CN106497850A - A kind of method of purification mycoplasma hyopneumoniae - Google Patents
A kind of method of purification mycoplasma hyopneumoniae Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 58
- 241000204045 Mycoplasma hyopneumoniae Species 0.000 title claims abstract description 45
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- 102000036639 antigens Human genes 0.000 claims abstract description 91
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- 238000000108 ultra-filtration Methods 0.000 claims abstract description 27
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- 241000204031 Mycoplasma Species 0.000 claims 1
- 238000001914 filtration Methods 0.000 claims 1
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- 238000011084 recovery Methods 0.000 abstract description 12
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- 102000004169 proteins and genes Human genes 0.000 abstract description 8
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- 238000004519 manufacturing process Methods 0.000 abstract description 5
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Abstract
The invention provides a kind of method of purification mycoplasma hyopneumoniae, the technical scheme is with antigen culture fluid directly as raw material, successively clarified process, ultrafiltration concentration, filter wash three step process are accused and are completed, adopted process unit is devised on this basis based on laboratory facilities, using hollow-fibre membrane shearing force low, dust containing capacity is high the characteristics of, and operating condition is optimized, antigen homeostasis ensure that by the restriction of transmembrane pressure, shear rate, and ensure that removal of impurity.Application this method Purification of Pig mycoplasma pneumoniae antigen, in the case of not concentrated, rate of recovery of antigen reaches more than 87.8%, and in antigen liquid, porcine blood serum removes 60.7%；After 5 times of concentrations, up to 98%, rate of recovery of antigen 62%, porcine blood serum content reduce by 90.3% with Proantigen liquid phase ratio to foreign protein clearance.Simultaneously as this method treating capacity is big, processing speed is fast, simple to operate, process stabilizing, automatization or semi-automatic production therefore can be realized.
Description
Technical field
The present invention relates to antigenic substance technical field of purification, further to the purification technique of the mycoplasma for vaccine,
Specifically related to a kind of method of purification mycoplasma hyopneumoniae.
Background technology
Mycoplasmal pneumonia, is that one kind is drawn by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp)
The Chronic exposure respiratory infectious disease for rising, to pant as cardinal symptom, also known as swine enzootic pneumonia.The disease has a very wide distribution, infects
Rate is high, mortality rate is low, and ill pig decreased growth, feed conversion rate reduce, easily causes the Secondary cases or mixed of his antibacterial or virus
Close sexy dye, such as porcine reproductive and respiratory syndrome virus, porcine circovirus, streptococcus pneumoniae, haemophilus parasuises, mycoplasma hyorhinises
And pasteurella multocida etc..
Vaccine is the important biomolecule product of Mycoplasmal pneumonia preventing and treating, and with antigen performance in the development process of vaccine
The most key, from the point of view of using effect aspect, inactivation antigen or it is attenuated or antigen should all have significant immunogen
Property, while body is stimulated, and little, untoward reaction rate is low, for the immunogenicity for obtaining, the purification of antigen is most important.At present
The wider inactivated vaccine of application is that auspicious times of the mycoplasma hyopneumoniae inactivated vaccine of Pfizer's production is fitted, its adjuvant that applies
Amphigen has preferable immunostimulatory potency.Domestic using more be the weak poison of the Mhp168 strains developed by Jiangsu academy of agricultural sciences
Live vaccine, using the immunity of pulmonary injection mode.The culture of mycoplasma hyopneumoniae and preservation difficulty are big, traditional culture freeze drying process
Can cause after serum used or other protein injections and seriously cause allergic reaction, therefore prepare high-purity antigen, remove culture medium
The foreign proteins such as middle serum, optimization production of vaccine downstream process are extremely urgent.
In the vaccine preparation technology of prior art, the purification effect of mycoplasma hyopneumoniae antigen have to be hoisted, on the one hand its
Rate of recovery of antigen is relatively low, while also there is foreign protein removes incomplete phenomenon, additionally, the correlation technique flow process of prior art
Loaded down with trivial details, it is not easy to production-scale amplification.
Content of the invention
It is contemplated that for the technological deficiency of prior art, there is provided a kind of method of purification mycoplasma hyopneumoniae, to solve
The certainly low technical problem of the mycoplasma hyopneumoniae purifying process rate of recovery of antigen of prior art.
The invention solves the problems that another technical problem be that the mycoplasma hyopneumoniae purifying process of prior art is difficult to fully go
The removal of impurity.
The invention solves the problems that another technical problem be prior art mycoplasma hyopneumoniae purifying process treating capacity limited,
It is unfavorable for production-scale amplification.
For realizing that above technical purpose, the present invention are employed the following technical solutions：
A kind of method of purification mycoplasma hyopneumoniae, comprises the following steps：
1) mycoplasma hyopneumoniae antigen culture fluid is taken, and clarifying treatment is collected product and is clarification antigen liquid；
2) by step 1) the clarification antigen liquid that obtains is concentrated by ultrafiltration：
3) by step 2) after products therefrom buffer carries out 3～7 times of volume filter washes, collect product.
Preferably, step 1) contained antigen in the mycoplasma hyopneumoniae antigen culture fluid is mycoplasma hyopneumoniae
The mycoplasma hyopneumoniae antigen of active antigen or inactivation.
Preferably, step 1) clarifying treatment be using filter membrane, film bag or hollow fiber ultrafiltration membrane realize.
Preferably, step 2) described be concentrated by ultrafiltration be using doughnut membrane filtration system realize.
Preferably, hollow fiber column aperture is 300～700KD in the doughnut membrane filtration system.
Preferably, step 2) multiple that is concentrated by ultrafiltration is 2～5 times of volumes.
Preferably, step 2) during the ultrafiltration concentration, shear rate is 2000～6000sec-1, more excellent, be
4000sec-1.
Preferably, step 2) during the ultrafiltration concentration, transmembrane pressure is 5～10psi, more excellent, is 7.5psi.
Preferably, step 3) described in buffer be PBS solution that pH value is 7.2～7.4.
Preferably, step 3) filter wash is equal-volume filter wash.
Preferably, step 2) described in ultrafiltration concentration, step 3) the filter wash process be collection backflow, discard
Cross liquid.
In above technical scheme, step 2) described in ultrafiltration concentration antigen liquid can be carried out 2～20 times, even more big
The concentration of volume.
The invention provides a kind of method of purification mycoplasma hyopneumoniae, the technical scheme with antigen culture fluid directly as
Raw material, successively clarified process, ultrafiltration concentration, filter wash three step process are accused and are completed, and are designed based on laboratory facilities on this basis
The process unit for being adopted, using hollow-fibre membrane shearing force low, dust containing capacity is high the characteristics of, and optimize operating condition, lead to
Cross transmembrane pressure, the restriction of shear rate ensure that antigen homeostasis, and ensure that removal of impurity.
Application this method Purification of Pig mycoplasma pneumoniae antigen, in the case of not concentrated, rate of recovery of antigen reaches
More than 87.8%, in antigen liquid, porcine blood serum removes 60.7%；After 5 times of concentrations, foreign protein clearance is up to 98%, rate of recovery of antigen
62%, porcine blood serum content is with Proantigen liquid phase than reducing by 90.3%.Simultaneously as this method treating capacity is big, processing speed is fast, behaviour
Make simple, process stabilizing, therefore can realize automatization or semi-automatic production.The mycoplasma hyopneumoniae of application this method purification
Vaccine prepared by antigen, for reducing, i (mycoplasma hyopneumoniae) vaccine product side reaction, Improving The Quality of Products are significant.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessively unnecessary details,
Will not be described in detail to belonging to known structure or function in following examples.
Approximating language used in following examples can be used for quantitative expression, show in the feelings for not changing basic function
Quantity can be allowed under condition to have certain variation.Therefore, with " about ", that the numerical value revised of the language such as " left and right " is not limited to this is accurate
Numerical value itself.In certain embodiments, " about " represent allow its revise numerical value positive and negative 10 (10%) scope
Interior change, such as, what " about 100 " represented can be any numerical value between 90 to 110.Additionally, " the about first numerical value is arrived
In the statement of second value ", the first and second numerical value two values are at about revised.In some cases, approximating language
May be relevant with the precision of measuring instrument.
In addition to being defined, in following examples, technology used and scientific terminology have and art technology people of the present invention
The identical meanings that member is commonly understood by.
In following examples, test reagent consumptive material used, if no special instructions, is routine biochemistry reagent；The experiment
Method, if no special instructions, is conventional method；Quantitative test in following examples, is respectively provided with three times and repeats to test, as a result
Average；% in following examples, if no special instructions, is weight/mass percentage composition.
Embodiment 1
The preparation of mycoplasma hyopneumoniae antigen
Pressing fermenter volume 70% adds mycoplasma hyopneumoniae fluid medium, 121 DEG C of sterilizing 20min to treat culture medium certainly
When being so cooled to 37 DEG C, 20% porcine blood serum is added, is stirred；The 10% access production hyopneumoniae original by culture medium total amount
Body seed liquor, 37 DEG C of stir cultures 3～7 days, harvests bacterium solution when 7.5 drop to 6.8 whne pH value.
Embodiment 2
The purification of mycoplasma hyopneumoniae antigen
Mycoplasma hyopneumoniae antigen liquid 3～6um of application filter membranes clarification of results；
The Hollow fiber systems of application 300KD carry out purification to the antigen liquid that clarifies, and shearing force is controlled in 4000s-1, cross-film
Pressure TMP is controlled in 5～10psi, antigen liquid is concentrated into original volume 1/2, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrated solution is diluted to original volume with aseptic PBS (PH7.2), shearing force is controlled in 4000s-1, cross-film
Pressure TMP is controlled in 5～10psi, and antigen liquid is concentrated into original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrated solution is diluted to original volume with aseptic PBS (PH7.2), shearing force is controlled in 4000s-1, cross-film
Pressure TMP is controlled in 5～10psi, and antigen liquid is concentrated into original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrated solution is diluted to original volume with aseptic PBS (PH7.2), the trapped fluid of results is the pig of purification
Mycoplasma pneumoniae antigen.
Total protein content after determining before purification below by way of BCA methods, and albumen clearance, experimental result such as table are calculated with this
Shown in 1.Rate of recovery of antigen after being determined before purification by double-antibody sandwich Elisa methods, experimental result are as shown in table 2.By Sanguis sus domestica
Pure protein residue kit measurement before purification after porcine blood serum residual volume, experimental result is as shown in table 3.
Rear before purification total protein content and corresponding albumen clearance that table 1 is detected with BCA methods
| Before purification (mg/ml) | After purification (mg/ml) | Albumen clearance |
| 16.4 | 13.9 | 15.2% |
The rear before purification rate of recovery of antigen that table 2 is detected with double-antibody sandwich Elisa methods
The rear before purification porcine blood serum residual volume that table 3 is detected with porcine hemoglobin remaining reagent box
| Sample ID | Concentration ug/ml | Volume ml | Quality mg | Porcine blood serum clearance |
| Before purification | 315.9 | 200 | 63.18 | - |
| After purification | 132.8 | 200 | 26.56 | 58% |
Embodiment 3
The purification of mycoplasma hyopneumoniae inactivation antigen
Harvest mycoplasma hyopneumoniae antigen liquid by final concentration 0.2% add formaldehyde, 37 DEG C inactivation 24h；
The mycoplasma hyopneumoniae antigen liquid of inactivation is clarified with 3～6um filter membranes；
The Hollow fiber systems of application 300KD carry out purification to the antigen liquid that clarifies, and shearing force is controlled in 4000s-1, cross-film
Pressure TMP is controlled in 5～10psi, antigen liquid is concentrated into original volume 1/2, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrated solution is diluted to original volume with aseptic PBS (PH7.2), shearing force is controlled in 4000s-1, cross-film
Pressure TMP is controlled in 5～10psi, and antigen liquid is concentrated into original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrated solution is diluted to original volume with aseptic PBS (PH7.2), shearing force is controlled in 4000s-1, cross-film
Pressure TMP is controlled in 5～10psi, and antigen liquid is concentrated into original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrated solution is diluted to original volume with aseptic PBS (PH7.2), the trapped fluid of results is the pig of purification
Mycoplasma pneumoniae antigen.
Total protein content after determining before purification below by way of BCA methods, and albumen clearance, experimental result such as table are calculated with this
Shown in 4.Rate of recovery of antigen after being determined before purification by double-antibody sandwich Elisa methods, experimental result are as shown in table 5.By Sanguis sus domestica
Pure protein residue kit measurement before purification after porcine blood serum residual volume, experimental result is as shown in table 6.
Rear before purification total protein content and corresponding albumen clearance that table 4 is detected with BCA methods
| Before purification (mg/ml) | After purification (mg/ml) | Albumen clearance |
| 16.4 | 14.2 | 13.4% |
The rear before purification rate of recovery of antigen that table 5 is detected with double-antibody sandwich Elisa methods
The rear before purification porcine blood serum residual volume that table 6 is detected with porcine hemoglobin remaining reagent box
| Sample ID | Concentration ug/ml | Volume ml | Quality mg | Porcine blood serum clearance |
| Before purification | 315.9 | 200 | 63.18 | - |
| After purification | 124.1 | 200 | 24.82 | 60.7% |
Embodiment 4
The concentration of mycoplasma hyopneumoniae inactivation antigen
Mycoplasma hyopneumoniae antigen liquid 3～6um of application filter membranes clarification of results；
The Hollow fiber systems of application 300KD are concentrated and purified to the antigen liquid that clarifies, and shearing force is controlled in 4000s-1,
Transmembrane pressure TMP is controlled in 5～10psi, antigen liquid is concentrated into original volume 1/5, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrated solution twice is diluted with aseptic PBS (PH7.2), shearing force is controlled in 4000s-1, transmembrane pressure TMP
Antigen liquid is concentrated into original volume in 5～10psi by control, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrated solution twice is diluted with aseptic PBS (PH7.2), shearing force is controlled in 4000s-1, transmembrane pressure TMP
Antigen liquid is concentrated into original volume in 5～10psi by control, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrated solution twice is diluted with aseptic PBS (PH7.2), shearing force is controlled in 4000s-1, transmembrane pressure TMP
Antigen liquid is concentrated into original volume in 5～10psi by control, abandons permeate, harvests the hyopneumoniae that trapped fluid is purified concentration
Pathogen inactivation antigen.
Total protein content after determining before purification below by way of BCA methods, and albumen clearance, experimental result such as table are calculated with this
Shown in 7.Rate of recovery of antigen after being determined before purification by double-antibody sandwich Elisa methods, experimental result are as shown in table 8.By Sanguis sus domestica
Pure protein residue kit measurement before purification after porcine blood serum residual volume, experimental result is as shown in table 9.
Rear before purification total protein content and corresponding albumen clearance that table 7 is detected with BCA methods
The rear before purification rate of recovery of antigen that table 8 is detected with double-antibody sandwich Elisa methods
The rear before purification porcine blood serum residual volume that table 9 is detected with porcine hemoglobin remaining reagent box
| Sample ID | Concentration ug/ml | Volume ml | Quality mg | Porcine blood serum clearance |
| Stock solution | 315.9 | 200 | 63.18 | - |
| After concentration | 152.5 | 40 | 6.1 | 90.3% |
Embodiment 5
Efficacy test after before purification
(mycoplasma hyopneumoniae serum antibody IHA potency is not higher than 1 to body weight 1.5～2.0kg healthy rabbits:5) 15, wherein
5 each hind leg muscles inject the inactivation antigen 0.5ml before purification of embodiment 4, and 5 hind leg muscles injection embodiments 4 are inactivated after purification
Antigen 0.5ml, 14 days after injection, identical approach, same dose two are exempted from.Remaining 5 are not inoculated with as control.Two exempt from 30 days afterwards,
Take a blood sample together with matched group, rabbit anteserum mycoplasma hyopneumoniae antibody titer is detected with IHA methods.Effect testing result such as 10 institute of table
Show：
The forward and backward impact to antibody titer after immunity inoculation of 10 mycoplasma hyopneumoniae antigen purification of table
Embodiment 6
A kind of method of purification mycoplasma hyopneumoniae, comprises the following steps：
1) mycoplasma hyopneumoniae antigen culture fluid is taken, and clarifying treatment is collected product and is clarification antigen liquid；
2) by step 1) the clarification antigen liquid that obtains is concentrated by ultrafiltration：
3) by step 2) after products therefrom buffer carries out 3 times of volume filter washes, collect product.
On the basis of above technical scheme, following condition is met：
Step 1) contained antigen in the mycoplasma hyopneumoniae antigen culture fluid be mycoplasma hyopneumoniae active antigen or
The mycoplasma hyopneumoniae antigen of inactivation.
Step 1) clarifying treatment be using filter membrane, film bag or hollow fiber ultrafiltration membrane realize.
Step 2) described be concentrated by ultrafiltration be using doughnut membrane filtration system realize.
In the doughnut membrane filtration system, hollow fiber column aperture is 300KD.
Step 2) multiple that is concentrated by ultrafiltration is 2 times of volumes.
Step 2) during the ultrafiltration concentration, shear rate is 2000sec-1.
Step 2) during the ultrafiltration concentration, transmembrane pressure is 5psi.
Step 3) described in buffer be PBS solution that pH value is 7.2.
Step 3) filter wash is equal-volume filter wash.
Embodiment 7
A kind of method of purification mycoplasma hyopneumoniae, comprises the following steps：
1) mycoplasma hyopneumoniae antigen culture fluid is taken, and clarifying treatment is collected product and is clarification antigen liquid；
2) by step 1) the clarification antigen liquid that obtains is concentrated by ultrafiltration：
3) by step 2) after products therefrom buffer carries out 7 times of volume filter washes, collect product.
On the basis of above technical scheme, following condition is met：
In the doughnut membrane filtration system, hollow fiber column aperture is 700KD.
Step 2) multiple that is concentrated by ultrafiltration is 5 times of volumes.
Step 2) during the ultrafiltration concentration, shear rate is 6000sec-1.
Step 2) during the ultrafiltration concentration, transmembrane pressure is 10psi.
Step 3) described in buffer be PBS solution that pH value is 7.4.
Embodiment 7
A kind of method of purification mycoplasma hyopneumoniae, comprises the following steps：
1) mycoplasma hyopneumoniae antigen culture fluid is taken, and clarifying treatment is collected product and is clarification antigen liquid；
2) by step 1) the clarification antigen liquid that obtains is concentrated by ultrafiltration：
3) by step 2) after products therefrom buffer carries out 5 times of volume filter washes, collect product.
Above embodiments of the invention are described in detail, but the content have been only presently preferred embodiments of the present invention,
Not in order to limiting the present invention.All any modification, equivalent and improvement that is made in the application range of the present invention etc., all should
It is included within protection scope of the present invention.
Claims (10)
1. a kind of method of purification mycoplasma hyopneumoniae, it is characterised in that comprise the following steps：
1) mycoplasma hyopneumoniae antigen culture fluid is taken, and clarifying treatment is collected product and is clarification antigen liquid；
2) by step 1) the clarification antigen liquid that obtains is concentrated by ultrafiltration：
3) by step 2) after products therefrom buffer carries out 3～7 times of volume filter washes, collect product.
2. a kind of method of purification mycoplasma hyopneumoniae according to claim 1, it is characterised in that step 1) Pulmonis Sus domestica
In scorching mycoplasma antigen culture fluid, contained antigen is the mycoplasma hyopneumoniae antigen of mycoplasma hyopneumoniae active antigen or inactivation.
3. a kind of method of purification mycoplasma hyopneumoniae according to claim 1, it is characterised in that step 1) clarification
Process is realized using filter membrane, film bag or hollow fiber ultrafiltration membrane.
4. a kind of method of purification mycoplasma hyopneumoniae according to claim 1, it is characterised in that step 2) ultrafiltration
Concentration is realized using doughnut membrane filtration system.
5. a kind of method of purification mycoplasma hyopneumoniae according to claim 4, it is characterised in that the hollow-fibre membrane
In filtration system, hollow fiber column aperture is 300～700KD.
6. a kind of method of purification mycoplasma hyopneumoniae according to claim 1, it is characterised in that step 2) it is concentrated by ultrafiltration
Multiple be 2～5 times of volumes.
7. a kind of method of purification mycoplasma hyopneumoniae according to claim 4, it is characterised in that step 2) ultrafiltration
In concentration process, shear rate is 2000～6000sec-1.
8. a kind of method of purification mycoplasma hyopneumoniae according to claim 4, it is characterised in that step 2) ultrafiltration
In concentration process, transmembrane pressure is 5～10psi.
9. a kind of method of purification mycoplasma hyopneumoniae according to claim 1, it is characterised in that step 3) described in slow
Rush the PBS solution that liquid is that pH value is 7.2～7.4.
10. a kind of method of purification mycoplasma hyopneumoniae according to claim 1, it is characterised in that step 3) filter wash
It is equal-volume filter wash.
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| CN110157652A (en) * | 2019-06-28 | 2019-08-23 | 江苏南农高科技股份有限公司 | A kind of method that mycoplasma hyopneumoniae antigen concentrates and purifies |
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| CN110157652A (en) * | 2019-06-28 | 2019-08-23 | 江苏南农高科技股份有限公司 | A kind of method that mycoplasma hyopneumoniae antigen concentrates and purifies |
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