CN1546172A - Hyper-immune serum for resisting SARS virus and its preparation - Google Patents
Hyper-immune serum for resisting SARS virus and its preparation Download PDFInfo
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- CN1546172A CN1546172A CNA2003101123725A CN200310112372A CN1546172A CN 1546172 A CN1546172 A CN 1546172A CN A2003101123725 A CNA2003101123725 A CN A2003101123725A CN 200310112372 A CN200310112372 A CN 200310112372A CN 1546172 A CN1546172 A CN 1546172A
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Abstract
The invention discloses a SARS virus hyper-immune serum and method for preparing it, wherein the process according to the invention comprises the steps of, deactivation of SARS virus, injecting the mixture of deactivated SARS virus and immunologic adjuvant into the body of animals, collecting the animal blood after immunization, separating plasma from the collected blood.
Description
Technical field
The present invention relates to a kind of anti-" SARS " viral hyper-immune serum and preparation method thereof, more particularly, the present invention relates to a kind of " SARS " virus and be that antigen makes resists " SARS " viral hyper-immune serum and preparation method thereof with deactivation.
Background technology
SARS virus is the arch-criminal of the SARS disease of wreaking havoc in China and even the world wide in last year.Its propagation has had a strong impact on people's order orthobiosis." SARS " virus is a kind of brand-new coronavirus, the recent mutation of non-certain known coronavirus.Coronavirus is a kind of RNA viruses.Since in March, 2003, reported the gene order that causes sars coronavirus both at home and abroad in succession, this virus contains 5 ' complete end sequence, coded sequence and 3 ' end sequence, full rna gene group length is 29727 nucleotide, composition structure and other coronavirus of gene coding region are similar, comprise a plurality of open reading frames (ORFS), respectively the poly synthase protein of coding virus (polymerase 1a, 1b) and structural protein.
Globulin preparation is modal immune serum, and globulin preparation has tetanus antitoxin, diphtheria antitoxin, gas gangrene toxin, botulinum Antitoxin, AAS etc.At present, common globulin preparation has rabies antiserum, antivenin on the market.Rabies antiserum is by rabies virus immunity horse, the liquid that makes with ammonium sulfate salting-out process behind pepsin digestion or lyophilizing immunoglobulin preparation.Be used to cooperate antirabic vaccine to sting the wounded as head, face, cervical region or multi-section position and inoculate seriously being bitten by crazy animal, The sooner the bettern by injection after the crazy animal bite, sting injection within back 48 hours, can reduce sickness rate.Horse blood slurry refining form of antivenin after with snake venom immunity horse is for treatment venom person's usefulness.
The patent application 03128064.1 of China discloses a kind of anti-severe acute respiratory syndrome serum of horse that is used for the treatment of and prevents severe acute respiratory syndrome (SARS), it is to obtain immune blood plasma by the SARS of tissue culture inactivation of viruses immunity horses, the liquid or the lyophilizing immunoglobulin preparation that after gastric enzyme digestion, make with ammonium sulfate salting-out process, have in the specificity and the effect of SARS virus, be used to be subjected to the treatment of SARS virus infected patient, also can be used for high-risk group's prevention.Usually adopt intravenous injection, also can do intramuscular or subcutaneous injection.
The patent application 03128116.8 of China discloses a kind of immune formulation that is used to prevent and treat SARS, it is by patient's SARS convalescent period blood plasma or contains the tire human normal plasma of SARS virus antibody of height and extract and through the people SARS immunoglobulin of viral inactivation treatment preparation through cold ethanol Protein Separation method, can treat the patient who infected by SARS virus effectively, also can be used for high-risk group's prevention.
So far, people do not have a kind of medicine that can treat SARS effectively yet.SARS virus might be staged a comeback in next year, threaten human beings'health.Therefore, the task of the medicine of effective treatment of research and prevention SARS is extremely urgent.
Summary of the invention
One of the object of the invention has provided the method for the hyper-immune serum of a kind of preparation treatment SARS virus (SARSV).
The method of the present invention prepares anti-" SARS " viral hyper-immune serum may further comprise the steps:
(1) with the method for formalin-inactivated or the method for temperature deactivation " SARS " virus is carried out deactivation, make " SARS " virus of deactivation;
(2) with " SARS " of deactivation virus be expelled in the animal body after immunological adjuvant mixes;
(3) blood of collection immunity back animal;
(4) from the blood that step (3) is gathered, isolate serum.
This method can further include following steps:
(5) from the resulting serum of step (4), further isolate effective ingredient;
(6) the resulting effective ingredient of step (5) is made with extra care and purification.
Above-mentioned preparation method is based on following principle: the antigenic component in " SARS " virus of deactivation can induce and stimulate animal to produce high titre neutralizing antibody, and this neutralizing antibody can combine with " SARS " virus-specific in human body, removes the purpose that enters intravital " SARS " virus and reach treatment " SARS ".
The present inventor is according to this principle, utilizes in Guangzhou and separates " SARS " virus that obtains, and makes inactivated vaccine after deactivation, is expelled in the animal bodies such as horse, pig with adjuvant that can the booster immunization stimulating effect then.After confirming that the generation height is exempted from antibody, from blood, extract serum composition.The innovative point of this project is to exempt from antibody with newfound " SARS " virus preparation therapeutic type height.
In the step (1) of above-mentioned preparation method before, can also comprise the step that " SARS " virus is increased.
In step (1), the ablation method of employing can be the formalin-inactivated method, or the temperature ablation method.To detect after the deactivation, to guarantee the no pathogenecity of " SARS " virus of deactivation.The control key of deactivation condition is to control deactivation temperature, time and concentration of formaldehyde.
If select for use formaldehyde to carry out deactivation, then the concentration of formaldehyde can be controlled in the scope of 0.10%-1.00%, is preferably the formaldehyde of 0.15%-0.60%, and the time of deactivation is that a few hours were to tens of hours.For example, the formalin-inactivated SARS virus of employing 0.2% is more than 24 hours, and perhaps 0.4% formalin-inactivated SARS virus can make SARS virus feeling of loss metachromia more than 2 hours, and inactivation ratio is 100%, and antigenicity is unaffected before and after the inactivation of virus.Although 0.2% formaldehyde effect can inactivation of viruses more than 24 hours, can detect the nucleic acid of high copy, therefore more preferably adopt 24 hours conditions of formalin-inactivated of 0.4% as deactivation SARS vaccine.This ablation method is inactivation of viruses effectively, and antigenicity is unaffected before and after the deactivation.
If select the temperature deactivation for use, then deactivation temperature generally is controlled in the 46-66 ℃ of scope, and inactivation time generally is controlled in 10-100 minute scope.The temperature deactivation can be carried out in water-bath.
When using " SARS " virus immunity animal of deactivation in step (2), be booster immunization effect and acquisition high-load antibody, intramuscular injection is in animal body after preferably " SARS " virus of immunological adjuvant and deactivation being mixed.Wherein, the animal of immunity can be mice, rat, rabbit, pig, cattle, monkey or horse, is preferably horse or pig.
The method of immunity both can be to use " SARS " virus (" SARS " inactivated vaccine) immune animal of deactivation separately, also can adopt " SARS " inactivated vaccine and influenza vaccines to unite animal is carried out immunity.In order to improve the immunologic function of human body specifically to " SARS " virus and influenza virus, preferably adopt " SARS " inactivated vaccine and influenza vaccines to unite animal is carried out immunity, more preferably immunological adjuvant and " SARS " inactivated vaccine are mixed the back intramuscular injection in animal body with influenza vaccines.
Immunological adjuvant used in the present invention can be one or more in the following material: and freund adjuvant (Freund ' s adjuvant, comprise Fo Shi Freund's complete adjuvant (FCA) and Freund incomplete adjunvant (FIA)), aluminium hydroxide and CpG, be preferably Fo Shi Freund's complete adjuvant or Freund incomplete adjunvant, more preferably Fo Shi Freund's complete adjuvant and Freund incomplete adjunvant.
Immune programme for children divides fundamental immunity and booster immunization.Twice antigen of the general injection of fundamental immunity use FCA+SARSV for the first time, second time and use FCA+SARSV later on.Booster immunization carries out inoculation method after a couple of days of fundamental immunity can be back multiple spot subcutaneous injection, back multiple spot intramuscular injection, intragluteal injection.Select the interval and the length of suitable immunity can reach immune effect preferably.
In step (4), the method for the separation of serum that is adopted is room temperature nature coacervation or lead extruding process.
Can consider that the reason of carrying out step (5) and (6) is: the serum direct injection of animal may cause side reaction for the people.Therefore can carry out purification and extract active ingredient and isolate immunoglobulin G (IgG) in the serum serum.The separation of IgG, method refining and purification are: use chromatographic column IgG.Way with enzyme action is digested to fragment with active ingredient IgG, and the reuse gel chromatography extracts effective fragment, removes invalid element.Enzyme is preferably papain.Carry out the post affinity purification then.
Finish (4) in step or finish (5) and (6) can identify afterwards, the active detection, for example use the albuminometry detectable concentration, detect serum, IgG and Fab fragment with the neutralizing antibody test, obtain tiring of neutralization " SARS " virus.Can also be to product packing under aseptic condition.
What the present invention adopted is the animal serum composition.Though the animal serum composition is used for human body not as popularity serum, present domestic and international technology all can't be the animal serum humanization, and can't prepare anti-" SARS " viral hyper-immune serum in large quantities by collection " SARS " patient's serum.Therefore the present invention is the technology that is fit to suitability for industrialized production.The hyper-immune serum that makes in this way has positive effect to the ability that promotes anti-" SARS " viral infection of human body, to other viruses or microorganism no cross reaction, to the advantage that human body has no side effect, filled up the domestic and international blank of the no effective and special medicine of " SARS " treatment.
Below in conjunction with embodiment, further specify the present invention, but the present invention is not limited to these embodiment, any according to essence spirit of the present invention improvement or substitute, still belong to scope required for protection in claims of the present invention.
The specific embodiment
The animal of using among the embodiment:
1. animal is selected
Select 3-5 year in good condition, without the dark horse of immunity (rufous or black, white, cyan horse need not), raise before the immunity more than 90 days, carry out equine infectious anemia, glanders, brucellosis inspection, select healthy horse and be used for animal immune.Used horses comprise Changchun horse and Guangdong horse.
2. the animal feeding place is selected and management
As the raising farm, animal carries out disinfection before entering a week away from the colony house in other animal feeding place in selection.The special messenger raises, and apparatus and apparatus special use are put warning sign on every side in one's power at feed lot and avoided the idler to enter.Animal excrements sterilization back is buried.
Embodiment 1. antigen preparation
With 250ml log 10
-6.7TCID
50The SARS virus cell culture of/ml is an antigen, carries out deactivation with 0.4% formaldehyde, 5000g * 30 minute centrifuging and taking supernatant, and 20000-25000g ultracentrifugation 1.5h gets precipitation, sterile purified water 16 * dilution.Preparation Fo Shi Freund's complete adjuvant (FCA) (for the product of Sigma) is used for immunity with Freund (FIA) (for the product of Sigma).
Embodiment 2. immune programme for children
3 Guangdong horses of antigen immune with embodiment 1 preparation.Immunity can be divided into fundamental immunity and booster immunization.
Twice antigen of the general injection of fundamental immunity use FCA+SARSV for the first time, second time and use FIA+SARSV later on.Fundamental immunity (seeing Table 1) is carried out booster immunization after 10-14 days for the second time, and booster immunization carries out (seeing Table 2) behind the 24th day of fundamental immunity.
Table 1 fundamental immunity
| Natural law d | ????1 | ????10 | ????24 |
| Pin | ????1 | ????2 | |
| Injected dose (ml) | ????1 | ????2 | |
| The vaccine kind | FCA+SARSV | ?FIA+SARSV |
Table 2 booster immunization
| Natural law (d) | ????1 | ????7 | ????14 | ????21 | ????28 | ????41 | ????43 |
| Pin | ????1 | ????2 | ????3 | ????4 | ????5 | Blood sampling | Blood sampling |
| Dosage (ml) | ????2.5 | ????2.5 | ????3.0 | ????3.0 | ????3.0 | ||
| Vaccine | ??????????????????????????FIA+SARSV | ||||||
Embodiment 3. serum separate and preserve
Press the horse of step immunity back 3 Guangdong inoculation of embodiment 2 immunity, exempt from 7 days in the end after blood sampling, and blood sampling for the second time every one day, the preceding 12 hours not feed material of taking a blood sample, but do not limit drinking-water, blood sampling volume is pressed the 17-19ml/Kg body weight and is calculated, and adopts 10-20ml blood separation serum before each immunity and is equipped with inspection.
Adopt in horse whole blood to 1000 or the 2000ml graduated cylinder, with lead extruding process separation of serum ,-20 ℃ of preservations.
The separation of embodiment 4.IgG
Weighing embodiment 3 isolated horse serum 100ml put on the magnetic stirring apparatus and stir.The sodium-acetate buffer (pH4.0) that adds the 60Mm of 200ml is adjusted pH to 4.8.It is sad slowly to drip, and continues under the room temperature to stir 30 minutes, and every 10ml adds sad 1.5ml.Centrifugal 30 minutes of 5000g keeps supernatant.Regulate pH to 5.7 with 1M NaOH.The supernatant that contains IgG is dialysed to PBS, and-20 ℃ of preservations are put in packing then.
Embodiment 5. horse immunity inoculation SARS inactivated vaccine ELISA detect antibody and adopt indirect elisa method:
(1) carbonic acid buffer with Ph9.6 wraps 1: 100 dilution back of SARS inactivated vaccine by 96 orifice plates, and 4 ℃ are spent the night;
(2) seal with 15% calf serum PBST (1 ‰ tween);
(3) sample serum is with containing application of sample after 100 times of the 5% horse serum PBST dilutions;
(4) add the monoclonal antibody Mus IgG antibody of HRP labelling;
(5) add o-phenylenediamine substrate solution and H
2O
2, the lucifuge colour developing;
(6) H of usefulness 2M
2SO
4Cessation reaction;
(7) survey the light absorption value of each hole 490nm with the Bio-Rad505 microplate reader.
Indirect elisa method detects the antibody result: 3 dry goods of Guangdong inoculation, immunity inoculation the after two months, the antibody titer that ELISA detects was up to 1: 40960.
Embodiment 6. immune post neutralization antibody tests
1. the viral dilution of viral dilution after with titration becomes 100TCID
50/ 25ul.
2. the serum of animal serum SARS virus immunity inoculation horse different times, with serum on 96 aseptic orifice plates since 1: 10, doubly be diluted to 1: 10240 continuously, each dilution factor 2 hole, the various animals of gathering simultaneously before the immunity inoculation are used in contrast.
3. neutralization test adds 25ul 100TCID in above serum dilution holes
50The Z2-Y3 virus applications liquid of/25ul shakes up rearmounted 36 ℃ of 5%CO2 incubators gently and cultivates, and establishes normal cell contrast and the experiment of virus titer residual titration simultaneously.
4. result's observation is dripped day (pathological changes appears in cell 100%) result of determination from the 4th day observed result.
The neutralizing antibody testing result: 3 dry goods of Guangdong inoculation, after the immunity inoculation 60, NAT is 1: 6400.
The antibody test of embodiment 7.6 coupe spring horse immunity post neutralization
Press 6 dry goods of the method immunity of embodiment 2-7 in the Changchun immunity, immunity inoculation in the time of 20 days after, NAT is 1: 2560-5120.
Embodiment 6 and 7 result show, all can produce higher protection antibody (neutralizing antibody) with SARS inactivated vaccine immunity horse.
Embodiment 8. extracts the method for hyper-immune serum in the body of pig
Immune swine, step is with reference to embodiment 2-7.
Claims (10)
1. method for preparing anti-" SARS " viral hyper-immune serum, described method may further comprise the steps:
(1) with the method for formalin-inactivated or the method for temperature deactivation " SARS " virus is carried out deactivation, make " SARS " virus of deactivation;
(2) with " SARS " of deactivation virus be expelled in the animal body after immunological adjuvant mixes;
(3) blood of collection immunity back animal;
(4) from the blood that step (3) is gathered, isolate serum.
2, the method for claim 1 is characterized in that, described method also further comprises the steps:
(5) from the resulting serum of step (4), further isolate effective ingredient;
(6) the resulting effective ingredient of step (5) is made with extra care and purification.
3, method as claimed in claim 1 or 2 is characterized in that, in step (1), the concentration range of the formalin-inactivated that is adopted is 0.10%-1.00%, and the temperature range of the temperature deactivation of being adopted is 46-66 ℃.
4, method as claimed in claim 1 or 2 is characterized in that, in step (2), the immunological adjuvant that is adopted is Fo Shi Freund's complete adjuvant and/or Freund incomplete adjunvant.
5, method as claimed in claim 1 or 2 is characterized in that, in step (2), the animal of being adopted is horse or pig.
6, method as claimed in claim 1 or 2 is characterized in that, in step (4), the method for the separation of serum that is adopted is room temperature nature coacervation or lead extruding process.
7, method as claimed in claim 2 is characterized in that, in step (5), isolated effective ingredient is an immunoglobulin G.
8, method as claimed in claim 7 is characterized in that, in step (6), the method for the refining and purification that is adopted is digested to fragment for first method with enzymic digestion with immunoglobulin G, extracts its effective fragment then, removes invalid element.
9, method as claimed in claim 8 is characterized in that, the method for described enzymic digestion is the papain enzyme cutting method.
10, a kind of anti-" SARS " viral hyper-immune serum according to the described method preparation of one of aforesaid right requirement is characterized in that, described anti-" SARS " viral hyper-immune serum is that antigen makes with deactivation " SARS " virus.
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| CNA2003101123725A CN1546172A (en) | 2003-11-28 | 2003-11-28 | Hyper-immune serum for resisting SARS virus and its preparation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103705918A (en) * | 2013-12-24 | 2014-04-09 | 北京大北农科技集团股份有限公司动物医学研究中心 | Porcine epidemic diarrhea virus resistant hyper-immune serum and preparation method thereof |
| CN111184741A (en) * | 2020-02-20 | 2020-05-22 | 张宏亮 | Preparation method of biological agent for treating virus infected person, related biological agent and application |
-
2003
- 2003-11-28 CN CNA2003101123725A patent/CN1546172A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103705918A (en) * | 2013-12-24 | 2014-04-09 | 北京大北农科技集团股份有限公司动物医学研究中心 | Porcine epidemic diarrhea virus resistant hyper-immune serum and preparation method thereof |
| CN111184741A (en) * | 2020-02-20 | 2020-05-22 | 张宏亮 | Preparation method of biological agent for treating virus infected person, related biological agent and application |
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