CN101974490B - Method for purifying swine fever viruses - Google Patents
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- CN101974490B CN101974490B CN2010105527319A CN201010552731A CN101974490B CN 101974490 B CN101974490 B CN 101974490B CN 2010105527319 A CN2010105527319 A CN 2010105527319A CN 201010552731 A CN201010552731 A CN 201010552731A CN 101974490 B CN101974490 B CN 101974490B
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Abstract
The invention discloses a method for purifying swine fever viruses from the cell sap of swine fever viruses reproduced in primary calf testicular cells, which comprises the following steps: 1) filtering the cell culture fluid of swine fever viruses; 2) concentrating the cell fluid (subjected to primary filtering) of swine fever viruses by way of transverse-cutting ultrafiltration concentration; and 3) purifying the swine fever viruses by using an anion exchange chromatography column. By using the method to purify swine fever viruses, the recovery rate of virus is 71.6 percent, the removing rate of hybrid protein is 91.06 percent, and the activity of virus has no obvious loss.
Description
Technical field
The present invention relates to a kind of purifying mode of Pestivirus suis, be specifically related to the method for purifying Pestivirus suis from the enchylema of former generation bull testis cell proliferation Pestivirus suis.
Background technology
Swine fever is a kind of crushing transmissible disease of serious harm pig industry.The 1950's, China initiated hog cholera lapinised virus vaccine world-famous, had formulated different vaccine manufacturing process subsequently, organized seedling, ox precursor reactant to organize seedling and swine fever bull testis cell vaccine etc. as newborn rabbit, and using commonplace is the bovine testicle cell seedling.The swine fever attenuated vaccine is a strain attenuated vaccine as safe as a house; pig to various ages and kind all is free from side effects; and good immune efficacy is arranged, and it can induce humoral immunization and cellullar immunologic response simultaneously, all can provide effective immunoprotection to the Pestivirus suis strain of different genotype.But the viral vaccine that generally is used for animal contains the impurity components such as protein, carbohydrate, lipid and nucleic acid that derive from animal tissues, cell and substratum, and the cellular metabolism by product etc., it is irritated that the inoculation animal is produced, side effects such as persistent fever.
The method purifying of international monopoly WO 98/22588 chromatography comes from the adenovirus of cell cultures; The U.S. uses the method that patent US20090123989 has described a kind of method purification of adenoviral from the solution that contains adenovirus of cell proliferation by ultrafiltration; Adopt method purified virus granular adenovirus carrier (Peixoto et al., 2008 of ion-exchange and film affinity chromatography; Sellick, 2006), purifying avian influenza virus (Kalbfuss et al., 2007; Opitzet al., 2009; Opitz et al., 2007).United States Patent (USP) 4725546 adopts the Mierocrystalline cellulose of sulphating or the mode purifying japanese encephalitis virus that crosslinked lipopolysaccharides passes through chromatography, thereby provides good highly purified vaccine for treating japanese encephalitis virus.United States Patent (USP) 6207439 adopts the Mierocrystalline cellulose of sulphating or crosslinked lipopolysaccharides to pass through the mode influenza virus purification of chromatography, obtains good vaccine thereby improve viral purity.United States Patent (USP) 3874999 passes through MgSO
4Precipitation contains the foreign protein in the viral liquid, and reaches the purpose of purified virus.Liu Jingsong etc. prepare affinity column with the IgG sepharose 4B coupling of the anti-bull testis cell culture of rabbit, remove residual bull testis cell culture composition in the PEG precipitator method partial purification antigen by the affinity chromatography immunosorption then.Its result shows that by the purifying antigen of 3 reverse affinity columns, its total protein clearance reaches 76.9% (Liu Jingsong etc., 1992).
Though the purification process of virus is had a variety of, all has some shortcomings, for example MgSO
4Its activity influence to virus of the precipitator method is very big, at the MgSO of high density
4Virus is inactivated in the solution; Though and the method for utilizing affinity chromatography can obtain the higher virus of purity, the filler cost of affinity chromatography is too high, can not be extensive use of in live vaccine.Pestivirus suis is a single-stranded RNA virus, and relative molecular weight is 4 * 10
6Dalton.This viral rounded particulate state, its diameter is 38～44nm, nucleocapsid is symmetric 20 body three-dimensional arrangements.The resistibility of Pestivirus suis is stronger, and the virus in the blood can be survived 7 days at 37 ℃, and 60 ℃ of Pestivirus suis will lose activity in 10 minutes in cell culture.Virus is stable in the environment of pH5-10.At present, the swine Fever Vaccine of use is the swine fever attenuated vaccine that Pestivirus suis forms through weakization of rabbit.In the process of suitability for industrialized production, all not through Pestivirus suis is carried out purifying.Have only part producer to concentrate through virus, for example mode such as membrane filtration is removed materials such as small portion foreign protein or cell debris.Like this, produce the viral vaccine that is used for animal and contain the impurity components such as protein, carbohydrate, lipid and nucleic acid that derive from animal tissues, cell and substratum, and the cellular metabolism by product etc., it is irritated that the inoculation animal is produced, side effects such as persistent fever.
Summary of the invention
The purpose of this invention is to provide a kind of Pestivirus suis purifying mode.
This Pestivirus suis purification process comprises the steps:
1) the Pestivirus suis cell culture fluid is filtered; 2) stay the ultrafiltration and concentration mode that the Pestivirus suis enchylema of first filter is concentrated by laterally cutting; 3) use the anion-exchange chromatography post that Pestivirus suis is carried out purifying.
Described Pestivirus suis cell culture fluid is the cell culture fluid of former generation bull testis cell proliferation Pestivirus suis.
Wherein in the step 1) Pestivirus suis enchylema being carried out filtering filter membrane aperture is 0.2～1.2 μ m.The preferred above filter membrane of 1.0 μ m that adopts earlier adopts 0.2～0.45 μ m again, more is preferably the filter membrane in 0.45 μ m aperture.
Step 2 wherein) to carry out spissated multiple be 10～40 times to described Pestivirus suis enchylema to first filter, is preferably 20 times.Describedly cut laterally that to stay the concentrated membrane pore size of ultrafiltration and concentration be 1K～100K, be preferably 30～50K.
Wherein the described anion chromatography post of step 3) is selected from following chromatographic resin: UNOsphere Q, Marcro-prep High Q, Qsepharose
TMFast Flow HiPrep16/DEAEFF, DEAE-SephadexA-25, CMSepharoseFastFlow are preferably Marcro-prep High Q.
The present invention also provides the method for anion-exchange chromatography purifying Pestivirus suis, comprises the steps:
1) dress chromatography column
Chromatography column is vertically put surely, closed the fluid mouth of pipe, the column top connects dress post container.The mixed solution of 50% (V/V) Marcro-prep High Q medium is stirred gently, add the uniform medium mixed solution of 40ml toward dress post container.After leaving standstill 3h, open the fluid mouth of pipe, the unnecessary liquid that flows away is separated dress post container gently.
2) balance chromatography column
Each pipette of chromatograph is inserted in the respective liquid bottle, started chromatograph, preheating 30min.Start low-pressure pump, bubble in the pipeline discharged, and with corresponding solution with each pipe flushing.Suspend low-pressure pump, chromatography column and the chromatograph that installs connected.3CV (column volume) ultrapure water (filtering); Follow 2CV 1.5mol/L NaCl+0.025mol/L Tris (pH 8.4), NaCl concentration is by the per-cent realization by adjustment A2 liquid and B2 liquid, i.e. 25%A2+75%B2; Last 4CV A2 liquid flushing chromatography column.Wherein said A2 liquid is that A2 is 0.025mol/L Tris; PH 8.4; B2 liquid is 2.0mol/L NaCl+0.025mol/L Tris, and pH 8.4.
3) balance swine fever cell venom
Add 0.5mol/L Tris (pH 7.5) in concentrated 20 times swine fever cell venom, the volume ratio of swine fever cell venom and 0.5mol/L Tris is 2: 38.22 μ m filtering with microporous membrane 2 times.Packing and to be stored in-20 ℃ of refrigerators standby.
4) carry out chromatography automatically.
The initialization system automated procedure, it is 2ml that injection chromatography pillar cell venom is set, and flow velocity is set is: 8ml/min (272cm/h), set linear salt concentration gradient elution process, 0M NaCl, 0.4M NaCl, 1.5M NaCl, 2.0M NaCl, the automatic chromatography of start-up system.
5) artificially collect the chromatography sample liquid
The iso-electric point of Pestivirus suis particle is about about 4.5, and (pH value 7.5) is electronegative in this chromatographic system, with Marcro-prep High Q chromatography column stronger adsorption is arranged, and is washed out when improving the ionic concn of elutriant gradually.Detect 9 elution peaks altogether, reclaim 5,6 elution peaks, the Pestivirus suis liquid behind the acquisition purifying is deposited in-20 refrigerators.
6) chromatography column aftertreatment
The chromatography column of regenerating on request injects 20% ethanol and seals chromatography column up for safekeeping, prevents microorganism reproduction in the post.
The present invention is directed to the virus vaccines of bull testis passage; by the transverse flow ultra-filtration membrane concentrate, method such as reinforcing yin essence ion exchange chromatography; removed most of biogenic impurity component; purification enrichment has the virus antigen composition of immanoprotection action, makes the purified virus vaccine safer than traditional rough vaccine.The method according to this invention purifying Pestivirus suis, the viral rate of recovery are 71.6%, and the foreign protein clearance is 91.06%, and virus activity does not have significantly sacrificing.
Description of drawings
Fig. 1 is Marcro-prep High Q anion-exchange chromatography figure.
Fig. 2 is the ELISA typical curve.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The Pestivirus suis cell culture fluid of bull testis passage is the membrane filtration of 0.45 μ m by the aperture, removes big impurity such as cell debris.Then the Pestivirus suis cell culture fluid of first filter by laterally cutting the mode of staying ultrafiltration and concentration, viral liquid is concentrated 20 times, wherein laterally cutting and staying the ultrafiltration and concentration membrane pore size is 30K.Use Marcro-prep High Q anion-exchange chromatography that viral liquid is carried out purifying, step is as follows:
1) dress chromatography column
Chromatography column is vertically put surely, closed the fluid mouth of pipe, the column top connects dress post container.The mixed solution of 50% (V/V) Marcro-prep High Q medium is stirred gently, add the uniform medium mixed solution of 40ml toward dress post container.After leaving standstill 3h, open the fluid mouth of pipe, the unnecessary liquid that flows away is separated dress post container gently.
2) balance chromatography column
Each pipette of chromatograph is inserted in the respective liquid bottle, started chromatograph, preheating 30min.Start low-pressure pump, bubble in the pipeline discharged, and with corresponding solution with each pipe flushing.Suspend low-pressure pump, chromatography column and the chromatograph that installs connected.3CV (column volume) ultrapure water (filtering); Follow 2CV 1.5mol/L NaCl+0.025mol/L Tris (pH 8.4), NaCl concentration is by the per-cent realization by adjustment A2 liquid and B2 liquid, i.e. 25%A2+75%B2; Last 4CV A2 liquid flushing chromatography column.Wherein said A2 liquid is that A2 is 0.025mol/L Tris; PH 8.4; B2 liquid is 2.0mol/L NaCl+0.025mol/L Tris, and pH 8.4.
3) balance swine fever cell venom
Get 38ml swine fever cell venom, add 2ml 0.5mol/L Tris (pH 7.5) sample buffer.22 μ m filtering with microporous membrane 2 times ,-20 ℃ of refrigerators are standby.
4) carry out chromatography automatically.
Initialization system automated procedure, implantation step 3) the swine fever cell venom 12ml that crosses of balance is provided with flow velocity and is: 8ml/min (272cm/h), set linear salt concentration gradient elution process.The automatic chromatography of start-up system.
5) artificially collect the chromatography sample liquid
The iso-electric point of Pestivirus suis particle is about about 4.5, and (pH value 7.5) is electronegative in this chromatographic system, with Marcro-prep High Q chromatography column stronger adsorption is arranged, and is washed out when improving the ionic concn of elutriant gradually.Detect 9 elution peaks (Fig. 1) altogether, reclaim the sample liquid of each absorption peak, and carry out record, deposit in-20 refrigerators after the collection.
6) chromatography column aftertreatment
The chromatography column of regenerating on request injects 20% ethanol and seals chromatography column up for safekeeping, prevents microorganism reproduction in the post.
Sample behind the chromatography is carried out the ELISA immunodetection, and step is as follows:
1) standard model preparation
With sterile saline by 10 times, 20 times, 40 times, 80 times, dilute aforesaid 20 times of viral concentrated solutions, prepare a series of concentration standard samples: 10 times of diluents of swine fever cell toxicant concentrated solution, 20 times of diluents of swine fever cell toxicant concentrated solution, at 40 times of diluents of swine fever cell toxicant concentrated solution, 80 times of diluents of swine fever cell toxicant concentrated solution, physiological saline.
2) standard model and chromatography sample ELISA detect
(1) detect the adding of antibody: take out micro-reaction plate with antibody sandwich, and on recorder the position of good each sample of mark; The detection antibody that in each reacting hole, adds 50 μ l.Can use the pipettor application of sample.
(2) adding of sample: in the diplopore of negative control, respectively add 50 μ l negative controls; In the diplopore of positive control, respectively add 50 μ l positive controls; In remaining reacting hole, add 50 μ l test samples respectively.To change hair washing for different test samples; Flick micro-reaction plate or with vibrator vibration, with the solution mixing in the Sptting plate.
(3) hatching of sample: under 37 ℃ condition, hatched 2 hours, and in the process of hatching, should hatch, in case the liquid evaporation in the reacting hole with the micro-reaction plate sealing or in wet box.
(4) wash plate: the liquid substance in the sucking-off reacting hole is also abandoned in the waste liquid cylinder; The washings that each reacting hole adds about 300 μ l washs, and washs five times.Each washing back sucking-off the liquid in porose.When washing and before adding ELIAS secondary antibody, to prevent that dry-out phenomenon from appearring in Sptting plate.After having washed the last time, Sptting plate is patted dry on bibulous material.
(5) add the horseradish peroxidase-labeled thing:
In each reacting hole, add 100 μ l horseradish peroxidase-labeled things.
(6) the horseradish peroxidase-labeled thing is hatched:
Hatched 30 minutes in room temperature (18-25 ℃).
(7) repeating step (4)
(8) add substrate: in each reacting hole, add 100 μ l tmb substrates.
(9) substrate is hatched: at room temperature (dark place) (18-25 ℃) hatched 10 minutes.Timing begins when adding first hole to calculate.
(10) termination reaction: the stop buffer termination reaction that in each reacting hole, adds 100 μ l.Addition sequence is with the application of sample order of substrate, with step 9.
(11) read plate: use ultramicron ultraviolet photometer, the air zeroing; Absorbance in wavelength 450nm reading and recording sample and contrast; Calculation result.
The ELISA detection method of setting up according to the present invention is set up the ELISA typical curve and is found that light absorption value and the virus concentration of ELISA have good linear relationship (Fig. 2).Straight-line equation by the typical curve match calculates, the light absorption value of the sample of 9 elution peaks is as shown in table 1, calculates the viral rate of recovery and is respectively 21.7% and 49.9% (calculation formula: the Pestivirus suis rate of recovery=(sample OD value-negative control OD value) * recovery sample volume/concentrated back sample OD value * last sample volume).Merging the actual viral rate of recovery of 5,6 samples is 71.6%.
Table 1 ELISA detected result
Detect through ELISA, virus particle mainly is arranged in sample 5,6 these elution peaks (table 1), and separating effect is better, and other sample contains extremely few virion, the overwhelming majority is the serum in the substratum, products of cellular metabolism, harmful secretory product, little cell debris etc.Why also there is virion in other sample in the sepn process, mainly be because: 1) large protein such as a small amount of virion and little cell debris or lipid material are closely linked; 2) a small amount of virolysis, and ELISA still can detect when detecting; 3) the Pestivirus suis particle is originally with regard to big or small heterogeneity, therefore, and electrically charged also heterogeneity.
By the coomassie brilliant blue staining method 9 elution samples are carried out the mensuration of protein content, the result is as shown in table 2.
Table 2 pH7.5 sample protein content and protein recovery
As can be seen from Table 2, with chromatography sample 5,6 merge as the protein content in this sample of recovery sample of this purifying be before the purifying provirus liquid 8.94%, promptly the sample foreign protein clearance behind the purifying is that 91.06% (method of calculation are: the protein content x100% that sample protein content=contained Tot Prot/sample solution of each sample is total).
Embodiment 2
(1) sterile saline dilute sample
Use sterile saline, swine fever cell venom is diluted to 100,000 times and 300,000 times.Sample (5,6) amalgamation liquid is diluted to 10,000 times and 10 times.
(2) sample injection
Get 15 new zealand rabbits (Zhongshan University's Experimental Animal Center) (body weight 2kg ± 0.2kg), on the volume 1-15 number.According to the form below is to each new zealand rabbit injection respective sample.Injecting method: the 1ml syringe, inhale the 1ml sample, inject gently from each new zealand rabbit auricular vein.
As shown in table 3 by the generate heat active result of viral sample that identifies recovery of the typing of rabbit body.
The rabbit health check-up of virus survival is surveyed behind table 3 purifying
Virus stock solution used dilutes 100,000 times can make rabbit that typing heat takes place, and sample 5,6 amalgamation liquids dilute 100,000 times can cause rabbit generation typing thermal response.This result shows that the virus quantity of recovery is significantly loss not, and has kept viral activity.
In sum, by this patent method purified virus vaccine, reclaim the elutriant of 5 and 6 chromatographic peaks, the foreign protein clearance is 91.06%, the viral rate of recovery 71.6%, the no obvious loss of activity of virus.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. a Pestivirus suis purification process is characterized in that, comprises the steps:
1) adopt the filter membrane of 0.2～1.2 μ m, the cell culture fluid of bull testis cell proliferation Pestivirus suis filters to former generation; 2) stay the ultrafiltration and concentration mode that the Pestivirus suis liquid of first filter is concentrated 10～40 times by laterally cutting, wherein laterally cutting and staying the ultrafiltration and concentration membrane pore size is 1K～100K; 3) use Marcro-prep High Q anion-exchange chromatography post that Pestivirus suis liquid is carried out purifying.
2. purification process according to claim 1 is characterized in that, described step 2) cycles of concentration be 20 times.
3. purification process according to claim 1 is characterized in that, described step 2) cut laterally that to stay the ultrafiltration and concentration membrane pore size be 30K～50K.
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| WO2018161858A1 (en) | 2017-03-06 | 2018-09-13 | Guangzhou Realbenefitspot Pharmaceutical Co., Ltd. | Methods of producing and characterizing virus vaccine and virus vaccine composition |
| CN108524929B (en) * | 2017-03-06 | 2019-05-07 | 广州瑞贝斯药业有限公司 | A kind of production method of rabies vacciness |
| EP3420076B1 (en) | 2017-03-06 | 2024-02-21 | Guangzhou Realbenefitspot Pharmaceutical Co., Ltd. | Methods of producing and characterizing virus vaccine and virus vaccine composition |
| CN107384878A (en) * | 2017-09-04 | 2017-11-24 | 肇庆大华农生物药品有限公司 | A kind of method of consummate pig circular ring virus |
| CN107936116A (en) * | 2018-01-16 | 2018-04-20 | 河南牧业经济学院 | The preparation method of the anti-CSFV monoclonal antibodies of high-titer |
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Effective date of registration: 20170516 Address after: 527400 Yunfu City, Xinxing County Province, the new town of North East embankment road, No. 6 Co-patentee after: Zhaoqing Dahuanong Biological Pharmaceutical Co., Ltd. Patentee after: GUANGDONG WENS DAHUANONG BIOTECHNOLOGY CO., LTD. Address before: 527400 Guangdong Xinxing County north embankment road Temperature Science Park Co-patentee before: Zhaoqing Dahuanong Biological Pharmaceutical Co., Ltd. Patentee before: Guangdong Dahuanong Animal Health Products Co., Ltd. |