CN100432096C - Transfer factor specific for SARS virus and method for preparing same - Google Patents
Transfer factor specific for SARS virus and method for preparing same Download PDFInfo
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- CN100432096C CN100432096C CNB2003101123744A CN200310112374A CN100432096C CN 100432096 C CN100432096 C CN 100432096C CN B2003101123744 A CNB2003101123744 A CN B2003101123744A CN 200310112374 A CN200310112374 A CN 200310112374A CN 100432096 C CN100432096 C CN 100432096C
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Abstract
The present invention discloses a 'SARS' virus transspecific factor and a preparation method thereof. The method of the present invention mainly comprises the following steps of 'SARS' virus inactivation, animal immunity, transspecific factor extraction and transspecific factor refining and purification. The 'SARS' virus transspecific factor of the present invention can be prepared by using inactivated 'SARS' viruses as antigens, and the obtained product can combine 'SARS' viruses with specificity. The 'SARS' virus transspecific factor can also be prepared by using inactivated 'SARS' viruses and an inactivated vaccine for influenza viruses as antigens, and the obtained product has specific efficiency on 'SARS' viruses and influenza viruses so as to perform effective therapeutic effect on 'SARS' and influenza.
Description
Technical field
The present invention relates to a kind of " SARS " virus specific transfer factor and preparation method thereof, specifically, the present invention relates to a kind of " SARS " virus and be " SARS " virus specific transfer factor that antigen makes 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 this year.Its propagation has had a strong impact on people's orthobiosis order.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, encoding 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 frame (ORF
S), respectively the poly synthase protein of coding virus (polymerase 1a, 1b) and structural protein.
Transfer factor (Transfer factor) is a kind of low molecular core thuja acid and the polypeptide that has immunocompetent lymphocyte to discharge in the white corpuscle, and its essence is the mixture of a small amount of nucleic acid and micromolecule polypeptide.It can improve receptor's immunologic function with being transferred to another individuality by the specific immune response of certain antigen (cause of disease) immunity from a certain individuality non-specificly, promotes immune cell factor to discharge.
Transfer factor has dialyzability, does not contain albumen, contains polypeptide, nucleic acid in the rough transfer factor, and its activity is not destroyed by trypsinase, DNA enzyme and RNA enzyme, but under-20 ℃ of conditions prolonged preservation, molecular weight is less than 5000.
Transfer factor also has the ability of transitional cell immunity.Using very little dosage just can make acceptor produce part or systemic reaction, action time is fast, and 3-4h just can activate host's lymphocyte, makes acceptor sensitization, and the time length was 1 year.Can make unsensitized lymphocyte sensitization in external transfer factor, thymus pyrimidine infiltrates to be increased, and forfeiture and the lymphocyte that sheep red blood cell (SRBC) forms the Rose ability are partly recovered.Transfer factor can not be intersected between planting and shifted, now proves, and between the animal kind, become the human-animal all can shift mutually, only be that metastasis degree is variant.
Transfer factor produce at home with clinical application for many years, comprise non-specific transfer factor and specific transfer factor.Non-specific transfer factor is mainly used in immunologic function and the resistance against diseases that improves human body, and specific transfer factor can also excite at certain antigenic immune response except the function that possesses non-specific transfer factor.Transfer factor is generally extracted from immune organs such as spleen and lymphoglandula and white corpuscle.
China's patent application 03108006.5 discloses a kind of anti-sars virus transfer factor preparation method; at first; SARS virus is through Hep-2 cell, HEKC, human embryonic lung cell or chicken embryo culture propagation, the protective antigen for preparing SARS virus antigen and express preparation according to the gene order of SARS virus with gene engineering method.Secondly, with discontinuous gradient density centrifugation purified virus antigen or with the heavy layer of 20% sucrose concentrated antigen.Then, use the virus antigen immune animal of being extracted.At last, from immune animal, extract the anti-sars virus transfer factor.
So far, people do not develop a kind of medicine that can treat SARS effectively yet.SARS virus might be staged a comeback in next year, threaten human beings'health.Research is extremely urgent to the task of the medicine of effective treatment and prevention SARS.
Summary of the invention
One of purpose of the present invention provides the method for a kind of preparation " SARS " virus specific transfer factor.
In the method for preparation of the present invention " SARS " virus-specific immune ribonucleic acid, comprise the steps:
(1) " 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) extract " SARS " virus specific transfer factor the tissue of the animal after immunity;
" SARS " virus specific transfer factor that (4) will extract is made with extra care, purifying and packing.
Above-mentioned preparation method is based on following principle: the antigenic component in " SARS " virus of deactivation can induce and stimulate animal to produce immune response, sharp in vivo immunologically competent cell etc., and these cells can be secreted transfer factor, thereby activate the specific cell immunoreaction at " SARS " virus.
The present inventor is according to this principle, utilization separates " SARS " virus that obtains in Guangzhou, after deactivation, make inactivated vaccine, be expelled in the animal bodies such as pig with adjuvant that can the booster immunization stimulatory effect then, bring out animal and produce specific immune response.After confirming to produce immune response, extract the transfer factor in the tissues such as liver, spleen, lymphoglandula, produce effective medicine of a kind of anti-" SARS " from the angle of specific immunity.
In step (1) before, can pass through the pcr amplification SARS virus.Deactivation can be formalin-inactivated or temperature deactivation in the step (1), also can adopt other ablation method.Influenza virus is adopted commercially available inactivation of viruses, and " SARS " virus will be passed through deactivation.The key of the control of deactivation condition is the control to deactivation temperature, time or concentration of formaldehyde.Temperature is controlled at 46-66 ℃ of scope when adopting the temperature deactivation, and the time was controlled in 10-100 minute.For example can adopt 56 ℃ 60 minutes.Concentration of formaldehyde is at 0.10%-1.00%.The time of deactivation is that a few hours were to tens of hours.For example, can be more than 24 hours with 0.2% formalin-inactivated SARS virus, perhaps 0.4% formalin-inactivated SARS virus can make SARS virus forfeiture perception more than 2 hours, and inactivation ratio is 100%; Antigen 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 preferred 0.4% 24 hours the conditions of formalin-inactivated that adopt as deactivation SARS vaccine.This ablation method is inactivation of viruses effectively, and antigen is unaffected before and after the deactivation.
In step (2) except " SARS " of deactivation virus and immunological adjuvant, also adopt the inactivated vaccine of influenza virus to mix together after, come the combined immunization animal.
Wherein, adjuvant can the enhancing immunity effect, the adjuvant that uses can be that freund adjuvant comprises one or more in Fo Shi Freund's complete adjuvant (FCA) and Freund incomplete adjunvant (FIA), aluminum hydroxide adjuvant, the CpG adjuvant, is preferably use freund adjuvant Freund's complete adjuvant or Freund.More preferably use freund adjuvant Freund's complete adjuvant and Freund.
The animal of immunity can be mouse, rat, rabbit, pig, ox, monkey or horse, is preferably pig.
The method of immunity can be to use SARS inactivated vaccine immune animal separately, also can be simultaneously animal to be carried out immunity with SARS inactivated vaccine and influenza vaccines.Because with " SARS " and influenza antigen combined immunization animal, produced transfer factor all has specificity to " SARS " and influenza, is key point of the present invention, therefore preferred this scheme that adopts.
Immunity (inoculation) can be a single injection, also can be multiple injection.Owing to generally want multiple injection just can reach hyperimmune state, so be preferably multiple injection.Inoculation method is preferably back multiple spot intramuscular injection.Select the timed interval and the length of suitable immunity can reach immune effect preferably.The time that generally needs a couple of days.Be preferably 0 day, inoculated in 15 days, 21 days, 40 days.
In step (3), gather liver, spleen and/or the lymph node tissue of immunity back animal,, extract " SARS " virus specific transfer factor then its cytoclasis.Wherein, cytoclastic method can adopt that tissue mashing machine smashs to pieces, ultrasonic disruption etc., but tissue mashing machine commonly used smashs to pieces, makes homogenate.The method of extracting " SARS " virus specific transfer factor is the freeze thawing dialysis method, hatch dialysis method or ethanol extraction method.
In step (4), can adopt methods such as filtering with microporous membrane, ultrafiltration, dialysis to make with extra care and purifying according to prior art.Can identify as required afterwards in step (4), the active detection, for example use nucleic acid determination method detectable level, measure specificity and effect with the biological activity method." SARS " virus specific transfer factor that obtains behind the refining and purifying can carry out packing under aseptic condition.
On the other hand, the present invention also provides a kind of " SARS " virus specific transfer factor by the method preparation, can be that antigen makes with deactivation " SARS " virus both, can be that antigen makes with deactivation " SARS " virus and inactivated influenza virus vaccine also." SARS " virus and influenza virus all there are specificity, thereby can all play effective result of treatment " SARS " and influenza.
Detection through strictness, " SARS " virus specific transfer factor that is made by this method meets national biological product standards, the transfer factor of this specific immunity has positive effect to the ability that promotes anti-" SARS " virus infection of human body, " SARS " and treatment of influenza there is the specific therapy effect, the advantage that human body is had no side effect, filled up the domestic and international blank of the no effective and special medicine of " SARS " treatment, Special Significance arranged preventing and treating " SARS ".
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.
Embodiment
Employed material is in the following example:
1. influenza vaccines: the Shanghai biological factory is produced.
2. adjuvant: Fo Shi Freund's complete adjuvant and Freund incomplete adjunvant, Sigma product.
3.HRP anti-pig IgG of mark rabbit and IgM enzyme labelled antibody, U.S.'s product.
4. ultra-fine filter: U.S. Millipore company product.
The preparation of embodiment 1 SARS inactivated vaccine
In floorage is 275cm
2Cultivate VeroE6 (China national institute of viruses) in the cell bottle, treat that cell grows up to fine and close individual layer after, wash cell 3 times with serum-free medium, add the 100ml serum-free medium, inoculate F69 and Z respectively
2-Z
3SARS-Cov virus strain, cytopathy be during to 75%-100%, titration virus, TCID
50Be log10 * 10
7/ ml.Culturing bottle is put-20 ℃ of refrigerator freeze thawing 3 times, shaken up and put 2-8 ℃ of refrigerator overnight, behind 0.4% formalin-inactivated 24hr, 5000rpm * 30min discards precipitation, and supernatant is as the SARS inactivated vaccine.
Embodiment 2 immune programme for children (the SARS inactivated vaccine is immunity separately)
Healthy castration boar 17 (body weight 50Kg, breeding pig farms, Guangzhou) is used embodiment SARS inactivated vaccine (TCID50Log107/ml).Totally 4 times, the 1st time is Fo Shi Freund's complete adjuvant+SARS inactivated vaccine; The 2nd time is Freund incomplete adjunvant+SARS inactivated vaccine, and the 3rd time is the immunity of simple SARS inactivated vaccine with the 4th; Dosage 3 * TCID50; The multiple spot intramuscular injection of inoculation method back.The timed interval of immunity is 0d, 15d, 21d, 40d.Gather whole blood 1-3ml from ear vein, the timed interval of serum collection is 0d, 5d, 7d, 10d, 15d, 21d, 28d, 35d and 42d.Centrifugation serum then ,-20 ℃ of preservations are standby.
Embodiment 2. immune programme for children (SARS inactivated vaccine and influenza vaccines combined immunization)
Healthy 13 of boars of castration (body weight 50Kg, breeding pig farm, Guangzhou).With SARS inactivated vaccine (TCID50Log107/ml)+influenza vaccines immunity, totally 4 times, the 1st time is Fo Shi Freund's complete adjuvant+SARS inactivated vaccine+influenza vaccines immunity; The 2nd Freund incomplete adjunvant+SARS inactivated vaccine+influenza vaccines immunity, the 3rd time is SARS inactivated vaccine+influenza vaccines immunity with the 4th.Dosage: SARS is 3 * TCID
50Influenza virus is 3 person-portions/pig the 1st time, and later 3 times is 1 person-portion/pig; The multiple spot intramuscular injection of inoculation method back.The timed interval of immunity is 0d, 15d, 21d, 40d.Gather whole blood 1-3ml from ear vein, the timed interval of serum collection is 0d, 5d, 7d, 10d, 15d, 21d, 28d, 35d and 42d.Centrifugation serum then ,-20 ℃ of preservations are standby.
The check neutralizing antibody of embodiment 3. immune effects detects
Immune swine in embodiment 1 and 2 is carried out according to the method that the evaluation of inactivated vaccine immune effect detects.
1. the viral dilution of viral dilution after with titration becomes 100TCID
50/ 25ul.
2. the serum of animal serum SARS virus immunization pig different times, with serum on 96 aseptic orifice plates since 1: 10, doubly be diluted to 1: 10240 continuously, each extent of dilution 2 hole, the various animals of gathering simultaneously before the immunization 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%CO gently
2Incubator is cultivated, and establishes normal cell contrast and the experiment of virus titer residual titration simultaneously.
4. result's observation is from the 4th day observations, the 7th day (pathology appears in cell 100%) result of determination.
The result shows: the pig of enforcement in example 2 and 3 all detected the neutralizing antibody of anti-SARS virus at the 5th day, after the 4th immunization, reached 1: 240 with antibody titer in it, met the requirement of immune ribonucleic acid preparation.
The check of embodiment 4. immune effects: IgG detects
Adopting indirect elisa method to carry out IgG the immune swine in embodiment 1 and 2 detects:
(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% calf serum PBST dilutions;
(4) add the monoclonal antibody mouse IgG antibody of HRP mark;
(5) add O-Phenylene Diamine substrate solution and H
2O
2, the lucifuge colour developing;
(6) H of usefulness 2M
2SO
4Termination reaction;
(7) survey the light absorption value of each hole 490nm with the Bio-Rad505 microplate reader.
The extracting method of embodiment 5. specific transfer factors (TF) (freeze thawing dialysis method)
Lawrence method (freeze thawing dialysis method): as becoming raw material with spleen or lymph, method is: get the spleen of fresh or stored frozen, remove its surperficial tunicle, reticular tissue such as fat, shred, put in the high-speed tissue mashing machine and smash (10000-12000rpm, start 3min to pieces, stop 5min, 3 times repeatedly), no complete lymphocyte in the microscopy tissue homogenate is put-40 ℃ of freezing 72h then, dialyse after the thawing
Following steps are the same
Embodiment 6.The extracting method of specific transfer factor (hatching dialysis method)
Hatch dialysis method: get the lymphoglandula or the spleen of sensitized animal, remove tunicle, reticular tissue such as fat shred, and make the lymphocyte suspension, with stainless steel sift cell are filtered in the Hanks liquid, and transferring pH is 7.3; Cell concn is 1 * 109/15ml, puts in 37 ℃ of waters bath with thermostatic control, hatches 4h, and constantly stirs, and its supernatant liquor is got in back centrifugal (1500-2000rpm), and with the deionized water of 10 times of amounts, under 4 ℃ of conditions, dialysis 16-18h gets its extracellular fluid dialysis, is rough TF.
The extracting method (ethanol extraction method) of embodiment 7. specific transfer factors
Ethanol extraction method: get fresh Lymphoid tissue (comprising spleen, lymphoglandula and peripheral blood leucocyte), be cut into small pieces, add 40% ethanol of 1 times of volume refrigerative 80% ethanol and 3 times of volumes, smash 3min at a high speed to pieces, transfer pH5.0-5.1, in ice bath, place 30-60min subsequently, back centrifugal (2500rpm) 20min under 0-4 ℃ of condition, get its supernatant liquor, add the equal-volume deionized water, lyophilize is extracted thing by every gram Lymphoid tissue and is added the 2ml deionized water dissolving, carry out ultrafiltration, degerming packing then.External evidence adds corresponding antigens or adds micro-transfer factor (TF) in lymphocyte culture fluid, all can stimulate lymphocyte to produce a large amount of TF.
Claims (3)
1, the method for a kind of preparation " SARS " virus specific transfer factor, this method comprises the steps:
(1) " SARS " virus is carried out formalin-inactivated or temperature deactivation, make " SARS " virus of deactivation; The concentration of formaldehyde is 0.10%-1.00% when wherein, adopting formalin-inactivated; Temperature is controlled at 46-66 ℃ of scope when adopting the temperature deactivation, and the time was controlled in 10-100 minute;
(2) with the influenza virus of " SARS " of deactivation virus, deactivation be expelled in the animal body after immunological adjuvant mixes, come the combined immunization animal; Wherein, described immunological adjuvant is Fo Shi Freund's complete adjuvant and/or Freund incomplete adjunvant;
(3) extract " SARS " virus specific transfer factor the tissue of the animal after immunity;
" SARS " virus specific transfer factor that (4) will extract is made with extra care, purifying and packing.
2, the method for claim 1 is characterized in that, in step (3), gathers liver, spleen and/or the lymph node tissue of immunity back animal, with its cytoclasis, extracts " SARS " virus specific transfer factor then.
3, the method for claim 1 is characterized in that, the method for extracting " SARS " virus specific transfer factor is the freeze thawing dialysis method, hatch dialysis method or ethanol extraction method.
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| CN1439424A (en) * | 2003-04-30 | 2003-09-03 | 中国人民解放军第四军医大学第一附属医院 | Anti-SARS virus transfer factor preparation method |
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| CN1439424A (en) * | 2003-04-30 | 2003-09-03 | 中国人民解放军第四军医大学第一附属医院 | Anti-SARS virus transfer factor preparation method |
Non-Patent Citations (6)
| Title |
|---|
| SARS冠状病毒灭活疫苗研究初报. 广东省防治非典型肺炎科技攻关疫苗专辑组.广东医学，第24卷第SARS专辑（I）期. 2003 |
| SARS冠状病毒灭活疫苗研究初报. 广东省防治非典型肺炎科技攻关疫苗专题组.广东医学，第24卷第SARS专辑（I）期. 2003 |
| SARS冠状病毒灭活疫苗研究初报. 广东省防治非典型肺炎科技攻关疫苗专辑组.广东医学，第24卷第SARS专辑（I）期. 2003 * |
| SARS冠状病毒灭活疫苗研究初报. 广东省防治非典型肺炎科技攻关疫苗专题组.广东医学，第24卷第SARS专辑（I）期. 2003 * |
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