CN104558166B - The method for reducing non-specific antibody in anti-GCRV polyclonal antibody - Google Patents
The method for reducing non-specific antibody in anti-GCRV polyclonal antibody Download PDFInfo
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- CN104558166B CN104558166B CN201510050935.5A CN201510050935A CN104558166B CN 104558166 B CN104558166 B CN 104558166B CN 201510050935 A CN201510050935 A CN 201510050935A CN 104558166 B CN104558166 B CN 104558166B
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Abstract
The invention discloses a kind of method for reducing non-specific antibody in anti-GCRV polyclonal antibody, comprise the following steps successively:GCRV is concentrated using film bag；2) GCRV is tentatively sieved using sieve chromatography；3) cation exchange chromatography GCRV is used；4) rabbit-anti GCRV polyclonal antibody is prepared；5) non-specific antibody in anti-GCRV polyclonal antibody is removed.By the method for purifying, the purity and titre of grass carp hemorrhage virus are improved, reduces non-specific polyclonal antibody caused by other non-viral albumen.Combined by polyclonal antibody with CIK cell fragment, remove the non-specific antibody in the polyclonal antibody of part.
Description
Technical field
The present invention relates to veterinary biologics field, and it is more to reduce anti-GCRV more specifically to one kind
The method of non-specific antibody in clonal antibody.
Background technology
Antigenic stimulus body, immunological response is produced, what is synthesized and secreted by the thick liquid cell of body has specificity with antigen
One Histaglobin of binding ability, here it is immunoglobulin, this immunoglobulin for having specific binding capacity with antigen
It is exactly antibody.Antigen is typically what is be made up of multiple antigenic determinants, stimulates body by a kind of antigenic determinant, is drenched by a B
Bar cell receive the antigen caused by antibody be referred to as monoclonal antibody.Body is stimulated by a variety of antigenic determinants, correspondingly
With regard to producing various monoclonal antibodies, these monoclonal antibodies mixed in together are exactly polyclonal antibody, are produced in body
Raw antibody is exactly polyclonal antibody.
Hemorrhagic disease of grass carp is a kind of communicable disease for seriously endangering Grass Carp Juveniles, and the disease has each organ of grass carp, tissue
Different degrees of hyperemia, bleeding.The pathogen of hemorrhagic disease of grass carp is arc reovirus virus.At present, hemorrhagic disease of grass carp uses grass carp
Bleeding disease vaccine carries out artificial immunity prevention.
Meeting when whether hemorrhagic disease of grass carp attenuated live vaccines pollute other exogenous viruses is detected in China's veterinary biologicses
It is using the polyclonal antibody to anti-grass carp hemorrhage virus, i.e., thin using kind of CIK is followed by with grass carp hemorrhage virus in polyclonal antibody
Whether born of the same parents, observation CIK cell there is lesion.CIK cell is epithelioid cell, adherent not too strong；And prepared by conventional method
Polyclonal antibody contains very more non-specific antibodies, and serum that these antibody are not only able in anti-culture, to be also resistant to CIK thin
A series of materials of born of the same parents' fragment etc..Using in this polyclonal antibody and after GCHV, inoculation CIK cell can cause
There is lesion caused by similar virus in cell.Therefore, the potency of polyclonal antibody prepared by conventional method is relatively low.
The content of the invention
The technical problem to be solved in the present invention is, for the drawbacks described above of prior art, there is provided one kind reduces anti-grass carp
The method of non-specific antibody in reovirus polyclonal antibody.
The technical solution adopted for the present invention to solve the technical problems is：It is more to provide a kind of anti-GCRV of reduction
The method of non-specific antibody, comprises the following steps successively in clonal antibody:
2) GCRV is tentatively sieved using sieve chromatography；
3) cation exchange chromatography GCRV is used；
4) rabbit-anti GCRV polyclonal antibody is prepared；
5) non-specific antibody in anti-GCRV polyclonal antibody is removed.
The method of the present invention for reducing non-specific antibody in anti-GCRV polyclonal antibody, wherein,
Sieve chromatography in the step 2) uses Sepharose 6fast flow gel columns；By GCRV loading extremely
Gel column, with 0.005-0.02mol/L, pH=6.0-8.0 Tris-HCl buffer solutions are eluted, and collect first eluting peak
Sample.
The method of the present invention for reducing non-specific antibody in anti-GCRV polyclonal antibody, wherein,
Sepharose is balanced with 0.02-0.03mol/L, pH=6.0-8.0 phosphate buffer before loading in the step 2)
6fast flow gel columns.
The method of the present invention for reducing non-specific antibody in anti-GCRV polyclonal antibody, wherein,
The DEAE anion-exchange columns that ion-exchange chromatography in the step 3) uses, the grass carp of primary dcreening operation in step 2) is exhaled into the lonely disease of intestines
Venom loading is eluted to DEAE anion-exchange columns with 0.1-1.0M NaCl solution.
The method of the present invention for reducing non-specific antibody in anti-GCRV polyclonal antibody, wherein,
The 0.01-0.3mol/L before loading in the step 3), pH=6.0-8.0 Tri s-HCl buffer solutions balance DEAE anion
Exchange column.
The method of the present invention for reducing non-specific antibody in anti-GCRV polyclonal antibody, wherein,
The step 5) comprises the following steps：
51) CIK cell is cultivated on Gibco M199 culture mediums, after CIK cell growth is fine and close, outwells Gibco's
M199 culture mediums, 0.2-0.3% pancreatin is added, after CIK cell digests well, outwell pancreatin and add 15-20mLPBS solution and blow
Dissipate and reclaim CIK cell, then centrifuge 10-30 minutes at 1500-3000 revs/min, remove supernatant, it is molten to add 15-20mL PBS
Liquid, 8000 revs/min of centrifugation 10-30 minutes, remove supernatant, obtain CIK cell fragment after -20 DEG C of multigelations；
52) it is polyclonal that the GCRV prepared in step 4) is added in the CIK cell fragment that step 51) obtains
Antibody serum 10mL, suspend precipitation, and 8000 revs/min centrifuge 20 minutes after 37 DEG C of incubation 1h, collect supernatant solution.
The method of the present invention for reducing non-specific antibody in anti-GCRV polyclonal antibody, wherein,
Also include the step 1) concentrated using film bag to GCRV before step 2).
The method of the present invention for reducing non-specific antibody in anti-GCRV polyclonal antibody, wherein,
The step 1) is as follows：By the 3000-6000 revs/min of centrifugation of GCRV liquid, centrifugation time is 20-50 minutes, is removed
The bulk matter such as cell fragment in GCRV liquid, then concentrated by retaining aperture for 30-300K film bags.
Implement the method for non-specific antibody in the anti-GCRV polyclonal antibody of reduction of the present invention and have with
Lower beneficial effect：
(1) GCRV is concentrated by film bag, recycles the method progress of molecular sieve, ion exchange pure
Change, remove in nutrient solution more than 85% foreign protein, virus can reclaim 64%, the titre and purity improved.Wherein, film bag
What is removed is less than the protein in film bag aperture, while has the function that concentration；And what sieve chromatography removed is exhaled with grass carp
The protein that the lonely virus of intestines can separate very well in chromatography process；And ion exchange remove be and GCRV
The negatively charged protein to differ greatly.
(2) combined by the anti-GCRV polyclonal antibody of preparation with CIK cell fragment, remove the anti-grass in part
Non-specific antibody in fish reovirus polyclonal antibody, reduce the part in anti-GCRV polyclonal antibody
Non-specific antibody, and then reduce its toxicity and its antibody titer of raising to CIK cell.The virales of purifying is that reduction is more
Non-specific antibody in clonal antibody, and the anti-GCRV polyclonal antibody of rabbit anteserum has anti-CIK cell broken
The non-specific antibody of the various albumen such as piece；Polyclonal antibody is prepared using the GCRV of purifying and uses CIK thin
Non-specific antibody etc. is for lowering the toxicity and raising Anti-TNF-α to CIK cell in born of the same parents' fragment and in polyclonal antibody
The potency of body.
Brief description of the drawings
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing：
Fig. 1 is the separating effect by Sepharose 6fast flow sieve chromatographies for GCRV liquid；
Fig. 2 is the separating effect by DEAE- glucan ion-exchange chromatographies for GCRV liquid.
Embodiment
Below, with reference to accompanying drawing and embodiment, the present invention is described further：
The present invention provides a kind of method for reducing non-specific antibody in anti-GCRV polyclonal antibody, successively
Comprise the following steps:
1) GCRV is concentrated using film bag：
By the 3000-6000 revs/min of centrifugation of GCRV liquid, centrifugation time is 20-50 minutes, removes grass carp and exhales intestines
The bulk matter such as cell fragment in lonely virus liquid, wherein, preferable centrifugal condition is 3500-5000 revs/min, and centrifugation time should
More than 30 minutes；Then concentrated by retaining aperture for 30-300K film bags, wherein, retention aperture is more preferably less than 100K,
The GCRV liquid concentrated.
2) GCRV is tentatively sieved using sieve chromatography：
Sieve chromatography uses Sepharose 6fas t flow gel columns；With 0.02-0.03mol/L, pH=before loading
6.0-8.0 phosphate buffer balance Sepharose 6fast flow gel columns, specifically, with 0.025mol/L, pH=
7.0 phosphate buffer balance Sepharose 6fast flow gel columns；Then grass carp step 1) being concentrated to give exhales
Intestines orphan's virus liquid loading is to gel column, and with 0.005-0.02mol/L, pH=6.0-8.0 Tris-HCl buffer solutions are eluted,
Specifically, eluted with 0.01mol/L, pH=7.0 Tris-HCl buffer solutions, collect first eluting peak sample, i.e., preliminary sieve
The GCRV liquid divided.
3) cation exchange chromatography GCRV is used：
The DEAE anion-exchange columns that ion-exchange chromatography uses, with 0.01-0.3mol/L, pH=6.0-8.0 before loading
Tris-HCl buffer solutions balance DEAE anion-exchange columns, specifically, with 0.01mol/L, pH=7.0 Tris-HCl is buffered
Liquid balances DEAE anion-exchange columns；The GCRV liquid loading for the preliminary screening that step 2) is obtained to DEAE the moon from
Sub- exchange column, eluted with 0.1-1.0M NaCl solution, collect the GCRV liquid that eluent produces purifying.
4) rabbit-anti GCRV polyclonal antibody is prepared：
The GCHV liquid of the purifying obtained using step 3), conventionally prepares rabbit-anti hemorrhagic disease of grass carp
The polyclonal antibody of virus.
5) non-specific antibody in anti-GCRV polyclonal antibody is removed：
Wherein, the step 5) comprises the following steps：
51) CIK cell is cultivated on the T25 for the M199 culture mediums for filling Gibco flat bottle, treats that CIK cell growth causes
After close, Gibco M199 culture mediums are outwelled, 0.2-0.3% pancreatin is added, specifically, adds 0.25% pancreatin, treat that CIK is thin
Born of the same parents digest it is good after, outwell pancreatin and add 15-20mLPBS solution and dispel and reclaim CIK cell, specifically, add 20mL PBS
Solution；Then 10-30 minutes are centrifuged at 1500-3000 revs/min, removes supernatant, added 15-20mL PBS solutions, specifically, add
Enter 20mL PBS solutions；8000 revs/min of centrifugation 10-30 minutes, remove supernatant, it is broken to obtain CIK cell after -20 DEG C of multigelations
Piece；
52) more grams of the anti-GCRV prepared in step 4) is added in the CIK cell fragment that step 51) obtains
Grand antibody serum 10mL, suspend precipitation, and 8000 revs/min centrifuge 20 minutes after 37 DEG C of incubation 1h, collect supernatant solution, repeat to walk
Rapid 52) 1 time, just eliminating can be combined with each other and shadow in anti-GCRV polyclonal antibody with CIK cell
The material of Hemapoiesis is rung, produces the anti-GCRV polyclonal antibody for removing part non-specific antibody.
In this step with can be with CIK cell in CIK cell fragment and in anti-GCRV polyclonal antibody
The non-specific antibody of fragment reaction, to reduce the toxicity of CIK cell；It is adherent shaky mainly due to CIK cell, it is polyclonal
Antibody influences its growth.
Below, the anti-GCRV polyclonal antibody of removal non-specific antibody prepared by the detection present invention is dividing
Situation is removed from viral recovering state in purge process and albumen.
Polyclonal antibody is prepared using the method for the present invention：3500 revs/min of GCRV liquid centrifuges 20 minutes, goes
Fall the bulk matter such as cell fragment in GCRV liquid.Then concentrated by retaining aperture for 30K film bags.Concentration
Sample afterwards uses for lower following experiment.Sieve chromatography is carried out first tentatively to sieve GCRV, is used
Filler is Sepharose 6fast flow；Purification condition is to be balanced with 0.025mol/L, pH=7.0 phosphate buffer
Sepharose 6fast flow gel columns.Concentrating virus liquid is splined on Sepharose 6fast flow gel columns, used
0.025mol/L, pH=7.0 phosphate buffer elution.First eluting peak sample is collected, sees Fig. 1, grass carp exhales the lonely disease of intestines
Venom by the separating effects of Sepharose 6fast flow sieve chromatographies, wherein, curve represents GCRV
Protein concentration in liquid；Black arrow represents the eluting peak of GCRV.
Above-mentioned eluting peak carries out consummate, purification condition 0.01mol/L using DEAE anion-exchange columns, pH=7.0's
Tri s-HCl buffer solutions balance DEAE anion-exchange columns, and the virus liquid that previous step purifies is splined on into DEAE anion exchanges
Post, eluted with 0.5M NaCl solution, it is that GCRV liquid passes through DEAE- glucan ion exchange layers to see Fig. 2, Fig. 2
The separating effect of analysis, wherein, curve represents protein concentration in GCRV liquid；Arrow represents fish reovirus
Eluting peak.Finally concentrated using 30K film bags.
Each step Virus Sample is taken to carry out virus titer measure, measurement result is shown in Table 1.Wherein, the sample that each step obtains
The measure of protein content is detected using Coomassie brilliant G-250 method in product.
The GCRV rate of recovery=(sample virus TCID after processing50Volume after × processing)/(before processing sample disease
Malicious TCID50 × processing front volume) × 100%, it is shown in Table 1.
Foreign protein clearance=(total protein content after sample total protein content-sample treatment)/sample total protein in sample
Content × 100%.
The GCRV of table 1 viral recovering state and albumen removal situation during isolating and purifying
| Separating step | Protein content | Viral level | Volume |
| Step 1)：Film bag concentrates (30K) | 93% | 98% | 25ml |
| Step 2)：Gel column | 40% | 85% | 75ml |
| Step 3)：Ion exchange column | 20% | 65% | 50ml |
| Step 5)：Film bag concentrates (30K) | 13% | 64% | 15ml |
From table 1, during GCRV of the invention isolates and purifies, the purity of GCRV and
Titre greatly improves, and reduces non-specific polyclonal antibody caused by other non-viral albumen.
Below, the neutralization titer for the polyclonal antibody that the detection polyclonal antibody for preparing of the present invention is prepared with conventional method and
Both toxicity to CIK cell.
1st, detect the rabbit-anti GCRV polyclonal antibody of conventional method preparation and prepared using inventive embodiments
Polyclonal antibody to the toxicity of CIK cell, both toxicity to CIK cell of contrast.
Cell toxicity determination method is to dilute the polyclonal positive using M199 culture mediums (directly buying in Gibco companies)
Serum, extension rate are 2 times, 4 times, 8 times and 16 times dilutions.Then add CIK cell and culture 1 hour is carried out at 28 DEG C, point
Cell growth condition was not observed in the 1-7 days, polyclonal serum is shown in Table 2 to the toxicity profile of CIK cell.
The conventional method prepares polyclonal antibody and referred to, after breeding grass carp hemorrhage virus, without purifying, direct immunization
Rabbit, and the polyclonal antibody prepared.
Toxicity profile of the polyclonal serum of table 2 to CIK cell
Note："+" represents that cell growth state is good；"-" represents cell growth abnormity or death.
From table 2, when polyclonal serum concentration is higher, after polyclonal antibody addition CIK cell prepared by conventional method,
There is the phenomenon of growth failure or death in CIK cell, illustrate the polyclonal antibody of conventional method preparation to the toxicity of CIK cell compared with
Greatly；And the inventive method prepare polyclonal antibody add CIK cell after, CIK cell do not occur growth failure or death
Phenomenon, illustrate that polyclonal antibody prepared by conventional method is smaller to the toxicity of CIK cell.
2nd, neutralization titer determines
Neutralization titer assay method is that 37 DEG C of the ratio such as virus-culturing fluid and polyclonal serum mixing is incubated 30 minutes, its virus
Content is 107.5TCID50/ 0.1mL, then determine its TCID50。
Neutralization situation of the polyclonal antibody of table 3 to GCRV
Note："+" represents to neutralize completely；"-" represents not exclusively to neutralize
Neutralization titer measure is to evaluate the potency of polyclonal antibody prepared by distinct methods, as shown in Table 3, is passed through in the present invention
Polyclonal antibody prepared by GCRV after purification is higher than the polyclonal antibody potency prepared in conventional method.
It will be apparent to those skilled in the art that technical scheme that can be as described above and design, make other various
Corresponding change and deformation, and all these changes and deformation should all belong to the protection domain of the claims in the present invention
Within.
Claims (7)
- A kind of 1. method for reducing non-specific antibody in anti-GCRV polyclonal antibody, it is characterised in that successively Comprise the following steps:2）GCRV is tentatively sieved using sieve chromatography；3）Using cation exchange chromatography GCRV；4）Prepare rabbit-anti GCRV polyclonal antibody；5）Remove the non-specific antibody in anti-GCRV polyclonal antibody;The step 5）Comprise the following steps：51）CIK cell is cultivated on Gibco M199 culture mediums, after CIK cell growth is fine and close, outwells Gibco M199 Culture medium, add 0.2-0.3% pancreatin, after CIK cell digest it is good after, outwell pancreatin and add 15-20mLPBS solution and dispel simultaneously CIK cell is reclaimed, then centrifuges 10-30 minutes at 1500-3000 revs/min, removes supernatant, adds 15-20mL PBS solutions ,- 8000 revs/min of centrifugation 10-30 minutes, remove supernatant, obtain CIK cell fragment after 20 DEG C of multigelations；52）In step 51）Step 4 is added in obtained CIK cell fragment）The anti-GCRV Anti-TNF-α of middle preparation Body serum 10mL, suspend precipitation, and 8000 revs/min centrifuge 20 minutes after 37 DEG C of incubation 1h, collect supernatant solution.
- 2. the method according to claim 1 for reducing non-specific antibody in anti-GCRV polyclonal antibody, Characterized in that, the step 2）In sieve chromatography use the fast flow gel columns of Sepharose 6；Grass carp is exhaled into intestines To gel column, with 0.005-0.02mol/L, pH=6.0-8.0 Tris-HCl buffer solutions are eluted lonely viral loading, are collected First eluting peak sample.
- 3. the method according to claim 2 for reducing non-specific antibody in anti-GCRV polyclonal antibody, Characterized in that, it is described by GCRV loading to gel column before with 0.02-0.03mol/L, pH=6.0-8.0's Phosphate buffer balances the fast flow gel columns of Sepharose 6.
- 4. the method according to claim 1 for reducing non-specific antibody in anti-GCRV polyclonal antibody, Characterized in that, the step 3）In ion-exchange chromatography use DEAE anion-exchange columns, by step 2）Middle primary dcreening operation GCRV liquid loading is eluted to DEAE anion-exchange columns with 0.1-1.0M NaCl solution.
- 5. the method according to claim 4 for reducing non-specific antibody in anti-GCRV polyclonal antibody, Characterized in that, described by step 2）Before the GCRV liquid loading of middle primary dcreening operation to DEAE anion-exchange columns with 0.01-0.3mol/L, pH=6.0-8.0 Tris-HCl buffer solutions balance DEAE anion-exchange columns.
- 6. the method according to claim 1 for reducing non-specific antibody in anti-GCRV polyclonal antibody, Characterized in that, in step 2）Also include the step 1 concentrated using film bag to GCRV before）.
- 7. the method according to claim 6 for reducing non-specific antibody in anti-GCRV polyclonal antibody, Characterized in that, the step 1）It is as follows：By the 3000-6000 revs/min of centrifugation of GCRV liquid, centrifugation time 20- 50 minutes, then concentrated by retaining aperture for 30-300K film bags.
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| CN101638427A (en) * | 2008-08-01 | 2010-02-03 | 上海泽润生物科技有限公司 | Method for purifying virus antigens |
| CN104193817A (en) * | 2014-09-05 | 2014-12-10 | 桂林英美特生物技术有限公司 | Process for purifying human retinol binding protein and preparation process of polyclonal antibody thereof |
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| CN101638427A (en) * | 2008-08-01 | 2010-02-03 | 上海泽润生物科技有限公司 | Method for purifying virus antigens |
| CN104193817A (en) * | 2014-09-05 | 2014-12-10 | 桂林英美特生物技术有限公司 | Process for purifying human retinol binding protein and preparation process of polyclonal antibody thereof |
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