CN101695569A - Univalent and bivalent gene engineered subunit vaccine for hand-foot-and-mouth disease and preparation method thereof - Google Patents

Abstract

The invention discloses a univalent and bivalent gene engineered subunit vaccine for preventing Enterovirus 71 (EV71) and Coxsackie virus A16 (Cox.A16) of hand-foot-and-mouth disease, and a preparation method thereof. The preparation method comprises the following steps: respectively obtaining recombinant baculovirus Bac-EV71-P1-3CD and Bac-Cox.A16-P1-3CD by gene engineering means, respectively efficiently coexpressing similar SeQ ID No.1 EV71 P1 and Se Q ID No.2 Cox.A16 P1 and 3CD proteins in insect cells, and respectively self-assembling into EV71 VLP and Cox.A16 VLP; establishing and verifying a vaccine strain third-stage seed lot library; culturing cells; inoculating and propagating virus; lysing the cells, ultra-filtering and purifying virus suspension; and further preparing the univalent and bivalent vaccine. The vaccine has good application prospect for preventing the hand-foot-and-mouth disease.

Description

Single, double bivalent gene engineered subunit vaccine of a kind of hand-foot-mouth disease and preparation method thereof

Technical field

The invention belongs to field of biological pharmacy, relate to a kind of vaccine and preparation method thereof, single, double bivalent gene engineered subunit vaccine of particularly a kind of hand-foot-mouth disease and preparation method thereof.

Technical background

Hand-foot-mouth disease is by Picornaviridae, and enterovirus genus virus causes, has now become the important infectious disease of harm infantile health and public health security.Hand-foot-mouth disease is mainly caused by enterovirns type 71 (EV71) and coxsackie virus A 16-type (Cox.A16).Infect EV71 and the caused clinical symptoms of Cox.A16 and be difficult to difference.Infect the usually concurrent serious neural inflammation of EV71, comprise viral meningitis, encephalitis, and the paralysis of similar poliomyelitis, the pulmonary edema that causes, pneumorrhagia often cause the quick death of infant.Cox.A16 is the important cause of disease that causes myocarditis, pericarditis, cardiomyopathy and other serious disease.Hand-foot-mouth disease symptom from infecting to occurring is generally 3-6 days.Patient is everlasting and skin ulcer occurs in the mouth, and the skin erythema appears in palm, sole.Some case mainly is the infant less than 3 years old, severe complications can occur, and worsen suddenly short-term fever back, and dead fast.

Still do not have effective vaccine or medicine at present and can prevent and treat hand-foot-mouth disease.

Since reported first EV71 in 1974, EV71 worldwide causes repeatedly eruption and prevalence.Over nearly 10 years, not only number of the infected is many in Asian-Pacific area wildness for EV71 virus popular, and death is many.CONTINENTAL AREA OF CHINA in 1981 by Shanghai reported first hand-foot-mouth disease, after this, all there is the report of primary disease outbreak of epidemic in tens provinces such as Beijing, Shandong, Guangdong.It is popular that hand-foot-mouth disease takes place in area such as Linyi, Shandong in 2007,39606 examples of falling ill, dead 14 examples.2008, the inland of China reported the hand-foot-mouth disease case 488955 examples altogether, dead 126 examples.From on May 2nd, 2008, Ministry of Public Health was included hand-foot-mouth disease the management of in Class C Notifiable disease.The hand-foot-mouth disease epidemic situation still spread in the China's Mainland in 2009.

Epidemiologic data shows that Cox.A16 is general and EV71 is popular together, in epidemiological process, considers that Cox.A16 causes symptomless infection more, and is may shared in the air actual ratio higher.After Fuyang hand-foot-mouth disease epidemic situation took place in 2008, the CDC laboratory detection result of case specimen in China showed that in 582 hand-foot-mouth disease case positive samples, EV71 accounts for 54.5%, and Cox.A16 accounts for 17.4%.

It may be due to new genotype is worldwide propagated that the hand-foot-mouth disease that is caused by EV71 or Cox.A16 breaks out repeatedly.Molecular epidemiology analysis to 1970-2004 whole world EV71 separated strain shows that EV71 virus is divided into 3 genotype, i.e. A, B, C type, 8 gene hypotypes, i.e. B1-B4 and C1-C4 hypotype.To 1983-2003 Fukushima area (Fukushima, Japan) the Cox.A16 separated strain carries out genetic remodeling and shows, the Cox.A16 separated strain formed 3 different heredity bunch (A, B, C).1999-2004 China Shenzhen area EV71 and Cox.A16 separated strain are carried out the genetic remodeling analysis, show the main popular C4 hypotype of EV71 virus, the main popular C type of Cox.A16 virus.Advance the EV71 virus that was separated in several years and be mainly the C4 hypotype from light disease case of China's Mainland hand-foot-mouth disease and severe cases specimen, Cox.A16 virus is mainly the C type.

EV71 and Cox.A16 virion are the three-dimensional symmetric spherical structure of icosahedron, no peplos and projection.The capsid of virion is made of 60 subunits, and the latter is again the pentamer spline structure that is assembled into by 4 kinds of capsid proteins (VP1-VP4).Antigenic determinant is located substantially on the VP1-VP3, and VP4 is embedded in the inboard of virus coat.EV71 and Cox.A16 viral genome are the sub-thread positive chain RNA of 7400-7420 nucleotide, an open reading frame is only arranged in the genome, coding contains about 2190-2200 amino acid whose polyprotein, in its both sides is 5 ' and 3 '-noncoding region (UTRs), and the end in its 3 '-UTRs district contains an adjustable length poly-A.Polyprotein can further be hydrolyzed into P1, P2, P3 precursor protein.P1 precursor protein coding VP1, VP2, VP3, VP4 virus capsid protein; P2 and P3 precursor protein coding 7 non-structural proteins (2A-2C and 3A-3D).

Under the inspiration of poliomyelitis vaccine,Salk and attenuated live vaccine, there has been laboratory to carry out the research of hand-foot-and-mouth disease inactivated vaccine and attenuated live vaccine, obtained some progress, but still had a segment distance apart from clinical practice.Once there was the EV71 formalin-inactivated vaccine successfully to control the report that EV71 propagates in Bulgaria, but do not see the report that reuses inactivated vaccine.TaiWan, China He Mei has screened in the township Strain YN3-4a that a strain is suitable for preparing the EV71 inactivated vaccine, viral generation height, strong, wide, the progeny virus stable height of cross protection spectrum of immunogenicity.Aspect the development of attenuated live vaccines, the surplus person of outstanding talent of TaiWan, China forces with EV71 and Cox.A16 low virulent strain immune mouse and has obtained immunoprotection to EV71 virulent strain, shows to have the cross protection function.Japan Minetaro Arita adopts the reverse genetic rescue low virulent strain EV71 (S1-3 ') that learns a skill, and behind the immune stump-tailed macaque, neurovirulence reduces, and has broad-spectrum cross protection activity.Attenuated live vaccines because EV71 and Cox.A16 virulence decision group are unclear so far, is difficult in a period of time study successfully.

In traditional vaccine research, the laboratory research of subunit vaccine has obtained gratifying progress.Adopt Pichia sp., escherichia coli, insect cell, Salmonella, pertussis antibacterial etc. to express the structural protein VP1 of EV71 and Cox.A16, and utilize patient's positive serum to confirm that the VP1 albumen of expressing has immunogenicity.Can induce protection behind the dna vaccination immune mouse of VP1 structural protein that insect cell and Salmonella are expressed and the development of use VP1 gene to EV71 or Cox.A16 infection.

The virion ghost that does not contain viral nucleic acid that virus-like particle (VLP) is made up of viral capsid proteins.Because the virus antigen on VLP surface and polypeptide epitope are almost as broad as long with real virus, so can cause strong immunoreation, and do not have potential side effect, are to think the most potential vaccine research direction at present.(Recombivax HB, Merck) (Gardisil's hepatitis B virus VLP vaccine Merck) goes on the market with human papillomavirus VLP vaccine.Taiwan's scholars Hu Yucheng expresses, purification EV71 VLP, and immune mouse shows that the VLP vaccine can stimulate body to produce stronger immunoprotection than inactivated vaccine, VP1 protein vaccine, VP1 dna vaccination, has showed application promise in clinical practice.

Although carried out relevant EV71 and the work of Cox.A16 developing vaccines, the present still untapped vaccine that goes out to have medical commercial application value.

Summary of the invention

The purpose of this invention is to provide a kind of enterovirns type 71 (EV71) of hand-foot-mouth disease and single, double bivalent gene engineered subunit vaccine of coxsackie virus A 16-type (Cox.A16) of preventing; The present invention also aims to disclose the preparation method of above-mentioned vaccine.

The present invention seeks to be achieved by the following scheme.

Unit price subunit vaccine of the present invention is by EV71 VLP antigen or Cox.A16 VLP antigen and Al (OH) ₃Adjuvant is formulated by following weight percent content:

EV71 VLP antigen or Cox.A16 VLP antigen 0.0015-0.003 weight portion

Al (OH) ₃Adjuvant 0.25-0.5 weight portion.

The two valency subunit vaccines of the present invention are by EV71VLP antigen and Cox.A16VLP antigen and Al (OH) ₃Adjuvant is formulated by following weight percent content:

EV71VLP antigen and Cox.A16VLP antigen 0.0015-0.002 weight portion

Al (OH) ₃Adjuvant 0.25-0.5 weight portion;

Wherein the antigenic ratio of EV71VLP antigen and Cox.A16VLP is 1: 1.

The preparation method of the single, double valency vaccine of the present invention comprises the steps:

A, obtain recombinant baculovirus Bac-EV71-P1-3CD and Bac-Cox.A16-P1-3CD respectively by genetic engineering means, the efficient similar SeQ ID of coexpression NO.1EV71P1, SeQ IDNO.2Cox.A16P1 and 3CD albumen in insect cell respectively, and be self-assembled into EV71VLP and Cox.A16VLP respectively;

B sets up and three grades of seed lot storehouses of calibrating vaccine strain;

C adopts the insect cell of serum-free medium high density suspension culture from Sf-9/Hi-5 cell line;

D grows up to 1.5-2 * 10 at cell ⁶During cell/ml, the inoculation recombinant baculovirus;

E, virus is propagation and efficient coexpression P1 and 3CD albumen in cell, produces VLP;

F, the suspension that cracking insect cell and collecting cell are produced, clarification filtration is removed the insect cell residue;

G, ultrafiltration and concentration virus vlps suspension;

H, the viral suspension behind the ultrafiltration and concentration by gel permeation chromatography and ion-exchange chromatography purified virus VLP, perhaps by sucrose density gradient centrifugation purified virus VLP, gets the virus stock solution used of purification, i.e. antigen;

I, preparation and calibrating vaccine semi-finished product and finished product, safety, effectiveness and the stability of measurement vaccine.

Wherein, P1 and 3CD gene order adopt RT-PCR method amplification hand-foot-mouth disease Strain to obtain in a step: SeQ IDNO.1 EV71 P1 and 3CD gene order utilize RT-PCR method amplification EV71 (H13-Henan-08) viral RNA to obtain, SeQ ID NO.2Cox.A16P1 and 3CD gene order utilize RT-PCR method amplification Cox.A16 (S10-shandong-08) viral RNA to obtain, and order-checking showed correct after gene all was cloned into the pMD18-T carrier; P1 and 3CD gene order also can adopt the synthetic mode of full gene to obtain, and are optimized according to the insecticide preference codon.

Set up in the b step and three grades of seed lot storehouses of calibrating vaccine strain, promptly primordial seed is criticized, the method for main seed lot and work seed lot is: primordial seed criticize into through on the Sf-9 cell of plaque purification 4 generation seed culture of viruses; Identify record, history, source and biological characteristics that primordial seed is criticized; Criticize at the Sf-9 cell from primordial seed and to be uploaded to the 5th on behalf of main seed lot; The main seed lot total degree that continues to go down to posterity on the Sf-9 cell is no more than 12 generations preparation work seed lot.

Carry out clarification filtration by filter behind the suspension that the harvesting cracking is produced in the f step, the filter aperture is preferably 0.45 μ m.

The preferred 100-300KD of molecular cut off of the ultrafilter membrane of ultrafiltration and concentration virus vlps suspension in the g step, preferred 100 times of cycles of concentration.

The concrete steps of viral liquid purification are in the h step:

Gel permeation chromatography and ion-exchange chromatography purification: carry out gel permeation chromatography, medium adopts suitable gel filtration medium, as: the Fractolgel that German Merk company produces, the PBS of the preferred pH6.5-7.5 of balance liquid; Carry out ion-exchange chromatography again, preferably use the weak anionic exchange media, for example: the Fractogel EMD DEAE that Merck company produces, buffer salt solution preferably phosphate buffer system, level pad preferred salt concentration range 0.05-0.1M, pH6.5-7.5 contains the sodium chloride of 0.06-0.12M; Elution buffer preferred salt concentration 0.05-0.1M, pH6.5-7.5 contains the sodium chloride of 0.2-0.4M;

Or sucrose density gradient centrifugation purification: preferably using the discontinuous sucrose density gradient is 15%, 30% and 65%, and the 30000g ultracentrifugation was collected the milky band on the 15-35% sucrose interface after 3 hours;

Through behind the above-mentioned purification, the cell rests dna content is lower than 100pg/ml, gets the EV71VLP virus stock solution used or the Cox.A16VLP virus stock solution used of purification, i.e. EV71VLP antigen or Cox.A16VLP antigen.

Vaccine semi-finished product in the i step and finished product are meant that the VLP virus stock solution used with above-mentioned purification is mixed with the carrier that pharmacy is accepted: the buffer of standard, stabilizing agent, diluent, antiseptic, solubilizing agent; Or be mixed with the dosage form of being convenient to slow release, or add aluminum salt absorption adjuvant, or injection type; Preferably with the EV71VLP virus stock solution used and the Cox.A16VLP virus stock solution used of above-mentioned purification, mixed is prepared into the form of bivalence routinely.

Wherein, the using dosage of vaccine of the present invention is:

Unit price: a person-portion is 0.5ml, contains EV71VLP antigen or Cox.A16VLP antigen 1 .5-3 μ g, Al (OH) ₃Adjuvant 0.25-0.5mg;

Two valencys: a person-portion is 0.5ml, contains EV71VLP antigen and Cox.A16VLP antigen 1.5-3 μ g altogether, Al (OH) ₃Adjuvant 0.25-0.5mg.

Following experimental example or embodiment further specify but are not limited to the present invention.

The specific embodiment

Embodiment 1: prepare hand-foot-mouth disease subunit vaccine of the present invention

1, the preparation of recombinant baculovirus Bac-EV71-P1-3CD and Bac-Cox.A16-P1-3CD

Cell: adopt African green monkey kidney continuous cell line (Vero cell line) and insect cell line Sf-9.

Virus: strain EV71 uses H13-Henan-08, and strain Cox.A16 uses S10-Shandong-08.

Cell culture: the recovery cell, in cell recovery to a Tissue Culture Flask with a peace bottle, cultivate after 4-6 days, spread out of 4 Tissue Culture Flasks according to 1: 4 branch kind ratio.

Virus inoculation: cell culture discarded cell culture supernatant after 3 to 5 days, inoculation hand-foot-mouth disease virus.The virus inoculation amount is 0.1MOI, 37 ℃ of cultivation temperature, and incubation time 30-40 hour, it was the DMEM culture medium that contains 2% hyclone that virus is kept liquid.

Results virus: when obvious pathological changes appears in cell, collect freeze thawing 3 times, results virus by conventional method.

Purified virus RNA: use TaKaRa MiniBEST Viral RNA Extraction Kit Ver.3.0 test kit purified virus RNA.

Amplification EV71 and Cox.A16P1 and 3CD gene:

Get the RNA template 5 μ l of said extracted, add each 1 μ l of primer, 2.5mM dNTPs 1 μ l, 65 ℃ of degeneration 5 minutes ice bath 1-3 minute, add 5 * Buffer, 4 μ l, RNasin 1 μ l, AMV RT 1 μ l (9U) then; DEPC water is mended to 20 μ l, and 42 ℃ were reacted 60 minutes then, and 95 ℃ were reacted 10 minutes, added RNasin 1 μ l again, and 37 ℃ were reacted 20 minutes, and product is carried out pcr amplification immediately.The pcr amplification system is 10 * Extaq Buffer, 10 μ l, dNTP (2.5mM) 8 μ l, and each 2 μ l of each gene upstream and downstream primer, Extaq enzyme 4 μ l (20U), cDNA 1 μ l mends to 100 μ l with deionized water.Employed primer is as follows:

EV71p1u：gcgcgcatgggttcgcaagtgtctacaca

Ev71p1d：aagcttttaaagagtggtgatcgctgtgcgact

Ev713cdu：ctcgagatgggcccgagccttgactttgctctc

Ev713cdd：gcatgcttaaaataactcgagcca

Coxa16p1u：gcgcgcatggggtcacaagtctc

Coxa16p1d：aagcttttacaatgttgttatcttgtc

Coxa163cdu：ctcgagatggaccgagcttagactttgc

Coxa163cdd：gcatgcttaaaataattcgagccaatt

Amplified production is connected in the pMD18-T carrier after the glue recovery respectively, obtains pTEV71-P1, pTEV71-3CD, pTCA16-P1, pTCA16-3CD, order-checking shows correct (seeing sequence table SeQ ID NO.1 and SeQ ID NO.2).

With pTEV71-P1, pTEV71-3CD, pTCA16-P1, pTCA16-3CD, pFastBac ^TMDUAL is double digestion respectively, and utilizes the TaKaRa gel to reclaim test kit and reclaim the purpose fragment, utilizes the T4 ligase will reclaim fragment respectively and connects.To connect product and transform JM109 competence antibacterial, obtain transfer vector pDual-EV71-P1-3CD and pDual-Cox.A16-P1-3CD, identify correct through enzyme action and PCR.

According to the Bac-to-Bac Baculovirus Expression Systems of Invitrogen company operating instruction, pDual-EV71-P1-3CD and pDual-Cox.A16-P1-3CD are transformed DH10BAC competence antibacterial respectively, 42 ℃ of heat shocks 60 move, competent cell is added among the 1mlLB 37 ℃ of concussions to be cultivated 4 hours, get 100 μ l and coat the three anti-flat boards that contain kanamycin (50 μ g/ml), gentamycin (7 μ g/ml) and tetracycline (10 μ g/ml), cultivate more than 24 hours for 37 ℃.Through blue white macula screening, the streak culture again phenotypic evaluation of carrying out of white colony of picking reorganization is confirmed as the colony inoculation of white and is cultivated, and the single white colony of picking connects bacterium and cultivated 24 hours, and PCR identifies and obtained correct reorganization bacterium.

Receive bacterium and extract Bacmid, get 1.5ml bacterium liquid, the centrifugal supernatant that goes adds 0.3mlSolution I[15mM TrisHCl (pH 8.0), 10mM EDTA, 100 μ g/mlRNase A]; Add 0.3mlSolutionII (0.2M NaOH, 1%SDS), mixing gently, room temperature was placed 5 minutes; Slowly add the 0.3m13M potassium acetate, ice bath 5-10 minute; Centrifugal 10 minutes of 13000rpm, supernatant is drawn onto another pipe, adds the 0.8ml isopropyl alcohol; Ice bath 5-10 minute; Centrifugal 15 minutes of 13000rpm; Outwell supernatant, add 1ml70% ethanol, put upside down for several times, centrifugal 5 minutes of room temperature is inhaled and is abandoned supernatant, and room temperature was placed 5-10 minute, add 30 μ lTE dissolution precipitations, the dissolving back is frozen in-20 ℃, perhaps adopts Invitrogen company rod granule to extract test kit and extracts rod granule, determines correctness, purity, the concentration of Bacmid through agarose gel electrophoresis, PCR evaluation, spectrophotometric determination.

With the CellFECTIN reagent respectively Sf9 insect cell (coverage rate of 70%-80%) in the transfection 3ml Tissue Culture Flask of Bacmid of will recombinating.Inoculation about 1 * 10 in the 3ml Tissue Culture Flask ⁶Individual cell, the room temperature adhere-wall culture is more than 1 hour; Bacmid DNA uses centrifugal 5 minutes of maximum speed; Get 5 μ l plasmids and join in the unparalleled anti-Grace of 100 μ l serum-frees (hereinafter to be referred as the two no Grace) culture fluid mixing gently; Get 6 μ l liposomees and join in 1 pair of no Grace culture fluid of 100 μ mixing gently, the culture fluid that will contain plasmid joins in the culture fluid that contains liposome, and mixing was placed 30 minutes for 37 ℃ gently; It is inferior that cell is given a baby a bath on the third day after its birth with two no Grace, and the room temperature sense is done 15 minutes; The lipid-DNA complex is added among the two no Grace of 0.8ml, above-mentioned 1ml culture fluid is added in the 3ml Tissue Culture Flask, cultivated 12-16 hour for 28 ℃, add the fresh culture fluid of 3ml, 72 hours visible significantly cytopathys, collect cleer and peaceful cell precipitation on the sick cell, frozen respectively in-80 ℃ standby, recombinant virus called after Bac-EV71-P1-3CD and Bac-Cox.A16-P1-3CD.But the recombinant virus direct infection Sf9 insect cell of collecting carries out virus amplification and the VLP product is expressed.

2, the foundation of viral seed lot and calibrating

Production of vaccine should be that the triode reason is carried out on the basis with viral seed lot system with seed culture of viruses, promptly primordial seed criticize, main seed lot and work seed lot.Primordial seed criticize on the Sf-9 4 generation seed culture of viruses.Primordial seed is criticized and should be identified its record, history, source and biological characteristics.Criticize at the Sf-9 cell to be uploaded to the 5th from primordial seed, examine and determine comprehensively on behalf of main seed lot, the main seed lot preparation work seed lot that on the Sf-9 cell, continues to go down to posterity, the total degree that goes down to posterity on the Sf-9 cell must not surpass for 12 generations.Seed culture of viruses is kept at below-60 ℃.The seed lot calibrating comprises projects such as discrimination test, sterility test, titration of virus, viral exogenous factor inspection, immunogenicity inspection, and is standby behind the assay approval.

3, the preparation of EV71 and Cox.A16 VLP

The high density suspension culture grows up to 1.5-2 * 10 from the insect cell of Sf-9 cell line at cell ⁶During cell/ml, the inoculation recombinant baculovirus, virus is propagation and coexpression P1 and 3CD albumen in cell, and spontaneous assembling produces VLP, and the cracking insect cell is also collected the suspension that is produced.

Clarification filtration: the filter with 0.45 μ m filters all results supernatants.

Ultrafiltration and concentration virus: with molecular cut off 200KD ultrafilter membrane, 100 times of cycles of concentration.

Sucrose density gradient centrifugation: use discontinuous sucrose density gradient (15%, 30%, 65% sucrose density gradient), the 30000g ultracentrifugation was collected the milky band on the 15-35% sucrose interface after 3 hours, and ultracentrifugation is removed sucrose and concentrated and reclaims virus.

Gel permeation chromatography: the PBS balance Sepharose6FFTM medium with pH6.5-7.5 carries out viral purification, collects void volume stream and wears the peak.Foreign protein is removed and can be reached more than 95%.

Ion-exchange chromatography: adopt DEAE-SepharoseFF ^TMMedium, balance liquid are pH6.5-7.5, contain the PBS of 0.05-0.15M sodium chloride that eluent is the PBS that contains 0.2-0.5M sodium chloride, pH6.5-7.5.Foreign protein is removed and can be reached more than 99%, and the VLP virus stock solution used behind the purification is VLP antigen.

Preparation: get EV71 and Cox.A16VLP virus stock solution used behind the purification, add adjuvant again, the preparation injection type, the configuration proportion of two kinds of VLP virus stock solution useds is 1: 1.

4, vaccine purity analysis

According to " Chinese Pharmacopoeia (the 3rd edition) " and " Chinese biological goods rules " requirement, measure the outward appearance of vaccine finished product, sterility test, dna content, protein content, pH value etc. about " passage cell is as biological product cellular matrix rules ".(seeing Table 1)

Table 1 vaccine finished product verification result

5, vaccine potency analysis

Adopt Reed﹠amp; The Muench method is measured the serum neutralization and is tired.

With 56 ℃ of deactivations in 30 minutes of antiserum to be measured, not contain the 199 culture medium dilution 1-2 of Ox blood serum ¹¹Doubly.To dilute back sample adding and contain 100TCID ₅₀Virus (64-160TCID ₅₀/ 0.1ml), 2% hyclone was hatched 12 hours for 4 ℃ in 199 culture medium of equal volume.

Mixed liquor after hatching is added in the 96 hole tissue culturing plates of monolayer Vero cell (2 * 10 ⁴Cells/well), hatched 1 hour for 37 ℃.

Remove culture medium in the culture plate, add fresh 199 culture medium of 100 μ L2% hyclones, 5%CO ₂, 37 ℃ of cultivations.

With microscopic examination, when the Vero cytopathy does not increase in being cultured to plate (the 7th day), adopt Reed﹠amp; The Muench method is calculated the antiserum neutralization and is tired.

With vaccine immunity SPF mice of the present invention and Rhesus Macacus, every each mice 0.1mL, Rhesus Macacus 0.5mL, immunity in 14 days is 3 times at interval, last immunity blood sampling in back 15 days, separation of serum.After 56 ℃ of deactivations in 30 minutes, carry out neutralization test, measure serum neutralization tire (seeing Table 2).

The serum that table 2 vaccine finished product immunity Rhesus Macacus produces is to the NAT of enterovirus clinical separation strain

6, safety analysis

According to " Chinese Pharmacopoeia (the 3rd edition) " and " Chinese biological goods rules " subunit vaccine of preparation is carried out abnormal toxicity test.

Use the mice and the Cavia porcellus of cleaning grade standard to carry out abnormal toxicity test (seeing Table 3).

Table 3 vaccine finished product abnormal toxicity test result

Get body weight 18-22g mice, take by weighing every mice body weight before the injection.Every batch sample is injected the 0.5mL test vaccine with 5 mices, every mouse peritoneal, observes 14 days.

Get body weight 250-350g Cavia porcellus, take by weighing every Cavia porcellus body weight before the injection.Every batch sample is with 5 Cavia porcelluss, and every guinea pig intraperitoneal injection 5.0mL test vaccine was observed 14 days.

Carrying out allergenicity test (seeing Table 4), is the positive control of hypersensitive test with the Ox blood serum, the negative contrast of normal saline.Every group of 5 Cavia porcelluss are respectively at abdominal part hypodermic 0.5mL.The next day once, totally 3 times, back 24 days of the 3rd injection, ear vein gives same substance 0.5mL, observes the injection afterreaction immediately.

Table 4 vaccine finished product allergenicity result of the test

7, vaccine stability analysis

The vaccine finished product is put in 4 ℃ for a long time, and room temperature (20-25 ℃), is observed and is renderd a service stability, Antigen Stability, pH stability, appearance stability by 37 ℃.Result of the test shows that vaccine deposits stable under 4 ℃.The effectiveness stability test that hand-foot-and-mouth disease inactivated vaccine is 4 ℃ the results are shown in Table 5.

The effectiveness stability test result that table 5 vaccine finished product is 4 ℃

。

Sequence table

SeQ ID NO.1:EV71 P1 and 3CD gene order:

P1：

atgggttcgcaagtgtctacacagcgctccggttctcacgaaaactcaaactcagccactgagggttctaccataaactacaccac

cattaattactacaaagactcctatgctgccacagcaggcaaacagagtctcaagcaggatccagacaagtttgcaaatcctgtta

aagacatcttcactgaaatggcagcgccactgaagtccccatccgctgaggcatgtggatacagtgatcgagtggcgcaattaact

attggcaactccaccatcaccacgcaagaagcggctaatatcatagtcggttatggtgagtggccttcctactgctcagattctga

cgctacagcagtggataaaccaacgcgcccggatgtttcagtgaacaggttttacacattggacactaaattgtgggagaaatcgt

ccaagggatggtactggaagttcccggatgtgttaactgaaactggggtttttgggcaaaatgcacaattccactacctctaccga

tcagggttctgcatccacgtgcagtgcaatgccagtaaattccaccaaggagcactcctagtcgctgtcctaccagagtatgtcat

tgggacagtggcaggcggtacagggacggaagatacccaccccccttacaagcagactcaacccggcgccgatggcttcgagttgc

aacacccgtacgtgcttgatgctggcatcccaatatcacagttaacagtgtgcccacaccagtggattaatttgaggaccaacaac

tgtgctacaataatagtgccatacattaacgcactgccttttgattctgccttgaaccattgcaactttggcctgttggttgtgcc

tattagcccactagactacgaccaaggagcgacgccagtaatccctataactatcacattggccccaatgtgttctgaattcgcag

gtcttaggcaggcagtcacgcaagggttccccaccgagctaaaacctggcacaaatcaatttttaaccaccgatgatggcgtttca

gcacctattctaccgaacttccaccccaccccgtgtatccacatacctggtgaagttaggaacttgctagagttatgccaggtgga

gaccattctggaggttaacaatgtgcccacgaatgccactagcttaatggagagactgcgcttcccggtctcagcacaagcaggga

aaggtgagctgtgtgcggtgtttagagccgatcctgggcgaaatggaccatggcaatccaccttactgggtcagttgtgcgggtac

tacacccaatggtcaggatcattggaagtcaccttcatgtttactggatccttcatggctaccggcaagatgctcatagcctatac

accgccaggaggtcctctgcccaaggaccgggcgaccgccatgttgggcacgcacgtcatctgggattttgggctgcaatcgtctg

ttacccttgtaataccatggatcagcaacactcattatagagcacatgcccgagatggagtgtttgactactacaccacagggtta

gtcagtatatggtatcagacaaattacgtggttccaatcggtgcgcccaacacagcctatataatagcactagcggcagcccaaaa

gaacttcactatgaaattgtgcaaggatgctagtgatatcctgcagacgggcaccatccagggagatagggtggcagatgtaattg

aaagttccataggagatagcgtgagcagagccctcactcacgctctaccagcacccacaggccagaacacacaggtgagcagtcat

cgactggatacaggcaaggttccagcactccaagctgctgaaattggagcatcatcaaatgctagtgacgagagcatgattgagac

acgctgtgttcttaactcgcacagtacagctgagaccactcttgatagtttcttcagcagggcgggattagttggagagatagatc

tccctcttaagggcacaactaacccaaatggttatgccaactgggacatagacataacaggttacgcgcaaatgcgtagaaaggta

gagctattcacctacatgcgctttgatgcagagttcacttttgttgcgtgcacacccaccggggaagttgtcccacaattgctcca

atatatgtttgtgccacctggagcccctaagccagattctagggaatcccttgcatggcaaaccgccactaacccctcagtttttg

tcaagctgtcagaccctccagcgcaggtttcagtgccattcatgtcacctgcgagtgcttatcaatggttttatgacggatatccc

acattcggagaacacaaacaggagaaagatcttgaatacggggcatgtcctaataacatgatgggcacgttctcagtgcggactgt

ggggacctccaagtctaagtaccctttagtggttaggatttacatgagaatgaagcacgtcagggcgtggatacctcgcccgatgc

gtaaccagaactacctattcaaagccaacccaaattatgctggcaactccattaagccaactggtgccagtcgcacagcgatcacc

actctt

3CD：

ggcccgagccttgactttgctctctccctactgagaaggaacatcaggcaagtccaaacagaccaggggcatttcaccatgttggg

tgttagggatcgcttagcagtcctcccacgccactcacaacctggcaaaactatttggattgagcacaaactcgtgaacgtccttg

atgcagttgaactggtggatgagcaaggagtcaacctggaattaaccctcatcactcttgacaccaacgaaaagtttagggatatc

accaaattcatcccagaaaatattagcactgctagtgatgccaccctagtgatcaacacggagcacatgccatcaatgtttgtccc

ggtgggtgacgttgtgcagtatggctttttgaatctcagtggtaagcctacccatcgcaccatgatgtacaactttcctactaaag

caggacagtgtggaggagtggtgacatctgttgggaaggttgtcggtattcacattggtggcaatggcagacaaggtttttgcgca

ggcctcaaaaggagttactttgctagtgaacaaggggagatccagtgggttaagcccaataaagaaactggaagactcaacatcaa

tggaccaacccgcaccaagttagaacctagtgtattccatgacatcttcgagggaaataaagaaccagctgtcttgcacagtaaag

acccccgacttgaggtagattttgaacaggccctgttctctaagtatgtgggaaacacactacatgagcctgacgagtacatcaag

gaggcagctcttcattatgcaaaccaattaaagcaactagaaattaatacctctcaaatgagcatggaggaggcctgctacggtac

tgagaatcttgaggctattgatcttcacactagtgcaggttacccctatagtgccctggggataaagaaaagagacatcttagacc

ctaccaccagggacgtgagtagaatgaagttctacatggacaagtatggtcttgatcttccttactccacttatgtcaaggacgag

ctgcgctcgattgataaaattaagaaagggaagtcccgtctgatcgaggccagtagtctaaatgattcagtgtacctcagaatggc

tttcggacatttgtatgaggctttccacgcaaatcctgggacgataactggatcagccgtggggtgtaaccctgacacattctgga

gcaagctgccaattttgctccctggctcactctttgcctttgactactcaggttatgatgccagccttagccctgtctggttcaga

gcattagaattggttcttagggagatagggtatagtgaagaggcaatctcactcattgagggaatcaaccacacacatcatgtgta

tcgtaataagacctattgtgtgcttggtgggatgccctcaggctgttcgggaacatccattttcaactcaatgatcaacaacatta

ttatcagagcactgctcataaaaacatttaagggcattgatttggatgaactcaacatggtcgcttatggagacgatgtgctcgct

agctatcccttcccaattgattgcttggaactagcaaagactggtaaggagtacggtctgaccatgactcctgctgataaatctcc

ttgctttaatgaggtcaattggggtaatgcgaccttcctcaaaaggggctttttgcccgatgagcagtttccatttttgattcacc

ctactatgccaatgagggagatccatgagtccattcgatggaccaaggacgcacgaaacactcaagatcatgtgcggtccttgtgc

ctcctagcatggcataatggtaagcaagaatatgagaagtttgtgagcacaattaggtctgtcccagtaggaagagcgttggctat

cccaaattatgaaaatcttagacgcaattggctcgagttattt

SeQ ID NO.2:Cox.A16P1 and 3CD gene order:

P1：

atggggtcacaagtctcaacccaacgatcgggttcccacgaaaattcgaactcagcatcagaaggatctactataaactacaccac

catcaactattacaaggatgcatatgctgccagcgcgggtcgccaagatatgtctcaggaccctaagaaatttacagaccctgtga

tggatgtcatacacgagatggctcctcccttgaaatcacccagtgctgaagcttgtggttatagtgatcgagttgcccaactcaca

attggaaactccacaatcactacacaagaagctgcaaacattataatagcatatggggaatggcccgagtattgcaaggacgctga

tgccacagctgttgacaagcccaccagacccgatgtgtcggtaaataggttcttcacccttgatactaaatcgtgggctaaagact

cgaagggatggtactggaaattcccggacgttttgacggaggtgggcgtgtttgggcagaatgcgcaattccattatctgtataga

tccggattctgtgtgcacgtgcagtgcaatgccagcaaattccaccagggtgctctcttggttgccatactgcctgagtacgtgct

gggcaccattgccgggggcgatggtaacgagaactcacatcccccgtacgtcaccacccagccaggacaggtgggtgctgtactta

caaatccttatgttttggatgctggggtaccccttagtcaattgacggtgtgtccgcatcagtggattaacctacgaaccaacaac

tgtgcgaccatcatagtaccatacatgaatactgtaccattcgattcggccctaaaccattgcaactttggcttaattgtagtgcc

cgtagtaccactcgactttaacgctggagctacatcagaaataccaataactgtcaccatcgcacccatgtgcgctgaatttgcag

gtttgcgacaggcaatcaaacaggggatacctaccgagttgaagcccggtactaatcagtttctcactactgatgatggtgtctca

gctcccattttgcccggattccaccctactccagccatacacatacctggtgaagtgcgcaatctgttggagatttgcagggtaga

gaccatattggaggtgaacaatctacagagtaatgagacaacccccatgcaacgactatgcttccctgtctcggtgcagagtaaga

cgggggaattgtgtgctgtttttagggccgaccctggtaggaatggaccgtggcagtcgactattttaggacagttgtgtaggtac

tatactcaatggtctggatctctggaagtcactttcatgtttgctggatcgttcatggcaacaggaaagatgctaatcgcatacac

acctccggggggtggggtcccagcagatcggctcactgcaatgctgggaacccatgtgatatgggattttggcctccaatcctcag

tcacgctagttataccatggataagcaacacacactatagggcgcacgccaaggacggttattttgattattacaccactggcacg

atcactatatggtatcagacaaattatgtcgtacctattggagcccccacaacagcctatattgtggccctcgcagccgctcagga

caactttaccatgaaactgtgcaaagacactgaggatattgagcaatctgcaaacatccagggtgatggaattgcagacatgattg

accaggctgtcacttcccgagttggtcgtgcgctgacatccttacaggtagaacctaccgccgccaacaccaatgctagtgagcac

agattgggcaccgggctcgtccccgccttgcaggctgcagagaccggcgcctcttctaatgcacaggatgagaatcttatagaaac

ccggtgtgtgttgaaccatcactccactcaagagaccacgattggcaactttttcagtcgagcaggactagtgagtattattacca

tgcccaccacaggtacccaaaacaccgatgggtatgtgaactgggatattgacttgatgggttatgctcaaatgaggcgtaagtgt

gagctattcacatacatgcgctttgatgcagagtttacatttgtagctgccaaaccaaacggtgagctagtaccacaattgttgca

gtacatgtatgtgcctcccggagctccaaaacctacgtcccgggattcctttgcctggcagactgctaccaatccttccatcttcg

tcaagttgactgaccccccggcacaagtgtcagtacccttcatgtctcccgccagcgcctaccagtggttttacgatggctatcca

acgtttggagcccatccacaatcgaatgacgcagactatggccaatgtccaaacaacatgatgggcacctttagcatcagaactgt

aggcactgagaaatccccccattctatcacccttcgggtgtatatgagaatcaagcatgtcagggcctggataccccggccactca

gaaaccaaccgtacctttttaagacaaatccaaattataaaggcaacgacatcaaatgcaccagcacaagtagggacaaaataaca

acacta

3CD：

ggcccaagtcttgattttgctctctcattgcttaggaggaacatcagacaagtccagacagaccaaggccacttcactatgctagg

tgttagagatcgcttggctgtcctcccgcgacactcgcagcctgggaagacaatatgggtagaacacaagcttataaacatcttag

atgctgttgagttggtggatgaacaaggggtcaacttagaactgaccctggttactcttgataccaatgagaagttcagagacatc

accaagttcatcccagaaaacattagcgctgccagtgatgccaccctagtgatcaacacagagcacatgccctccatgttcgtgcc

agtgggtgacgttgtgcaatacggtttcctaaacctcagcggaaaacccacccatcgtaccatgatgtacaactttcccactaagg

ctggacaatgtggtggagtggtgacatctgttggaaaggttatcggcatacatattggtggcaatggcaggcagggcttttgtgca

ggactcaagaggagttattttgccagtgagcaaggagagatccagtgggtcaagcccaataaagagacaggcagactcaacatcaa

tgggccgactcgcactaagctcgaacccagtgtgttccatgatgtctttgaggggaacaaagaaccagcagtcttgcatagcagag

acccacgccttgaagtggactttgagcaggctttattctccaagtatgtagggaacacactgcatgagcctgacgagtacatcaaa

gaggcggccctccactatgcaaatcagttgaagcaactggacatcaacacttctcaaatgagtatggaggaagcttgctatggtac

tgagaaccttgaggccattgatctccacaccagcgcgggctacccctacagtgctctgggaataaagaagagagatattttagatc

ccactaccagagatgtgagtaagatgaaattctatatggacaagtatggccttgaccttccttactctacctatgtcaaggatgag

ctccgctcgatagataagattaagaaaggaaaatcccgcctgatcgaggctagtagtctaaatgactcagtgtacctcagaatggc

ctttggtcacttgtacgagactttccacgcaaatccaggaaccataactggctcagctgtgggatgcaacccggatacattctgga

gtaaactaccaatcctgcttccaggttcactctttgcatttgattactcaggctatgatgctagtctcagtccagtctggtttaga

gcactggagctggtcctcagggaggtgggctatagtgaggaggcagtctcccttatagaggggattaaccatacacaccatgtata

ccgcaataaaacttactgtgtacttggtggaatgccctcaggctgttcaggaacatccattttcaactcaatgatcaacaacatca

taattaggacattgctcataaaaacattcaagggcattgatttggacgagcttaacatggtcgcctacggggatgatgtgcttgct

agttatcctttcccaatcgattgcctggaattggcaagaacaggcaaagagtatggtttgactatgactcctgcagacaaatctcc

ttgcttcaatgaggtaaattggggcaatgcaacctttctcaaaagaggcttcttgcccgatgagcaattccctttcttgatccatc

ctaccatgccaatgaaggagattcacgaatctattcgatggaccaaggacgcacgaaacactcaagatcacgtacgatccctgtgt

cttttggcgtggcacaatggtaagcaggagtatgaaaaatttgtgagcacaattaggtctgtcccagtaggaaaagctttggctat

accgaattatgaaaatctgagacgcaattggctcgaattattt

SEQ?ID?NO：1.txt

SEQUENCE?LISTING

＜110〉Academy of Military Medicine, PLA's microorganism is popular

Sick institute

＜120〉the single, double bivalent gene engineered subunit vaccine of a kind of hand-foot-mouth disease

And preparation method thereof

<130>000

<160>2

<170>PatentIn?version?3.3

<210>1

<211>2586

<212>DNA

＜213〉artificial sequence

<400>1

atgggttcgc?aagtgtctac?acagcgctcc?ggttctcacg

aaaactcaaa?ctcagccact 60

gagggttcta?ccataaacta?caccaccatt?aattactaca

aagactccta?tgctgccaca 120

gcaggcaaac?agagtctcaa?gcaggatcca?gacaagtttg

caaatcctgt?taaagacatc 180

ttcactgaaa?tggcagcgcc?actgaagtcc?ccatccgctg

aggcatgtgg?atacagtgat 240

SEQ?ID?NO：1.txt

cgagtggcgc?aattaactat?tggcaactcc?accatcacca

cgcaagaagc?ggctaatatc 300

atagtcggtt?atggtgagtg?gccttcctac?tgctcagatt

ctgacgctac?agcagtggat 360

aaaccaacgc?gcccggatgt?ttcagtgaac?aggttttaca

cattggacac?taaattgtgg 420

gagaaatcgt?ccaagggatg?gtactggaag?ttcccggatg

tgttaactga?aactggggtt 480

tttgggcaaa?atgcacaatt?ccactacctc?taccgatcag

ggttctgcat ccacgtgcag 540

tgcaatgcca?gtaaattcca?ccaaggagca?ctcctagtcg

ctgtcctacc?agagtatgtc 600

attgggacag?tggcaggcgg?tacagggacg?gaagataccc

acccccctta?caagcagact 660

caacccggcg?ccgatggctt?cgagttgcaa?cacccgtacg

tgcttgatgc tggcatccca 720

atatcacagt?taacagtgtg?cccacaccag?tggattaatt

tgaggaccaa?caactgtgct 780

acaataatag?tgccatacat?taacgcactg?ccttttgatt

ctgccttgaa?ccattgcaac 840

SEQ?ID?NO：1.txt

tttggcctgt?tggttgtgcc?tattagccca?ctagactacg

accaaggagc?gacgccagta 900

atccctataa?ctatcacatt?ggccccaatg?tgttctgaat

tcgcaggtct?taggcaggca 960

gtcacgcaag?ggttccccac?cgagctaaaa?cctggcacaa

atcaattttt?aaccaccgat 1020

gatggcgttt?cagcacctat?tctaccgaac?ttccacccca

ccccgtgtat?ccacatacct 1080

ggtgaagtta?ggaacttgct?agagttatgc?caggtggaga

ccattctgga?ggttaacaat 1140

gtgcccacga?atgccactag?cttaatggag?agactgcgct

tcccggtctc?agcacaagca 1200

gggaaaggtg?agctgtgtgc?ggtgtttaga?gccgatcctg

ggcgaaatgg?accatggcaa 1260

tccaccttac?tgggtcagtt?gtgcgggtac?tacacccaat

ggtcaggatc?attggaagtc 1320

accttcatgt?ttactggatc?cttcatggct?accggcaaga

tgctcatagc?ctatacaccg 1380

ccaggaggtc?ctctgcccaa?ggaccgggcg?accgccatgt

tgggcacgca?cgtcatctgg 1440

gattttgggc?tgcaatcgtc?tgttaccctt?gtaataccat

SEQ?ID?NO：1.t?xt

ggatcagcaa?cactcattat 1500

agagcacatg?cccgagatgg?agtgtttgac?tactacacca

cagggttagt?cagtatatgg 1560

tatcagacaa?attacgtggt?tccaatcggt?gcgcccaaca

cagcctatat?aatagcacta 1620

gcggcagccc?aaaagaactt?cactatgaaa?ttgtgcaagg

atgctagtga?tatcctgcag 1680

acgggcacca?tccagggaga?tagggtggca?gatgtaattg

aaagttccat?aggagatagc 1740

gtgagcagag?ccctcactca?cgctctacca?gcacccacag

gccagaacac?acaggtgagc 1800

agtcatcgac?tggatacagg?caaggttcca?gcactccaag

ctgctgaaat?tggagcatca 1860

tcaaatgcta?gtgacgagag?catgattgag?acacgctgtg

ttcttaactc?gcacagtaca 1920

gctgagacca?ctcttgatag?tttcttcagc?agggcgggat

tagttggaga?gatagatctc 1980

cctcttaagg?gcacaactaa?cccaaatggt?tatgccaact

gggacataga?cataacaggt 2040

tacgcgcaaa?tgcgtagaaa?ggtagagcta?ttcacctaca

tgcgctttga?tgcagagttc 2100

SEQ?ID?NO：1.txt

acttttgttg?cgtgcacacc?caccggggaa?gttgtcccac

aattgctcca?atatatgttt 2160

gtgccacctg?gagcccctaa?gccagattct?agggaatccc

ttgcatggca?aaccgccact 2220

aacccctcag?tttttgtcaa?gctgtcagac?cctccagcgc

aggtttcagt?gccattcatg 2280

tcacctgcga?gtgcttatca?atggttttat?gacggatatc

ccacattcgg?agaacacaaa 2340

caggagaaag?atcttgaata?cggggcatgt?cctaataaca

tgatgggcac?gttctcagtg 2400

cggactgtgg?ggacctccaa?gtctaagtac?cctttagtgg

ttaggattta?catgagaatg 2460

aagcacgtca?gggcgtggat?acctcgcccg?atgcgtaacc

agaactacct?attcaaagcc 2520

aacccaaatt?atgctggcaa?ctccattaag?ccaactggtg

ccagtcgcac?agcgatcacc 2580

actctt

2586

<210>2

<211>1935

SEQ?ID?NO：1.txt

<212>DNA

＜213〉artificial sequence

<400>2

ggcccgagcc?ttgactttgc?tctctcccta?ctgagaagga

acatcaggca?agtccaaaca 60

gaccaggggc?atttcaccat?gttgggtgtt?agggatcgct

tagcagtcct?cccacgccac 120

tcacaacctg?gcaaaactat?ttggattgag?cacaaactcg

tgaacgtcct?tgatgcagtt 180

gaactggtgg?atgagcaagg?agtcaacctg?gaattaaccc

tcatcactct?tgacaccaac 240

gaaaagttta?gggatatcac?caaattcatc?ccagaaaata

ttagcactgc?tagtgatgcc 300

accctagtga?tcaacacgga?gcacatgcca?tcaatgtttg

tcccggtggg?tgacgttgtg 360

cagtatggct?ttttgaatct?cagtggtaag?cctacccatc

gcaccatgat?gtacaacttt 420

cctactaaag?caggacagtg?tggaggagtg?gtgacatctg

ttgggaaggt?tgtcggtatt 480

cacattggtg?gcaatggcag?acaaggtttt?tgcgcaggcc

tcaaaaggag?ttactttgct 540

SEQ?ID?NO：1.txt

agtgaacaag?gggagatcca?gtgggttaag?cccaataaag

aaactggaag?actcaacatc 600

aatggaccaa?cccgcaccaa?gttagaacct?agtgtattcc

atgacatctt?cgagggaaat 660

aaagaaccag?ctgtcttgca?cagtaaagac?ccccgacttg

aggtagattt?tgaacaggcc 720

ctgttctcta?agtatgtggg?aaacacacta?catgagcctg

acgagtacat?caaggaggca 780

gctcttcatt?atgcaaacca?attaaagcaa?ctagaaatta

atacctctca?aatgagcatg 840

gaggaggcct?gctacggtac?tgagaatctt?gaggctattg

atcttcacac?tagtgcaggt 900

tacccctata?gtgccctggg?gataaagaaa?agagacatct

tagaccctac?caccagggac 960

gtgagtagaa?tgaagttcta?catggacaag?tatggtcttg

atcttcctta?ctccacttat 1020

gtcaaggacg?agctgcgctc?gattgataaa?attaagaaag

ggaagtcccg?tctgatcgag 1080

gccagtagtc?taaatgattc?agtgtacctc?agaatggctt

tcggacattt?gtatgaggct 1140

ttccacgcaa?atcctgggac?gataactgga?tcagccgtgg

SEQ?ID?NO：1.txt

ggtgtaaccc?tgacacattc 1200

tggagcaagc?tgccaatttt?gctccctggc?tcactctttg

cctttgacta?ctcaggttat 1260

gatgccagcc?ttagccctgt?ctggttcaga?gcattagaat

tggttcttag?ggagataggg 1320

tatagtgaag?aggcaatctc?actcattgag?ggaatcaacc

acacacatca?tgtgtatcgt 1380

aataagacct?attgtgtgct?tggtgggatg?ccctcaggct

gttcgggaac atccattttc 1440

aactcaatga?tcaacaacat?tattatcaga?gcactgctca

taaaaacatt?taagggcatt 1500

gatttggatg?aactcaacat?ggtcgcttat?ggagacgatg

tgctcgctag?ctatcccttc 1560

ccaattgatt?gcttggaact?agcaaagact?ggtaaggagt

acggtctgac?catgactcct 1620

gctgataaat?ctccttgctt?taatgaggtc?aattggggta

atgcgacctt?cctcaaaagg 1680

ggctttttgc?ccgatgagca?gtttccattt?ttgattcacc

ctactatgcc?aatgagggag 1740

atccatgagt?ccattcgatg?gaccaaggac?gcacgaaaca

ctcaagatca?tgtgcggtcc 1800

SEQ?ID?NO：1.txt

ttgtgcctcc?tagcatggca?taatggtaag?caagaatatg

agaagtttgt?gagcacaatt 1860

aggtctgtcc?cagtaggaag?agcgttggct?atcccaaatt

atgaaaatct?tagacgcaat 1920

tggctcgagt?tattt

1935

SEQ?ID?NO：2.txt

SEQUENCE?LISTING

＜110〉Academy of Military Medicine, PLA's microorganism is popular

Sick institute

＜120〉the single, double bivalent gene engineered subunit vaccine of a kind of hand-foot-mouth disease

And preparation method thereof

<130>000

<160>2

<170>PatentIn?version?3.3

<210>1

<211>2586

<212>DNA

＜213〉artificial sequence

<400>1

atggggtcac?aagtctcaac?ccaacgatcg?ggttcccacg

aaaattcgaa?ctcagcatca 60

gaaggatcta?ctataaacta?caccaccatc?aactattaca

aggatgcata?tgctgccagc 120

gcgggtcgcc?aagatatgtc?tcaggaccct?aagaaattta

cagaccctgt?gatggatgtc 180

atacacgaga?tggctcctcc?cttgaaatca?cccagtgctg

aagcttgtgg?ttatagtgat 240

SEQ?ID?NO：2.txt

cgagttgccc?aactcacaat?tggaaactcc?acaatcacta

cacaagaagc?tgcaaacatt 300

ataatagcat?atggggaatg?gcccgagtat?tgcaaggacg

ctgatgccac?agctgttgac 360

aagcccacca?gacccgatgt?gtcggtaaat?aggttcttca

cccttgatac?taaatcgtgg 420

gctaaagact?cgaagggatg?gtactggaaa?ttcccggacg

ttttgacgga?ggtgggcgtg 480

tttgggcaga?atgcgcaatt?ccattatctg?tatagatccg

gattctgtgt?gcacgtgcag 540

tgcaatgcca?gcaaattcca?ccagggtgct?ctcttggttg

ccatactgcc?tgagtacgtg 600

ctgggcacca?ttgccggggg?cgatggtaac?gagaactcac

atcccccgta?cgtcaccacc 660

cagccaggac?aggtgggtgc?tgtacttaca?aatccttatg

ttttggatgc?tggggtaccc 720

cttagtcaat?tgacggtgtg?tccgcatcag?tggattaacc

tacgaaccaa?caactgtgcg 780

accatcatag?taccatacat?gaatactgta?ccattcgatt

cggccctaaa?ccattgcaac 840

SEQ?ID?NO：2.txt

tttggcttaa?ttgtagtgcc?cgtagtacca?ctcgacttta

acgctggagc?tacatcagaa 900

ataccaataa?ctgtcaccat?cgcacccatg?tgcgctgaat

ttgcaggttt?gcgacaggca 960

atcaaacagg?ggatacctac?cgagttgaag?cccggtacta

atcagtttct?cactactgat 1020

gatggtgtct?cagctcccat?tttgcccgga?ttccacccta

ctccagccat?acacatacct 1080

ggtgaagtgc?gcaatctgtt?ggagatttgc?agggtagaga

ccatattgga?ggtgaacaat 1140

ctacagagta?atgagacaac?ccccatgcaa?cgactatgct

tccctgtctc?ggtgcagagt 1200

aagacggggg?aattgtgtgc?tgtttttagg?gccgaccctg

gtaggaatgg?accgtggcag 1260

tcgactattt?taggacagtt?gtgtaggtac?tatactcaat

ggtctggatc?tctggaagtc 1320

actttcatgt?ttgctggatc?gttcatggca?acaggaaaga

tgctaatcgc?atacacacct 1380

ccggggggtg?gggtcccagc?agatcggctc?actgcaatgc

tgggaaccca?tgtgatatgg 1440

gattttggcc?tccaatcctc?agtcacgcta?gttataccat

SEQ?ID?NO：2.t?xt

ggataagcaa?cacacactat 1500

agggcgcacg?ccaaggacgg?ttattttgat?tattacacca

ctggcacgat?cactatatgg 1560

tatcagacaa?attatgtcgt?acctattgga?gcccccacaa

cagcctatat?tgtggccctc 1620

gcagccgctc?aggacaactt?taccatgaaa?ctgtgcaaag

acactgagga?tattgagcaa 1680

tctgcaaaca?tccagggtga?tggaattgca?gacatgattg

accaggctgt?cacttcccga 1740

gttggtcgtg?cgctgacatc?cttacaggta?gaacctaccg

ccgccaacac?caatgctagt 1800

gagcacagat?tgggcaccgg?gctcgtcccc?gccttgcagg

ctgcagagac?cggcgcctct 1860

tctaatgcac?aggatgagaa?tcttatagaa?acccggtgtg

tgttgaacca?tcactccact 1920

caagagacca?cgattggcaa?ctttttcagt?cgagcaggac

tagtgagtat?tattaccatg 1980

cccaccacag?gtacccaaaa?caccgatggg?tatgtgaact

gggatattga?cttgatgggt 2040

tatgctcaaa?tgaggcgtaa?gtgtgagcta?ttcacataca

tgcgctttga?tgcagagttt 2100

SEQ?ID?NO：2.txt

acatttgtag?ctgccaaacc?aaacggtgag?ctagtaccac

aattgttgca?gtacatgtat 2160

gtgcctcccg?gagctccaaa?acctacgtcc?cgggattcct

ttgcctggca?gactgctacc 2220

aatccttcca?tcttcgtcaa?gttgactgac?cccccggcac

aagtgtcagt?acccttcatg 2280

tctcccgcca?gcgcctacca?gtggttttac?gatggctatc

caacgtttgg?agcccatcca 2340

caatcgaatg?acgcagacta?tggccaatgt?ccaaacaaca

tgatgggcac?ctttagcatc 2400

agaactgtag?gcactgagaa?atccccccat?tctatcaccc

ttcgggtgta?tatgagaatc 2460

aagcatgtca?gggcctggat?accccggcca?ctcagaaacc

aaccgtacct?ttttaagaca 2520

aatccaaatt?ataaaggcaa?cgacatcaaa?tgcaccagca

caagtaggga?caaaataaca 2580

acacta

2586

<210>2

<211>1935

SEQ?ID?NO：2.t?xt

<212>DNA

＜213〉artificial sequence

<400>2

ggcccaagtc?ttgattttgc?tctctcattg?cttaggagga

acatcagaca?agtccagaca 60

gaccaaggcc?acttcactat?gctaggtgtt?agagatcgct

tggctgtcct?cccgcgacac 120

tcgcagcctg?ggaagacaat?atgggtagaa?cacaagctta

taaacatctt?agatgctgtt 180

gagttggtgg?atgaacaagg?ggtcaactta?gaactgaccc

tggttactct?tgataccaat 240

gagaagttca?gagacatcac?caagttcatc?ccagaaaaca

ttagcgctgc?cagtgatgcc 300

accctagtga?tcaacacaga?gcacatgccc?tccatgttcg

tgccagtggg?tgacgttgtg 360

caatacggtt?tcctaaacct?cagcggaaaa?cccacccatc

gtaccatgat?gtacaacttt 420

cccactaagg?ctggacaatg?tggtggagtg?gtgacatctg

ttggaaaggt?tatcggcata 480

catattggtg?gcaatggcag?gcagggcttt?tgtgcaggac

tcaagaggag?ttattttgcc 540

SEQ?ID?NO：2.txt

agtgagcaag?gagagatcca?gtgggtcaag?cccaataaag

agacaggcag?actcaacatc 600

aatgggccga?ctcgcactaa?gctcgaaccc?agtgtgttcc

atgatgtctt?tgaggggaac 660

aaagaaccag?cagtcttgca?tagcagagac?ccacgccttg

aagtggactt?tgagcaggct 720

ttattctcca?agtatgtagg?gaacacactg?catgagcctg

acgagtacat?caaagaggcg 780

gccctccact?atgcaaatca?gttgaagcaa?ctggacatca

acacttctca?aatgagtatg 840

gaggaagctt?gctatggtac?tgagaacctt?gaggccattg

atctccacac cagcgcgggc 900

tacccctaca?gtgctctggg?aataaagaag?agagatattt

tagatcccac?taccagagat 960

gtgagtaaga?tgaaattcta?tatggacaag?tatggccttg

accttcctta?ctctacctat 1020

gtcaaggatg?agctccgctc?gatagataag?attaagaaag

gaaaatcccg?cctgatcgag 1080

gctagtagtc?taaatgactc?agtgtacctc?agaatggcct

ttggtcactt?gtacgagact 1140

ttccacgcaa?atccaggaac?cataactggc?tcagctgtgg

SEQ?ID?NO：2.txt

gatgcaaccc?ggatacattc 1200

tggagtaaac?taccaatcct?gcttccaggt?tcactctttg

catttgatta?ctcaggctat 1260

gatgctagtc?tcagtccagt?ctggtttaga?gcactggagc

tggtcctcag?ggaggtgggc 1320

tatagtgagg?aggcagtctc?ccttatagag?gggattaacc

atacacacca?tgtataccgc 1380

aataaaactt?actgtgtact?tggtggaatg?ccctcaggct

gttcaggaac?atccattttc 1440

aactcaatga?tcaacaacat?cataattagg?acattgctca

taaaaacatt?caagggcatt 1500

gatttggacg?agcttaacat?ggtcgcctac?ggggatgatg

tgcttgctag?ttatcctttc 1560

ccaatcgatt?gcctggaatt?ggcaagaaca?ggcaaagagt

atggtttgac?tatgactcct 1620

gcagacaaat?ctccttgctt?caatgaggta?aattggggca

atgcaacctt?tctcaaaaga 1680

ggcttcttgc?ccgatgagca?attccctttc?ttgatccatc

ctaccatgcc?aatgaaggag 1740

attcacgaat?ctattcgatg?gaccaaggac?gcacgaaaca

ctcaagatca?cgtacgatcc 1800

SEQ?ID?NO：2.txt

ctgtgtcttt?tggcgtggca?caatggtaag?caggagtatg

aaaaatttgt?gagcacaatt 1860

aggtctgtcc?cagtaggaaa?agctttggct ataccgaatt

atgaaaatct?gagacgcaat 1920

tggctcgaat tattt

1935

Claims (10)

1. single, double valency subunit vaccine of preventing hand-foot-mouth disease is characterized in that:

This unit price subunit vaccine is by EV71 VLP antigen or Cox.A16 VLP antigen and Al (OH) ₃Adjuvant is formulated by following weight percent content:

EV71 VLP antigen or Cox.A16 VLP antigen 0.0015-0.003 weight portion

Al (OH) ₃Adjuvant 0.25-0.5 weight portion;

This pair valency subunit vaccine is by EV71 VLP antigen and Cox.A16 VLP antigen and Al (OH) ₃Adjuvant is formulated by following weight percent content:

EV71 VLP antigen and Cox.A16 VLP antigen 0.0015-0.002 weight portion

Al (OH) ₃Adjuvant 0.25-0.5 weight portion;

Wherein EV71 VLP antigen and the antigenic ratio of Cox.A16 VLP are 1: 1.

2. the preparation method of single, double valency subunit vaccine as claimed in claim 1 is characterized in that this method comprises the steps:

A, obtain recombinant baculovirus Bac-EV71-P1-3CD and Bac-Cox.A16-P1-3CD respectively by genetic engineering means, the efficient similar SeQ ID of coexpression NO.1 EV71 P1, SeQ IDNO.2 Cox.A16 P1 and 3CD albumen in insect cell respectively, and be self-assembled into EV71 VLP and Cox.A16 VLP respectively;

B sets up and three grades of seed lot storehouses of calibrating vaccine strain;

C adopts the insect cell of serum-free medium high density suspension culture from Sf-9/Hi-5 cell line;

D grows up to 1.5-2 * 10 at cell ⁶During cell/ml, the inoculation recombinant baculovirus;

E, virus is propagation and efficient coexpression P1 and 3CD albumen in cell, produces VLP;

F, the suspension that cracking insect cell and collecting cell are produced, clarification filtration is removed the insect cell residue;

G, ultrafiltration and concentration virus vlps suspension;

H, the viral suspension behind the ultrafiltration and concentration by gel permeation chromatography and ion-exchange chromatography purified virus VLP, perhaps by sucrose density gradient centrifugation purified virus VLP, gets the virus stock solution used of purification, i.e. antigen;

I prepares and examines and determine single, double valency vaccine semi-finished product and finished product, measures safety, effectiveness and the stability of vaccine.

3. the preparation method of single, double valency subunit vaccine as claimed in claim 2 is characterized in that:

Wherein P1 and 3CD gene order adopt RT-PCR method amplification hand-foot-mouth disease Strain to obtain in a step: SeQ IDNO.1 EV71 P1 and 3CD gene order utilize RT-PCR method amplification EV71 (H13-Henan-08) viral RNA to obtain, SeQ ID NO.2 Cox.A16 P1 and 3CD gene order utilize RT-PCR method amplification Cox.A16 (S10-shandong-08) viral RNA to obtain, and order-checking showed correct after gene all was cloned into the pMD18-T carrier; P1 and 3CD gene order also can adopt the synthetic mode of full gene to obtain, and are optimized according to the insecticide preference codon.

4. as the preparation method of claim 2 or 3 described single, double valency subunit vaccines, it is characterized in that:

Wherein set up in the b step and three grades of seed lot storehouses of calibrating vaccine strain, promptly primordial seed is criticized, the method for main seed lot and work seed lot is: primordial seed criticize into through on the Sf-9 cell of plaque purification 4 generation seed culture of viruses; Identify record, history, source and biological characteristics that primordial seed is criticized; Criticize at the Sf-9 cell from primordial seed and to be uploaded to the 5th on behalf of main seed lot; The main seed lot total degree that continues to go down to posterity on the Sf-9 cell is no more than 12 generations preparation work seed lot.

5. as the preparation method of the arbitrary described single, double valency subunit vaccine of claim 2-4, it is characterized in that:

Wherein carry out clarification filtration by filter behind the suspension that the harvesting cracking is produced in the f step, the filter aperture is 0.45 μ m.

6. as the preparation method of the arbitrary described single, double valency subunit vaccine of claim 2-5, it is characterized in that:

Wherein the molecular cut off of the ultrafilter membrane of ultrafiltration and concentration virus vlps suspension is 100-300KD in the g step, and cycles of concentration is 100 times.

7. as the preparation method of the arbitrary described single, double valency subunit vaccine of claim 2-5, it is characterized in that:

Wherein the concrete steps of viral liquid purification are in the h step:

Gel permeation chromatography and ion-exchange chromatography purification: carry out gel permeation chromatography, medium adopts suitable gel filtration medium, and balance liquid is the PBS of pH6.5-7.5; Carry out ion-exchange chromatography again, use the weak anionic exchange media, buffer salt solution is a phosphatebuffer buffer system, and level pad is salinity scope 0.05-0.1M, and pH6.5-7.5 contains the sodium chloride of 0.06-0.12M; Elution buffer is salinity 0.05-0.1M, and pH6.5-7.5 contains the sodium chloride of 0.2-0.4M;

Or sucrose density gradient centrifugation purification: the discontinuous sucrose density gradient of use is 15%, 30% and 65%, and the 30000g ultracentrifugation was collected the milky band on the 15-35% sucrose interface after 3 hours;

Through behind the above-mentioned purification, the cell rests dna content is lower than 100pg/ml, gets the EV71 VLP virus stock solution used or the Cox.A16 VLP virus stock solution used of purification, i.e. EV71 VLP antigen or Cox.A16 VLP antigen.

8. as the preparation method of the arbitrary described single, double valency subunit vaccine of claim 2-7, it is characterized in that:

Wherein vaccine semi-finished product in the i step and finished product are meant that VLP virus stock solution used with above-mentioned purification is mixed with and comprise pharmaceutically acceptable carrier: the buffer of standard, stabilizing agent, diluent, antiseptic, solubilizing agent; Or be mixed with the dosage form of being convenient to slow release, or add aluminum salt absorption adjuvant, or injection type;

With the EV71 VLP virus stock solution used and the Cox.A16 VLP virus stock solution used of above-mentioned purification, mixed is prepared into the form of bivalence routinely.

9. as the application of the arbitrary described single, double valency subunit vaccine of claim 1-8 in preparation prevention hand-foot-mouth disease medicine.

10. application as claimed in claim 9 is characterized in that the using dosage of this vaccine is:

Unit price: a person-portion is 0.5ml, contains EV71 VLP antigen or Cox.A16 VLP antigen 1 .5-3 μ g, Al (OH) ₃Adjuvant 0.25-0.5mg;

Two valencys: a person-portion is 0.5ml, contains EV71 VLP antigen and Cox.A16 VLP antigen 1.5-3 μ g altogether, Al (OH) ₃Adjuvant 0.25-0.5mg.