CN104829710A - An anti-Ebola-virus immunoglobulin F(ab')2 and a preparing method thereof - Google Patents
An anti-Ebola-virus immunoglobulin F(ab')2 and a preparing method thereof Download PDFInfo
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Abstract
An anti-Ebola-virus immunoglobulin F(ab')2 and a preparing method thereof are disclosed. Three trivalent EBOV vaccine antigens, namely an EBOV nucleic acid vaccine, an EBOV subunit vaccine and an EBOV VLP vaccine, are utilized to immunize a healthy animal to produce a high-valence EBOV antiserum, and an Fc segment capable of causing side effects is removed to prepare the anti-EBOV specific immunoglobulin F(ab')2. Tests prove that the anti-EBOV specific immunoglobulin F(ab')2 prepared by the method is high in neutralization valence, purity and safety and stable, meets requirements of biological products in China, can specifically neutralize EBOV, has specific treatment effects for EBOV infection, can effectively treat Ebola virus disease and is a therapeutic drug with high value.
Description
Technical field
The present invention relates to the IgF (ab ') of a kind of anti-Ebola virus
2and preparation method thereof, belong to biotechnological pharmaceutics field.
Background technology
EBOV (Ebola virus, EBOV) be one of the most strong zoonosis, often cause that patient has a fever, the change of nausea,vomiting,diarrhea, the colour of skin, sore all over, body internal hemorrhage, the external multiple symptom such as hemorrhage, often again cause death because of apoplexy, myocardial infarction, hypovolemic shock or Multiple Organ Failure, the mortality ratio of human infection reaches as high as 90%.EBOV comprises five kinds of hypotypes, Ebola-Zaire (Ebola-Zaire, Z-EBOV), Ebola-Sudan (Ebola-Sudan, S-EBOV), Ebola-Cote d'lvoire (Ebola-Coted ' Ivoire, C-EBOV), Ebola-Christopher Eccleston (Ebola-Reston, and Ebola-Ben Dibujiao (Ebola-bundibugyo, B-EBOLA) R-EBOV).Wherein strong with the virulence of Z-EBOV, it is the highest that people infects rear mortality ratio; S-EBOV takes second place; C-EBOV has pathogenic to chimpanzee, to people, then virulence is the most weak; R-EBOV is not pathogenic to people, but has lethality to the primate beyond people.2014, EBOV wreaks havoc in Africa, causes the west African states such as Sierra Leone to enter " emergency state ", and WHO advises global groupcontrol EBOV, EBOV becomes " public health emergency of international concern ", and prevention and control EBOV has become the important scientific problems that current international community is badly in need of solving.
So far the whole world there is no the effective ways of prevention and therapy EBOV.As everyone knows, therapeutic antibodies is one of most effective measure for the treatment of virus, and direct injection is in patient body, rapid-action, good effect, and side reaction is low, is current great control and prevention of disease " trump card ", becomes the choice drug of control and prevention of disease.At present; NIH (NIH) and defence threaten the mouse monoclonal antibody ZMapp of the Mapp Biopharmaceutical company research and development under the alleviation council (Defense Threat Reduction Agency); cure Liang Ming U.S. patient with severe symptoms (helping non-medical staff) that EBOV infects; simultaneously; even if infect in 5 days at EBOV and inject ZMapp; also non-human primate under fire can be protected completely; make its symptom obviously improve and return to one's perfect health, directly demonstrate the critical therapeutic effect of therapeutic antibodies at prevention and control EBOV.
According to the experience that human history fights back the disease, specific antibody therapy has been one of important means of Prevention of Infectious Diseases.Along with the continuous progress of Protocols in Molecular Biology, antibody preparation experienced by several developmental stage, the first stage, direct infusion rehabilitation clients blood plasma thereupon; Subordinate phase, the antiserum(antisera) prepared after infusion immunity heterologous animal; Phase III, infusion monoclonal antibody; Fourth stage, infusion humanized antibody, at present, above-mentioned all kinds of antibody production techniques is still in industrial community widespread use.And prepare antiserum(antisera) technology after immune heterologous animal and also experienced by traditional whole antibody molecule, develop into and effectively remove the Fc fragment antibody molecule that can cause toxic side effect and other blood plasma exogenous factor today, obtain having and the high purity F of pathogenic agent specific combination (ab ')
2treatment antibody, security improves further.
Summary of the invention
An object of the present invention is to provide a kind of IgF for the preparation of anti-Ebola virus (ab ')
2complete immunogen.
IgF for the preparation of anti-Ebola virus provided by the invention (ab ')
2complete immunogen be made up of the virus sample particle vaccines of the nucleic acid vaccine of Ebola virus, the subunit vaccine of Ebola virus and Ebola virus;
The nucleic acid vaccine of described Ebola virus is following at least one: Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III;
The subunit vaccine of described Ebola virus is following at least one: Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and Cote d'lvoire type EBOV subunit vaccine VI;
The virus sample particle vaccines of described Ebola virus is following at least one: Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and Cote d'lvoire type EBOV VLP vaccine Ⅸ;
Described Zaire type EBOV nucleic acid vaccine I is the recombinant vectors of expressing Zaire type EBOV G-protein 1 encoding gene;
Described the Sudan type EBOV nucleic acid vaccine II is the recombinant vectors of expressing the Sudan type EBOV G-protein 1 encoding gene;
Described Cote d'lvoire type EBOV nucleic acid vaccine III is the recombinant vectors of expressing Cote d'lvoire type EBOV G-protein 1 encoding gene;
Described Zaire type EBOV subunit vaccine IV is Zaire type EBOV G-protein 2;
Described the Sudan type EBOV subunit vaccine V is the Sudan type EBOV G-protein 2;
Described Cote d'lvoire type EBOV subunit vaccine VI is Cote d'lvoire type EBOV G-protein 2;
Described Zaire type EBOV VLP vaccine VII is the virus-like particle of expressing Zaire type EBOV G-protein 1 encoding gene and Zaire type EBOV VP40 albumen 3 encoding gene;
Described the Sudan type EBOV VLP vaccine VIII is the virus-like particle of expressing the Sudan type EBOV G-protein 1 encoding gene and the Sudan type EBOV VP40 albumen 3 encoding gene;
Described Cote d'lvoire type EBOV VLP vaccine Ⅸ is the virus-like particle of expressing Cote d'lvoire type EBOV G-protein 1 encoding gene and Cote d'lvoire type EBOV VP40 albumen 3;
The aminoacid sequence of described Zaire type EBOV G-protein 1 is as shown in sequence in sequence table 10;
The aminoacid sequence of described the Sudan type EBOV G-protein 1 is as shown in sequence in sequence table 11;
The aminoacid sequence of described Cote d'lvoire type EBOV G-protein 1 is as shown in sequence in sequence table 12;
The aminoacid sequence of described Zaire type EBOV G-protein 2 is as shown in sequence in sequence table 13;
The aminoacid sequence of described the Sudan type EBOV G-protein 2 is as shown in sequence in sequence 14;
The aminoacid sequence of described Cote d'lvoire type EBOV G-protein 2 is as shown in sequence 15;
The aminoacid sequence of described Zaire type EBOV VP40 albumen 3 is as shown in sequence in sequence table 16;
The aminoacid sequence of described the Sudan type EBOV VP40 albumen 3 is as shown in sequence in sequence table 17;
The aminoacid sequence of described Cote d'lvoire type EBOV VP40 albumen 3 is as shown in sequence in sequence table 18.
In above-mentioned complete immunogen,
Described Ebola virus nucleic acid vaccine is made up of described Zaire type EBOV nucleic acid vaccine I, described the Sudan type EBOV nucleic acid vaccine II and described Cote d'lvoire type EBOV nucleic acid vaccine III;
The mass ratio of described Zaire type EBOV nucleic acid vaccine I, described the Sudan type EBOV nucleic acid vaccine II and described Cote d'lvoire type EBOV nucleic acid vaccine III is 1:1:1;
Described Ebola virus subunit vaccine is for be made up of described Zaire type EBOV subunit vaccine IV, described the Sudan type EBOV subunit vaccine V and described Cote d'lvoire type EBOV subunit vaccine VI;
Described Zaire type EBOV subunit vaccine IV; Described the Sudan type EBOV subunit vaccine V; The mass ratio of described Cote d'lvoire type EBOV subunit vaccine VI is 1:1:1;
Described research of Ebola vaccine is for be made up of described Zaire type EBOV VLP vaccine VII, described the Sudan type EBOVVLP vaccine VIII and described Cote d'lvoire type EBOV VLP vaccine Ⅸ;
The mass ratio of described Zaire type EBOV VLP vaccine VII, described the Sudan type EBOV VLP vaccine VIII and described Cote d'lvoire type EBOV VLP vaccine Ⅸ is 1:1:1.
In above-mentioned complete immunogen,
The recombinant vectors of described expression Zaire type EBOV G-protein 1 is the carrier obtained by the encoding gene of described Zaire type EBOV G-protein 1 insertion pVAX1 carrier;
The recombinant vectors of described expression the Sudan type EBOV G-protein 1 is the carrier obtained by the encoding gene of described the Sudan type EBOV G-protein 1 insertion pVAX1 carrier;
The recombinant vectors of described expression Cote d'lvoire type EBOV G-protein 1 is the carrier obtained by the encoding gene of described Cote d'lvoire type EBOVG albumen 1 insertion pVAX1 carrier;
In above-mentioned complete immunogen, described Zaire type EBOV G-protein 2 is that the encoding gene of Zaire type EBOV G-protein 2 is inserted pFastBac1 carrier, obtain recombinant vectors A, then recombinant vectors A is imported in object cell, express the Zaire type EBOV G-protein 2 obtained;
Described the Sudan type EBOV G-protein 2 is that the encoding gene of the Sudan type EBOV G-protein 2 is inserted pFastBac1 carrier, obtains recombinant vectors B, then imports in object cell by recombinant vectors B, expresses the Sudan type EBOV G-protein 2 obtained;
Described Cote d'lvoire type EBOV G-protein 2 is that the encoding gene of Cote d'lvoire type EBOV G-protein 2 is inserted pFastBac1 carrier, obtains recombinant vectors C, then imports in object cell by recombinant vectors C, expresses the Cote d'lvoire type EBOV G-protein 2 obtained.
In above-mentioned complete immunogen,
Described Zaire type EBOV G-protein 1 encoding gene and Zaire type EBOV VP40 albumen 3 encoding gene are called the recombinant vectors transfection object cell of A by name, namely obtain Zaire type EBOV VLP vaccine VII;
Described name is called that the recombinant vectors of A is that Zaire type EBOV G-protein 1 encoding gene and Zaire type EBOV VP40 albumen 3 encoding gene are inserted the carrier obtained in pFastBacDUAL carrier;
Described the Sudan type EBOV G-protein 1 encoding gene and the Sudan type EBOV VP40 albumen 3 encoding gene are called the recombinant vectors transfection object cell of B by name, namely obtain the Sudan type EBOV VLP vaccine VIII;
Described name is called that the recombinant vectors of B is that the Sudan type EBOV G-protein 1 encoding gene and the Sudan type EBOVVP40 albumen 3 encoding gene are inserted the carrier obtained in pFastBacDUAL carrier;
Described Cote d'lvoire type EBOV G-protein 1 encoding gene and Cote d'lvoire type EBOV VP40 albumen 3 encoding gene are called the recombinant vectors transfection object cell of C by name, namely obtain Cote d'lvoire type EBOV VLP vaccine Ⅸ;
Described name is called that the recombinant vectors of C is that Cote d'lvoire type EBOV G-protein 1 encoding gene and Cote d'lvoire type EBOV VP40 albumen 3 encoding gene are inserted the carrier obtained in pFastBacDUAL carrier.
In above-mentioned complete immunogen,
The coding gene sequence of described Zaire type EBOV G-protein 1 is as shown in sequence in sequence table 1;
The coding gene sequence of described the Sudan type EBOV G-protein 1 is as shown in sequence in sequence table 2;
The coding gene sequence of described Cote d'lvoire type EBOV G-protein 1 is as shown in sequence in sequence table 3;
The coding gene sequence of described Zaire type EBOV subunit vaccine IV is as shown in sequence in sequence table 4;
The coding gene sequence of described the Sudan type EBOV subunit vaccine V is as shown in sequence in sequence table 5;
The coding gene sequence of described Cote d'lvoire type EBOV subunit vaccine VI is as shown in sequence in sequence table 6;
The coding gene sequence of described Zaire type EBOV VP40 albumen 3 is as shown in sequence in sequence table 7;
The coding gene sequence of described the Sudan type EBOV VP40 albumen 3 is as shown in sequence in sequence table 8;
The coding gene sequence of described Cote d'lvoire type EBOV VP40 albumen 3 is as shown in sequence in sequence table 9.
In above-mentioned complete immunogen, described object cell is Sf9 insect cell.
Another object of the present invention is to provide the IgF (ab ') of the anti-Ebola virus that above-mentioned immunogen prepares
2, anti-Ebola virus antibody or in and the product of Ebola virus.
Above-mentioned immunogen is at the IgF (ab ') of the anti-Ebola virus of preparation
2in application; Or the application of above-mentioned immunogen in the antibody of the anti-Ebola virus of preparation; Or the application of above-mentioned immunogen in preparation and in the product of Ebola virus also belongs to object of the present invention.
The IgF (ab ') of above-mentioned anti-Ebola virus
2, anti-Ebola virus antibody or in and Ebola virus product preparation the application prevented and/or treated in the product of the disease that Ebola virus causes also belong to protection scope of the present invention.
Last object of the present invention be a kind of prepare anti-Ebola virus antibody or in and the test kit of product of Ebola virus.
The antibody of the anti-Ebola virus of preparation provided by the invention or in and the test kit of product of Ebola virus comprise above-mentioned immunogen.
In mentioned reagent box, described test kit also comprises the immunization method be documented in readable carrier:
Described immunization method is: first time immunity Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and each 3mg of Cote d'lvoire type EBOV nucleic acid vaccine III mix the trivalent nucleic acid vaccine obtained, second time immunity Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and each 6mg of Cote d'lvoire type EBOV nucleic acid vaccine III mix the trivalent nucleic acid vaccine obtained, third time immunity Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and each 12mg of Cote d'lvoire type EBOV nucleic acid vaccine III mix the trivalent nucleic acid vaccine obtained, 4th immunity mixes with each 24mg of Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III the trivalent nucleic acid vaccine obtained,
Or first time immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V, each 1mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained; Second time immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V, each 2mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained; Third time immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and each 3mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained; 4th immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and each 4mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained;
Or first time immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 1mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained; Second time immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 2mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained; Third time immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII, each 3mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained; 4th immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 4mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained;
Or first time immunity nucleic acid vaccine Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and each 1mg of Cote d'lvoire type EBOV nucleic acid vaccine III mix the trivalent nucleic acid vaccine obtained; Second time immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and each 1mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained; Third time immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 2mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained; 4th immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 3mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained;
Or the EBOV nucleic acid vaccine I of first time immune 1mg; The EBOV subunit vaccine IV of the immune 1mg of second time; The EBOV VLP vaccine VII of third time immune 2mg; The EBOV VLP vaccine VII of the 4th immune 3mg.
In mentioned reagent box, the animal of described immunity is horse; The weight of described horse is 300-500kg.
The present invention utilizes three kinds of trivalent EBOV vaccine antigens (EBOV nucleic acid vaccine, EBOV subunit vaccine and EBOV VLP vaccine) immune health animal, produce the antiserum(antisera) of high-titer EBOV, removal can cause the Fc fragment of side effect, prepares anti-EBOV specific immunoglobulin F (ab ')
2.Prove by experiment: the anti-EBOV specific immunoglobulin F (ab ') that the present invention prepares
2not only Neutralizing titer, purity, security are all higher and stable, meet the requirement of Products in China code, and can special in and EBOV, to EBOV infect have specific treatment effect, can effectively treat ebola disease viral disease, be very valuable curative drug.
Accompanying drawing explanation
Fig. 1 is the EBOV pseudovirus result of fluorescent microscope test experience example 1 preparation.Figure 1A is Zaire Ebola virus pseudovirus (1:1000 dilution) fluorescent microscope result; Figure 1B is Sudan Ebola virus pseudovirus (1:1000 dilution) fluorescent microscope result; Fig. 1 C is Coted ' Ivoire Ebola virus pseudovirus (1:1000 dilution) fluorescent microscope result; Fig. 1 D is blank fluorescent microscope result; Fig. 1 E is observations under the red exciting light of Zaire Ebola virus pseudovirus.
Fig. 2 is the qualification result of EBOV nucleic acid vaccine prepared by embodiment 1.M1, M2 are molecular weight Marker; Z, S, C are respectively Zaire, Sudan, Coted ' Ivoire Ebola virus nucleic acid vaccine EcoR I and Xba I double digestion result; Z1 is Zaire Ebola virus nucleic acid vaccine purity assay result.
Fig. 3 is the verification result of EBOV G-protein subunit vaccine prepared by embodiment 2.M is molecular weight Marker; ZG is Zaire Ebola virus subunit vaccine SDS-PAGE result.
Fig. 4 is the verification result of EBOV VLP vaccine prepared by embodiment 3.Fig. 4 A is Zaire Ebola virus VLP SDS-PAGE representative result; Fig. 4 B is Zaire Ebola virus VLP transmission electron microscope picture.
Fig. 5 is the anti-Ebola virus IgF (ab ') of horse prepared by embodiment 1,3,5
2the SDS-PAGE representative result of finished product.M is molecular weight Marker; IgG is blood plasma; 1,3,5 be respectively embodiment 1,3,5 prepare the anti-Ebola virus IgF (ab ') of horse
2.
Embodiment
Following used experimental technique if no special instructions, is ordinary method.
Following material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Following experimental example and embodiment further illustrate, but are not limited to the present invention.
Embodiment 1, EBOV nucleic acid vaccine and anti-EBOV specific immunoglobulin F (ab ')
2preparation
One, the preparation of EBOV nucleic acid vaccine
1, the acquisition of goal gene
Download EBOV G-protein complete genome sequence (2031bp) from NCBI website, utilize DNAMAN software to carry out sequence alignment, the homology of analysis of nucleotide and aminoacid sequence, obtain the nucleotide sequence that can represent modern popular strain hereditary feature; Analyze G-protein nucleotide sequence, commercialization nucleic acid vaccine carrier pVAX1 sequence endonuclease digestion site; Restriction endonuclease (EcoR I) sequence (GAATTC) and KozaK sequence (ACTATGG) is added in G-protein nucleotide sequence upstream, downstream adds termination codon subsequence (TAA) and restriction endonuclease (Xba I) sequence (TCTAGA), commercial company full genome synthesis EBOV nucleic acid vaccine target-gene sequence: the target-gene sequence of Zaire type EBOV G-protein is as shown in sequence in sequence table 1; The target-gene sequence of the Sudan type EBOV G-protein is as shown in sequence in sequence table 2; The target-gene sequence of Cote d'lvoire type EBOV G-protein is as shown in sequence in sequence table 3.
2, the acquisition of EBOV nucleic acid vaccine
(1) structure of recombinant vectors
Use the coding gene sequence (coding gene sequence of the Zaire type EBOV G-protein as shown in sequence in sequence table 1 of restriction enzyme EcoR I and Xba I (TAKARA company) double digestion EBOV nucleic acid vaccine carrier pVAX1 (Invitrogen company, article No.: V260-20) and above-mentioned three kinds of EBOV nucleic acid vaccine target gene G-protein respectively; The coding gene sequence of the Sudan type EBOV G-protein as shown in sequence in sequence table 2; The coding gene sequence of the Cote d'lvoire type EBOV G-protein as shown in sequence in sequence table 3), reclaim and obtain skeleton carrier and three object fragments, utilize ligase enzyme connecting framework carrier and object fragment respectively, obtain recombinant vectors pVZG, pVSG, pVCG.
Respectively sequence verification is carried out to three recombinant vectorss pVZG, pVSG, pVCG.Result shows: recombinant vectors pVZG is for replacing with the coding gene sequence of the Zaire type EBOV G-protein as shown in 1-2031 position in sequence in sequence table 1 by the DNA between the EcoR I of pVAX1 carrier and Xba I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pVAX1 carrier, the aminoacid sequence of the Zaire type EBOV G-protein of expression is as shown in sequence in sequence table 10; Recombinant vectors pVSG is for replacing with the coding gene sequence of the Sudan type EBOV G-protein as shown in 1-2031 position in sequence in sequence table 2 by the DNA between the EcoR I of pVAX1 carrier and Xba I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pVAX1 carrier, express the aminoacid sequence of Zaire type EBOV G-protein as shown in sequence in sequence table 11; Recombinant vectors pVCG is for replacing with the coding gene sequence of the Cote d'lvoire type EBOV G-protein as shown in 1-2031 position in sequence in sequence table 3 by the DNA between the EcoR I of pVAX1 carrier and Xba I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pVAX1 carrier, express the aminoacid sequence of Cote d'lvoire type EBOV G-protein as shown in sequence in sequence table 12.
(2) acquisition of recombinant bacterium
Three kinds of recombinant vectorss pVZG, pVSG, pVCG step (1) being obtained are transform competent bacteria DH5 α respectively, and coated plate, selects mono-clonal.
Recombinant bacterium DH5 α/pVZG, DH5 α/pVSG, DH5 α/pVCG plasmid is carried with the little extraction reagent kit of plasmid is little, and double digestion qualification (see Fig. 2) is carried out to the plasmid extracted, enzyme is cut and obtained the bacterium that size is about 3000bp and 2000bp two band is positive bacteria, is DH5 α/pVZG, DH5 α/pVSG, DH5 α/pVCG.
(3) acquisition of EBOV nucleic acid vaccine
Respectively DH5 α/pVZG, DH5 α/pVSG, DH5 α/pVCG again large scale fermentation are cultured to OD600 and are about 14, with the large upgrading grain of the large extraction reagent kit of plasmid, obtain pVZG (being Zaire type EBOV nucleic acid vaccine I), pVSG (being the Sudan type EBOV nucleic acid vaccine II) and pVCG (being Cote d'lvoire type EBOV nucleic acid vaccine III).
(4) concentration and purity detecting
The concentration of three kinds of nucleic acid vaccine Zaire type EBOV nucleic acid vaccines I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III that step (3) obtains and purity (see Fig. 2) are detected.Result shows: the concentration of three kinds of nucleic acid vaccines is all at 17mg/ml; A260/A280 is 2.05, A260/A230 and is 2.24, and purity is all more than 95%, and all meet the requirement of biological products code to nucleic acid vaccine, less than-70 DEG C save backup.
Two, the preparation of EBOV pseudovirus
EBOV pseudovirus is the ENV envelope protein replacing HIV by slow virus packaging system with the envelope protein G-protein of EBOV, form the pseudovirus being cyst membrane with the envelope protein G-protein of EBOV, it single-wheel can infect responsive target cell, but can not superinfection target cell, after infecting, its EGFP gene carried will enter target cell and expressing green fluorescent protein (EGFP), under fluorescent microscope, can measure its fluorescence stove number, the number of fluorescence stove number represents tiring of pseudovirus.Specific as follows:
Buy the three plasmid slow virus packaging systems of Inovogen Tech Co., Ltd., 293V slow virus packing cell and 293T lentivirus titers measure cell.Prepare pseudovirus according to slow virus process specifications, one-step optimization of going forward side by side, step is as follows:
(1) cell is prepared before transfection
In the Tissue Culture Flask of 75 square centimeters, adjustment 293V cell, to suitable concn, adds 20ml and comprises 10% heat-inactivated foetal calf serum, and 1% dual anti-DMEM substratum, is put in 37 DEG C, 5%CO
2incubator is cultivated;
(2) the abundant removal of culturing cell serum
Cell reaches 70 ~ 80% when being paved with, and changes 16ml and comprises 1% dual anti-DMEM substratum, be put in 37 DEG C, 5%CO
2incubator cultivates 2 hours;
(3) prepare before transfection
Prepare the cell pipe of a 15ml before transfection, add 4ml Opti-MEM, 80 μ l transfectionreagent (Lipofectamine successively
tM2000), 40 μ g pLV, 40 μ g pHelper1.0 and 40 μ g above-mentioned one preparation nucleic acid vaccine, mix 30 seconds, static 20 minutes of room temperature, obtains mixture;
The nucleic acid vaccine of an above-mentioned preparation is respectively Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III.
(4) transfection
Mixture is added 293V cell, be put in 37 DEG C, 5%CO
2incubator;
(5) after transfection 12 hours, comprise 1% dual anti-DMEM substratum with 20ml and replace above transfection media, be put in 37 DEG C, 5%CO
2incubator continues cultivation 60 hours;
(6) collect supernatant, and through 0.2 μm of membrane filtration, packing, is put in-76 DEG C, stand-by; Be Zaire type EBOV pseudovirus Ⅹ (Zaire), the Sudan type EBOV pseudovirus Ⅺ (Sudan), Cote d'lvoire type EBOV pseudovirus Ⅻ (Coted ' Ivoire).
(7) use 293T cell, measure pseudovirus titre, the results are shown in Figure 1 and table 1 according to Reed-Muench method, detect in 6 months, three kinds of pseudovirus titres are all more than 1 × 10
4fFU/ml.
Table 1, Ebola virus pseudovirus titre results
Three, immune health animal
Adopt to surpass and exempt from strategy, all adopt subcutaneous, the horses (horses weight be 400kg) of muscle, inguinal lymph nodes multiple spot immunity through quarantining qualified at every turn, 18 ~ 23 days, interval, first time immune 9mg trivalent nucleic acid vaccine (each 3mg mixing of Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III obtains trivalent nucleic acid vaccine); The immune 18mg trivalent nucleic acid vaccine of second time (each 6mg mixing of Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III obtains trivalent nucleic acid vaccine); Third time immune 36mg trivalent nucleic acid vaccine (each 12mg mixing of Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III obtains trivalent nucleic acid vaccine); 4th immune 72mg trivalent nucleic acid vaccine (each 24mg mixing of Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III obtains trivalent nucleic acid vaccine); Gather blood plasma when immune animal NAT reaches 3200, less than-70 DEG C save backup.
Four, anti-EBOV specific immunoglobulin F (ab ')
2preparation
1, anti-EBOV specific immunoglobulin F (ab ')
2preparation method
According to granted patent " a kind of IgF (ab ') of anti-Staphylococcus aureus enterotoxin B
2and preparation method thereof (application number: 201110202539.1; Application publication number CN102286100A; The patent No.: ZL201110202539.1) " in F (ab ')
2fragment preparation method refines anti-EBOV specific immunoglobulin F (ab ')
2, by above-mentioned three blood plasma obtained with according to granted patent " a kind of IgF (ab ') of anti-Staphylococcus aureus enterotoxin B
2and preparation method thereof (application number: 201110202539.1; Application publication number CN102286100A; The patent No.: ZL201110202539.1) " in F (ab ')
2prepared by fragment approach, collect F (ab ')
2fragment peak, obtains anti-EBOV specific immunoglobulin F (ab ')
2.
2, anti-EBOV specific immunoglobulin F (ab ')
2purity and NAT detect
1) purity
Show through HPLC measurement result: F (ab ')
2purity is more than 90%.
2) NAT
Responsive target cell is infected in the single-wheel of EBOV pseudovirus energy, but can not superinfection target cell, and after infecting, its EGFP gene carried will enter target cell and expressing green fluorescent protein EGFP, and use fluorescent microscope, can measure its fluorescence stove number; After specificity neutralizing antibody is combined with pseudovirus, pseudovirion can be made to lose infectivity, suppress the expression of EGFP, thus fluorescence stove number is reduced.Remaining pseudovirus fluorescence stove number after being neutralized by mensuration measuring samples, determines the EBOV antibody titer in sample, i.e. micro-rapid fluorescence stove inhibition test method.
Adopt micro-rapid fluorescence stove inhibition test method to the anti-EBOV specific immunoglobulin F (ab ') of above-mentioned 1 preparation
2nAT measures, and concrete grammar is as follows:
(1) get one piece of 96 orifice plate laterally to use, every block plate can do 6 parts of test serums, carries out serial dilution to serum to be checked.The 96 every holes of orifice plate add 50 μ l diluents; A1-A12: every hole adds respective sample 50 μ l, inhales the capable mixing of 50 μ l to B by after the capable mixing of A, mixes that to be diluted to G capable successively, and the antibody 50 μ l that absorption G is capable discards; H1-H6 adds corresponding test serum sample 50 μ l, mixing; Cell control well adds antibody diluent 50 μ l, mixing;
(2) by viral dilution to 100FFU/0.05ml, get the vertical hanging drop of 50 μ l and enter in each hole of 96 orifice plates (except serum, cell controls); Separately get 1.5ml 25000FFU/0.05ml virus in little centrifuge tube, keep in for 4 DEG C, treat back to survey virus titer;
(3) Tissue Culture Plate is patted mixing, in 37 DEG C and 1h;
(4) virus titer returns and measures 4 tubules, and often pipe adds 0.9ml diluent, and the 100FFU/0.05ml virus drawing 0.1ml adds in the 1st pipe, and as 10FFU/0.05ml after mixing, 10 times are diluted to 1FFU/0.05ml successively; Separately get one 96 orifice plates, each every hole of dilution virus is added 0.1ml, and do 8 multiple holes; Reserve 4 holes as cell control well, every hole adds 0.1ml viral dilution liquid simultaneously;
(5) use Digestive system peptic cell, be prepared into 2 × 10
5the cell suspension of individual/ml, every hole adds 0.1ml cell suspension respectively, and mixing, puts into 35 DEG C, 5%CO
2cultivation is hatched in incubator; Put fluorescent microscope counting fluorescence stove value, and record titration of virus result, to suppress the inverse of the most high dilution of the serum of 50% fluorescence stove for endpoint titers, after 2 days, judge net result.
(6) neutralization test result judgment basis: have 1 hole to occur fluorescence stove in 2 holes of most high dilution serum, fluorescence stove does not appear in another hole, and this dilution inverse is the NAT of this serum specimen; When fluorescence stove appears in high dilution 2 hole, there is not fluorescence stove in adjacent low extent of dilution 2 hole completely, then the inverse of both Average dilutions is the NAT of this serum specimen; When 1 hole fluorescence stove all appears in two adjacent extent of dilution serum, there is not fluorescence stove in another 1 hole, then the inverse of both Average dilutions is the NAT of this serum specimen.Attention: if viral residual titration result is not in the scope of 32 ~ 320FFU/0.05ml, it is invalid to test, repeats experiment.
Result shows: the anti-EBOV specific immunoglobulin F (ab ') of above-mentioned 1 preparation
2to the NAT of Zaire type EBOV pseudovirus Ⅹ, the Sudan type EBOV pseudovirus Ⅺ, Cote d'lvoire type EBOV pseudovirus Ⅻ in table 2.
Table 2, anti-EBOV IgF (ab ')
2nAT result
Embodiment 2, EBOV subunit vaccine and anti-EBOV specific immunoglobulin F (ab ')
2preparation
One, the acquisition of EBOV subunit vaccine
1, the acquisition of goal gene
Optimization can represent the nucleotide sequence of modern popular strain hereditary feature, makes codon be suitable in expressed in insect cells; Analyze G-protein nucleotide sequence, commercialization insect expression vector pFastBac1 (Invitrogen company, article No.: 10360-014) sequence endonuclease digestion site; Restriction endonuclease (EcoR I) sequence (GAATTC) is added in G-protein nucleotide sequence upstream, downstream adds polyhistidine sequence label (CACCACCACCACCACCAC), termination codon subsequence (TAA) and restriction endonuclease (Xba I) sequence (TCTAGA), commercial company full genome synthesis EBOV subunit vaccine target-gene sequence: the target-gene sequence of Zaire type EBOV G-protein is as shown in sequence in sequence table 4; The target-gene sequence of the Sudan type EBOV G-protein is as shown in sequence in sequence table 5; The target-gene sequence of Cote d'lvoire type EBOV G-protein is as shown in sequence in sequence table 6.
2, the acquisition of EBOV subunit vaccine
(1) acquisition of strain
According to Bac to Bac Baculovirus EXpression Systems operational manual (Invitrogen company of Invitrogen company, operational manual article No.: 10359-016) standard operating procedure, obtain EBOV subunit vaccine strain (Zaire type EBOV subunit vaccine strain IV; The Sudan type EBOV subunit vaccine strain V; Cote d'lvoire type EBOV subunit vaccine strain VI).Specific as follows:
Use the coding gene sequence (coding gene sequence of the Zaire type EBOV G-protein as shown in sequence in sequence table 4 of restriction enzyme EcoR I and Xba I (TAKARA company) double digestion carrier pFastBac1 (Invitrogen company, article No.: 10360-014) and above-mentioned three kinds of EBOV subunit vaccine target gene G-protein respectively; The coding gene sequence of the Sudan type EBOV G-protein as shown in sequence in sequence table 5; The coding gene sequence of the Cote d'lvoire type EBOV G-protein as shown in sequence in sequence table 6), reclaim and obtain skeleton carrier and three object fragments, utilize ligase enzyme connecting framework carrier and object fragment respectively, obtain recombinant vectors pFZG, pFSG, pFCG.
Respectively sequence verification is carried out to three recombinant vectorss pFZG, pFSG, pFCG.Result shows: recombinant vectors pFZG is for replacing with the coding gene sequence of the Zaire type EBOV G-protein as shown in 1-2049 position in sequence in sequence table 4 by the DNA between the EcoR I of pFastBac1 carrier and Xba I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pFastBac1 carrier, the aminoacid sequence of the Zaire type EBOV G-protein of expression is as shown in sequence in sequence table 13; Recombinant vectors pFSG is for replacing with the coding gene sequence of the Sudan type EBOV G-protein as shown in 1-2049 position in sequence in sequence table 5 by the DNA between the EcoR I of pFastBac1 carrier and Xba I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pFastBac1 carrier, express the aminoacid sequence of Zaire type EBOV G-protein as shown in sequence in sequence table 14; Recombinant vectors pFCG is for replacing with the coding gene sequence of the Cote d'lvoire type EBOV G-protein as shown in 1-2049 position in sequence in sequence table 6 by the DNA between the EcoR I of pFastBac1 carrier and Xba I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pFastBac1 carrier, express the aminoacid sequence of Cote d'lvoire type EBOV G-protein as shown in sequence in sequence table 15.
After conventionally, with pFZG, pFSG, pFCG respectively transfection Sf 9 insect cell, packaging, obtains recombinant baculovirus, i.e. EBOV subunit vaccine strain (Zaire type EBOV subunit vaccine strain IV; The Sudan type EBOV subunit vaccine strain V; Cote d'lvoire type EBOV subunit vaccine strain VI).
(2) acquisition of EBOV G-protein and purifying
Large scale fermentation Sf9 insect cell (cell concn 5 × 10
6cells/ml) (Sf9 insect cell is purchased from Invitrogen company, catalog number: 11496-015), the three kinds of EBOV subunit vaccine strains (0.1 ~ 1mol) using step (1) to prepare respectively infect Sf9 cell, fermented liquid is gathered in the crops after lysis, with Millipore300kD mwco membrane bag filter wash, the nickel post purchased from GE company is added after concentrated, the EBOV G-protein of AKTA Purifier100 purifying instrument automatic purifying band His label, obtain Zaire type EBOV subunit protein respectively, the Sudan type EBOV subunit protein, Cote d'lvoire type EBOV subunit protein (size of albumen is 120kD).
(3) detection of purity
The purity of three kinds of EBOV G-protein that step (2) obtains is detected (the results are shown in Figure 3 of Zaire type EBOV subunit protein).Result shows: the purity of three kinds of EBOV G-protein is all more than 95%.
(4) acquisition of EBOV subunit vaccine
Three kinds of EBOV G-protein step (2) obtained add aluminium adjuvant absorption respectively and prepare subunit vaccine (mass ratio of albumen and aluminium adjuvant is 1:1): obtain Zaire type EBOV subunit vaccine IV; The Sudan type EBOV subunit vaccine V; Cote d'lvoire type EBOV subunit vaccine VI (1ml vaccine liquid is containing 1.0mg aluminium salt and 1.0mg albumen).Through calibrating, EBOV G-protein adsorption rate is all more than 95%, and 4 ~ 8 DEG C save backup.
Two, the preparation of EBOV pseudovirus
Prepare pseudovirus with reference to two of experimental example 1, and detection is tired, obtain type EBOV subunit pseudovirus of Zaire (Zaire); Type EBOV subunit of the Sudan pseudovirus (Sudan); Type EBOV subunit of Cote d'lvoire pseudovirus (Coted ' Ivoire), tire consistent with result shown in table 1.
Three, immune health animal
Adopt to surpass and exempt from strategy, all adopt subcutaneous, the horses (horses weight be 400kg) of muscle, inguinal lymph nodes multiple spot immunity through quarantining qualified at every turn, 18 ~ 23 days, interval, first time immune 3mg trivalent subunit vaccine (each 1mg mixing of Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and Cote d'lvoire type EBOV subunit vaccine VI obtains trivalent subunit vaccine); The immune 6mg trivalent subunit vaccine of second time (each 2mg mixing of Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and Cote d'lvoire type EBOV subunit vaccine VI obtains trivalent subunit vaccine); Third time immune 9mg trivalent subunit vaccine (each 3mg mixing of Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and Cote d'lvoire type EBOV subunit vaccine VI obtains trivalent subunit vaccine); 4th immune 12mg trivalent subunit grain vaccine (each 4mg mixing of Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and Cote d'lvoire type EBOV subunit vaccine VI obtains trivalent subunit vaccine); Gather blood plasma when immune animal NAT reaches 3200, less than-70 DEG C save backup.
Four, anti-EBOV specific immunoglobulin F (ab ')
2preparation
1, anti-EBOV specific immunoglobulin F (ab ')
2preparation method
By above-mentioned three blood plasma obtained with according to granted patent " a kind of IgF (ab ') of anti-Staphylococcus aureus enterotoxin B
2and preparation method thereof (application number: 201110202539.1; Application publication number CN102286100A; The patent No.: ZL201110202539.1) " in F (ab ')
2prepared by fragment approach, collect F (ab ')
2fragment, obtains anti-EBOV specific immunoglobulin F (ab ')
2.
2, anti-EBOV specific immunoglobulin F (ab ')
2purity and NAT detect
1) purity
Show through HPLC measurement result: F (ab ')
2purity is more than 90%.
2) NAT
With reference to experimental example 1 four 2 2) method measure the anti-EBOV specific immunoglobulin F (ab ') of embodiment 2
2nAT, 4 ~ 8 DEG C save backup.
Result is as shown in table 2.
Embodiment 3, EBOV VLP vaccine and anti-EBOV specific immunoglobulin F (ab ')
2preparation
One, the preparation of EBOV VLP vaccine
1, the acquisition of goal gene
Optimization can represent the nucleotide sequence of modern popular strain hereditary feature, makes codon be suitable in expressed in insect cells; Analyze G-protein nucleotide sequence, VP40 protein nucleotide sequence, commercialization insect expression vector pFastBacDUAL (purchased from Invitrogen company, article No.: 10712-024) sequence endonuclease digestion site; Add restriction endonuclease (EcoR I) sequence (GAATTC) in G-protein nucleotide sequence upstream, downstream adds termination codon subsequence (TAA) and restriction endonuclease (Xba I) sequence (TCTAGA); Restriction endonuclease (Xho I) sequence (CTCGAG) is added in VP40 protein nucleotide sequence upstream, downstream adds termination codon subsequence (TAA) and restriction endonuclease (Kpn I) sequence (GGTACC), commercial company full genome synthesis EBOV VLP vaccine target gene: the target-gene sequence of Zaire type EBOV VP40 albumen is as shown in sequence in sequence table 7; The target-gene sequence of the Sudan type EBOVVP40 albumen is as shown in sequence in sequence table 8; The target-gene sequence of Cote d'lvoire type EBOV VP40 albumen is as shown in sequence in sequence table 9.
2, the acquisition of EBOV VLP vaccine
(1) acquisition of strain
According to Invitrogen company Bac to Bac Baculovirus EXpression Systems operational manual standard operating procedure, obtain EBOV VLP vaccine strain (Zaire type EBOV VLP vaccine strain VII; The Sudan type EBOV VLP vaccine strain VIII; Cote d'lvoire type EBOV VLP vaccine strain Ⅸ), specific as follows:
Use restriction enzyme EcoR I and Xba I (TAKARA company) double digestion carrier pFastBacDUAL and above-mentioned three kinds of EBOV nucleic acid vaccine target-gene sequences (coding gene sequence of the Zaire type EBOV G-protein as shown in sequence in sequence table 1 respectively; The coding gene sequence of the Sudan type EBOVG albumen as shown in sequence in sequence table 2; The coding gene sequence of the Cote d'lvoire type EBOV G-protein as shown in sequence in sequence table 3), reclaim and obtain skeleton carrier and object fragment; Utilize ligase enzyme connecting framework carrier and object fragment respectively, obtain recombinant vectors pDZG, pDSG, pDCG.
Respectively sequence verification is carried out to three recombinant vectorss pDZG, pDSG, pDCG.Result shows: recombinant vectors pDZG is for replacing with the coding gene sequence of the Zaire type EBOV G-protein as shown in 1-2031 position in sequence in sequence table 1 by the DNA between the EcoR I of pFastBacDUAL carrier and Xba I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pFastBacDUAL carrier, the aminoacid sequence of the Zaire type EBOVG albumen of expression is as shown in sequence in sequence table 10; Recombinant vectors pDSG is for replacing with the coding gene sequence of the Sudan type EBOV G-protein as shown in 1-2031 position in sequence in sequence table 2 by the DNA between the EcoR I of pFastBacDUAL carrier and Xba I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pFastBacDUAL carrier, express the aminoacid sequence of Zaire type EBOV G-protein as shown in sequence in sequence table 11; Recombinant vectors pDCG is for replacing with the coding gene sequence of the Cote d'lvoire type EBOV G-protein as shown in 1-2031 position in sequence in sequence table 3 by the DNA between the EcoR I of pFastBacDUAL carrier and Xba I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pFastBacDUAL carrier, express the aminoacid sequence of Cote d'lvoire type EBOV G-protein as shown in sequence in sequence table 12.
Use restriction enzyme Xho I and Kpn I (TAKARA company) double digestion carrier pDZG, pDSG, pDCG respectively, and the coding gene sequence (coding gene sequence of the Zaire type EBOV VP40 albumen as shown in sequence in sequence table 7 of above-mentioned three kinds of EBOV VLP vaccine target gene VP40 albumen; The coding gene sequence of the Sudan type EBOV VP40 albumen as shown in sequence in sequence table 8; The coding gene sequence of the Cote d'lvoire type EBOV VP40 albumen as shown in sequence in sequence table 9), reclaim and obtain three skeleton carriers and three object fragments; Utilize ligase enzyme to connect the coding gene sequence of the Zaire type EBOVVP40 albumen shown in sequence 7 in pDZG skeleton carrier and sequence table, obtain recombinant vectors pFDZG; Utilize ligase enzyme to connect the coding gene sequence of the Sudan type EBOV VP40 albumen shown in sequence 8 in pDSG skeleton carrier and sequence table, obtain recombinant vectors pFDSG; Utilize ligase enzyme to connect the coding gene sequence of the Cote d'lvoire type EBOV VP40 albumen shown in sequence 9 in pDCG skeleton carrier and sequence table, obtain recombinant vectors pFDCG.
Respectively sequence verification is carried out to three recombinant vectorss pFDZG, pFDSG, pFDCG.Result shows: recombinant vectors pFDZG is for replacing with the coding gene sequence of the Zaire type EBOV VP40 albumen as shown in 1-981 position in sequence in sequence table 7 by the DNA between the Xho I of pDZG carrier and Kpn I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pDZG carrier, the aminoacid sequence of the Zaire type EBOV VP40 of expression is as shown in sequence in sequence table 16; Recombinant vectors pFDSG is for replacing with the coding gene sequence of the Sudan type EBOV VP40 albumen as shown in 1-981 position in sequence in sequence table 8 by the DNA between the Xho I of pDSG carrier and Kpn I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pDSG carrier, express the aminoacid sequence of Zaire type EBOV VP40 albumen as shown in sequence in sequence table 17; Recombinant vectors pFDCG is for replacing with the coding gene sequence of the Cote d'lvoire type EBOV VP40 albumen as shown in 1-981 position in sequence in sequence table 9 by the DNA between the Xho I of pDCG carrier and Kpn I restriction enzyme site, keep the constant recombinant vectors obtained of other sequences of pDCG carrier, express the aminoacid sequence of Cote d'lvoire type EBOV VP40 albumen as shown in sequence in sequence table 18.
After conventionally, with recombinant vectors pFDZG, pFDSG, pFDCG respectively transfection Sf 9 insect cell, packaging, obtains recombinant baculovirus, i.e. EBOV VLP vaccine strain (Zaire type EBOV VLP vaccine strain IV; The Sudan type EBOV VLP vaccine strain V; Cote d'lvoire type EBOV VLP vaccine strain VI).
(2) acquisition of EBOV G-protein and purifying
Large scale fermentation Sf9 insect cell (cell concn 5 × 10
6cells/ml), three kinds of EBOV VLP vaccine strains (the Zaire type EBOV VLP vaccine strain IV using step (1) to prepare respectively; The Sudan type EBOV VLP vaccine strain V; Cote d'lvoire type EBOVVLP vaccine strain VI) (0.1 ~ 1mol) infect Sf9 cell, fermented liquid is gathered in the crops after lysis, with Millipore 300kD mwco membrane bag filter wash, concentrated after carry out sucrose density gradient centrifugation, the commercial 4FF gel column of GE is added after obtaining sample, the automatic purifying of AKTA Purifier100 purifying instrument, desugar, sample concentrates through Millipore 300kD mwco membrane bag, obtains Zaire type EBOV VLP vaccine VII antigen; The Sudan type EBOV VLP vaccine VIII antigen; Cote d'lvoire type EBOV VLP vaccine Ⅸ antigen.
(3) detection of purity
To Zaire type EBOV VLP vaccine VII antigen that step (2) obtains; The Sudan type EBOV VLP vaccine VIII antigen; The purity of Cote d'lvoire type EBOV VLP vaccine Ⅸ antigen carries out detecting (see Fig. 4), and result shows: the purity of three kinds of EBOV VLP vaccine antigens is all more than 95%.
(4) acquisition of EBOV VLP vaccine
Zaire type EBOV VLP vaccine VII antigen step (3) obtained, the Sudan type EBOV VLP vaccine VIII antigen and Cote d'lvoire type EBOV VLP vaccine Ⅸ antigen add white-oil adjuvant emulsification (proportioning of white-oil adjuvant and VLP vaccine antigen is 1ml:10mg) respectively and prepare VLP vaccine: obtain Zaire type EBOV VLP vaccine VII; The Sudan type EBOV VLP vaccine VIII; Cote d'lvoire type EBOV VLP vaccine Ⅸ.Through calibrating, EBOVVLP vaccine antigen embedding rate is more than 95%, and 4 ~ 8 DEG C save backup.
Two, the preparation of EBOV pseudovirus
Prepare pseudovirus with reference to two of experimental example 1, and detection is tired, obtain type EBOV subunit pseudovirus of Zaire (Zaire); Type EBOV subunit of the Sudan pseudovirus (Sudan); Type EBOV subunit of Cote d'lvoire pseudovirus (Coted ' Ivoire), tire consistent with result shown in table 1.
Three, immune health animal
Adopt to surpass and exempt from strategy, all adopt subcutaneous, the horses (horses weight be 400kg) of muscle, inguinal lymph nodes multiple spot immunity through quarantining qualified at every turn, 18 ~ 23 days, interval, first time immune 3mg trivalent VLP vaccine (each 1mg mixing of Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and Cote d'lvoire type EBOV VLP vaccine Ⅸ obtains trivalent VLP vaccine); The immune 6mg trivalent VLP vaccine of second time (each 2mg mixing of Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and Cote d'lvoire type EBOV VLP vaccine Ⅸ obtains trivalent VLP vaccine); Third time immune 9mg trivalent VLP vaccine (each 3mg mixing of Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and Cote d'lvoire type EBOV VLP vaccine Ⅸ obtains trivalent VLP vaccine); 4th immune 12mg trivalent VLP vaccine (each 4mg mixing of Zaire type EBOVVLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and Cote d'lvoire type EBOV VLP vaccine Ⅸ obtains trivalent VLP vaccine); Gather blood plasma when immune animal NAT reaches 3200, less than-70 DEG C save backup.
Four, anti-EBOV specific immunoglobulin F (ab ')
2preparation
1, anti-EBOV specific immunoglobulin F (ab ')
2preparation method
By above-mentioned three blood plasma obtained with according to granted patent " a kind of IgF (ab ') of anti-Staphylococcus aureus enterotoxin B
2and preparation method thereof (application number: 201110202539.1; Application publication number CN102286100A; The patent No.: ZL201110202539.1) " in F (ab ')
2prepared by fragment approach, collect F (ab ')
2fragment, obtains anti-EBOV specific immunoglobulin F (ab ')
2.
2, anti-EBOV specific immunoglobulin F (ab ')
2purity and NAT detect
1) purity
Show through HPLC measurement result: F (ab ')
2purity more than 90%,
2) NAT
With reference to experimental example 1 four 2 2) method measure the anti-EBOV specific immunoglobulin F (ab ') of embodiment 2
2nAT, 4 ~ 8 DEG C save backup.
Result is as shown in table 2.
Embodiment 4, use EBOV nucleic acid vaccine, EBOV subunit vaccine, EBOV VLP vaccine immunity healthy animal prepare anti-EBOV blood plasma and anti-EBOV specific immunoglobulin F (ab ')
2
One, immune health animal prepares anti-EBOV blood plasma
Use the EBOV nucleic acid vaccine prepared by embodiment 1,2,3, EBOV subunit vaccine, EBOV VLP vaccine as antigen, adopt Prime ~ Boost immunization strategy, all adopt subcutaneous, the horses (horses weight be 400kg) of muscle, inguinal lymph nodes multiple spot immunity through quarantining qualified at every turn, 18 ~ 23 days, interval, first time immune 3mg trivalent nucleic acid vaccine (each 1mg mixing of Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III obtains trivalent nucleic acid vaccine); The immune 3mg trivalent subunit vaccine of second time (each 1mg mixing of Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and Cote d'lvoire type EBOV subunit vaccine VI obtains trivalent subunit vaccine); Third time immune 6mg trivalent VLP vaccine (each 2mg mixing of Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and Cote d'lvoire type EBOV VLP vaccine Ⅸ obtains trivalent VLP vaccine); 4th immune 9mg trivalent VLP vaccine (each 3mg mixing of Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and Cote d'lvoire type EBOV VLP vaccine Ⅸ obtains trivalent VLP vaccine); Gather blood plasma when immune animal NAT reaches 3200, less than-70 DEG C save backup.
Two, the preparation of EBOV pseudovirus
Prepare pseudovirus with reference to two of experimental example 1, and detection is tired, obtain type EBOV subunit pseudovirus of Zaire (Zaire); Type EBOV subunit of the Sudan pseudovirus (Sudan); Type EBOV subunit of Cote d'lvoire pseudovirus (Coted ' Ivoire), tire consistent with result shown in table 1.
Three, anti-EBOV specific immunoglobulin F (ab ')
2preparation
1, anti-EBOV specific immunoglobulin F (ab ')
2preparation method
By above-mentioned three blood plasma obtained with according to granted patent " a kind of IgF (ab ') of anti-Staphylococcus aureus enterotoxin B
2and preparation method thereof (application number: 201110202539.1; Application publication number CN102286100A; The patent No.: ZL201110202539.1) " in F (ab ')
2prepared by fragment approach, collect F (ab ')
2fragment, obtains anti-EBOV specific immunoglobulin F (ab ')
2.
2, anti-EBOV specific immunoglobulin F (ab ')
2purity and NAT detect
1) purity
Show through HPLC measurement result: F (ab ')
2purity is more than 90%.
2) NAT
With reference to experimental example 1 four 2 2) method measure the anti-EBOV specific immunoglobulin F (ab ') of embodiment 2
2nAT, 4 ~ 8 DEG C save backup.
Result is as shown in table 2.
Embodiment 5, use Zaire type EBOV nucleic acid vaccine, Zaire type EBOV subunit vaccine, Zaire type EBOV VLP vaccine immunity healthy animal prepare anti-Zaire type EBOV blood plasma and anti-Zaire type EBOV specific immunoglobulin F (ab ')
2
One, immune health animal prepares anti-Zaire type EBOV blood plasma
Use the Zaire type EBOV nucleic acid vaccine prepared by embodiment 1,2,3, Zaire type EBOV subunit vaccine, Zaire's type EBOV VLP vaccine as antigen, adopt Prime ~ Boost immunization strategy, all adopt subcutaneous, the horses (horses weight be 400kg) of muscle, inguinal lymph nodes multiple spot immunity through quarantining qualified at every turn, 18 ~ 23 days, interval, the EBOV nucleic acid vaccine I of first time immune 1mg; The EBOV subunit vaccine IV of the immune 1mg of second time; The EBOV VLP vaccine VII of third time immune 2mg; The EBOV VLP vaccine VII of the 4th immune 3mg; Gather blood plasma when immune animal NAT reaches 3200, less than-70 DEG C save backup.
Two, the preparation of EBOV pseudovirus
Prepare pseudovirus with reference to two of experimental example 1, and detection is tired, obtain type EBOV subunit pseudovirus of Zaire (Zaire); Type EBOV subunit of the Sudan pseudovirus (Sudan); Type EBOV subunit of Cote d'lvoire pseudovirus (Coted ' Ivoire), tire consistent with result shown in table 1.
Three, anti-Zaire type EBOV specific immunoglobulin F (ab ')
2preparation
1, anti-EBOV specific immunoglobulin F (ab ')
2preparation method
By above-mentioned three blood plasma obtained with according to granted patent " a kind of IgF (ab ') of anti-Staphylococcus aureus enterotoxin B
2and preparation method thereof (application number: 201110202539.1; Application publication number CN102286100A; The patent No.: ZL201110202539.1) " in F (ab ')
2prepared by fragment approach, collect F (ab ')
2fragment, obtains anti-EBOV specific immunoglobulin F (ab ')
2.
2, anti-EBOV specific immunoglobulin F (ab ')
2purity and NAT detect
1) purity
Show through HPLC measurement result: F (ab ')
2purity is more than 90%.
2) NAT
With reference to experimental example 1 four 2 2) method measure the anti-EBOV specific immunoglobulin F (ab ') of embodiment 2
2nAT, 4 ~ 8 DEG C save backup.
Result is as shown in table 2.
Experimental example 6, anti-EBOV specific immunoglobulin F (ab ')
2security detect
According to 2010 version " Chinese Pharmacopoeia " requirement, part entrusts Beijing Zhaoyan New Drug Research Center, in strict accordance with the step of each Detection of content in version " Chinese Pharmacopoeia " annex in 2010, to 5 batches of anti-EBOV specific immunoglobulin F (ab ') that embodiment 1 ~ 5 prepares
2security detects.
Detected result table 3 shows: 5 batches of anti-EBOV specific immunoglobulin F (ab ') that embodiment 1 ~ 5 prepares
2be colourless or light yellow transparent liquid, purity higher than 90%, toxicity without exception, aseptic, without thermal source, class blood group A substance content is lower than 4 μ g/ml; Confirm do not have cross reaction with the normal popular feeling, liver, spleen, lung, kidney, brain, lymphoglandula, intestinal tissue organ by immunohistochemical methods, proving that goods do not have the composition of anti-human tissue, is safe for patient treatment.
Anti-EBOV IgF (ab ') prepared by table 3, embodiment 1-5
2security detected result
Experimental example 7, anti-EBOV specific immunoglobulin F (ab ')
2detection of Stability
To 5 batches of anti-EBOV specific immunoglobulin F (ab ') that embodiment 1 ~ 5 prepares
2stability detects.Concrete steps are as follows: 5 batches that embodiment 1 ~ 5 are prepared anti-EBOV specific immunoglobulin F (ab ')
2the environment that stoste is placed in 4 ~ 8 DEG C and 36 ~ 38 DEG C respectively observes stability.
Table 4,5 results show: the 5 batches of anti-EBOV specific immunoglobulin F (ab ') being placed in 4 ~ 8 DEG C
2stoste is placed more than 6 months Neutralizing titer and is not subtracted, and has good stability; Be placed in 5 batches of anti-EBOV specific immunoglobulin F (ab ') of 36 ~ 38 DEG C
2stoste is placed more than 3 months Neutralizing titer and is not subtracted, and slightly declines when 6 months.
Anti-EBOV IgF (ab ') prepared by table 4, embodiment 1-5
24 DEG C of Detection of Stability results
Anti-EBOV IgF (ab ') prepared by table 5, embodiment 1-5
237 DEG C of Detection of Stability results
Claims (10)
1. for the preparation of the IgF (ab ') of anti-Ebola virus
2complete immunogen, be made up of the virus sample particle vaccines of the nucleic acid vaccine of Ebola virus, the subunit vaccine of Ebola virus and Ebola virus;
The nucleic acid vaccine of described Ebola virus is following at least one: Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III;
The subunit vaccine of described Ebola virus is following at least one: Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and Cote d'lvoire type EBOV subunit vaccine VI;
The virus sample particle vaccines of described Ebola virus is following at least one: Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and Cote d'lvoire type EBOV VLP vaccine Ⅸ;
Described Zaire type EBOV nucleic acid vaccine I is the recombinant vectors of expressing Zaire type EBOV G-protein 1 encoding gene;
Described the Sudan type EBOV nucleic acid vaccine II is the recombinant vectors of expressing the Sudan type EBOV G-protein 1 encoding gene;
Described Cote d'lvoire type EBOV nucleic acid vaccine III is the recombinant vectors of expressing Cote d'lvoire type EBOV G-protein 1 encoding gene;
Described Zaire type EBOV subunit vaccine IV is Zaire type EBOV G-protein 2;
Described the Sudan type EBOV subunit vaccine V is the Sudan type EBOV G-protein 2;
Described Cote d'lvoire type EBOV subunit vaccine VI is Cote d'lvoire type EBOV G-protein 2;
Described Zaire type EBOV VLP vaccine VII is the virus-like particle of expressing Zaire type EBOV G-protein 1 encoding gene and Zaire type EBOV VP40 albumen 3 encoding gene;
Described the Sudan type EBOV VLP vaccine VIII is the virus-like particle of expressing the Sudan type EBOV G-protein 1 encoding gene and the Sudan type EBOVVP40 albumen 3 encoding gene;
Described Cote d'lvoire type EBOV VLP vaccine Ⅸ is the virus-like particle of expressing Cote d'lvoire type EBOV G-protein 1 encoding gene and Cote d'lvoire type EBOV VP40 albumen 3;
The aminoacid sequence of described Zaire type EBOV G-protein 1 is as shown in sequence in sequence table 10;
The aminoacid sequence of described the Sudan type EBOV G-protein 1 is as shown in sequence in sequence table 11;
The aminoacid sequence of described Cote d'lvoire type EBOV G-protein 1 is as shown in sequence in sequence table 12;
The aminoacid sequence of described Zaire type EBOV G-protein 2 is as shown in sequence in sequence table 13;
The aminoacid sequence of described the Sudan type EBOV G-protein 2 is as shown in sequence in sequence 14;
The aminoacid sequence of described Cote d'lvoire type EBOV G-protein 2 is as shown in sequence 15;
The aminoacid sequence of described Zaire type EBOV VP40 albumen 3 is as shown in sequence in sequence table 16;
The aminoacid sequence of described the Sudan type EBOV VP40 albumen 3 is as shown in sequence in sequence table 17;
The aminoacid sequence of described Cote d'lvoire type EBOV VP40 albumen 3 is as shown in sequence in sequence table 18.
2. complete immunogen according to claim 1, is characterized in that:
Described Ebola virus nucleic acid vaccine is made up of described Zaire type EBOV nucleic acid vaccine I, described the Sudan type EBOV nucleic acid vaccine II and described Cote d'lvoire type EBOV nucleic acid vaccine III;
The mass ratio of described Zaire type EBOV nucleic acid vaccine I, described the Sudan type EBOV nucleic acid vaccine II and described Cote d'lvoire type EBOV nucleic acid vaccine III is 1:1:1;
Described Ebola virus subunit vaccine is for be made up of described Zaire type EBOV subunit vaccine IV, described the Sudan type EBOV subunit vaccine V and described Cote d'lvoire type EBOV subunit vaccine VI;
Described Zaire type EBOV subunit vaccine IV; Described the Sudan type EBOV subunit vaccine V; The mass ratio of described Cote d'lvoire type EBOV subunit vaccine VI is 1:1:1;
Described research of Ebola vaccine is for be made up of described Zaire type EBOV VLP vaccine VII, described the Sudan type EBOV VLP vaccine VIII and described Cote d'lvoire type EBOV VLP vaccine Ⅸ;
The mass ratio of described Zaire type EBOV VLP vaccine VII, described the Sudan type EBOV VLP vaccine VIII and described Cote d'lvoire type EBOV VLP vaccine Ⅸ is 1:1:1.
3. complete immunogen according to claim 1 and 2, is characterized in that:
The recombinant vectors of described expression Zaire type EBOV G-protein 1 is the carrier obtained by the encoding gene of described Zaire type EBOV G-protein 1 insertion pVAX1 carrier;
The recombinant vectors of described expression the Sudan type EBOV G-protein 1 is the carrier obtained by the encoding gene of described the Sudan type EBOV G-protein 1 insertion pVAX1 carrier;
The recombinant vectors of described expression Cote d'lvoire type EBOV G-protein 1 is the carrier obtained by the encoding gene of described Cote d'lvoire type EBOV G-protein 1 insertion pVAX1 carrier.
4., according to described complete immunogen arbitrary in claim 1-3, it is characterized in that:
Described Zaire type EBOV G-protein 1 encoding gene and Zaire type EBOV VP40 albumen 3 encoding gene are called the recombinant vectors transfection object cell of A by name, namely obtain Zaire type EBOV VLP vaccine VII;
Described name is called that the recombinant vectors of A is that Zaire type EBOV G-protein 1 encoding gene and Zaire type EBOV VP40 albumen 3 encoding gene are inserted the carrier obtained in pFastBacDUAL carrier;
Described the Sudan type EBOV G-protein 1 encoding gene and the Sudan type EBOV VP40 albumen 3 encoding gene are called the recombinant vectors transfection object cell of B by name, namely obtain the Sudan type EBOV VLP vaccine VIII;
Described name is called that the recombinant vectors of B is that the Sudan type EBOV G-protein 1 encoding gene and the Sudan type EBOV VP40 albumen 3 encoding gene are inserted the carrier obtained in pFastBacDUAL carrier;
Described Cote d'lvoire type EBOV G-protein 1 encoding gene and Cote d'lvoire type EBOV VP40 albumen 3 encoding gene are called the recombinant vectors transfection object cell of C by name, namely obtain Cote d'lvoire type EBOV VLP vaccine Ⅸ;
Described name is called that the recombinant vectors of C is that Cote d'lvoire type EBOV G-protein 1 encoding gene and Cote d'lvoire type EBOVVP40 albumen 3 encoding gene are inserted the carrier obtained in pFastBacDUAL carrier.
5., according to described complete immunogen arbitrary in claim 1-4, it is characterized in that:
The coding gene sequence of described Zaire type EBOV G-protein 1 is as shown in sequence in sequence table 1;
The coding gene sequence of described the Sudan type EBOV G-protein 1 is as shown in sequence in sequence table 2;
The coding gene sequence of described Cote d'lvoire type EBOV G-protein 1 is as shown in sequence in sequence table 3;
The coding gene sequence of described Zaire type EBOV subunit vaccine IV is as shown in sequence in sequence table 4;
The coding gene sequence of described the Sudan type EBOV subunit vaccine V is as shown in sequence in sequence table 5;
The coding gene sequence of described Cote d'lvoire type EBOV subunit vaccine VI is as shown in sequence in sequence table 6;
The coding gene sequence of described Zaire type EBOV VP40 albumen 3 is as shown in sequence in sequence table 7;
The coding gene sequence of described the Sudan type EBOV VP40 albumen 3 is as shown in sequence in sequence table 8;
The coding gene sequence of described Cote d'lvoire type EBOV VP40 albumen 3 is as shown in sequence in sequence table 9.
6. the IgF (ab ') of the anti-Ebola virus prepared by described immunogen arbitrary in claim 1-5
2, anti-Ebola virus antibody or in and the product of Ebola virus.
7. in claim 1-5 arbitrary described immunogen at the IgF (ab ') of the anti-Ebola virus of preparation
2in application;
Or the application of arbitrary described immunogen in the antibody of the anti-Ebola virus of preparation in claim 1-5;
Or the application of arbitrary described immunogen in preparation and in the product of Ebola virus in claim 1-5.
8. the IgF (ab ') of the described anti-Ebola virus in claim 7
2, described anti-Ebola virus antibody or described in and the product of Ebola virus preparing the application prevented and/or treated in the product of the disease that Ebola virus causes.
9. prepare anti-Ebola virus antibody or in and the test kit of product of Ebola virus, comprise arbitrary described immunogen in claim 1-5.
10. test kit according to claim 9, is characterized in that: described test kit also comprises the immunization method be documented in readable carrier:
Described immunization method is: first time immunity Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and each 3mg of Cote d'lvoire type EBOV nucleic acid vaccine III mix the trivalent nucleic acid vaccine obtained, second time immunity Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and each 6mg of Cote d'lvoire type EBOV nucleic acid vaccine III mix the trivalent nucleic acid vaccine obtained, third time immunity Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and each 12mg of Cote d'lvoire type EBOV nucleic acid vaccine III mix the trivalent nucleic acid vaccine obtained, 4th immunity mixes with each 24mg of Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and Cote d'lvoire type EBOV nucleic acid vaccine III the trivalent nucleic acid vaccine obtained,
Or first time immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V, each 1mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained; Second time immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V, each 2mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained; Third time immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and each 3mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained; 4th immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and each 4mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained;
Or first time immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 1mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained; Second time immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 2mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained; Third time immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII, each 3mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained; 4th immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 4mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained;
Or first time immunity nucleic acid vaccine Zaire type EBOV nucleic acid vaccine I, the Sudan type EBOV nucleic acid vaccine II and each 1mg of Cote d'lvoire type EBOV nucleic acid vaccine III mix the trivalent nucleic acid vaccine obtained; Second time immunity mixes with Zaire type EBOV subunit vaccine IV, the Sudan type EBOV subunit vaccine V and each 1mg of Cote d'lvoire type EBOV subunit vaccine VI trivalent subunit vaccine obtained; Third time immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 2mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained; 4th immunity mixes with Zaire type EBOV VLP vaccine VII, the Sudan type EBOV VLP vaccine VIII and each 3mg of Cote d'lvoire type EBOV VLP vaccine Ⅸ the trivalent VLP vaccine obtained;
Or the EBOV nucleic acid vaccine I of first time immune 1mg; The EBOV subunit vaccine IV of the immune 1mg of second time; The EBOV VLP vaccine VII of third time immune 2mg; The EBOV VLP vaccine VII of the 4th immune 3mg;
The animal of described immunity is horse; The weight of described horse is 300-500kg.
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