CN103045630A - Encephalitis virus protein and encoding gene and application thereof - Google Patents

Abstract

The invention discloses an encephalitis virus protein and an encoding gene and application thereof. The encephalitis virus protein is a protein of the following 1) or 2): 1) a protein consisting of an amino acid sequence shown as a sequence 2 in a sequence table; and 2) a protein derived from the 1) by substituting and/ or deleting and/ or adding one or more amino acid residues on the basis of the amino acid sequence shown as the sequence 2 and having the same function as the 1). Experiments show that the obtained recombinant adenovirus VAD-E2E3 has a prospect of becoming a vaccine candidate for an encephalitis genetic engineering vaccine.

Description

Encephalitis albumen and encoding gene thereof and application

The application is that application number is 201010506163.9, the applying date is on October 9th, 2010, invention and created name is divided an application for " encephalitis albumen and encoding gene thereof and application ".

Technical field

The present invention relates to a kind of encephalitis albumen and encoding gene and application.

Background technology

Encephalitis is by the direct infringement of virus or by the brain acute inflammatory disease due to the allergy of virus or the initiation of other foreign proteins.Encephalitis can be the clinical manifestation of former of viral infection, or the clinical manifestation of secondary, the virus that causes former encephalitis has poliovirus, the epidemic virus such as Echo virus, Coxsackie virus, or sporadic by the forest encephalitis of the arthropod-borne Flavivirus such as mosquito, tick and the eastern equine encephalitis virus of batch film Viraceae alphavirus etc.

Alphavirus is Togaviridae (Togaviridae) alphavirus (Alphavirus) member, take hematophagous buges such as mosquito ticks as communication media, can cause multiple infectious diseases common to human beings and animals.In recent years, abroad to the part or all of sequence order-checking of many strains Alphavirus.Domestic molecular biology research to this viroid is also obtained certain result.Existing result of study shows: this Tobamovirus neutralizing antibody determinant mainly concentrates on the glycoprotein such as structural protein E1 and E2, and wherein proportion is higher on the E2 albumen.In addition in recent years adenovirus carrier because having the multiple advantages such as security is good, host range is wide, efficiency of infection is high, be widely used in the research of gene therapy and recombinant vaccine, we analyze and have compared many strains Alphavirus nucleotide sequence for this reason, synthetic the Nucleotide of the higher 3kb of homology, utilize adenovirus vector construct recombination engineered vaccine, for preventing and treating disease and lay a good foundation.

Summary of the invention

An object of the present invention is to provide a kind of and encephalitis vaccine associated protein and encoding gene thereof.

Albumen E2E3 provided by the invention, synthetic is following 1) or 2) albumen:

1) albumen that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;

2) with the aminoacid sequence of sequence 2 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have identical function by 1) protein of deriving.

Sequence 2 in the sequence table is comprised of 529 amino-acid residues, the replacement of described one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.

The encoding gene of above-mentioned protein E2E3 also is the scope of protection of the invention, and described encoding gene is following 1)-4) in the gene shown in arbitrary:

1) dna molecular shown in the sequence 1 in the sequence table;

2) in the sequence table sequence 1 from the dna molecular shown in 5 ' the terminal 1-1589 position Nucleotide;

3) under stringent condition with 1) or 2) dna molecule hybridize that limits and the dna molecular with identical function;

4) with 1) or 2) dna sequence dna that limits has at least 90% homology and have the dna molecular of identical function.

Recombinant vectors, transgenic cell line, recombinant bacterium, expression cassette or the recombinant virus that contains above-mentioned encoding gene also is the scope of protection of the invention.

Described recombinant vectors is that pAdeasy-1 and recombinant shuttle plasmid obtain through homologous recombination; Described recombinant shuttle plasmid is to insert described encoding gene to obtain between the KpNI of carrier pShuttle-CMV and HindIII restriction enzyme site.

Described recombinant virus is recombinant adenovirus; Described recombinant adenovirus is the recombinant adenovirus with the encoding gene transfection host cell acquisition of above-mentioned albumen.

Described host cell is eukaryotic cell, is preferably stripped mammalian cell, especially is preferably the HEK-293 cell.

Another object of the present invention provides a kind of vaccine.

Vaccine provided by the invention, its activeconstituents be following any one:

1) described albumen; 2) described encoding gene; 3) described recombinant adenovirus.

The application of above-mentioned recombinant adenovirus in the preparation vaccine also is the scope of protection of the invention.

Described vaccine is the encephalitis vaccine.

The application of above-mentioned recombinant adenovirus in the toughener of preparation raising IFN-gamma activity also is the scope of protection of the invention.

The synthetic E2E3 that experiment showed, of the present invention is assembled into the recombinant adenovirus vAd-E2E3 that carries the target antigen gene by transfection HEK-293 cell.Recombinant adenovirus vAd-E2E3 infects 293 cells energy effective expression target protein; By collunarium approach inoculation BalB/C mouse; utilize IiT detection specificity antibody horizontal; the result shows; although it is different with the level of keeping that constructed recombinant adenovirus produces the time of specific antibody; but all produced better humoral immune reaction, provide important Data support for further carrying out the immunoprotection experiment.Because virus protein expression of denier in body can evoke immune response, so the recombinant adenovirus vAd-E2E3 that carries encephalitis structure gene that makes up, promise to be the candidate vaccine strain of encephalitis recombinant vaccine.

Description of drawings

Fig. 1 is that the pAd-E2E3 enzyme is cut evaluation figure

Fig. 2 is that recombinant plasmid Pac I enzyme is cut with PCR and identified

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Eastern equine encephalitis virus (Eastern Equine Encephalomyelitis virus) (HE Jing; CHANG GuoHui; WU Jin Song; LI ZhongDuo; The ZHU QingYu:Cloning and expression of E2gene of easternequine encephalomyelitis virus.Bulletin of The Academy of Military MedicalSciences.2002.02. public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.）

The HEK-293 cell (Invitrogen company product, Cat.No.R750-07); Test required restriction enzyme respectively available from TaKaRa and Biolabs company; Liposome Lipofectamin 2000 is available from Invitrogen company; Platinum pfx Taqase is available from Invitrogen company.

The acquisition of embodiment 1, recombinant adenovirus (vAd-E2E3)

1, contains structure and the evaluation of goal gene pAdtrack-E2E3

According to Eastern equine encephalitis virus (Eastern Equine Encephalomyelitis virus) genome sequence (NC-001547), the sequence 1 in the sequence table is designed in a plurality of sites in the mutant nucleotide sequence, and synthetic goes out sequence 1.

The gene of design primer amplification coding E2E3 albumen, primer sequence (primer contains respectively KpNI and HindIII restriction enzyme site) is seen lower:

E2E3-F:5’-ACGGggtaccATGTCACTAGTGACCACCATGTGTCT-3’

E2E3-R:5’-ACCCaagcttACTAAAAAAGGCACGACACAG-3’

The DNA that goes out sequence 1 take synthetic is template, utilizes E2E3-F and E2E3-R primer to carrying out the PCR reaction, and the PCR product that obtains is identified through 1% agarose gel electrophoresis, has obtained expection fragment of the same size, is about 1.5Kb.

Through order-checking, this PCR product has sequence 1 in the sequence table from 5 ' terminal 1-1589 position Nucleotide, unnamed gene E2E3 with this PCR product, its OFR be sequence 1 from 5 ' terminal 1-1589 position Nucleotide, the albumen called after E2E3 of this genes encoding, the aminoacid sequence of this albumen is the sequence 2 in the sequence table, and sequence 2 is comprised of 529 amino-acid residues.

Reclaim the PCR product, the PCR product is added " A " to be processed, PCR sample after the processing and pMD-18T carrier (TaKaRa company product, Code:D101A.Lot CK3201A) links to each other, transform the DH5a competent cell, the positive bacterium colony of picking extracts sequence verification behind the plasmid, the result for this plasmid for sequence in the sequence table 1 obtained called after pTE2E3 from 5 ' terminal 1-1589 position Nucleotide insertion pMD-18T.

With KpNI and HindIII double digestion recombinant plasmid pTE2E3 and plasmid pShuttle-CMV(Invitrogen company product, Catalog#240007), enzyme is cut product and is cut the glue recovery with gel recovery test kit respectively, and under the effect of T4DNA ligase enzyme, spend the night in 16 ℃ of connections, connect product and transform intestinal bacteria DH10B(Invitrogen company product, Cat.No.18290-015) select positive colony after, extracting plasmid checks order, the result for this plasmid for sequence in the sequence table 1 inserted the carrier that obtains between the KpnI of pShuttle-CMV and HindIII restriction enzyme site, called after pAdtrack-E2E3 from 5 ' terminal 1-1589 position Nucleotide.

2, the structure of recombinant adenoviral vector

Bacterium BJ5183 is available from the outstanding U.S. gene in Shanghai Pharmaceutical Technology Co., Ltd, and article No. is: GMS12223.2.

With adenovirus skeleton plasmid pAdeasy-1(Invitrogen company product, Catalog#240005, contain the GFP reporter gene) pass through PacI (available from Biolabs company, article No. is: after R05647S) enzyme is cut, change among the Host Strains BJ5183, obtain containing the Host Strains BJ5183 of adenovirus skeleton plasmid pAdeasy-1.

Use PmeI(available from Biolabs company above-mentioned pAdtrack-E2E3 and shuttle plasmid pShuttle-CMV, article No. is: R0560S) through 37 ℃ of fully digestion, (available from OMGEA company, article No. is: D2500-01) reclaim linearizing pAdtrack-E2E3 and pShuttle-CMV to use Gel Extraction kit test kit.

Linearizing plasmid pAdtrack-E2E3 electricity is transformed in the Host Strains BJ5183 competent cell that contains adenovirus skeleton plasmid pAdeasy-1, because E.coli BJ5183 has the Sm resistance, pAdeasy-1 has the Amp resistance, and recombinant plasmid pAdtrack-E2E3 and shuttling expression plasmid vector pShuttle-CMV have the Kan resistance, Kan resistance in regrouping process on the shuttle plasmid has replaced the Amp resistance on the pAdeasy-1, therefore come screening positive clone, the positive colony that obtains according to Kan and the dual resistance of Sm.

The plasmid that extraction changes the positive colony of linearizing plasmid pAdtrack-E2E3 over to carries out the PacI enzyme and cuts evaluation, the 1%DNA gel electrophoresis the results are shown in 1:pAdTrack-E2E3 2 shown in Figure 1: positive colony plasmid 3:pShuttle-CMV4:pAdeasy-1 5:DL15000Marker, after as can be seen from the figure swimming lane 1 recombinant shuttle plasmid pAdTack-QE cuts through Pac I enzyme, produce 2 fragments, one of them contains the about 7Kb of goal gene E2E3, another fragment contains replicon and the about 3Kb of kalamycin resistance gene size on the shuttle plasmid pShuttle-CMV, after swimming lane 2 is cut through Pac I enzyme, produce 2 fragments, the size of the small segment that produces is 4.5Kb, contain replicon and kalamycin resistance gene, large stretch of degree is that the viral skeleton size that contains the E2E3 of goal gene is about the 35Kb(virus skeleton about 33.5Kb of size and goal gene E2E3 1.5Kb).With this recombinant plasmid called after recombinant plasmid pAd-E2E3.

The plasmid that extraction changes the positive colony of linearizing plasmid pAdtrack-E2E3 over to carries out the PCR evaluation, primer is E2E3-F, E2E3-R, the results are shown in shown in Figure 2,1:pShuttle-CMV 2:pAdTrack-E2E3 3:pAd-E2E3 4:pAdeasy-1 5:DL15000Marker, can find out, recombinant plasmid pAd-E2E3 can amplify and the purpose sheet degree 1.5Kb that expects, further specifies explanation and has successfully made up the recombinant adenovirus skeleton plasmid pAd-E2E3 that carries goal gene E2E3.

3, be assembled into recombinant adenovirus

The HEK-293 cell is inoculated in 6 orifice plates, 37 ℃ of cultivations, Growth of Cells is to 60%-70%, with liposome Lipofectamin 2000(available from Invitrogen company) mediation recombinant adenovirus skeleton plasmid pAd-E2E3 is transfected in the cell, after transfection was finished 7-10 days, collecting cell was taken turns multigelation through 3, centrifugal collection supernatant liquor is the recombinant adenovirus vAd-E2E3 that contains goal gene.Transfection experiment carries out to specifications, cultivates 7-10 days, regularly detects whether produce fluorescence under fluorescent microscope, judges whether transfection is successful, owing to contain the reporter gene of green fluorescent protein (GFP) on this recombinant virus.

4, the expression of the amplification of recombinant adenovirus vAd-E2E3 and target protein

1) amplification of recombinant adenovirus vAd-E2E3

After above-mentioned 3 transfections that obtain were finished 7-10 days, collecting cell was taken turns multigelation through 3, and centrifugal collection supernatant liquor is recombinant adenovirus vAd-E2E3, carries out next generation amplification, increases continuously for 3 generations, collects supernatant liquor the second time, than the first time quantity increase.

2) expression of target protein

(recombinant adenovirus vAd-E2E3, the concentration of virus is LgTCID to get the supernatant liquor of collecting for the second time of the above-mentioned acquisition of 200ul ₅₀/ 0.2ml=8.0) infect 5x10 ⁶The HEK-293 cell, after 4 days, collecting cell.Cell carries out the SDS-PAGE electrophoresis after treatment.The result is: through the SDS electrophoresis, obtain size and be the albumen of 110KD (E2E3 albumen), with the in the same size of expection albumen.Illustrate that recombinant adenovirus effectively mediates E2E3 at cells.

Adopting uses the same method changes above-mentioned linearizing pShuttle-CMV and pAdeasy-1 in the Host Strains BJ5183 competent cell jointly, obtains recombinant adenoviral vector, is denoted as pAd-GFP; With pAd-GFP transfection HEK-293 cell, obtain negative control adenovirus vAd-GFP.Contrast among the adenovirus vAd-GFP without the E2E3 protein expression.

The collunarium inoculation neutralizing antibody of embodiment 2, recombinant adenovirus (vAd-E2E3) is measured

The BALB/c mouse in age in 6-7 week is divided into 3 groups at random, is respectively vAd-GFP control group and vAd-E2E3 immune group, every group of 20 mouse.

VAd-GFP control group: wipe away the nose mode and infect 20ul by embodiment 1 acquisition contrast adenovirus vAd-GFP (10 ⁸Pfu).

VAd-E2E3 immune group: wipe away the nose mode and infect the recombinant adenovirus (vAd-E2E3) (10 that 20ul is obtained by embodiment 1 ⁸Pfu).

PBS control group: wipe away the nose mode and infect 20ulPBS solution (0.01M/L pH7.2)

One, serum and lymphocyte detect

With the 4th all booster immunization of 3 groups after the immunity first time once.Every interval week age gathers mouse blood by the tail vein after immunity, centrifugal rear serum and the lymphocyte precipitation of collecting respectively.

1, indirect immunofluorescence assay is measured the antibody response of virus in the immune serum

The HEK-293 that infects with Eastern equine encephalitis virus (Eastern Equine Encephalomyelitis virus) prepares the antigen sheet, and the method for preparation is as described below:

Prepare before the experiment: the antigen sheet is placed on the antigen frame, discharging 2 rows on each frame, 4 of 1 rows, complete rear covering with cover prevents entering of dust.Carry out according to following step afterwards:

1) treats 75cm ²In the time of about the HEK-293 Growth of Cells to 80% of square vase, after 1 hour, the sucking-off adsorption liquid is added fresh cell maintenance medium, puts into incubator and cultivates with Eastern equine encephalitis virus 2ml cells infected;

2) (about 25%-50%) cytopathic the time appears when cell, method peptic cell according to passage, nutrient solution (the DMEM cell growth medium that contains 10% foetal calf serum with an amount of volume, concrete prescription: in 500mlDMEM liquid, add 50ml foetal calf serum, 100X Antibiotic-Antimycotic, 100X NEAA, 100ulHEPES, the 7.5%(quality percentage composition through the degerming of 0.22um membrane filtration of preparing with the laboratory afterwards) NaHCO ₃The pH value of regulator solution is to 7.0-7.2, and aforesaid liquid is GIBCO company product); 3ml) suspension cell, each antigen hole adds the 40ul cell suspension, puts into incubator behind the cover lid and continues to cultivate 8 hours;

3) get Sheng PBS solution (0.01mol/L pH=7.2) in the beaker, the antigen sheet after the absorption is slowly put into solution, soak about 1min, take out to be placed in the worktable and dries, need approximately 30min;

4) the antigen sheet is put into the beaker that fills acetone (Chemical Reagent Co., Ltd., Sinopharm Group's analytical pure concentration 99.5%) ,-20 ℃ of 30min or spend the night;

5) be stored in low temperature (below the 40 ℃) refrigerator for subsequent use after taking-up is dried.

Indirect immunofluorescence: be positioned over 37 ℃ of incubators behind the collection mouse whole blood and hatch 30min, behind the centrifugal 15min of 800rpm, draw supernatant and obtain serum.Afterwards serum is diluted the laggard immunofluorescence experiment that connects in the ranks according to requirement of experiment, concrete experimental procedure is as follows:

1) take out the antigen sheet from refrigerator, whether the cell above the visual inspection comes off, if the hole of coming off is arranged, abandoning need not;

2) in absorption the standardized circle on every side of drawing a circle of antigen is arranged with wax crayon at the antigen sheet, prevent that institute from adding the sample overflow;

3) experimentally need application of sample behind the serum that dilution detects, every hole 20-25ul generally need to do multiple hole, so need 50ul, does as required doubling dilution;

First concentration so need to add 10ul serum+90ul antibody diluent, fully mixing after is being taken out 50ul 1:10 diluent+50ul serum dilution (1:20) on the eddy mixer according to 1:10, and dilution is gone down according to this, generally is diluted to 1:160;

4) the antigen sheet of application of sample is put into the lunch box that is covered with plastic plate, put into 37 ℃, 5%CO ₂Hatch 30min in the incubator;

5) take out the antigen sheet, put into tank for washing plate, add washings (0.005M PBS+0.02% (volumn concentration) Tween 20) to the inside, wash 5min at horizontal oscillator tube, repeat 3 times;

6) after the antigen sheet is fully air-dry, add the fluorescence antibody 25ul/ hole of 1:50 dilution, put into behind the lunch box at 37 ℃, 5%CO ₂Hatch 30min in the incubator;

The dilution of fluorescence antibody: take out an amount of fluorescence antibody for serum source adding, dilute antibody with 0.02% (quality percentage composition) Yi Wensilan solution according to 1:50, this tests employed antibody is that FITC-conjugate-GoatAnti-Human IgG is available from Sigma company.

7) repeating step 5);

8) after air-dry, drip sterilized water in fluorescence microscopy Microscopic observation fluorescence situation and record the result.

Utilize indirect immunofluorescence assay to survey, the result is presented at first immunisation 2 all rear mice serum antibody horizontals and begins to raise, and tiring reaches to 1:80, is down to lower level after 3 weeks; Serum titer is 1:30.Behind the booster immunization, antibody horizontal raises rapidly again, and rising Amplitude Ratio first immunisation is whole wants high.Add exempt from after 1 Zhou Dazhi reach 1:160, and continue to about 4 weeks, and control group vAd-GFP and PBS do not detect specific antibody all the time.

2, CD in the flow cytometer showed lymphocyte ₄ ⁺, CD ₈ ⁺Number

CD in the flow cytometer showed lymphocyte ₄ ⁺, CD ₈ ⁺Number, the result is as follows:

In the immune mouse, CD after 1 week of vAd-E2E3 immunity ₄ ⁺Lymphocyte returns to normal level and accounts for 5% after accounting for 8%, 2 week of total lymphocyte, behind the booster immunization, and vAd-E2E3 immune group CD ₄ ⁺Lymphocyte begins continue to raise, and 5% rises to 25% by what account for total lymphocyte, and PBS control group and vAd-GFP group CD ₄ ⁺Lymphocyte accounts for 5% of total lymphocyte always, without significantly changing.

In the immune group mouse, vAd-E2E3 reaches to the peak CD after 1 week of immunity ₈ ⁺Lymphocyte accounts for 17% of total lymphocyte, returns to subsequently normal level and accounts for 2%, 3 all CD after head exempts from ₈ ⁺Lymphocyte quantity again raises and accounts for 3% of total lymphocyte; Behind the booster immunization, vAd-E2E3 immune group CD ₈ ⁺Lymphocyte begins continue to raise, and after reaching 12%, 3 week that accounts for total lymphocyte to peak value after immune 2 weeks, drop to normal level and slowly rise to again higher level after 2%, 4 week and account for 8%, and PBS control group and vAd-GFP group CD ₈ ⁺Lymphocyte accounts for 7% of total lymphocyte always.

Two, utilize enzyme linked immunological spot test (ELISPOT) that the cytokine founder cell is carried out quantitatively

Booster immunization is after 4 weeks, 4 mouse of every group of random choose, the aseptic spleen of taking immediately after the execution, adding 10ml contains the RPMI-1640 (RPMI-1640 that contains 10% foetal calf serum: add 50ml foetal calf serum, 100X Antibiotic-Antimycotic, 100X NEAA, 100ul HEPES in 500ml RPMIMEDIUM1640 liquid, the 7.5%NaHCO through filtration sterilization that disposes with the laboratory afterwards of 10% foetal calf serum ₃The pH value of (mass percent) regulator solution is 7.0-7.2; Above-mentioned RPMI MEDIUM1640, foetal calf serum, 100XAntibiotic-Antimycotic, 100X NEAA, HEPES are GIBCO company product; RPMI MEDIUM1640GIBCO company product, LOT:8109086), the spleen tissue is made cell suspension by 200 order copper mesh, collect in the aseptic centrifuge tube, the centrifugal 5min of 15000rpm/min, abandon after the supernatant liquor with 1640 substratum centrifuge washing cells twice, at last splenocyte is diluted to 2 * 10 ⁷Individual/ml, it is for subsequent use to make cell suspension.With the Eastern equine encephalitis virus of cell cultures as special stimulating factor (with 2 * 10 ⁷Individual/ml splenocyte 10 ⁸The Eastern equine encephalitis virus of pfu mixes, jointly hatched 48 hours, operation according to the ELISPOT experiment, splenocyte and special stimulating factor will join respectively in the hole, afterwards at the hole internal reaction, add in it after can not outside the hole, mixing), utilize enzyme linked immunological spot test (ELISPOT) that the cytokine founder cell is carried out quantitatively.

Concrete experimental procedure:

1.Coating antibody (coated antibody): carry out under the aseptic condition

With Coating buffer (1 * PBS pH7.2) dilution capture antibody (200 times), final concentration 5ug/ml be coated with plank, and every hole adds 4 ℃ of capture antibody that 100ul dilutes and spends the night.

2.Blocking(sealing): carry out under the aseptic condition

Blot coating buffer in the hole, wash plank, every hole adds 200ul Blocking solution, and hole flushing once.(buckleing dried at sterilization thieving paper)

3.Cell Activation (irritation cell): carry out under the aseptic condition

3.1 abandon Blocking solution, prepare to stimulate the antigen of usefulness, with organizing perfect medium dilution suitable concentration 50ug/ml, every hole adds 100ul; (not clappers)

3.2 prepare 1 * 10 ⁶Individual splenocyte suspension, every hole adds 100ul; (annotating: avoid vibrations)

3.3 cover lid, plank is placed 37 ℃, 5%CO2, and the incubator of 99% humidity was cultivated 48 hours according to the cell situation.(annotating: avoid collision constantly)

4.Detection antibody(detects antibody):

4.1 under aseptic condition, suck cell suspension, wash 2 times with deionized water (ice-cold deionized water, Low Osmotic Method lysing cell), every all over soaking 3-5min, following experimental implementation can be taken Bechtop and carry out outward;

4.2 every hole is washed 3 times with 200ul wash buffer I, abandons wash buffer I (patting dry after last time)

4.3 dilution Detection antibody is with dilution buffer (1 * PBS+10%FBS) 250 times of dilution, final concentration 2ug/ml.Every hole adds 100ul;

4.4 cover lid, incubated at room temperature 2 hours.

5. add enzyme and connect antibody:

5.1 abandon the Detection antibody-solutions, every hole is washed 3 times with 200ul wash buffer I.(last is all over patting dry);

5.2 with dilution buffer dilution Streptavidin-HRP(with 100 times of the dilutions of dilution buffer(1 * PBS+10%FBS).Matching while using is finished and unnecessary abandons it) every hole adds 100ul;

5.3 cover lid, incubated at room temperature 1 hour.

6. the result detects:

6.1 abandon Streptavidin-HRP solution, every hole is washed 4 times with 200ul wash buffer I;

6.2 every hole is washed (last is all over patting dry) 2 times with 200ul wash buffer II;

6.3 add substrate A EC, every hole adds 100ul(and notes lucifuge).The control maculiform is into about needs 15min;

6.4 stop substrate reactions with deionized water (washing 2 times);

6.5 room temperature lucifuge air blow drying plank 2 hours-spend the night, absolutely dry to plank, the plank that keeps in Dark Place is to be detected.Annotate: plank is not put in the baking box, prevents the film embrittlement, break.）

The result of result: ELISPOT determines by the number that last plank goes out spot, see that by naked eyes the vAd-E2E3 immune group goes out the spot number and obviously increases, explanation is after the stimulation of Eastern equine encephalitis virus, vAd-E2E3 immune group mouse boosting cell secretion of gamma-IFN activity obviously increases, vAd-QE immune group mouse boosting cell secretion of gamma-IFN activity is 4 times that contrast vAd-GFP organizes after the antigenic stimulation, is 11 times (adding up multiple according to going out the spot number) of PBS control group.

Three, attack the poison experiment

1, morbidity and survival condition

Booster immunization was put to death rear remaining mouse for every group of vAd-GFP control group, vAd-E2E3 immune group and PBS control group and is attacked the poison experiment in the BSL-3 laboratory after 4 weeks, was one group with 8 mouse again, was divided into two groups:

VAd-GFP control group A: 215 LD of abdominal injection ₅₀(mld) Eastern equine encephalitis virus

VAd-GFP control group B: 21 LD of abdominal injection ₅₀Eastern equine encephalitis virus

VAd-E2E3 immune group A: 215 LD of abdominal injection ₅₀(mld) Eastern equine encephalitis virus

VAd-E2E3 immune group B: 21 LD of abdominal injection ₅₀Eastern equine encephalitis virus

PBS control group A: 215 LD of abdominal injection ₅₀(mld) Eastern equine encephalitis virus

PBS control group B: 21 LD of abdominal injection ₅₀Eastern equine encephalitis virus

Continue 4 weeks to observe mouse invasion and survival condition.

The result is booster immunization after 4 weeks, and after the Eastern equine encephalitis virus of 215 LD50 was attacked, the protection efficient of vAd-E2E3 immune group A was that 0(8 mouse is all dead); When 21 LD50 Eastern equine encephalitis virus were attacked, the protection ratio of vAd-E2E3 immune group B only was 6 of 25%(8 dead mouses); All unprotect effects of control group (vAd-GFP control group A, vAd-GFP control group B, PBS control group A, PBS control group B) (8 mouse are all dead).Protection ratio is the quantity of last survival mice is attacked the front mouse of poison with it quantity ratio.

2, utilize the bhk cell individual layer to measure the mice serum neutralizing antibody

Collect the above-mentioned serum of respectively organizing mouse of attacking behind the poison, (Invitrogen company product, Cat.No.R760-07) individual layer is measured the mice serum neutralizing antibody to utilize bhk cell.After first immune serum suitably being diluted, put in 56 ℃ of water-baths and process 30min, destroy wherein complement and other heat labile non-specific toxicity factors, then with the collocation method of the cell maintenance medium of maintenance medium (containing the 2%(volume percent) foetal calf serum: in 500ml DMEM liquid, add 10ml foetal calf serum, 100X Antibiotic-Antimycotic, 100X NEAA, 100ul HEPES, afterwards with the 7.5%NaHCO through filtration sterilization of laboratory configuration ₃The pH value of (mass percent) regulator solution is 7.0-7.2; Above-mentioned DMEM, foetal calf serum, 100X Antibiotic-Antimycotic, 100X NEAA, HEPES are GIBCO company product) in aseptic centrifuge tube, carry out 2 times of serial dilutions.Get 100 TCID ₅₀The different dilution serum with equal-volume of Eastern equine encephalitis virus virus liquid, fully rearmounted 37 ℃ of mixing is hatched 1h, centrifuge tube mixing liquid 1-2 time turns upside down in the centre, take out 96 orifice plates after 1 hour, bhk cell in it has been paved with individual layer, suck the substratum on the cell monolayer, the virus of adding effect-antibody mixed solution (is above-mentioned mixing liquid of hatching, please examine), (positive control is the Eastern equine encephalitis virus virus liquid of 100 TCID50 for diluting with cell maintenance medium to establish simultaneously the positive and negative control hole on the culture plate, negative control is that blank is cell maintenance medium with the serum of the different concns of cell maintenance medium dilution).Put into 37 ℃ of incubators that contain 5%CO2 and cultivate, observe and record cytopathic situation every day.

The neutralization experiment detects serum titer, concrete grammar is: by the pathology situation (cell expands and becomes the circle gathering during pathology) of microscope observing cell, be designated as when hole inner cell pathology reaches more than 90% ++ ++, cytopathy reaches more than 75% and is designated as +++, cytopathy reaches more than 50% and is designated as ++, cytopathy reach more than 25% be designated as+, acellular pathology is designated as-, calculate at last the neutralization index of serum with the Reed-Muench method.

The result: vAd-E2E3 immune group A and vAd-E2E3 immune group B mice serum are tired and are 1:80 before the virus inoculation, after attacking malicious 4 weeks, the serum titer of the vAd-E2E3 immune group B survival mice after the Eastern equine encephalitis virus of 21 LD50 of inoculation is attacked is 1:300, and the survival mice serum titer of the vAd-E2E3 immune group A after 215 LD50 Eastern equine encephalitis virus are attacked then is 1:30.And control group (vAd-GFP control group A, vAd-GFP control group B, PBS control group A, PBS control group B) is all without the generation of specific antibody.

The result shows that constructed recombinant adenovirus vAd-E2E3 has produced better humoral immune reaction, provides important Data support for further carrying out the immunoprotection experiment.

Claims (4)

1. the recombinant vectors, transgenic cell line, recombinant bacterium or the expression cassette that contain the encoding gene of albumen;

Described albumen is following 1) or 2) albumen:

1) albumen that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;

2) with the aminoacid sequence of sequence 2 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have identical function by 1) albumen of deriving.

2. recombinant vectors according to claim 1, transgenic cell line, recombinant bacterium or expression cassette, it is characterized in that: described encoding gene is following a1), a2) a3) or a4) shown in gene:

A1) dna molecular shown in the sequence 1 in the sequence table;

A2) in the sequence table sequence 1 from the dna molecular shown in 5 ' the terminal 1-1589 position Nucleotide;

A3) under stringent condition with a1) or the dna molecule hybridize that a2) limits and the dna molecular with identical function;

A4) and a1) or a2) dna sequence dna that limits has at least 90% homology and has the dna molecular of identical function.

3. recombinant vectors according to claim 1 and 2 is characterized in that: described recombinant vectors is that pAdeasy-1 and recombinant shuttle plasmid obtain through homologous recombination; Described recombinant shuttle plasmid is that the encoding gene described in the claim 2 is inserted into the plasmid that obtains between the multiple clone site of carrier pShuttle-CMV.

4. vaccine, its activeconstituents be following any one:

A) albumen;

B) encoding gene of described albumen;

Described albumen is following 1) or 2) albumen:

1) albumen that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;

2) with the aminoacid sequence of sequence 2 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have identical function by 1) albumen of deriving;

The encoding gene of described albumen is following a1), a2) a3) or a4) shown in gene:

A1) dna molecular shown in the sequence 1 in the sequence table;

A2) in the sequence table sequence 1 from the dna molecular shown in 5 ' the terminal 1-1589 position Nucleotide;

A3) under stringent condition with a1) or the dna molecule hybridize that a2) limits and the dna molecular with identical function;

A4) and a1) or a2) dna sequence dna that limits has at least 90% homology and has the dna molecular of identical function.

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