CN106497850B - A method of purifying mycoplasma hyopneumoniae - Google Patents
A method of purifying mycoplasma hyopneumoniae Download PDFInfo
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Abstract
The present invention provides a kind of methods for purifying mycoplasma hyopneumoniae, the technical solution is with antigen culture solution directly as raw material, successively clarified processing, ultrafiltration concentration, filter wash three step process are accused and are completed, used process unit is devised based on laboratory facilities on this basis, the feature low using hollow-fibre membrane shearing force, dust containing capacity is high, and operating condition is optimized, antigen homeostasis ensure that by the restriction of transmembrane pressure, shear rate, and ensure removal of impurity.Using this method Purification of Pig mycoplasma pneumoniae antigen, in the case where being concentrated, rate of recovery of antigen reaches 87.8% or more, Swine serum removal 60.7% in antigen liquid；Removal rate of impure protein is up to 98% after 5 times of concentrations, and rate of recovery of antigen 62%, Swine serum content and Proantigen liquid phase are than reducing by 90.3%.Simultaneously as this method treating capacity is big, processing speed is fast, easy to operate, process stabilizing, therefore automation or semi-automatic production may be implemented.
Description
Technical field
The present invention relates to antigenic substance technical field of purification, further to the purification technique of the mycoplasma for vaccine,
More particularly to a kind of method for purifying mycoplasma hyopneumoniae.
Background technique
Mycoplasmal pneumonia is that one kind is drawn by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp)
The Chronic exposure respiratory infectious disease risen, to pant as cardinal symptom, also known as swine enzootic pneumonia.The disease has a very wide distribution, infects
Rate is high, the death rate is low, illness pig decreased growth, feed conversion rate reduce, easily cause his bacterium or virus it is secondary or mixed
Sexy dye is closed, such as porcine reproductive and respiratory syndrome virus, circovirus, streptococcus pneumonia, haemophilus parasuis, mycoplasma hyorhinis
And pasteurella multocida etc..
Vaccine is the important biomolecule product of Mycoplasmal pneumonia prevention and treatment, and with antigen performance in the development process of vaccine
It is the most key, from the point of view of using effect level, no matter inactivation antigen or attenuation or antigen should all have significant immunogene
Property, at the same to body stimulate it is small, adverse reaction rate is low, for the immunogenicity obtained, the purifying of antigen is most important.At present
It is auspicious times of mycoplasma hyopneumoniae inactivated vaccine suitable, the adjuvant of application of Pfizer's production using wider inactivated vaccine
Amphigen has preferable immunostimulatory potency.The country using it is more be the weak poison of Mhp168 strain developed by Jiangsu academy of agricultural sciences
Live vaccine, it is immune using pulmonary injection mode.The culture of mycoplasma hyopneumoniae and save big, the traditional culture freeze drying process of difficulty
It can cause seriously to cause allergic reaction after serum used or other protein injections, therefore prepare high-purity antigen, removal culture medium
The foreign proteins such as middle serum, optimization production of vaccine downstream process are extremely urgent.
In the vaccine preparation technique of the prior art, the purification effect of mycoplasma hyopneumoniae antigen have it is to be hoisted, on the one hand its
Rate of recovery of antigen is lower, while there is also foreign proteins to remove incomplete phenomenon, in addition, the correlation technique process of the prior art
It is cumbersome, it is not easy to production-scale amplification.
Summary of the invention
The present invention is directed to be directed to the technological deficiency of the prior art, a kind of method for purifying mycoplasma hyopneumoniae is provided, with solution
The certainly low technical problem of the mycoplasma hyopneumoniae purifying process rate of recovery of antigen of the prior art.
Another technical problem to be solved by the present invention is that the mycoplasma hyopneumoniae purifying process of the prior art is difficult to sufficiently go
Except impurity.
The invention solves another technical problem be the prior art mycoplasma hyopneumoniae purifying process treating capacity it is limited,
It is unfavorable for production-scale amplification.
To realize the above technical purpose, the invention adopts the following technical scheme:
A method of purifying mycoplasma hyopneumoniae, comprising the following steps:
1) mycoplasma hyopneumoniae antigen culture solution is taken, clarifying treatment, collecting product is to clarify antigen liquid；
2) the clarification antigen liquid that step 1) obtains is concentrated by ultrafiltration:
3) after step 2) products therefrom being carried out 3~7 times of volume filter washes with buffer, product is collected.
Preferably, antigen contained in step 1) the mycoplasma hyopneumoniae antigen culture solution is mycoplasma hyopneumoniae
Active antigen or the mycoplasma hyopneumoniae antigen of inactivation.
Preferably, the step 1) clarifying treatment is realized using filter membrane, film packet or hollow fiber ultrafiltration membrane.
Preferably, the step 2) ultrafiltration concentration is realized using doughnut membrane filtration system.
Preferably, hollow fiber column aperture is 300~700KD in the doughnut membrane filtration system.
Preferably, the multiple that step 2) is concentrated by ultrafiltration is 2~5 times of volumes.
Preferably, shear rate is 2000~6000sec during the step 2) ultrafiltration concentration-1, more preferably, it is
4000sec-1。
Preferably, transmembrane pressure is that 5~10psi is more preferably 7.5psi during the step 2) ultrafiltration concentration.
Preferably, buffer described in step 3) is the PBS solution that pH value is 7.2~7.4.
Preferably, the step 3) filter wash is isometric filter wash.
Preferably, ultrafiltration concentration described in step 2), step 3) the filter wash process are to collect phegma, discard
Cross liquid.
In above technical scheme, ultrafiltration concentration described in step 2) antigen liquid can be carried out 2~20 times, it is even more big
The concentration of volume.
The present invention provides it is a kind of purify mycoplasma hyopneumoniae method, the technical solution with antigen culture solution directly as
Raw material, successively clarified processing are concentrated by ultrafiltration, the i.e. announcement completion of filter wash three step process, are designed on this basis based on laboratory facilities
Used process unit, low using hollow-fibre membrane shearing force, dust containing capacity is high feature, and operating condition is optimized, lead to
Cross transmembrane pressure, the restriction of shear rate ensure that antigen homeostasis, and ensure removal of impurity.
Using this method Purification of Pig mycoplasma pneumoniae antigen, in the case where being concentrated, rate of recovery of antigen reaches
87.8% or more, Swine serum removal 60.7% in antigen liquid；Removal rate of impure protein is up to 98% after 5 times of concentrations, rate of recovery of antigen
62%, Swine serum content and Proantigen liquid phase are than reducing by 90.3%.Simultaneously as this method treating capacity is big, processing speed is fast, behaviour
Make simple, process stabilizing, therefore automation or semi-automatic production may be implemented.The mycoplasma hyopneumoniae purified using this method
The vaccine of antigen preparation, for reducing the side reaction of i (mycoplasma hyopneumoniae) vaccine product, Improving The Quality of Products is of great significance.
Specific embodiment
Below by specific embodiments of the present invention will be described in detail.In order to avoid excessive unnecessary details, In
It will not be described in detail in following embodiment to belonging to well known structure or function.
Approximating language used in following embodiment can be used for quantitative expression, show in the feelings for not changing basic function
Quantity is allowed to have certain variation under condition.Therefore, this is not limited to accurately with the modified numerical value of the language such as " about ", " left and right " institute
Numerical value itself.In some embodiments, the range for allowing its modified numerical value in positive and negative 10 (10%) " about " is indicated
Interior variation, for example, any numerical value that can be between 90 to 110 that " about 100 " indicate.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
It may be related with the precision of measuring instrument.
In addition to being defined, technical and scientific term used in following embodiment has and fields technology people of the present invention
The identical meanings that member is commonly understood by.
Test reagent consumptive material used in following embodiment is unless otherwise specified conventional biochemical reagent；The experiment
Method is unless otherwise specified conventional method；Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result
It is averaged；% in following embodiment is unless otherwise instructed mass percentage.
Embodiment 1
The preparation of mycoplasma hyopneumoniae antigen
By fermenter volume 70% be added mycoplasma hyopneumoniae fluid nutrient medium, 121 DEG C of sterilizing 20min, to culture medium oneself
When being so cooled to 37 DEG C, 20% Swine serum is added, stirs evenly；It is former with pig pneumonia branch by 10% access production of culture medium total amount
Body seed liquor, 37 DEG C stir culture 3~7 days, harvest bacterium solution when pH value drops to 6.8 by 7.5.
Embodiment 2
The purifying of mycoplasma hyopneumoniae antigen
The mycoplasma hyopneumoniae antigen liquid of harvest is clarified using 3~6um filter membrane；
Clear antigen liquid is purified using the Hollow fiber systems of 300KD, shearing force is controlled in 4000s-1, cross-film
It presses TMP control in 5~10psi, antigen liquid is concentrated into the 1/2 of original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrate is diluted to original volume with sterile PBS (PH7.2), shearing force is controlled in 4000s-1, cross-film
It presses TMP control in 5~10psi, antigen liquid is concentrated into original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrate is diluted to original volume with sterile PBS (PH7.2), shearing force is controlled in 4000s-1, cross-film
It presses TMP control in 5~10psi, antigen liquid is concentrated into original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrate is diluted to original volume with sterile PBS (PH7.2), the trapped fluid of harvest is the pig purified
Mycoplasma pneumoniae antigen.
Below by way of total protein content after the measurement before purification of BCA method, and albumen removal rate, experimental result such as table are calculated with this
Shown in 1.By rate of recovery of antigen after the measurement before purification of double-antibody sandwich Elisa method, experimental result is as shown in table 2.Pass through pig blood
Rear Swine serum residual volume, experimental result are as shown in table 3 before purification for pure protein residue kit measurement.
The total protein content rear before purification and corresponding albumen removal rate that table 1 is detected with BCA method
| (mg/ml) before purification | (mg/ml) after purification | Albumen removal rate |
| 16.4 | 13.9 | 15.2% |
The rate of recovery of antigen rear before purification that table 2 is detected with double-antibody sandwich Elisa method
The Swine serum residual volume rear before purification that table 3 is detected with porcine serum albumin remaining reagent box
| Sample ID | Concentration ug/ml | Volume ml | Quality mg | Swine serum removal rate |
| Before purification | 315.9 | 200 | 63.18 | - |
| After purification | 132.8 | 200 | 26.56 | 58% |
Embodiment 3
The purifying of mycoplasma hyopneumoniae inactivation antigen
Harvest mycoplasma hyopneumoniae antigen liquid by final concentration 0.2% be added formaldehyde, 37 DEG C inactivation for 24 hours；
The mycoplasma hyopneumoniae antigen liquid of inactivation 3~6um filter membrane is clarified；
Clear antigen liquid is purified using the Hollow fiber systems of 300KD, shearing force is controlled in 4000s-1, cross-film
It presses TMP control in 5~10psi, antigen liquid is concentrated into the 1/2 of original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrate is diluted to original volume with sterile PBS (PH7.2), shearing force is controlled in 4000s-1, cross-film
It presses TMP control in 5~10psi, antigen liquid is concentrated into original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrate is diluted to original volume with sterile PBS (PH7.2), shearing force is controlled in 4000s-1, cross-film
It presses TMP control in 5~10psi, antigen liquid is concentrated into original volume, abandons permeate, retains trapped fluid；
Above-mentioned antigen concentrate is diluted to original volume with sterile PBS (PH7.2), the trapped fluid of harvest is the pig purified
Mycoplasma pneumoniae antigen.
Below by way of total protein content after the measurement before purification of BCA method, and albumen removal rate, experimental result such as table are calculated with this
Shown in 4.By rate of recovery of antigen after the measurement before purification of double-antibody sandwich Elisa method, experimental result is as shown in table 5.Pass through pig blood
Rear Swine serum residual volume, experimental result are as shown in table 6 before purification for pure protein residue kit measurement.
The total protein content rear before purification and corresponding albumen removal rate that table 4 is detected with BCA method
| (mg/ml) before purification | (mg/ml) after purification | Albumen removal rate |
| 16.4 | 14.2 | 13.4% |
The rate of recovery of antigen rear before purification that table 5 is detected with double-antibody sandwich Elisa method
The Swine serum residual volume rear before purification that table 6 is detected with porcine serum albumin remaining reagent box
| Sample ID | Concentration ug/ml | Volume ml | Quality mg | Swine serum removal rate |
| 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
The mycoplasma hyopneumoniae antigen liquid of harvest is clarified using 3~6um filter membrane；
Clear antigen liquid is concentrated and purified using the Hollow fiber systems of 300KD, shearing force is controlled in 4000s-1,
Transmembrane pressure TMP control is concentrated into the 1/5 of original volume in 5~10psi, by antigen liquid, abandons permeate, retains trapped fluid；
Above-mentioned twice of antigen concentrate is diluted with sterile PBS (PH7.2), shearing force is controlled in 4000s-1, transmembrane pressure TMP
Control is concentrated into original volume in 5~10psi, by antigen liquid, abandons permeate, retains trapped fluid；
Above-mentioned twice of antigen concentrate is diluted with sterile PBS (PH7.2), shearing force is controlled in 4000s-1, transmembrane pressure TMP
Control is concentrated into original volume in 5~10psi, by antigen liquid, abandons permeate, retains trapped fluid；
Above-mentioned twice of antigen concentrate is diluted with sterile PBS (PH7.2), shearing force is controlled in 4000s-1, transmembrane pressure TMP
Control is concentrated into original volume in 5~10psi, by antigen liquid, abandons permeate, and harvest trapped fluid is the pig pneumonia branch for purifying concentration
Pathogen inactivation antigen.
Below by way of total protein content after the measurement before purification of BCA method, and albumen removal rate, experimental result such as table are calculated with this
Shown in 7.By rate of recovery of antigen after the measurement before purification of double-antibody sandwich Elisa method, experimental result is as shown in table 8.Pass through pig blood
Rear Swine serum residual volume, experimental result are as shown in table 9 before purification for pure protein residue kit measurement.
The total protein content rear before purification and corresponding albumen removal rate that table 7 is detected with BCA method
The rate of recovery of antigen rear before purification that table 8 is detected with double-antibody sandwich Elisa method
The Swine serum residual volume rear before purification that table 9 is detected with porcine serum albumin remaining reagent box
| Sample ID | Concentration ug/ml | Volume ml | Quality mg | Swine serum removal rate |
| Stoste | 315.9 | 200 | 63.18 | - |
| After concentration | 152.5 | 40 | 6.1 | 90.3% |
Embodiment 5
Efficacy test after before purification
1.5~2.0kg of weight healthy rabbits (mycoplasma hyopneumoniae serum antibody IHA potency is not higher than 1:5) 15, wherein
Inactivation antigen 0.5ml, 5 hind leg muscle injection embodiments 4 inactivate 5 each hind leg muscle injection embodiments 4 after purification before 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,
It takes a blood sample together with control group, detects rabbit anteserum mycoplasma hyopneumoniae antibody titer with IHA method.Effect testing result such as 10 institute of table
Show:
The forward and backward influence to antibody titer after immunity inoculation of 10 mycoplasma hyopneumoniae antigen purification of table
Embodiment 6
A method of purifying mycoplasma hyopneumoniae, comprising the following steps:
1) mycoplasma hyopneumoniae antigen culture solution is taken, clarifying treatment, collecting product is to clarify antigen liquid；
2) the clarification antigen liquid that step 1) obtains is concentrated by ultrafiltration:
3) after step 2) products therefrom being carried out 3 times of volume filter washes with buffer, product is collected.
On the basis of above technical scheme, meet the following conditions:
Antigen contained in step 1) the mycoplasma hyopneumoniae antigen culture solution be mycoplasma hyopneumoniae active antigen or
The mycoplasma hyopneumoniae antigen of inactivation.
Step 1) the clarifying treatment is realized using filter membrane, film packet or hollow fiber ultrafiltration membrane.
Step 2) the ultrafiltration concentration is realized using doughnut membrane filtration system.
Hollow fiber column aperture is 300KD in the doughnut membrane filtration system.
The multiple that step 2) is concentrated by ultrafiltration is 2 times of volumes.
During the step 2) ultrafiltration concentration, shear rate 2000sec-1。
During the step 2) ultrafiltration concentration, transmembrane pressure 5psi.
Buffer described in step 3) is the PBS solution that pH value is 7.2.
Step 3) the filter wash is isometric filter wash.
Embodiment 7
A method of purifying mycoplasma hyopneumoniae, comprising the following steps:
1) mycoplasma hyopneumoniae antigen culture solution is taken, clarifying treatment, collecting product is to clarify antigen liquid；
2) the clarification antigen liquid that step 1) obtains is concentrated by ultrafiltration:
3) after step 2) products therefrom being carried out 7 times of volume filter washes with buffer, product is collected.
On the basis of above technical scheme, meet the following conditions:
Hollow fiber column aperture is 700KD in the doughnut membrane filtration system.
The multiple that step 2) is concentrated by ultrafiltration is 5 times of volumes.
During the step 2) ultrafiltration concentration, shear rate 6000sec-1。
During the step 2) ultrafiltration concentration, transmembrane pressure 10psi.
Buffer described in step 3) is the PBS solution that pH value is 7.4.
Embodiment 7
A method of purifying mycoplasma hyopneumoniae, comprising the following steps:
1) mycoplasma hyopneumoniae antigen culture solution is taken, clarifying treatment, collecting product is to clarify antigen liquid；
2) the clarification antigen liquid that step 1) obtains is concentrated by ultrafiltration:
3) after step 2) products therefrom being carried out 5 times of volume filter washes with buffer, product is collected.
The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention,
It is not intended to limit the invention.All any modifications, equivalent replacements, and improvements etc. done in application range of the invention, should all
It is included within protection scope of the present invention.
Claims (6)
1. a kind of method of Purification of Pig mycoplasma pneumoniae antigen, it is characterised in that the following steps are included:
1) mycoplasma hyopneumoniae antigen culture solution is taken, clarifying treatment, collecting product is to clarify antigen liquid；
2) clarification antigen liquid is concentrated and purified using the Hollow fiber systems of 300KD, shearing force is controlled in 4000sec-1, across
Membrane pressure TMP control is concentrated into the 1/5 of clarification antigen liquid volume in 5-l0psi, by antigen liquid, abandons permeate, retains trapped fluid；
3) above-mentioned twice of antigen concentrate is diluted with the PBS of sterile PH7.2, shearing force is controlled in 4000sec-1, transmembrane pressure
TMP control is concentrated into the volume of above-mentioned antigen concentrate in 5-l0psi, abandons permeate, retains trapped fluid；
4) above-mentioned twice of antigen concentrate is diluted with the PBS of sterile PH7.2, shearing force is controlled in 4000sec-1, transmembrane pressure
TMP control is concentrated into the volume of above-mentioned antigen concentrate in 5-l0psi, abandons permeate, retains trapped fluid；
5) above-mentioned twice of antigen concentrate is diluted with the PBS of sterile PH7.2, shearing force is controlled in 4000sec-1, transmembrane pressure
TMP control is concentrated into the volume of above-mentioned antigen concentrate in 5-l0psi, abandons permeate, and harvest trapped fluid is to purify concentration
Mycoplasma hyopneumoniae antigen.
2. a kind of method of Purification of Pig mycoplasma pneumoniae antigen according to claim 1, which is characterized in that the pig of step 1)
3-6 μm of filter membrane clarification of pneumonia mycoplasma original fluid application.
3. a kind of method of Purification of Pig mycoplasma pneumoniae antigen according to claim 1, it is characterised in that step 1) is described
Antigen contained in mycoplasma hyopneumoniae antigen culture solution is the mycoplasma hyopneumoniae of mycoplasma hyopneumoniae active antigen or inactivation
Antigen.
4. a kind of method of Purification of Pig mycoplasma pneumoniae antigen according to claim 1, it is characterised in that step 1) is described
Clarifying treatment is realized using filter membrane.
5. a kind of method of Purification of Pig mycoplasma pneumoniae antigen according to claim 1, it is characterised in that step 1) is described
Clarifying treatment is realized using film packet.
6. a kind of method of Purification of Pig mycoplasma pneumoniae antigen according to claim 1, it is characterised in that step 1) is described
Clarifying treatment is realized using hollow fiber ultrafiltration membrane.
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| CN104271154A (en) * | 2012-04-04 | 2015-01-07 | 硕腾有限责任公司 | Mycoplasma hyopneumoniae vaccine |
| CN104338125A (en) * | 2013-07-29 | 2015-02-11 | 普莱柯生物工程股份有限公司 | Application of mycoplasma hyopneumoniae antigen in prevention and treatment of porcine respiratory disease complex |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104271154A (en) * | 2012-04-04 | 2015-01-07 | 硕腾有限责任公司 | Mycoplasma hyopneumoniae vaccine |
| CN104338125A (en) * | 2013-07-29 | 2015-02-11 | 普莱柯生物工程股份有限公司 | Application of mycoplasma hyopneumoniae antigen in prevention and treatment of porcine respiratory disease complex |
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| Title |
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| 一株猪肺炎支原体HN0613株的分离鉴定;廖永洪等;《中国兽药杂志》;20130331;第47卷(第3期);第4-6、10页 * |
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