CN101732716B - Antigen-antibody complex for preventing and/or treating avian influenza - Google Patents
Antigen-antibody complex for preventing and/or treating avian influenza Download PDFInfo
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- CN101732716B CN101732716B CN2008102260239A CN200810226023A CN101732716B CN 101732716 B CN101732716 B CN 101732716B CN 2008102260239 A CN2008102260239 A CN 2008102260239A CN 200810226023 A CN200810226023 A CN 200810226023A CN 101732716 B CN101732716 B CN 101732716B
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Abstract
The invention provides an antigen-antibody complex for preventing and/or treating avian influenza, which comprises inactivation avian influenza totiviruses which are used as antigen and an immune body thereof, and the mass concentration ratio of the antigen and the immune body is more than 1. After entering organisms to perform initial immunization, the antigen-antibody complex stimulates the organisms again to induce immunoreaction, and immune response is quick in speed and strong in reaction; the antigen-antibody complex used as a carrier is more favorable for capturing and presenting antigen presenting cells and can also strengthen a breeder reaction of T cells effectively; purified totiviruses used as the antigen increase the molecular weight of the complex, are more favorable for ingestion of immunocyte of the organisms on the antigen, cause the more extensive immunoreaction quickly, and have a significance for preventing avian influenza viruses of which the antigen is easy for variation. A preparation of the antigen-antibody complex does not need to use solid carriers, does not cause intense stimulus on the organisms or initiates the organisms to generate an adverse reaction, and is simple to prepare and safe and convenient to use.
Description
Technical field
The invention belongs to the immunomodulating field, relate to a kind of antigen antibody complex and comprise the preparation of this antigen antibody complex and their preparation method, also related to them and prevented and/or treated purposes in the medicine of bird flu in preparation.
Background technology
The popular of bird flu caused tremendous loss to the whole world, and at present it mainly taken preventive measures, and the Chinese government also adopts the route of migrating of satellite monitoring migratory bird in order to grasp bird flu epidemic situation comprehensively a situation arises.The avian influenza vaccine of whole world development roughly is divided into totivirus inactivated vaccine (inactivated influenza virus vaccines), genetic engineering subunit vaccine (subunitvaccines), dna vaccination (DNA vaccines), live recombinant vectors vaccine (recombine liveinfluenza virus vaccines), gene engineering for influenza virus live vaccine (genetically engineeredlive influenza virus vaccines), replication defect type influenza virus vaccine (replicationincompetent influenza virus vaccines), and actual use mostly is inactivated vaccine.The high pathogenic avian influenza vaccine that China uses at present mainly comprises 4 kinds: inactivated avian influenza vaccine (H5 hypotype, the N28 strain), recombinant fowl influenza virus inactivated vaccine (H5N1 hypotype, Re-1 strain), bird flu fowlpox virus genome engineered vaccine (H5 hypotype) and bird flu-newcastle reorganization bigeminal live vaccine.These vaccines can prevent body to avoid the infringement of virus, but individual invalid for what fallen ill.At present, oseltamivir phosphate capsule (Tamiflu), zanamivir (Zanamivir), amantadine (Amantadine) and rimantadine (Rimantadine) have certain curative effect.The clinical experiment data analysis shows that (influenza-like illness, ILI) protective rate with influenza A is respectively 25% and 61% to amantadine, and can make heating continuing time reduce 1 day to influenza sample disease.Rimantadine has similar curative effect, but its preventive effect is compared with placebo or other method and there was no significant difference.Simultaneously, there is result of study to show that amantadine and rimantadine all can cause tangible gastrointestinal side effect; Amantadine is compared with rimantadine, and it is more common to cause nervus centralis toxic and side effects and learning capacity to go down.In addition, this type of medicine is easy to generate drug resistance and crossing drug resistant, this with the stromatin gene order in single nucleotide undergo mutation relevant.Though four kinds of medicines that also have ion channel blocking agent and neuraminidase inhibitor two big apoplexy due to endogenous wind are official listing abroad.But, because existing medicine extensive use clinically, make influenza virus morph, these medicines have all been produced drug resistance in various degree, the ion channel blocking agent exists especially to defectives such as the invalid and neurotoxicityes of Type B influenza virus, therefore seeks new Therapeutic Method and be main developing direction from now on.
In order to regulate the immune function of body, enhancing body is the immunne response ability of unfavorable factor to external world, has all adopted the preparation of enhancing human body immunity power both at home and abroad, and they comprise: A, vaccine; B, natural synthetic immunostimulant; C, nonspecific immunopotentiating agent; D, hormone; E, specific antibody or specific immune ribonucleic acid.Whitfill (the patent No.: US5397568, US5397569) adopt the infections chicken cloacal bursa antigen antibody complex as vaccine, effectively prevent bursal disease, its preparation is widespread usage on market now, but said preparation can only be used for the prevention of bursal disease, and lacks the further research to its therapeutical effect; Wen Yumei (the patent No.: ZL93112409.3) prepared a kind of immunogenic antigen antibody complex preparation, be used for the treatment of the persistent infection of hepatitis B virus, entered clinical experimental stage now, but what adopt in the preparation is hepatitis B virus surface antigen, rather than totivirus.Fisher (patent No.: the immunization therapy method of the treatment acquired immune deficiency syndrome (AIDS) that WO88/08429) provides has adopted viral protective antigen or its corresponding peptide section, and gold-coloured staphylococci etc., neither antigen antibody complex.In sum, antigen antibody complex is obtained significant curative effect in some field as prevention and treatment means.
At present, bird flu brings crushing blow to aviculture, can also cause human and the rare animal morbidity, does not have the specific treatment medicine so far.Bird flu is the acute respiratory disease that is caused by bird flu virus.Bird flu virus is that 10 albumen of 8 sections RNA codings are formed, and wherein hemagglutinin (HA) and neuraminidase (NA) are main surface antigen, but antigenic drift and antigenic variation take place their gene easily, so above-mentioned several method is all inapplicable.
Summary of the invention
Therefore, the object of the present invention is to provide a kind of convenient to use, prevent and/or treat the antigen antibody complex of bird flu and preparation thereof, preparation method safely and effectively.This antigen antibody complex stimulates body to produce effective immunity by changing the antigenic path of offering at short notice, resists the invasion of popular virus.In initial infection, this antigen antibody complex can also play certain treatment effectiveness simultaneously.
Be used to realize that the technical scheme of above-mentioned purpose of the present invention is as follows:
A kind of being used for the treatment of and/or the antigen antibody complex of birds flu-preventing, wherein this antigen antibody complex comprises as antigenic deactivation bird flu totivirus and antibody thereof, and the mass concentration ratio of antigen and antibody is greater than 1.
In above-mentioned antigen antibody complex, described bird flu totivirus is the H5N1 subtype avian influenza virus; Described antibody is multivalent antibody IgY, for example the high immunity yolk antibody that repeatedly prepares behind the immune health laying hen of bird flu H5 hypotype inactivated vaccine.The mass concentration ratio of described antigen and antibody is preferably 1: 0.67.
A kind of preparation method of above-mentioned antigen antibody complex, comprising following steps:
(1) with the bird flu virus deactivation;
(2) blood source purification bird flu virus is as antigen;
(3) antigen and the antibody thereof with dilution is mixed in proportion, and places after 1～2 hour down for 37 ℃ and spends the night under 4 ℃; The centrifugal precipitation that obtains gets final product.
Above-mentioned preparation method specifically may further comprise the steps:
(1) inoculates the Embryo Gallus domesticus that 9 ages in days do not have specificity pathogenic microorganism (SPF) with the strain of H 5 N 1 avian influenza subtype virus, collect viral allantoic fluid under the aseptic condition of propagation back, add 0.4% formalin, 37 ℃ of following deactivations 72 hours;
(2) the healthy chicken erythrocyte purification bird flu virus of handling with the normal saline that contains formaldehyde is as antigen, and concentration is 1.38mg/ml behind 20 times of the antigen diluents, and concentration is 1.22mg/ml behind 20 times of the antibody dilutions;
(3) with 100 times of the antigen redilution of above-mentioned concentration, 150 times of antibody redilution are then according to 1: 1 ratio mixing of volume ratio, place after 1～2 hour down for 37 ℃ and spend the night under 4 ℃, take out next day after 12000 rev/mins centrifugal 30 minutes, abandon supernatant, precipitation is resuspended in 1000 times of physiological saline solution.
The present invention also provides and has comprised preparation of above-mentioned antigen antibody complex and preparation method thereof.This preparation method may further comprise the steps: above-mentioned antigen antibody complex is resuspended in the physiological saline solution.
The present invention also provides above-mentioned antigen antibody complex to prevent and/or treat purposes in the medicine of bird flu in preparation.
Antigen antibody complex provided by the invention carries excessive antigen as carrier, has changed the antigenic path of offering, thereby has promoted body's immunological function effectively.Simultaneously, the preparation that comprises this antigen antibody complex need not to use solid carrier, therefore can not cause intense stimulus or cause body body to produce untoward reaction such as inflammation, ulcer.
With respect to prior art, antigen antibody complex advantage provided by the invention is: after the antigen antibody complex of (1) antigen excess enters body, stimulate body to cause immunoreation once more after carrying out initial immunity under the organism physiology condition meeting, so immunne response speed is fast, reaction is strong; (2) antigen antibody complex more helps catching and offering of antigen presenting cell as carrier; (3) immune complex can also strengthen the breeder reaction of T cell effectively as carrier; (4) totivirus of purification has increased the molecular weight of complex as antigen, more helps immune cell to antigenic picked-up, causes immunoreation fast; (5) the bird flu totivirus with purification is an antigen, as antigen, can stimulate body to produce immunoreation widely than single albumen such as HA albumen, and this measure has more meaning for the bird flu virus that prevention antigen morphs easily.
The invention provides the compound formulation of its corresponding multivalent antibody of a kind of bird flu totivirus, it makes simple and fast, safe and convenient to use, can regulate the immunoreation of body, stimulate effectively that body is made comparatively fast, stronger immunoreation, in the prevention of bird flu and initial stage treatment, have effect preferably.
Description of drawings
Below, describe the present invention in conjunction with the accompanying drawings in detail, wherein:
Fig. 1: the antibody horizontal comparable situation of respectively organizing the chicken group after antigen antibody complex vaccine and the conventional vaccine immunity; The conventional vaccine group is meant recombinant fowl influenza virus inactivated vaccine (H5N1 hypotype, Re-1 strain); 1 group of avian influenza virus antigen and isopyknic antibody by purification of complex formulation is mixed and made into, and both mass concentration ratios are 1: 0.67; Complex formulation is that antigen and isopyknic antibody are mixed and made into both mass concentration ratios 1: 0.65 by bird flu HA albumen for 2 groups.
Fig. 2: the comparison of sickness rate, mortality rate and protective rate behind antigen antibody complex vaccine and the conventional vaccine vaccinated flock; The conventional vaccine group is meant recombinant fowl influenza virus inactivated vaccine (H5N1 hypotype, Re-1 strain); 1 group of avian influenza virus antigen and isopyknic antibody by purification of complex formulation is mixed and made into, and both mass concentration ratios are 1: 0.67; Complex formulation is that antigen and isopyknic antibody are mixed and made into both mass concentration ratios 1: 0.65 by bird flu HA albumen for 2 groups.
The specific embodiment
Embodiment 1: the preparation of bird flu antigen antibody complex preparation
Concrete preparation method is operated according to the following steps:
(1) inoculates 9 age in days SPF Embryo Gallus domesticus with the strain of H 5 N 1 avian influenza subtype virus, collect viral allantoic fluid under the aseptic condition of propagation back, add 0.4% formaldehyde, 37 ℃ of following deactivations 72 hours.
(2) blood source (containing the healthy chicken erythrocyte that the normal saline of formaldehyde was handled) purification bird flu virus, concentration is 1.38mg/ml behind 20 times of the antigen diluents after measured, concentration is 1.22mg/ml behind 20 times of the antibody dilutions.The IgY of described antibody for repeatedly preparing behind the immune health laying hen with conventional recombinant fowl influenza virus H5 hypotype bivalent inactivated vaccine (H5N1, Re-1 strain+Re-4 strain);
(3) with 100 times of the antigen redilution of above-mentioned concentration, 150 times of antibody redilution then according to 1: 1 ratio mixing of volume ratio, are placed after 1～2 hour down for 37 ℃ and are spent the night under 4 ℃.Take out next day after 12000 rev/mins centrifugal 30 minutes, abandoning supernatant.
(4) precipitation that will obtain is resuspended in the normal saline, carries out aseptic, safety, toxicity test, preserves standby down for after the assay was approved every-20 ℃.
Embodiment 2: immunne response and curative effect that bird flu antigen antibody complex preparation causes
(1) materials and methods
A. antigen: formalin-inactivated, the totivirus of the bird flu H 5 N 1 strain of formaldehyde normal saline purification is an antigen.
B. antibody: the multivalent antibody IgY of egg preparation that gives the repeatedly immune bird flu H5 of healthy laying hen hypotype inactivated vaccine output is as antibody.
C. in ELISA square formation burette test, influenza virus with purification is an antigen, IgY is an antibody, biphenyl diammonium (OPD) is that substrate is at 490nm place reading, get light absorption value and be 0.4 and be best proportioning, concentration is 1.38mg/ml behind 20 times of the antigen diluents, and concentration is 1.22mg/ml behind 20 times of the antibody dilutions, is used for the preparation of antigen antibody complex.With 100 times of the antigen redilution of above-mentioned concentration, 150 times of antibody redilution, 1: 1 by volume ratio mixing is placed after 1～2 hour down for 37 ℃ and is spent the night under 4 ℃ then.Take out next day after 12000 rev/mins centrifugal 30 minutes, abandon supernatant, the precipitation that obtains is resuspended in the physiological saline solution for 1000 times.
D. respectively by vein and intraperitoneal injection under jugular vein, the wing, took a blood sample at the 9th, 15,21 and 28 day respectively after the first pin immunity.
E, blank group adopt sterile saline, conventional recombinant fowl influenza virus H5 hypotype bivalent inactivated vaccine (H5N1, the Re-1 strain+Re-4 strain) immunity of test control group.
Embodiment 3: immunne response and curative effect that bird flu subunit complex formulation causes
(1) materials and methods
A. antigen: from the chick embryo allantoic liquid of bird flu virus, extract RNA, carry out RT-PCR, reclaim the HA genes of interest, connect the pET-21b carrier, change clonal expression in the BL21 competent cell over to.The ultrasonic degradation thalline, SDS-PAGE electrophoresis detection HA albumen.Ni-sepharose purification albumen.
B. antibody: the multivalent antibody IgY of egg preparation that gives the repeatedly immune bird flu H5 of healthy laying hen hypotype inactivated vaccine output is as antibody.
C. in ELISA square formation burette test, HA albumen with purification is antigen, IgY is an antibody, biphenyl diammonium (OPD) is that substrate is at the 490nm place, get light absorption value and be 0.4 and be best proportioning, concentration is 1.38mg/ml behind 20 times of the antigen diluents, and concentration is 1.22mg/ml behind 20 times of the antibody dilutions, is used for the preparation of antigen antibody complex.With 100 times of the antigen redilution of above-mentioned concentration, 150 times of antibody redilution, 1: 1 by volume ratio mixing is placed after 1～2 hour down for 37 ℃ and is spent the night under 4 ℃ then.Take out next day after 12000 rev/mins centrifugal 30 minutes, abandon supernatant, will precipitate 1000 times and be resuspended in the physiological saline solution.
D. respectively by vein and intraperitoneal injection under jugular vein, the wing, took a blood sample at the 9th, 15,21 and 28 day respectively after the first pin immunity.
E, blank group adopt sterile saline, conventional recombinant fowl influenza virus H5 hypotype bivalent inactivated vaccine (H5N1, the Re-1 strain+Re-4 strain) immunity of test control group.
Immunne response and efficacy result that the bird flu antigen antibody complex preparation of embodiment 2 preparations and embodiment 3 preparation bird flu subunit complex formulations cause see Fig. 1 and Fig. 2.Fig. 1 is an antibody horizontal comparable situation of respectively organizing the chicken group after two kinds of antigen antibody complex vaccines and the conventional vaccine immunity, utilize SPSS 11.5 software analysis, the result shows that the antibody horizontal of conventional vaccine group and complex formulation I group compares diversity extremely significantly (P＜0.01) with the blank group, and complex formulation II group and blank group do not have significant difference (P〉0.05).Fig. 2 is the comparison of sickness rate, mortality rate and protective rate behind two kinds of antigen antibody complex vaccines and the conventional vaccine vaccinated flock; Utilize SPSS 11.5 software analysis; the conventional vaccine group; complex formulation I group and complex formulation II group are compared with the blank group in sickness rate, mortality rate, the protective rate opposite sex extremely significantly (P＜0.01) that is wanting in, and complex formulation I group is organized with complex formulation II significant difference (P＜0.05).
Claims (7)
1. one kind is used for the treatment of and/or the antigen antibody complex of birds flu-preventing, wherein this antigen antibody complex is made up of antigen and antibody, described antigen is the H5N1 subtype avian influenza virus of deactivation, and described antibody is multivalent antibody IgY, and the mass concentration ratio of described antigen and antibody is 1: 0.67.
2. antigen antibody complex according to claim 1 is characterized in that, described multivalent antibody IgY is the high immunity yolk antibody that bird flu H5 hypotype inactivated vaccine repeatedly prepares behind the immune health laying hen.
3. the preparation method of claim 1 or 2 described antigen antibody complexs, comprising following steps:
(1) with the bird flu virus deactivation;
(2) blood source purification bird flu virus is as antigen;
(3) antigen and the antibody thereof with dilution is mixed in proportion, and places after 1～2 hour down for 37 ℃ and spends the night under 4 ℃; The centrifugal precipitation that obtains gets final product.
4. preparation method according to claim 3 is characterized in that, this method may further comprise the steps:
(1) inoculates the Embryo Gallus domesticus that 9 ages in days do not have the specificity pathogenic microorganism with the strain of H 5 N 1 avian influenza subtype virus, collect viral allantoic fluid under the aseptic condition of propagation back, add 0.4% formalin, 37 ℃ of following deactivations 72 hours;
(2) the healthy chicken erythrocyte purification bird flu virus of handling with the normal saline that contains formaldehyde is as antigen, and concentration is 1.38mg/ml behind 20 times of the antigen diluents, and concentration is 1.22mg/ml behind 20 times of the antibody dilutions;
(3) with 100 times of the antigen redilution of above-mentioned concentration, 150 times of antibody redilution are then according to 1: 1 ratio mixing of volume ratio, place after 1～2 hour down for 37 ℃ and spend the night under 4 ℃, take out next day after 12000 rev/mins centrifugal 30 minutes, abandon supernatant, precipitation is resuspended in 1000 times of physiological saline solution.
5. one kind is used for the treatment of and/or the preparation of the antigen antibody complex of birds flu-preventing, and its active component is claim 1 or 2 described antigen antibody complexs.
6. preparation according to claim 5 is characterized in that: described antigen antibody complex is suspended from the normal saline.
7. claim 1 or 2 described antigen antibody complexs prevent and/or treat purposes in the medicine of bird flu in preparation.
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| CN102526727B (en) * | 2012-01-04 | 2014-05-07 | 河北科星药业有限公司 | Anti-bird-flue egg yolk antibody injection and application thereof |
| US20180008702A1 (en) * | 2014-12-05 | 2018-01-11 | Celltrion Inc. | Adjuvant composition containing at least one influenza virus neutralizing and binding molecule and vaccine composition containing same |
| CN104984337B (en) * | 2014-12-31 | 2018-08-21 | 福州大北农生物技术有限公司 | A kind of newcastle disease, avian influenza antigen antibody complex inactivated vaccine and preparation method thereof |
| CN106267195B (en) * | 2016-08-30 | 2020-01-21 | 天津市中升挑战生物科技有限公司 | Triple antigen-antibody complex for porcine viral diarrhea and preparation method thereof |
| CN106729693A (en) * | 2016-12-20 | 2017-05-31 | 天津瑞普生物技术股份有限公司 | A kind of multipath synergetic immunity method for adenovirus |
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