CN102058881A - Gene recombinant vaccine for preventing enterovirus 71 infection and preparation method thereof - Google Patents

Abstract

The invention discloses a gene recombinant vaccine for preventing enterovirus 71 (EV71) infection and a preparation method thereof. The inflection comprises multiple diseases related with the nervous system such as hand-foot-and-mouth disease, paralytic diseases of sterile meningitis, cephalitis and poliomyelitis and the like. Escherichia coli labile enterotoxin B subunit (LTB) is used as an immunological enhancement adjuvant, two fragments of linear neutralizing epitope SP55 and SP70 in EV71 virus coat protein VP1 are used as antigens, prokaryotic expression plasmids of LTB-SP55-SP70 fusion genes are constructed by using gene engineering technology, the plasmids are expressed in escherichia coli, and a recombinant expression product is purified for preparing the EV71 virus gene engineering vaccine.

Description

Genetic vaccine that the prevention enterovirns type 71 infects and preparation method thereof

Technical field

The invention belongs to field of biological pharmacy, relate to a kind of vaccine and preparation method thereof.Be particularly related to be used to prevent enterovirns type 71 (EV71 virus) infect due to the genetic vaccine and preparation method thereof of the multiple disease relevant of the paralytic disease etc. of hand-foot-mouth disease, aseptic meningitis, encephalitis and poliomyelitis sample with nervous system.

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 (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 infant dead fast.Hand-foot-mouth disease symptom from infecting to occurring is generally 36 days.Patient is everlasting and finds skin ulcer 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 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 eruption and prevalence 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.Show according to up-to-date epidemic monitoring result, by May 16, whole nation accumulative total reported cases 553263 examples in 2010, sickness rate 41.66/10 ten thousand, serious symptom 7993 examples, dead 335 examples; Reported cases number (365630 example) is over the same period last year compared, and increases by 51.32%.From on May 2nd, 2008, Ministry of Public Health was included hand-foot-mouth disease in legal Class C communicable disease control.

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%.31 provinces, the whole nation in 2010 report the laboratory diagnosis case 14650 examples altogether, positive 7867 examples (53.70%) of EV71 wherein, positive 4579 examples (31.26/%) of CoxA16, positive 2204 examples (15.04%) of other enterovirus.In 7993 routine severe cases, 3249 examples are the laboratory diagnosis case, and wherein 86.21% for EV71 infects, and 3.76% for CoxA16 infects, and 10.03% is other enterovirus infections; In the 335 routine deaths, 256 examples are the laboratory diagnosis case, and wherein EV71 infects 239 examples, and CoxA16 infects 2 examples, other enterovirus infection 15 examples.

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 years global EV71 separated strains 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 year Fukushima area, nineteen eighty-three～2003 (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).The EV71 virus that is separated to from light disease case of China's Mainland hand-foot-mouth disease and severe cases specimen was mainly the C4 hypotype in recent years, and Cox.A16 virus is mainly the C type.

EV71 and Cox.A16 virion are the three-dimensional symmetric spherical junctions 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 opening code-reading frame is only arranged in the genome, coding contains about 2190～2200 amino acid whose polyproteins, 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, the research at the inactivated vaccine and the attenuated live vaccine of hand-foot-mouth disease has been arranged, and obtained some progress, but still immature 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 yield 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 the weak malicious post immune mouse of EV71 and Cox.A16 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.The attenuated live vaccine aspect, since unclear so far to EV71 and Cox.A16 virulence decision group, still get nowhere.

In traditional vaccine research, the laboratory research of subunit vaccine has obtained gratifying progress.Adopt Pichia sp., colon bacillus, 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.

Associate the successful Application of oral type poliovirus vaccine, and in recent years at mucosa-immune and oral vaccine progress of research, comprise the successful case of the oral recombinant vaccine of helicobacter pylori of the up-to-date approval listing of China, the development of visible EV71 oral type recombinant vaccine is not remote.

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

At the epidemic status of present hand-foot-mouth disease, for the better outburst of prevention hand-foot-mouth disease, the inventor designs and has made up EV71 amalgamation and expression albumen through unremitting effort.On this basis, the present inventor has developed the oral vaccine that is used to prevent the EV71 infection.The experiment proved that vaccine of the present invention or medicament have effective immunocompetence and/or counteracting toxic substances protective capability.

So one aspect of the present invention provides the vaccine that is used at the EV71 infection, comprise SP55 and two albumen of SP70 of EV71 in the described vaccine construct, and LTB albumen.

Further, the invention provides a kind of oral type vaccine that is used to prevent the EV71 infection.

Again on the one hand, the present invention also provides a kind of preparation to infect the method for oral type vaccine at EV71.

Again on the other hand, the invention provides the purposes of vaccine of the present invention in preparation prevention EV71 infection oral type vaccine.

Particularly, oral vaccine of recombinant gene of the multiple disease relevant such as paralytic disease that the invention provides a kind of hand-foot-mouth disease that can effectively prevent the EV71 viral infection to cause, aseptic meningitis, encephalitis and poliomyelitis sample and preparation method thereof with nervous system.

It is a kind of oral vaccine of recombinant gene that forms that merged by LTB-SP55-SP70 that the present invention prevents the reorganization oral vaccine of EV71 viral infection, it is that two sections polypeptide SP55 and SP70 among immunity enhancement adjuvant, the EV71 virus capsid protein VP1 is antigen with colon bacillus heat-labile toxin B subunit LTB, hobby according to colon bacillus is optimized codon, the prokaryotic expression system of technique for gene engineering structure rLTB-SP55-SP70, the genetic vaccine that obtains are adopted in the synthetic back of gene.Fusion gene pronucleus expression system and product thereof by the technique for gene engineering preparation, this vaccine is had contain reorganization adjuvant rLTB and rSP55 and rSP70 recombinant viral proteins antigen in the expression product simultaneously, thereby the relevant diseases such as paralytic disease of hand-foot-mouth disease, aseptic meningitis, encephalitis and the poliomyelitis sample that can cause the EV71 viral infection have the immunoprophylaxis effect, and it is easy to be reliable to have preparation method, characteristics such as production cost is comparatively cheap, and is easy to use, safe.

Its structure of vaccine of the present invention is represented that by LTB-SP55-SP70 its aminoacid sequence is listed in sequence table sequence 8 and vaccine construct corresponding nucleotide sequences of the present invention is listed in sequence table sequence 7,

Its aminoacid sequence separately of LTB-SP55-SP70 in the vaccine construct of the present invention is listed in the sequence table sequence 2,4,6, and its corresponding nucleotide sequences is listed in the sequence table sequence 1,3,5.

Technical scheme

The invention provides the preparation method and the effect authentication step thereof of the reorganization oral vaccine of EV71 viral infection: 1) codon optimized, the synthetic and order-checking of colon bacillus LTB gene, EV71 virus SP55 and SP70 gene; 2) the rLTB-SP55-SP70 gene synthetic after, carry out double digestion with Nde I and Xho I, downcut the fragment follow-up and gone the pET30a carrier of same double digestion and connect the structure prokaryotic expression plasmid; 3) recombiant protein rLTB-SP55-SP70 expresses, and SDS-PAGE electrophoresis and BioRad gel images analytical system are determined expression and output; 4) DEAE anion-exchange chromatography and Ni-NTA affinity chromatography purification rLTB-SP55-SP70; 5) the rLTB-SP55-SP70 aqueous solution can directly be made oral administration dripping pill; 6) rLTB-SP55-SP70 also can through lyophilization be prepared into oral capsule after medicinal excipient calcium phosphate mixes.

Beneficial effect

The present invention is by prokaryotic expression and the purification of recombiant protein rLTB-SP55-SP70, the oral recombinant vaccine of multivalence of the relevant diseases such as paralytic disease of hand-foot-mouth disease, aseptic meningitis, encephalitis and poliomyelitis sample that a kind of EV71 of prevention viral infection causes is provided, and adopt methods such as GM1-ELISA, the external neutralization experiment of immunity back mice serum antibody, confirm that rLTB maintains adjuvanticity in this vaccine, effectively the to neutralize infection of viral pair cell of the specific antibody that oral back produces.

The recombiant protein rLTB-SP55-SP70 of the inventive method preparation is a kind of antibacterial and virus amalgamation protein of harmless human body, and preparation method is safe and reliable, cost is lower, be easy to apply.

It is to take for people with oral administration dripping pill or oral capsule that the present invention prevents the oral vaccine of recombinant gene of EV71 viral infection, thus easy to use, can avoid injecting the pain and the discomfort that cause, also can avoid using syringe needle and syringe, reduced contamination of heavy; Low cost of manufacture is beneficial to and applies.

Description of drawings

Nucleotide sequence and aminoacid sequence after the optimization of Fig. 1 colon bacillus LTB gene codon:

Nucleotide sequence and aminoacid sequence after Fig. 2 EV71 virus SP55 and the optimization of SP70 gene codon

The LTB-SP55-SP70 fusion gene nucleotide and the aminoacid sequence of Fig. 3 colon bacillus LTB gene and EV71 virus SP55 and SP70 gene.Expression vector clone design.

Fig. 4 rLTB-SP55-SP70 protein SDS-PAGE glue.

The specific embodiment

Embodiment 1

It is a kind of reorganization oral vaccine of LTB-SP55-SP70 fusion gene that the present invention prevents the reorganization oral vaccine of EV71 viral infection, it is that two sections polypeptide SP55 and SP70 among immunity enhancement adjuvant, the EV71 virus capsid protein VP1 is antigen with colon bacillus heat-labile toxin B subunit LTB, hobby according to colon bacillus is optimized codon, the prokaryotic expression system of technique for gene engineering structure rLTB-SP55-SP70 is adopted in the synthetic back of gene, obtains the EV71 viral genetic engineering vaccine of high-purity recombination expression product.

The present invention develops the selection of recombinant vaccine to antigen and gene thereof: all EV71 viruses all have the gene of coding SP55 and SP70, characteristics such as have that sequence is conservative, expression is high, surface distributed, macromolecule and grain structure, antigenicity are strong, thus with these two albumen as EV71 recombinant vaccine first-selection antigen; This two fragment gene be in and antigenic determinant, wherein SP70 is stronger, can bring out body and produce and to have the protectiveness neutralizing antibody.

Recombinant vaccine of the present invention adopts the reason of oral vaccine to be: injecting immune normally induces serum antibody; And EV71 is because be intestinal infection virus, thus by oral vaccine when inducing serum antibody, can also induce body and produce mucosa-immune at EV71.Therefore, adopt the more effective generation immanoprotection action of EV71 virus oral vaccine energy.

Though recombinant vaccine has advantages such as antigenic component is clear and definite, side effect is little, the immune effect of vaccine that single proteantigen constitutes is often relatively poor.In recent years have many research datas to prove, colon bacillus heat-labile toxin (LT) has very strong mucosa-immune adjuvanticity.LT is made up of subunit A (LTA) and B (LTB) respectively, and wherein LTA is the toxicity position of LT, and the LTB function then is an adherent cell, and avirulence.Existing many bibliographical informations, LTB has the very strong activity of inducing mucosa-immune, is good mucosal adjuvant.

If adopt technique for gene engineering, two sections polypeptide SP55 among colon bacillus LTB, the EV71 virus capsid protein VP1 and SP70 is recombinant expressed respectively, and must make complex manufacturing increases production cost.Therefore, present embodiment becomes the such fusion gene of LTB-SP55-SP70 with LTB, SP55 and three gene series connection of SP70, makes up the LTB-SP55-SP70 prokaryotic expression system then.LTB-SP55-SP70 prokaryotic expression system recombination expression product is rLTB-SP55-SP70, contain mucosal adjuvant LTB and SP55 and SP70 antigen in its molecule simultaneously, not only can improve proteic immunogenicity, and can reduce production costs effectively and help industrialization because of the existence of this intramolecularly adjuvant of LTB.In addition, also to r LTB-SP55-SP70 immunogenicity, express prevention EV71 infects behind output and method of purification, adjuvanticity, the oral immunity mice effect etc. and verify, express back r LTB-SP55-SP70 output and can reach 24.3% of total bacterial protein, wherein rLTB still maintains adjuvanticity, can make mice produce specific antibody behind the oral immunity, the infection of the viral pair cell that can effectively neutralize in cell in vitro and in the experiment.Its result shows that rLTB-SP55-SP70 can be used for preventing the oral vaccine of recombinant gene of EV71 viral infection.

The preparation method of the oral vaccine of recombinant gene of present embodiment prevention EV71 viral infection comprises following step: 1) codon optimized, the synthetic and order-checking of colon bacillus LTB gene, EV71 virus SP55 and SP70 gene; 2) after the rLTB-SP55-SP70 gene synthesizes, digest with restricted enzyme Nde I and Xho I respectively, collect the purpose fragment and be connected, make up recombinant expression plasmid with the pET30a carrier of handling with restricted enzyme equally; 3) recombiant protein rLTB-SP55-SP70 expresses, and SDS-PAGE electrophoresis and BioRad gel images analytical system are determined expression and output; 4) DEAE anion-exchange chromatography and Ni-NTA affinity chromatography purification rLTB-SP55-SP70 albumen.Adopt the GMI-ELISA method that the rLTB adjuvanticity in the rLTB-SP55-SP70 molecule is identified in addition; With high-purity gene recombinant protein immunity BALB/c mouse, detect its specific antibody, and adopt in the external virus and this recombinant antigen of experimental evaluation to the preliminary immune protective efficiency of BALB/c mouse.

Colon bacillus LTB gene, EV71 virus SP55 and SP70 gene all obtain by gene is synthetic in the present embodiment; Used all experiments all can be from domestic and international related industry company or home sale agency of offshore company buy separately with material and reagent and relevant device in the present embodiment.The professional of technical fields such as microbiology, molecular biology, all can repeat the preparation method in the present embodiment and try out.

The preparation method of the fusion gene LTB-SP55-SP70 of above-mentioned synthetic is synthetic by precious biological engineering (Dalian) company limited direct gene.

Above-mentioned prokaryotic expression carrier pET30a is available from Novagen company, and E.coli BL21 (DE3) is available from the rich Deco skill Development Co., Ltd that steps in Beijing.The genes of interest LTB-SP55-SP70 and the pET30a carrier of above-mentioned synthetic gene are all carried out digestion process with Nde I and Xho I restricted enzyme,, obtain the purpose recombinant expression plasmid with 16 ℃ of connections of T4 dna ligase;

One, coupled reaction

1, the LTB-SP55-SP70 genetic fragment of pET30a carrier and synthetic is carried out digestion process with Nde I and two kinds of restricted enzyme of Xho I, collect the purpose fragment.

2, the target gene fragment with the pET30a carrier behind the 50ng double digestion and 5～8 times of moles joins in the aseptic Eppendorf pipe of 0.5ml.

3, add distilled water and replenish volume to 8 μ l, add 10 * T4 DNA Ligase buffer, 1 μ l then, T4 DNA Ligase 1 μ l all was thrown to the pipe end with microcentrifuge with liquid behind the mixing, in 16 ℃ of insulations 1 hour.

Do two groups of control reaction simultaneously, wherein matched group one only has the pET30a carrier segments behind the double digestion and does not have foreign DNA; Matched group two only has the exogenous dna fragment behind the double digestion and does not have carrier segments.

Two. the preparation of competent cell

1. the single colony inoculation of picking E.coli BL21 (DE3) is in the aseptic LB culture fluid of 1ml, and 37 ℃ 210 was changeed shaking table 2-3 hour.

2. the fresh bacterium liquid transferred species of getting 0.05～0.1ml cultivation is in the aseptic LB culture fluid of 50ml, and 37 ℃ of concuss reach 0.5 up to A600.

3. under aseptic condition, culture is transferred in the aseptic centrifuge tube of 50ml, 4 ℃ 4, the centrifugal 10min of 000rpm.

4. abandon supernatant, centrifuge tube is inverted on the filter paper, culture fluid is blotted, this is operating as under the aseptic condition.

5. get the resuspended precipitation of aseptic calcium chloride solution of 15ml 0.1M pre-cooling, place 10～15min in the ice-water bath, 4 ℃ 4, the centrifugal 10min of 000rpm.

6. abandon supernatant, precipitation is resuspended in soft mixing in 5ml (the aseptic calcium chloride solution that contains the 0.1M pre-cooling, 15% the aseptic glycerol) solution, places 10min in the ice-water bath.

7. bacterium liquid is sub-packed in the aseptic EP pipe-80 ℃ or liquid nitrogen cryopreservation.

8. get two pipe competent cells and add the aseptic ddH of 1 μ l respectively ₂O (negative control) and 1 μ l pure plasmid (positive control) transform (seeing below), to detect competent quality.The length of should asepticly being born on the negative control flat board, how many clump count purposes shows the height of competence efficient on the positive control flat board.

The preparation of LB:

Peptone 10g

Yeast extract 5g

NaCl 0.58g

1: 100 adjust pH of NaOH

Dissolve and add water and be settled to 1L, 121 ℃ * 20min high pressure steam sterilization

0.1M the preparation of calcium chloride solution:

0.1M?CaCl ₂

ddH ₂O?to?100ml

Annotate: above-mentioned solution all needs high pressure steam sterilization to handle

Three. transform

1. getting 100 μ l competent cells melts on ice-water bath.

2. add 10 μ l and connect product, dispel mixing gently, ice-water bath is placed 30min.

3. competent cell is put into 42 ℃ of water-bath heat shocks 90 seconds, after put into ice-water bath immediately and place 2min.

4. add the aseptic LB solution of 0.9ml, 150rpm on 37 ℃ of constant temperature shaking tables * 45min renaturation.

5. with the centrifugal 1min of competent cell 4000rpm, stay 200 μ l supernatants to precipitate softly and dispel, evenly coat the agar plate surface that contains penbritin, dull and stereotyped in 37 ℃ of inversion overnight incubation.

Four. the order-checking of recombiant plasmid is identified

1. above-mentioned conversion process can with the restricted enzyme screening, obtain to contain the bacterial plaque of reorganization exogenous dna fragment plasmid by extracting nucleic acid, carries out a small amount of and expresses.

2. through protein electrophoresis the correct sample of expection band of expression size will be arranged, and send to order-checking, determining whether the insertion of exogenous gene LTB-SP55-SP70, and whether insertion sequence is correct.

Expression and the method for purification of above-mentioned purpose recombination expression product rLTB-SP55-SP70 are:

One, the abduction delivering of recombinant protein

1. picking transforms single bacterium colony that plasmid is arranged, and be inoculated in 1.5ml and contain in the LB fluid medium of penbritin (50 μ g/ml), 37 ℃, 200rpm shaken cultivation 2-3 hour.

2. get 0.5ml bacterium liquid and preserve in 4 ℃ of refrigerators, add 1mol/L IPTG in the residue bacterium liquid, making the IPTG final concentration is 1mmol, 37 ℃, and 250rpm shaken cultivation 2 hours.Get the 1ml sample as inducing the back specimen, collect bacterial sediment.

4. will induce the back bacterial sediment resuspended with 20～40 μ l PBS (pH=8.0), add isopyknic 2 * SDS sample-loading buffer, boil heating 5min, sds page (SDS-PAGE) electrophoretic separation, behind the coomassie brilliant blue staining 1 hour, the decolouring observed result.

5. choose the bacterial clone of inducing success, enlarge the scale of inducing, collect bacterial sediment,, go to next step analysis and purification in-20 ℃ of preservations.

Two. recombinant protein is the proteic separation and purification of non-solubility

1. in the LB fluid medium 1L that contains penbritin (50 μ g/ml), inoculation contains the bacterium liquid of pET30a-LTB-SP55-SP70, and 37 ℃, the 200rpm overnight incubation, the back adds IPTG to final concentration 1mmol/L, continues to cultivate 4-5 hour by above-mentioned condition;

2.4000g centrifugal 10min collects bacterial precipitation, (0.05%Triton-X100,10mmol/L TrisCl pH8.0) blow afloat mixing, carry out ultrasonication (200 watts, act on 20 seconds, interval 20 seconds acts on 20 times) in ice-water bath with lysate;

3.9000g centrifugal 10min, precipitation blows afloat mixing with lysate, ultrasonication once more;

4.9000g centrifugal 10min, the precipitation with the base night (8mol/L carbamide, 10mmol/L TrisHCl, pH8.0) resuspended;

5.11000g centrifugal 10min, supernatant cross the HiS post, with 300mM imidazoles eluting destination protein;

6. with the destination protein reuse 10mmol/L TrisHCl of eluting, dialysed 2 hours for pH8.04 ℃;

7. DEAE anion chromatography post on Tou Xi the albumen is used 100mM, 200mM concentration NaCl eluting respectively, collects the purpose recombinant expression protein;

With the destination protein of 200mM NaCl eluting in-20 ℃ of preservations, go to next step analysis and purification.

Three, the renaturation of recombinant protein, lyophilizing and quantitative

Albumen behind the purification is slowly dialysed with the urea liquid that gradient reduces, and finally uses 0.01 * PBS dialysis, and the protein solution after the dialysis is through being lyophilized into powdery.With bovine serum albumin (BSA) is standard, adopts BIORAD company's quantification of protein reagent (protein assay) colorimetric determination Protein content.

Above-mentioned experimental result is as follows: 1) contain pET30a-LTB-SP55-SP70 E.coli BL21 (DE3) but engineering bacteria effective expression rLTB-SP55-SP70, output can reach 24.3% of total bacterial protein, sees Fig. 4; 2) through DEAE anion chromatography and Ni-NTA affinitive layer purification, rLTB-SP55-SP70 purity can reach 90%-95 %.

Be the immunogenicity of confirmation purpose recombination expression product rLTB-SP55-SP70 and the biologic activity of LTB, be respectively the antigen immune BALB/c mouse with rLTB-SP55-SP70, observe the binding ability of specific antibody production and rLTB-SP55-SP70 and ganglioside GM1.

1) immunogenicity detects: the injection group, at the subcutaneous multi-point injection rLTB-SP55-SP70 of mice, each dosage 5ug/ only, every injection in 14 days once, totally 3 times, gather mouse tail venous blood 0.5～1ml simultaneously respectively, inject for the third time and gathered mouse orbit venous blood 1ml in back 14 days, separation of serum detects specific antibody in the serum with test kit; Oral group, pass through the feed mode with rLTB-SP55-SP70, each dosage 2ug/ only, every 14 days feed once, totally 3 times, gather mouse tail venous blood 0.5～1ml simultaneously respectively, for the third time 14 days collection mouse orbit venous blood 1ml behind the feed, separation of serum detects specific antibody in the serum with test kit.

2) the LTB biologic activity detects: according to LTB can with the bonded characteristic of the specificity of GM1, available GM1-ELISA method detects recombinant expressed LT albumen and whether has biologic activity.

(1) GM1 powder 1mg is dissolved in 2ml PBS (pH7.4) solution, and final concentration is 0.5mg/ml, divides to install in the EP pipe;

(2) draw 12 μ l GM1 solution (0.5mg/ml) to 3.0ml carbonic acid buffer (1.59g Na ₂CO ₃, 2.93gNaHCO ₃Be dissolved in the 1L deionized water) in, be added to behind the mixing in the polystyrene coated slab, 100 μ l/ holes, 4 ℃ are spent the night;

(3) coating buffer is outwelled, every hole adds 170 μ l confining liquids (the PBS solution that contains 1.0%BSA), and 37 ℃ act on 1 hour;

(4) confining liquid is outwelled, washed 6 times with washing liquid (the PBS solution that contains 0.05%Tween-20), button is done;

(5) with PBS solution immune serum albumen is diluted to 100 μ g/ml, 10 μ g/ml and 1 μ g/ml, LTB albumen is diluted to 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 100ng/ml and 10ng/ml;

(6) every hole adds the sample 100 μ l of serial dilution, and each sample adds multiple hole, and as negative control, other gets not application of sample of 2 holes, as blank with PBS solution;

(7) outwell sample liquid, wash 6 times with washing liquid, button is done;

(8) every hole added 1: 6, the anti-CT holotoxin of the rabbit serum 100 μ l of 000 dilution, and 37 ℃ act on 1 hour;

(9) repeating step (7);

(10) every hole added 1: 2, goat anti-rabbit igg-HRP 100 μ l of 000 dilution, and 37 ℃ act on 1 hour;

(11) repeating step (7);

(12) every hole adds TMB colour developing A liquid, each 50 μ l of B liquid, and 37 ℃ were developed the color 15 minutes;

(13) every hole adds stop buffer 50 μ l, and mixing is measured each hole A with the blank zeroing ₄₅₀The nm value;

(14) marginal value (cutoff)=negative control A ₄₅₀Nm average+0.16, sample A ₄₅₀Nm value 〉=marginal value is judged to the positive; Sample A ₄₅₀Nm value＜marginal value is judged to feminine gender.

Above-mentioned experimental result is as follows:

1) can produce specific antibody behind the rLTB-SP55-SP70 immune mouse, with EV71 antibody assay kit tests positive.The results are shown in subordinate list 1.

2) GM1-ELISA detects: the rLTB-SP55-SP70 antiserum can combine with cattle ganglioside GM1, and its OD490 average is higher than negative control more than 20 times, so be judged to stronger ganglioside GM1 binding ability.The results are shown in subordinate list 2

For the neutralization of the specific antibody that confirms purpose recombinant products rLTB-SP55-SP70, carried out external virus neutralization experiment to virus.With the solution that contains rLTB-SP55-SP70, adopt subcutaneous multi-point injection (5ug/0.1ml/ time, immunity is 3 times altogether) and irritate stomach (2ug/1ml/ time, totally 3 times) dual mode immune mouse.Get mouse orbit venous blood after three immunity, separation of serum carries out external virus neutralization experiment.After the result showed for the third time immune mouse, injection group and oral group of mice specific serum had tangible neutralization to virus, and effectively the serum neutralization is tired more than 1: 32 dilution factor.The results are shown in subordinate list 3

The EV71 serum neutralization test:

Reagent is prepared: DMEM culture medium (having added 1%G, 3%PS), FBS (hyclone); 0.1% crystal violet solution, known titre viral suspension, test serum sample

Consumptive material is prepared: rifle and the rifle head of 24 well culture plates, 1000 μ L/200 μ L/20 μ L, 10mL/2mL suction pipe

Experimental procedure:

The going down to posterity and cultivating of cell: the neutralization experiment passes 24 orifice plates (5cm with the RD cell the previous day ²Little square vase passes 2 24 orifice plates) standby.Treat that the RD cell density grows at 80% o'clock in 24 orifice plates and can inoculate.

The dilution of virus: virus dilution suspension to 1/2 * 10 ^-5Titre.

The dilution of test serum sample: with the good viral liquid of dilution test serum was diluted by 1: 64,1: 128 or 1: 256 times in 24 new orifice plates, the dilution back is in 5%CO ₂, incubation 1h in 37 ℃ the incubator, during rock mixing once every 15min;

The inoculation of virus-serum mixed liquor: cells and supernatant suction in 24 orifice plates is gone, and every hole adds virus-serum mixed liquor (shown in the step 3) that 200 μ L dilution was hatched, in 5%CO ₂, incubation 1h in 37 ℃ the incubator, during rock mixing once every 15min; 1mL DMEM culture medium (containing 2%FBS) is added in every hole behind the 1h, continues to cultivate.Every plate is done the cell that the blank cell contrast in a hole and a hole only add viral suspension respectively and is contrasted.

The result observes: wait to leave standstill and cultivate about 72h (as too acid of cell liquid between culture period, can add appropriate culture medium), cells and supernatant is gone in suction, every hole adds an amount of 0.1% crystal violet solution (being advisable to cover hole surface), room temperature leaves standstill 10min, inhales and abandons supernatant, with distillation washing one time, blot observed result.

In sum, rLTB-SP55-SP70 can form a kind of oral EV71 recombinant vaccine product and the relevant disease that is used to prevent EV71 to infect.

Subordinate list 1: immune serum EV71 antibody test result

Embodiment 2:

The genetic vaccine that present embodiment prevention EV71 infects is that vaccine antigen LTB-SP55-SP70 aqueous solution directly is prepared into oral administration dripping pill, and the concentration of its aqueous solution can be 1mg/ml; Also can will be prepared into oral capsule after the LTB-SP55-SP70 lyophilization with after pharmaceutical excipient calcium phosphate mixes, calcium phosphate wherein also has more weak adjuvanticity.Every oral capsule includes 100～500 μ g, and the big or small visual different object of this amount is selected, the people of different sexes, different age brackets, its consumption difference; Even also can be in different animals with different dosage feed to.

Claims (10)

1. a genetic vaccine that prevents hand-foot-mouth disease is characterized in that, its aminoacid sequence is the sequence shown in the SeQ ID NO.8, and structure is LTB-SP55-SP70.

2. vaccine as claimed in claim 1, it is characterized in that, it is adjuvant oral vaccine of recombinant gene in a kind of cell, it is that two sections linear neutralizing epitope SP55 and SP70 among immunity enhancement adjuvant, the EV71 virus capsid protein VP1 is antigen with colon bacillus heat-labile toxin B subunit LTB, make up the prokaryotic expression plasmid of LTB-SP55-SP70 fusion gene with technique for gene engineering, in escherichia coli, express, and recombination expression product carried out purification, make EV71 viral genetic engineering vaccine.

3. vaccine as claimed in claim 1 is characterized in that said LTB-SP55-SP70 is prepared into oral administration dripping pill.

4. vaccine as claimed in claim 1 is characterized in that said LTB-SP55-SP70 is prepared into oral capsule.

5. the preparation method of vaccine according to claim 1 is characterized in that the method includes the steps of: 1) colon bacillus LTB gene, EV71 virus SP55 and SP70 gene codon optimize, synthetic and order-checking; 2) rLTB-SP55-SP70 prokaryotic expression system construction; 3) rLTB-SP55-SP70 Recombinant Protein Expression, SDS-PAGE electrophoresis and BioRad gel images analytical system are determined expression and output; 4) DEAE anion-exchange chromatography and Ni-NTA affinity chromatography purification rLTB-SP55-SP70; 5) adopt GM1-ELISA that the rLTB adjuvanticity in the rLTB-SP55-SP70 molecule is identified; 6) with rLTB-SP55-SP70 recombiant protein immunity BALB/c mouse, the antibody production of immune serum is detected, utilize external in and the immanoprotection action of the EV71 viral genetic engineering vaccine rLTB-SP55-SP70 of measuring oral immunity.

6. preparation method as claimed in claim 5 is characterized in that, wherein the said colon bacillus LTB of step 1) has the sequence shown in the SeQ ID NO.2 in the sequence table.

7. preparation method as claimed in claim 5, it is characterized in that: said EV71 virus SP55 and SP70 in the step 1), be that the synthetic EV71 virus gene sequence of gene is expressed according to the colon bacillus preference is codon optimized, have the sequence shown in the SeQ ID NO.4 and 6 in the sequence table.

8. preparation method as claimed in claim 5 is characterized in that: step 2) in the constructed expression vector, LTB strengthens adjuvant as molecular immune in a kind of cell.

9. a nucleotide sequence of expressing claim 1 vaccine is characterized in that, is the sequence shown in the SeQ ID NO.7.

10. express the nucleotide sequence of three subunits of LTB-SP55-SP70, it is characterized in that, be SeQ ID NO.1, the sequence shown in 3,5.

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