow fiber ultrafiltration column, discard the permeate, and leave 10L of retained liquid in the feed tank , recorded as the primary concentrated virus liquid; 1.2.6 Diafiltration of concentrate First diafiltration: Inject 10L of 0.1M PBS into the primary concentrate in the feed tank, resuspend, turn on the circulation pump, circulate at 400rpm for 30min, discard 10L of the permeate, and leave 10L of retained liquid in the feed tank as the secondary concentrated virus liquid ; Second diafiltration: Inject 10L of 0.1M PBS into the secondary concentrate in the feeding tank, resuspend, turn on the circulation pump, circulate at 400rpm for 30min, discard 10L of the wash filtrate, and leave 10L of retained liquid in the feeding tank, which is recorded as the third-level concentrated virus liquid; The third diafiltration: Inject 10L of 0.1M PBS into the tertiary concentrate in the feed tank, resuspend, turn on the circulation pump, circulate at 400rpm for 30min, discard 10L of the wash filtrate, and leave 10L of retained liquid in the feed tank, which is the finished virus concentrate; 2.2.7 Collection of virus liquid The finished virus concentrated solution obtained after the above treatment was collected and stored at -20°C.