CN101032621A - Preparing method of infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs - Google Patents
Preparing method of infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs Download PDFInfo
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- CN101032621A CN101032621A CN 200610013261 CN200610013261A CN101032621A CN 101032621 A CN101032621 A CN 101032621A CN 200610013261 CN200610013261 CN 200610013261 CN 200610013261 A CN200610013261 A CN 200610013261A CN 101032621 A CN101032621 A CN 101032621A
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Abstract
The present invention is preparation process of immunological monoclonal antibody preparation for pig transmissible gastroenteritis virus, and belongs to the field of biomedicine preparing technology. The preparation process includes preparing TGEV monoclonal antibody, preparing Chinese medicine extracting solution, and mixing TGEV monoclonal antibody and Chinese medicine extracting solution in the volume ratio of 1 to 0.8-1.2. The TGEV monoclonal antibody is prepared through injecting paraffin oil into mouse stomach cavity, injecting hybridoma cell in 6-9 days, drawing off ascites in 7-15 days, and centrifuging at 2500-3500 rpm for 8-12 min to obtain supernatant. The Chinese medicine extracting solution is prepared with isatis root and through soaking in water, decocting in water bath, filtering, concentrating filtrate, and adding alcohol to obtain supernatant. The present invention has high anti-infective effect, no medicine residue, no resistance and no toxic side effect.
Description
Technical field
The invention belongs to biological pharmacy technical field, is about a kind of infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method.
Background technology
Because the transmissible gastro-enteritis virus less immunogenic is cultivated relatively difficulty, the development of anti-swine infectious enterogastritis viral monoclonal antibodies is difficult.Though the report of successfully developing the anti-swine infectious enterogastritis viral monoclonal antibodies is arranged, the development the anti-swine infectious enterogastritis viral monoclonal antibodies do not neutralize virus activity, can not be used for clinical treatment.At present, also monoclonal antibody of transmissible gastro-enteritis virus is not made the report that immunity biological agent is used to prevent and treat transmissible gastroenteritis of swine clinically.
Summary of the invention
The technical problem of the present invention for existing in the solution known technology, and a kind of infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method is provided.
It is simple to the purpose of this invention is to provide a kind of technology, clinically transmissible gastroenteritis of swine is had the infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method of good prevention and therapeutic effect.
1. secrete the foundation of anti-TGEV monoclonal antibody hybridoma cell strain
Adopt the BALB/C mice in age 37 weeks of TGEV antigen immune of purification, immunizing dose is each 100ug/ mice.Antigen and equivalent Freund's complete adjuvant fully are mixed into emulsus, and lumbar injection head exempts from, and after 2 weeks, with antigen and the abundant mixing of equivalent incomplete Freund, injects one time two again with method and exempts from; 3d before the cell fusion, tail vein injection does not contain adjuvant antigen booster immunization.
Get immunized mice, extract eyeball blood-letting post-tensioning neck and put to death, be soaked in 5min in 75% the ethanol, aseptic taking-up spleen also divests connective tissue on every side, spleen is placed on the 200 order copper mesh, after pushing gently with the syringe heart, blow and beat gently, collect splenocyte suspension with serum-free DMEM culture fluid, the centrifugal 5min of 200r/min, abandon supernatant, after suspending with serum-free DMEM culture fluid, make splenocyte suspension.
Get the splenocyte suspension and the SP2/O cell suspension of above-mentioned preparation, ratio by cell number fully mixes at 5: 1, and the centrifugal 3min of 1000r/min discards supernatant as far as possible, use then at the bottom of the light finger bomb tube, make the loose one-tenth pasty state of sedimentation cell, put in 37 ℃ of water-baths, in 60S, slowly add the PEG (M:1500) of 0.7ml 50%, rotate simultaneously centrifuge tube gently, in 30S, cell suspension slowly in the suction tube, is left standstill 30S, in 30S, cell suspension slowly blown again and be back in the centrifuge tube.In 2min, slowly add an amount of serum-free DMEM culture fluid immediately, hanged cell gently, the centrifugal 5min of 500r/min abandons supernatant, adds 20ml DMEM-HAT with suspension cell.From CO
2Take out 2 culture plates that added feeder cells in the incubator; Every hole adds 100ul fused cell suspension, puts into 37 ℃, and saturated humidity contains 5%CO
2Incubator in cultivate, every day the observation of cell growing state.5d changes liquid with HAT culture fluid half amount, and 10d uses the HT culture fluid instead.When fused cell grows to covering culture hole 1/2-1/3 size, draw supernatant night work indirect ELISA and detect, be judged to positive hole with hole greater than 2 times of negative control hole OD values.It is high to pick out the OD value from positive hole, and cloning is carried out with limiting dilution assay in the eugonic hybridoma of cell hole.After treating that cell grows up to the clone, get supernatant and do the antibody ELISA detection, filter out the hybridoma cloning again in the positive hole that a clonal growth is only arranged, 3 times repeatedly, obtain the hybridoma cell strain of the anti-TGEV monoclonal antibody of 1 strain energy stably excreting, be numbered 2D6, the results back is frozen standby.
2. the monoclonal antibody of anti-TGEV preparation
(1) extracorporeal culture-ing: will build the hybridoma amplification culture of strain, and treat that cell concentration will reach 10
5Stop to change liquid when/ml is above, it is all dead to continue to cultivate cell.Collect medium centrifugal, 4 ℃ of preservations of supernatant are standby.
(2) induce the ascites method in the body: the autoclaved paraffin oil of 0.5mL is expelled to mouse peritoneal, a week back injection 10
6Individual hybridoma is in mouse peritoneal.After 7～10 days, when the mouse web portion extreme expansion, extract ascites, with the centrifugal 10min of ascites 3000rpm, the supernatant packing ,-20 ℃ of preservations are standby.
3. the preparation of Chinese medicine extraction liquid
Selecting for use has stronger inhibiting Chinese medicine Radix Isatidis to TGEV, adopts decoction and alcohol sedimentation technique to prepare extracting solution.Radix Isatidis is ground, get 1 part of its medicamental pulverata in conical flask, add 10 parts of distilled water, at room temperature after the soaked overnight, place 60 ℃ water-bath to heat 10 minutes, filter immediately, its filtrate is mixed concentrating under reduced pressure (temperature 60-70 ℃, negative pressure 7-8 kPa), concentrated solution adds ethanol, make to contain alcohol amount and reach 80%, to remove impurity such as deproteinize, polysaccharide and inorganic salts, supernatant is extracting solution.The 1ml extracting solution is equivalent to crude drug in whole 1g.Extracting solution is spray dried to powder, preserves standbyly, recovers original volume with preceding dilution.
4. the preparation of anti-TGEV monoclonal antibody immunity biological agent
Get a certain amount of anti-TGEV monoclonal antibody ascites, the dissolving Chinese medicine powder makes it revert to original volume, and adds oligofructose, makes its final concentration reach 10%, and this is the biological oral agents of anti-swine infectious enterogastritis virus monoclonal antibody immunity.For ease of preserving, can be made into lyophilized formulations, or the spray dried drying prescription, recover original volume with preceding dilution.
The present invention takes following technical scheme:
The infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method, comprise virus clone Antibody Preparation and Chinese medicine extraction liquid preparation process, be characterized in: preparation method comprises preparation, monoclonal antibody of transmissible gastro-enteritis virus and the Chinese medicine extraction liquid synthesis process of monoclonal antibody of transmissible gastro-enteritis virus preparation, Chinese medicine extraction liquid:
The preparation of monoclonal antibody of transmissible gastro-enteritis virus is adopted in the body and is induced the ascites method: the autoclaved paraffin oil of 0.3-0.7ml is expelled to mouse peritoneal, injected 10 in 6-9 days
5-10
7Individual hybridoma is in mouse peritoneal, and 7-15 days, extraction ascites at the centrifugal 8-12min of 2500-3500rpm, obtained the supernatant monoclonal antibody of transmissible gastro-enteritis virus with ascites when the mouse web portion extreme expansion.
Chinese medicine extraction liquid selects for use the Chinese medicine Radix Isatidis to adopt the decoction and alcohol sedimentation technique preparation; Radix Isatidis is ground, get 1 part of its medicamental pulverata, add distilled water 8-12 part, soaked 7-15 hour, and placed 50-70 ℃ water-bath to heat 8-12 minute, filter, filtrate is at 60-70 ℃, negative pressure 7-8 kPa condition concentrating under reduced pressure, concentrated solution adds ethanol, makes to contain the alcohol amount and reach 70-90%, and supernatant is extracting solution.
Monoclonal antibody of transmissible gastro-enteritis virus and Chinese medicine extraction liquid synthesis process: monoclonal antibody of transmissible gastro-enteritis virus and Chinese medicine extraction liquid by volume 1: 0.8-1.2 mixed infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs.
The present invention can also adopt following technical measures:
Described infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method is characterized in: Chinese medicine extraction liquid is made lyophilized formulations or is spray dried to powder.
Described infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method, be characterized in: monoclonal antibody of transmissible gastro-enteritis virus dissolving Chinese medicine powder, make it revert to original volume, and adding oligofructose, make its concentration reach 8-12%, obtain monoclonal antibody of transmissible gastro-enteritis virus biological preparation oral agents.
Described infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method is characterized in: preserve more than 2 years, but make lyophilized formulations or be spray dried to the powder long preservation for-10 ℃ to-20 ℃.
Advantage that the present invention has and good effect:
The anti-TGEV monoclonal antibody immunity biological agent of the present invention; merged in having and the strong Chinese medicine Radix Isatidis and the immunostimulant factor that resists absorption and direct deactivation arranged with the McAb of virus activity with to TGEV; strengthened protective effect that the anti-TGEV of piglet infects greatly and to the therapeutical effect of the sick pig of TGE; it is the new high efficiency medicine that prevention and treatment TGE infect; be a kind of pure natural, no drug residue, nuisanceless, the environment-friendly anti-infectives that has no drug resistance, have no side effect, overcome the toxic action and the drug resistance of existing antibiotic and synthetic chemical drugs.Middle pharmaceutically active ingredient wherein possess except self to inactivation of virus and the anti-adsorption, the immunoglobulin in the preparation is had good protective action, avoid the destruction of gastric acid when its oral back is held by gastric.Antibody, Chinese medicine and immunostimulant combined to be used to prevent and treat TGEV and to infect, and has significant advantage and positive effect.
The anti-TGEV immunity biological agent of the present invention preparation both can be oral, and injectable again can lower the M ﹠ M of piglet TGE greatly, has tangible economic benefit and social benefit.
The specific embodiment
For further understanding content of the present invention, characteristics and effect, exemplify following examples now and be elaborated:
The infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method comprises that monoclonal antibody of transmissible gastro-enteritis virus prepares, preparation, monoclonal antibody of transmissible gastro-enteritis virus and the Chinese medicine extraction liquid synthesis process of Chinese medicine extraction liquid:
The preparation of monoclonal antibody of transmissible gastro-enteritis virus:
Adopt in the body and induce the ascites method.The autoclaved paraffin oil of 0.5ml is expelled to mouse peritoneal, and inject 10 a day after tomorrow in week
6Individual hybridoma is in mouse peritoneal.7-10 days, when the mouse web portion extreme expansion, extract ascites, ascites at the centrifugal 10min of 3000rpm, is obtained the supernatant monoclonal antibody of transmissible gastro-enteritis virus.The supernatant packing ,-20 ℃ of preservations are standby.
The preparation of Chinese medicine extraction liquid:
Selecting for use has stronger inhibiting Chinese medicine Radix Isatidis to TGEV, adopts decoction and alcohol sedimentation technique to prepare extracting solution.Radix Isatidis is ground, get 1 part of its medicamental pulverata in conical flask, add 10 parts of distilled water, at room temperature after the soaked overnight, place 60 ℃ water-bath to heat 10 minutes, filter immediately, its filtrate is mixed concentrating under reduced pressure (temperature 60-70 ℃, negative pressure 7-8 kPa), concentrated solution adds ethanol, make to contain alcohol amount and reach 80%, to remove impurity such as deproteinize, polysaccharide and inorganic salts, supernatant is extracting solution.The 1ml extracting solution is equivalent to crude drug in whole 1g.Extracting solution is spray dried to powder, preserves standbyly, recovers original volume with preceding dilution.
The preparation of anti-TGEV monoclonal antibody immunity biological agent:
Get anti-TGEV monoclonal antibody ascites, the dissolving Chinese medicine powder makes it revert to original volume, and adds oligofructose, makes its final concentration reach 10%, and this is the biological oral agents of anti-swine infectious enterogastritis virus monoclonal antibody immunity.For ease of preserving, can be made into lyophilized formulations, or the spray dried drying prescription, recover original volume with preceding dilution.
Claims (4)
1. infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method, comprise virus clone Antibody Preparation and Chinese medicine extraction liquid preparation process, it is characterized in that: preparation method comprises preparation, monoclonal antibody of transmissible gastro-enteritis virus and the Chinese medicine extraction liquid synthesis process of monoclonal antibody of transmissible gastro-enteritis virus preparation, Chinese medicine extraction liquid; The preparation of monoclonal antibody of transmissible gastro-enteritis virus is adopted in the body and is induced the ascites method: the autoclaved paraffin oil of 0.3-0.7ml is expelled to mouse peritoneal, injected 10 in 6-9 days
5-10
7Individual hybridoma is in mouse peritoneal, and 7-15 days, extraction ascites at the centrifugal 8-12min of 2500-3500rpm, obtained the supernatant monoclonal antibody of transmissible gastro-enteritis virus with ascites when the mouse web portion extreme expansion;
Chinese medicine extraction liquid selects for use the Chinese medicine Radix Isatidis to adopt the decoction and alcohol sedimentation technique preparation; Radix Isatidis is ground, get 1 part of its medicamental pulverata, add distilled water 8-12 part, soaked 7-15 hour, and placed 50-70 ℃ water-bath to heat 8-12 minute, filter, filtrate is at 60-70 ℃, negative pressure 7-8 kPa condition concentrating under reduced pressure, concentrated solution adds ethanol, makes to contain the alcohol amount and reach 70-90%, and supernatant is extracting solution;
Monoclonal antibody of transmissible gastro-enteritis virus and Chinese medicine extraction liquid synthesis process: monoclonal antibody of transmissible gastro-enteritis virus and Chinese medicine extraction liquid by volume 1: 0.8-1.2 mixed infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs.
2. infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method according to claim 1 is characterized in that: Chinese medicine extraction liquid is made lyophilized formulations or is spray dried to powder.
3. infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method according to claim 2, it is characterized in that: monoclonal antibody of transmissible gastro-enteritis virus dissolving Chinese medicine powder, make it revert to original volume, and adding oligofructose, make its concentration reach 8-12%, obtain monoclonal antibody of transmissible gastro-enteritis virus biological preparation oral agents.
4. infectious gastroenteritis virus monoclonal antibody immunity biological agent of pigs preparation method according to claim 1 is characterized in that: preserve more than 2 years, but make lyophilized formulations or be spray dried to the powder long preservation for-10 ℃ to-20 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154516A (en) * | 2011-03-22 | 2011-08-17 | 上海交通大学 | Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof |
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2006
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154516A (en) * | 2011-03-22 | 2011-08-17 | 上海交通大学 | Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof |
| CN102154516B (en) * | 2011-03-22 | 2013-05-01 | 上海交通大学 | Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof |
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