CN102676583A - Recombinant adenovirus high expression vector for promoting effective presentation of hantavirus fusion protein G1S0.7 - Google Patents

Abstract

The invention relates to a ubiquitin Ub-containing recombinant adenovirus high expression vector for promoting effective presentation of a hantavirus fusion protein G1S0.7. Primarily, G1S0.7 mosaic genes of hantavirus-containing 76-118 strains and a G1S0.7-pCAG transfer vector of a CAG (Cytotoxin Associated Gene) promoter/enhancer are constructed for promoting effective presentation of highly expressed hantavirus fusion protein G1S0.7 by an organism. Compared with the recombinant adenovirus, partial humoral immunity response level and partial cell immunity response level of an animal organism can be effectively improved.

Description

Be used to the recombinant adenovirus high-expression vector that promotes that Hantaan virus fusion rotein G1S0.7 effectively offers

Technical field

The invention belongs to biological technical field, relate to association areas such as molecular biology, immunology and immunity application.The present invention relates to the reconstruction of adenovirus recombinant transfer vector and to hemorrhagic fever with renal syndrome (HFRS) the former a kind of recombinant adenovirus high-expression vector that promotes that Hantaan virus fusion rotein G1S0.7 effectively offers of being used to that causes a disease.

Background technology

Hemorrhagic fever with renal syndrome and recombinant vaccine present Research thereof:

Hemorrhagic fever with renal syndrome (hemorrhagic fever with renal syndrome; HFRS) be by Hantaan virus (Hantavirus; HV) cause that a kind of acute viral transmissible disease of being propagated by muroid etc. is a principal character with heating, hemorrhage and acute renal infringement clinically.China is the most serious country of HFRS epidemic situation in the world, has characteristics such as popular scope is wide, number of the infected is many, case fatality rate height, up to the present still lacks special efficacious therapy medicine clinically.

For Susceptible population's vaccination is the most effectively prevention and control measure of the extensive diffusion of infection prevention property disease.Therefore, the research to the HFRS vaccine is the focus in this field always.Though developed both at home and abroad the inactivated vaccine of HFRS in recent years, from the situation that the part crowd tries out, also there is obvious deficiency in such vaccine, mainly be a little less than it induces ability that body produces neutralizing antibody, irritation cell immunne response effectively.At present in the fundamental research of HFRS recombinant vaccine main use be virus envelope gp (Glycoprotein, GP), but the GP immunogenicity relatively a little less than, the antibody that stimulates body to produce occurs later, titre is also not high.The inventor is for many years to immunogenicity is the strongest among the HV nucleoprotein (Nucleocapsid Protein; NP) structure and function; The structure and the expression thereof of NP and GP different fragments mosaic gene (G1S0.7, G2S0.7), and the ability of fusion genes (albumen) stimulation immune response and influence factor thereof etc. have been carried out comparatively deep research.The inside and outside experimental result of a series of bodies shows that above-mentioned mosaic gene can stimulate the humoral immunization (comprising neutralizing antibody) of body to reply and cellullar immunologic response effectively, and its effect all is higher than non-chimeric group in the gland virus expression system.

Yet we have also found some problems under study for action, although as utilizing various systems all can give expression to complete fusion rotein, generally speaking expressing quantity still is on the low side.In addition, though all can stimulate body to produce body fluid and cellullar immunologic response with each expression system behind the fusion protein immunization animal, and the effect of gland virus expression system is better than other system, and the immune level of integral body is also not very good.Therefore, further select suitable expression vector, optimization expression system that the research of present HFRS recombinant vaccine is had great significance.

The applicant reconstructs the recombinant adenovirus transfer vector G1S0.7-pShuttle that contains mosaic gene G1S0.7 early stage; Its CMV promotor is replaced with CAG promotor/enhanser; Obtain containing the recombinant adenovirus transfer vector of the Hantaan virus fusion rotein G1S0.7 of CAG promotor/enhanser; Called after G1S0.7-pCAG is integrated among the adenovirus carrier DNA, obtains recombinant adenovirus rAd-G1S0.7-pCAG.Through with the recombinant adenovirus rAd-G1S0.7 that the does not replace promotor expression level of fusion rotein G1S0.7 relatively, the result shows that this recombinant adenovirus can effectively improve the expression of G1S0.7.

On this basis, the present invention is connected to the Ub gene on this transfer vector, utilizes recombinant gene that recombinant fragment is integrated among the adenovirus carrier DNA, obtains the recombinant adenovirus high-expression vector that can promote that Hantaan virus fusion rotein G1S0.7 effectively offers.

Optimize adenovirus expression carrier, promote body to offer antigenic:

Promote that vaccine component effectively offering in vivo is one of the approach that improves the efficient of HFRS recombinant vaccine, and Ub has outstanding advantage aspect angtigen presentation.Uiquitin-protease combined enzyme agent path is the main path of cell endogenous protein degraded, also is simultaneously the main path that endogenous antigen is processed, handled and present.Ubiquitin Ub is a kind of protein molecule of being made up of 76 amino acid, its C end for glycocoll (Glycine, Gly).After the substrate protein that will degrade is identified; Methionin (Lysine on ubiquitin end position Gly and the target protein; Lys) residue covalent attachment; On the 46th Lys of a part, form poly ubiquitin-substrate protein mixture before the Gly of other ubiquitin molecule is attached in order again, this mixture is finally degraded by proteasome.Have and discover that Ub and plasmid-encoded virus antigen transcribe jointly, translate; Can improve degraded in this antigenic cell; Making more, polypeptide segment gets into MHC-I class angtigen presentation approach; Increase the epitope peptide that gets into the MHC-I approach, thereby induce the CTL of intensive MHC-I restriction and the cell immune response of Thl class cell response.In addition; Research can prevent that fusion rotein is degraded to antigen and Ub monomer after also finding to change omega-Gly into Ala, and the antigen of this ubiquitinization can be degraded apace; Improved the probability that it is presented by the identification of MHC-I quasi-molecule, thereby it is active and to the protection efficient of mouse to have improved CTL greatly.

Summary of the invention

The objective of the invention is: a kind of recombinant adenovirus high-expression vector of promoting that Hantaan virus fusion rotein G1S0.7 effectively offers of being used to is provided, and it can effectively improve the part HI level and the cellullar immunologic response level of body.

Technical scheme of the present invention is: be designed for the recombinant adenovirus high-expression vector that promotes that Hantaan virus fusion rotein G1S0.7 effectively offers; Its preparation method is that the recombinant adenovirus transfer vector G1S0.7-pCAG that contains mosaic gene G1S0.7 is reconstructed; Ubiquitin antigen presentation molecular gene is connected on this carrier, and its complete genome sequence is shown in SEQ ID NO:1.

The present invention seeks to realize in the following manner: the G1S0.7 mosaic gene that contains Hantaan virus 76-118 strain that the applicant is made up in earlier stage and the recombinant adenovirus transfer vector G1S0.7-pCAG of CAG promotor/enhanser reconstruct; Ub antigen presentation molecular gene is connected on this carrier; And be integrated among the adenovirus carrier DNA, to obtain a kind of recombinant adenovirus high-expression vector of promoting that Hantaan virus fusion rotein G1S0.7 effectively offers of being used to.

Recombinant adenovirus transfer vector pCAG-G1S0.7 to containing mosaic gene G1S0.7 reconstructs; The Ub gene is connected on this carrier; The recombinant adenovirus transfer vector that obtains reconstructing, called after Ub-G1S0.7-pCAG, its complete genome sequence are shown in the SEQ ID NO:1.To recombinant transfer vector and adenovirus carrier double digestion, recombinant fragment Ub-G1S0.7-pCAG is integrated among the adenovirus carrier DNA, obtained recombinant adenoviral vector rAd-Ub-G1S0.7-pCAG.

The present invention utilizes recombinant gene; Mosaic gene and the CAG promotor/enhanser recombinant adenovirus transfer vector pCAG-G1S0.7 of the G1S0.7 that contains Hantaan virus 76-118 strain that the applicant is made up in earlier stage reconstruct; And recombinant fragment is integrated among the adenovirus carrier DNA, in the hope of obtaining a kind of recombinant adenovirus high-expression vector of promoting that Hantaan virus fusion rotein G1S0.7 effectively offers of being used to.With rAd-Ub-G1S0.7-pCAG of the present invention and do not integrate the recombinant adenovirus rAd-G1S0.7-pCAG of Ub gene early stage and the recombinant adenovirus rAd-G1S0.7 that does not replace the CAG promotor distinguishes the immunization experiment mouse; And carrying out the research of immunological characteristic, the result finds that rAd-Ub-G1S0.7-pCAG of the present invention can effectively improve the part HI level and the cellullar immunologic response level of body.

Description of drawings

Below in conjunction with the embodiment accompanying drawing the present invention is done into explanation down:

The pcr amplification result of Fig. 1 .Ub gene.Wherein: L:Ub; M:DL2000DNA maker;

Fig. 2 .Ub-S0.7-pCAG Nhe I and Sfi I double digestion qualification result.Wherein: L:Ub-S0.7-pCAG; M1:DNA wide range maker; M2:DL2000DNA maker;

Fig. 3. the PCR of recombinant dna identifies.Wherein: L1:Ub-G1S0.7-pCAG; M:200bp maker;

Fig. 4. the PCR of recombinant adenovirus identifies.Wherein: L1:Ub-G1S0.7-pCAG; M:200bpmaker;

The CPE phenomenon of Fig. 5 .HEK293 cell.A wherein: normal control; B: transfection is after 6 days;

The immunofluorescence detected result of Fig. 6 .Ub-G1S0.7-pCAG expression product.A wherein: detect the expression of NP; B: detect the expression of Ub; C:HEK 293 negative controls;

Fig. 7. each organizes the detected result of immune mouse cytokine IFN-γ;

Fig. 8. each organizes the detected result of immune mouse cytokine IL-2;

Fig. 9. contain the recombinant adenovirus immune mouse CTL detected result of G1S0.7 mosaic gene.

Embodiment

The used gland virus expression system of the present invention is available from the Adeno-X of CLONTECH company ^TMSystem (article No. K1650-1), its transfer vector is pShuttle.

The recombinant adenovirus transfer vector G1S0.7-pCAG that is adopted among the present invention is made up by the applicant in earlier stage.(seeing number of patent application 200910254561.3)

The recombinant adenovirus rAd-G1S0.7-pCAG that is adopted among the present invention, rAd-G1S0.7 by the applicant pack in earlier stage, purifying.

The two valency HFRS inactivated vaccines that adopted among the present invention are available from Zhejiang Tianyuan company.

Inserting the Ub gene order among the present invention is with Ub up primer, Ub down primer upstream and downstream primer, is template with pCMV-F3Ub, is that annealing temperature is carried out pcr amplification with 55 ℃, shown in accompanying drawing 1.The PCR product is connected for 14 ℃ with the T-easy carrier and spends the night, connect product electricity Transformed E .coli JM109 after coating contain Amp ⁺The agar plate of resistance is cultivated 12hr for 37 ℃, and select single bacterium colony and increase bacterium, be that primer carries out PCR and identifies that positive colony called after T easy-Ub send Jin Sirui biotechnology company to check order with Ub up primer, Ub down primer behind the upgrading grain.

The research contents of recombinant adenoviral vector construction process and recombinant adenovirus immunological characteristic is specific as follows among the present invention:

1. the structure of recombinant transfer vector Ub-G1S0.7-pCAG: adopt Sfi I and Nhe I double digestion S0.7-pCAG-pShuttle and T easy-Ub plasmid, adopt Takara glue to reclaim test kit recovery purpose fragment and carrier.Fragment is connected for 14 ℃ through the T4 ligase enzyme with carrier spends the night, and connects product electricity Transformed E .coli JM109, and coating contains Kana ⁺2 * YT agar plate of resistance is placed on 37 ℃ and cultivates 12hr.Select single bacterium colony and increase bacterium, enzyme is cut evaluation behind the upgrading grain, obtains positive colony, called after Ub-S0.7-pCAG.Adopt Nhe I, Not I double digestion Ub-S0.7-pCAG, obtain the Ub-pCAG skeleton.Adopt Xba I, Not I double digestion G1S0.7, obtain the G1S0.7 fragment.Fragment is connected for 14 ℃ with carrier spends the night Transformed E .coli JM109.Extract plasmid after picking list bacterium colony and the enlarged culturing, laggard through pcr amplification to the product row agarose gel electrophoresis analysis of advancing.The positive colony called after Ub-G1S0.7-pCAG (the Ub gene is positioned at the upper reaches of G1S0.7 mosaic gene) that obtains.Accompanying drawing 2. is Ub-S0.7-pCAG-pShuttle positive colony Nhe I and Sfi I double digestion qualification result.

2. the structure of recombinant adenoviral vector rAd-Ub-G1S0.7-pCAG, evaluation, packing and single spot purifying: recombinant transfer vector that above-mentioned structure is successful and adenoviral plasmid are used PI-Sce I and I-Ceu I double digestion respectively; Glue reclaims purpose fragment and carrier; 14 ℃ of connections of T4 ligase enzyme are spent the night, and connect the coating of product conversion (JM109 competence) back and contain Amp ⁺2 * YT solid medium, 37 ℃ of incubators are cultivated 12～14hr, enzyme is cut evaluation after selecting single bacterium colony and shaking bacterium upgrading grain, obtains positive colony.Accompanying drawing 3. is the PCR qualification result of rAd-Ub-G1S0.7-pCAG recombinant adenoviral vector.Recombinant adenoviral vector DNA is cut with linearization plasmid with Pac I enzyme list, and transfection HEK293 cell is waited to occur obvious CPE (Cytopathic Effect, cytopathy) back and is received cell, shown in accompanying drawing 5.After centrifugal deposition is resuspended in autoclaved PBS, centrifugal collection supernatant after multigelation should precipitate 3 times promptly obtains recombinant adenovirus.Accompanying drawing 4 is the PCR qualification result of rAd-Ub-G1S0.7-pCAG.Adopt plaque method purification of Recombinant adenovirus ,-80 ℃ of preservations.

3.rAd-Ub-G1S0.7-pCAG the detected result that fusion rotein G1S0.7 expresses: (concrete grammar is referring to CLONTECH Adeno-X to measure the recombinant adenovirus titre ^TMRapid Titer Kit, Cat.No.631028).Infect 293 cells with the MOI (multiplicity of infection, infection multiplicity) of 100pfu/cell, infect rAd-G1S0.7 recombinant adenovirus that the applicant packs in earlier stage simultaneously as contrast, the proteic expression of immunofluorescence testing goal behind the 48hr.

3.1NP the detection of expressing

24hr is with 1 * 10 before infecting ⁵The density of cells/well is laid on 24 orifice plates with the HEK293 cell, and the MOI with 100pfu/cell infected the HEK293 cell in second day, was replaced by fresh 10%S-DMEM nutrient solution behind the 4hr, every hole 500 μ L.Cell is placed CO ₂Cultivate 48hr in the incubator, make cell density reach 75%, use cold methanol-20 ℃ fixed cell 20min afterwards, PBS softly washes 3 times.The mAb-1A8 that adds dilution in 1: 1000 is (by the applicant's preparation and preservation; Can discern Hantaan virus specific antigen site, have higher combination activity), hatch 2hr for 37 ℃; PBS softly washes 3 times; The sheep anti mouse two that adds with the FITC mark of the blue liquid of 0.2 ‰ she Wens dilution in 1: 100 resists, and hatches 1h in 37 ℃, and PBS softly washes 3 times.Keep in Dark Place observations under the fluorescence inverted microscope.

3.2Ub the detection of expressing

Culturing cell and infective virus step such as preceding in 24 orifice plates; With 1: the 200 anti-Ub antibody of dilution commercialization specific anti, hatch 1hr for 37 ℃, it is anti-that PBS softly washes the goat-anti rabbit two that adds the FITC mark that dilutes at 1: 100 with the blue liquid of 0.2 ‰ she Wens after 3 times; Hatch 1hr in 37 ℃; PBS softly washes 3 times, keeps in Dark Place observations under the fluorescence inverted microscope.Accompanying drawing 6 is respectively the immunofluorescence detected result of fusion rotein S0.7 and Ub among the rAd-Ub-G1S0.7-pCAG.Wherein A is a S0.7 immunofluorescence detected result, and B is a Ub immunofluorescence detected result, and C is a HEK293 cell negative control.

4. recombinant adenovirus immunity C57BL/6 mouse: with each group recombinant adenovirus through behind propagation, purifying and titer determination, with 10 ⁸Pfu/0.5ml/ is in abdominal injection is gone into the mouse body (the female mouse of 4 week C57BL/6 mouse in age, 5 every group, available from experimentation on animals center, this school) only, at interval 2 all immunity once, totally three times; Normal mouse group (4 week C57BL/6 mouse in age female mouse, 5 every group, available from experimentation on animals center, this school) intraperitoneal injection of saline 0.5ml/ only, the Adenovirus group (the female mouse of 4 week C57BL/6 mouse in age, 5 every group, available from experimentation on animals center, this school; Adenovirus contains the lacZ gene available from Clotech company, through detecting the expression of beta-galactosidase enzymes, can be used as the adenovirus negative control) abdominal injection adenovirus 10 ⁸Pfu/ml/, inactivated vaccine group abdominal injection vaccine 10 μ l/, 2 weeks were injected once totally three times at interval.

5. the detected result of recombinant adenovirus immune serum specific antibody: at immune back 10 days for the third time each group immune mouse is plucked eyeball and get blood and separation of serum.Encapsulate HTNV NP 10 μ g/ml, 4 ℃ are spent the night; Each group immune serum is done 1: 10～1: 640 doubling dilution, and the vaccine control group is diluted to 1: 640, hatches 1hr for 37 ℃; The anti-mouse two of HRP-that each hole, washing back adds dilution in 1: 20000 resists, and hatches 1hr for 37 ℃; The washing back adds the colour developing of OPD substrate solution, and lucifuge adds stop buffer after placing 15min under the room temperature, measures the A490 value in each hole.Subordinate list 1 is each group immune serum specific antibody detected result.

6. the detected result of recombinant adenovirus immune serum neutralizing antibody: at immune back 10 days for the third time each group immune mouse is plucked eyeball and get blood and separation of serum.Infect and inoculate the Vero-E6 cell in 96 orifice plates previous day, make it grow up to individual layer.0.22 μ m filter filters the serum of respectively organizing immune mouse in advance; 1 * the PBS that crosses with high pressure then does 1: 5～1: 160 doubling dilution; Positive control mAb-3G1 (can discern NP/G2 and go up the specific antigen site; Have the active and blood clotting inhibition activity of higher neutralization) did doubling dilution since 1: 1000, negative control Sp2/0 ascites is done dilution in 1: 1000.With 100TCID ₅₀HTNV respectively with each experimental group serum and the abundant mixing of control sample, 37 ℃ of 5%CO ₂Hatch 1hr.Each porocyte supernatant of 96 orifice plates is exhausted, add the mixed liquid 100 μ l/ holes of each papova and serum (or ascites) respectively, do 4 multiple holes, set up virus control group and normal cell control group simultaneously.37 ℃ of 5%CO ₂After hatching 90min, exhaust each hole supernatant, add the complete RPIM-1640 nutrient solution that 200 μ l/ holes contain 2% calf serum, 37 ℃ of 5%CO ₂Hatched every nutrient solution that changed a time at a distance from 3 days 9 days.96 orifice plates are put into-80 ℃ of multigelations 3 times, detect the virus antigen in the freeze thawing liquid with the ELISA sandwich assay, concrete steps are following:

1: 1000 dilution mAb 1A8 and ascites Sp2/0,4 ℃ encapsulate and spend the night

↓

Add 100 μ l/ porocyte freeze thawing supernatants, hatch 1hr for 37 ℃

↓

Add the HRP-1A8 antibody of dilution in 1: 2000, hatch 1hr for 37 ℃

↓

Add 100 μ l/ hole OPD substrate solution colour developings, leave standstill 15min

↓

Add 50 μ l/ hole 2M H ₂SO ₄Stop buffer

↓

Measure each hole A ₄₉₀Value

The result judges: the P value is each immune group A ₄₉₀Value, the negative control group A of N ₄₉₀Value, positive with P/N value>=2.1, the serum greatest dilution that can suppress 50% cell infection is a NAT.Subordinate list 2 is each group immune serum NAT detected result.

7. the ELISPOT detected result of recombinant adenovirus immune mouse secrete cytokines:

7.1 immune mouse SPL preparation

Plucked eyeball execution at immune back 10 days for the third time and respectively organize mouse, aseptic separating spleen obtains splenocyte suspension.Add Gey ' s solution cracking red corpuscle wherein, centrifugal collection SPL adds incomplete RPIM-1640 washing 2 times, is resuspended in after centrifugal to contain in the complete RPIM-1640 nutrient solution of 2% calf serum, counts subsequent use.

7.2ELISPOT detect immune mouse cytokine IFN-γ, IL-2

Put to death mouse previous day; Encapsulate IL-2 check-out console (IFN-γ is for encapsulate check-out console in advance); Specific procedure is following: every hole adds 50 μ l/ holes, 70% ethanol under the aseptic condition, leaves standstill to abandon behind the 2min to the greatest extent, adds 200 μ l/ hole aseptic deionized waters washings 5 times; 100 μ l/ holes add specificity dilution antibody, and 4 ℃ encapsulate and spend the night.After aseptic 1 * PBS washing 5 times, 200 μ l/ holes add the complete RPIM-1640 nutrient solution room temperature sealing 30min that contains the 10%-calf serum, and after aseptic 1 * PBS washing 5 times, it is subsequent use to encapsulate completion.The above-mentioned experiment mice splenocyte of respectively organizing is diluted to 1 * 10 ⁷Cell/ml concentration, 100 μ l/ holes add a cytokines measurement plate, and add 10mg/ml HTNVNP as stimulator, and each sample is done 3 multiple holes.Add PHA (phytohemagglutinin, phytohaemagglutinin) in the positive control hole, final concentration is 4 μ g/ml, and negative control hole does not add stimulator, and the background control wells only adds substratum.37 ℃ of 5%CO ₂Hatch 18hr, aseptic 1 * PBS washs the mAb that adds the anti-mouse cytokine of biotinylation after 5 times, incubated at room 2hr; Aseptic 1 * PBS washs the HR labelled streptavidin that adds dilution in 1: 1000 after 5 times, and incubated at room 1hr adds tmb substrate colour developing liquid after high pressure 1 * PBS washs 5 times; Lucifuge leaves standstill, treat that spot occurs after, the tap water flushing; Lucifuge is dried in the shade, and measures the spot number with the ELISPOT readout instrument.Accompanying drawing 7 be each recombinant adenovirus cytokine IFN-γ detected result ( ^*P＜0.05, ^*P＜0.01).Accompanying drawing 8 be recombinant adenovirus cytokine IL-2 detected result ( ^*P＜0.05, ^*P＜0.01).

8. recombinant adenovirus immune mouse CTL cell killing laboratory test results:

8.1 effector cell's preparation

Pluck eyeball execution in back 10 days the 3rd immunity and respectively organize mouse; Aseptic separating spleen obtains splenocyte suspension (the same), is placed on to contain in the complete RIPM-1640 nutrient solution of 5% calf serum, and adding final concentration is the HTNV GP of 10 μ g/ml; The IL-2 of 25U/ml, 37 ℃ of 5%CO ₂, as CTL cell killing experimental effect cell.

8.2 the preparation of target cell

Carry a few days ago and infect the B16 cell as CTL cell killing experimental test target cell with recombinant adenovirus rAd-G1S0.7.

8.3CTL cell killing experiment

Counting effector cell and target cell are compared 50 μ l (2 * 10 according to 20: 1,50: 1,100: 1 effect targets ⁶/ hole, 1 * 10 ⁶/ hole, 4 * 10 ⁵/ hole) effector cell and 50 μ l (2 * 10 ⁴/ hole) target cell adds in 96 orifice plates, and 3 multiple holes are set, and with the normal mice splenocyte as negative control.Fabric swatch is established each item control wells (subordinate list 3 shows each control wells preparation program), detects as follows afterwards: 37 ℃ of 5%CO ₂Behind the incubated cell 4h, the centrifugal 5min of 250 * g room temperature, the maximum release aperture of centrifugal preceding 40min target cell adds 10 μ l/ hole lysates; Centrifugal back every hole sucking-off 50 μ l cell conditioned mediums add 50 μ l/ hole substrate solutions to new 96 orifice plates, and the room temperature lucifuge leaves standstill 30min; Add 50 μ l/ hole stop buffers, measure A ₄₉₀Value is calculated killing activity.Accompanying drawing 9 is a recombinant adenovirus immune mouse CTL detected result, is 50: 1 o'clock imitating the target ratio, and the killing activity of rAd-Ub-G1S0.7-pCAG group mouse boosting cell will be higher than other test group and control group (P＜0.05).

Table 1. is respectively organized immune serum specific antibody detected result

Table 2. is respectively organized immune serum NAT detected result

Each control wells preparation program of table 3.CTL fragmentation test

Sequence table

< 110>The Fourth Military Medical University of P.L.A

< 120>be used to the recombinant adenovirus high-expression vector that promotes that Hantaan virus fusion rotein G1S0.7 effectively offers

<160>1

<210>1

<211>7975

<212>DNA

<213>Artificial

<400>1

taactataac?ggtcctaagg?tagcgaaagc?tcagatctgg?atctcccgat?cccctatggt?60

cgactctcag?tacaatctgc?tctgatgccg?catagttaag?ccagtatctg?ctccctgctt?120

gtgtgttgga?ggtcgctgag?tagtgcgcga?gcaaaattta?agctacaaca?aggcaaggct?180

tgaccgacaa?ttgtcgacat?tgattattga?ctagttatta?atagtaatca?attacggggt?240

cattagttca?tagcccatat?atggagttcc?gcgttacata?acttacggta?aatggcccgc?300

ctggctgacc?gcccaacgac?ccccgcccat?tgacgtcaat?aatgacgtat?gttcccatag?360

taacgccaat?agggactttc?cattgacgtc?aatgggtgga?ctatttacgg?taaactgccc?420

acttggcagt?acatcaagtg?tatcatatgc?caagtacgcc?ccctattgac?gtcaatgacg?480

gtaaatggcc?cgcctggcat?tatgcccagt?acatgacctt?atgggacttt?cctacttggc?540

agtacatcta?cgtattagtc?atcgctatta?ccatgggtcg?aggtgagccc?cacgttctgc?600

ttcactctcc?ccatctcccc?cccctcccca?cccccaattt?tgtatttatt?tattttttaa?660

ttattttgtg?cagcgatggg?ggcggggggg?gggggggcgc?gcgccaggcg?gggcggggcg?720

gggcgagggg?cggggcgggg?cgaggcggag?aggtgcggcg?gcagccaatc?agagcggcgc?780

gctccgaaag?tttcctttta?tggcgaggcg?gcggcggcgg?cggccctata?aaaagcgaag?840

cgcgcggcgg?gcgggagtcg?ctgcgttgcc?ttcgccccgt?gccccgctcc?gcgccgcctc?900

gcgccgcccg?ccccggctct?gactgaccgc?gttactccca?caggtgagcg?ggcgggacgg?960

cccttctcct?ccgggctgta?attagcgctt?ggtttaatga?cggctcgttt?cttttctgtg?1020

gctgcgtgaa?agccttaaag?ggctccggga?gggccctttg?tgcggggggg?agcggctcgg?1080

ggggtgcgtg?cgtgtgtgtg?tgcgtgggga?gcgccgcgtg?cggcccgcgc?tgcccggcgg?1140

ctgtgagcgc?tgcgggcgcg?gcgcggggct?ttgtgcgctc?cgcgtgtgcg?cgaggggagc 1200

gcggccgggg?gcggtgcccc?gcggtgcggg?ggggctgcga?ggggaacaaa?ggctgcgtgc 1260

ggggtgtgtg?cgtggggggg?tgagcagggg?gtgtgggcgc?ggcggtcggg?ctgtaacccc 1320

cccctgcacc?cccctccccg?agttgctgag?cacggcccgg?cttcgggtgc?ggggctccgt 1380

gcggggcgtg?gcgcggggct?cgccgtgccg?ggcggggggt?ggcggcaggt?gggggtgccg 1440

ggcggggcgg?ggccgcctcg?ggccggggag?ggctcggggg?aggggcgcgg?cggccccgga 1500

gcgccggcgg?ctgtcgaggc?gcggcgagcc?gcagccattg?ccttttatgg?taatcgtgcg 1560

agagggcgca?gggacttcct?ttgtcccaaa?tctggcggag?ccgaaatctg?ggaggcgccg 1620

ccgcaccccc?tctagcgggc?gcgggcgaag?cggtgcggcg?ccggcaggaa?ggaaatgggc 1680

ggggagggcc?ttcgtgcgtc?gccgcgccgc?cgtccccttc?tccatctcca?gcctcggggc 1740

tgccgcaggg?ggacggctgc?cttcgggggg?gacggggcag?ggcggggttc?ggcttctggc 1800

gtgtgaccgg?cggctctaga?gcctctgcta?accatgttca?tgccttcttc?tttttcctac 1860

agctcctggg?caacgtgctg?gttgttgtgc?tgtctcatca?ttttggcaaa?gaattggcct 1920

cgacggccat?gcagattttc?gtgaagaccc?tgacaggcaa?gaccatcacc?ctggaggtcg 1980

agcccagtga?caccatagag?aatgtcaagg?caaagatcca?ggacaaggag?ggcatccccc 2040

ctgaccagca?gaggctgatc?tttgcaggca?agcagctgga?agatggccgc?accctgtcag 2100

actacaacat?ccagaaagag?tccaccctgc?acctggtcct?tcgcctgaga?ggtgcagatc 2160

agatcttaag?taagtaggct?agcgtttaaa?cgggccctct?agaatgggga?tatggaagtg 2220

gctagtgatg?gccagtttag?tatggcctgt?tttgacactg?agaaatgtct?atgacatgaa 2280

aattgagtgc?ccccatacag?taagttttgg?ggaaaacagt?gtgataggtt?atgtagaatt 2340

accccccgtg?ccattggccg?acacagcaca?gatggtgcct?gagagttctt?gtagcatgga 2400

taatcaccaa?tcgttgaata?caataacaaa?acatacccaa?gtaagttgga?gaggaaaggc 2460

tgatcagtca?cagtctagtc?aaaattcatt?tgagacagtg?tccactgaag?ttgacttgaa 2520

aggaacatgt?gttctaaaac?acaaaatggt?ggaagaatca?taccgtagta?ggaaatcagt 2580

aacctgttac?gacctgtctt?gcaatagcac?ttactgcaag?ccaacactat?acatgattgt 2640

accaattcat?gcatgcaata?tgatgaaaag?ctgtttgatt?gcattgggac?catacagagt 2700

acaggtggtt?tatgagagaa?cttactgtat?gacaggagtc?ctgattgaag?ggaaatgctt 2760

tgtcccagat?caaagtgtgg?tcagtattat?caagcatggg?atctttgata?ttgcaagtgt 2820

tcatattgta?tgtttctttg?ttgcagttaa?agggaatact?tataaaattt?ttgaacaggt 2880

taagaaatcc?tttgaatcaa?catgcaatga?tacagagaat?aaagtgcaag?gatattatat 2940

ttgtattgta?gggggaaact?ctgcaccaat?atatgttcca?acacttgatg?atttcagatc 3000

catggaagca?tttacaggaa?tcttcagatc?accacatggg?gaagatcatg?atctggctgg 3060

agaagaaatt?gcatcttatt?ctatagtcgg?acctgccaat?gcaaaagttc?ctcatagtgc 3120

tagctcagat?acattgagct?tgattgccta?ttcaggtata?ccatcttatt?cttcccttag 3180

catcctaaca?agttcaacag?aagctaagca?tgtattcagc?cctgggttgt?tcccaaaact 3240

taatcacaca?aattgtgata?aaagtgccat?accactcata?tggactggga?tgattgattt 3300

acctggatac?tacgaagctg?tccacccttg?tacagttttt?tgcgtattat?caggtcctgg 3360

ggcatcatgt?gaagcctttt?ctgaaggcgg?gattttcaac?ataacctctc?ccatgtgctt 3420

agtgtcaaaa?caaaatcgat?tccggttaac?agaacagcaa?gtgaattttg?tgtgtcagcg 3480

agtggacatg?gacattgttg?tgtactgcaa?cgggcagagg?aaagtaatat?taacaaaaac 3540

tctagttatt?ggacagtgta?tatatactat?aacaagctta?ttctcattac?tacctggagt 3600

agcacattct?attgctgttg?aattgtgtgt?acctgggttc?catggttggg?ccacagctgc 3660

tctgcttgtt?acattctgtt?tcggatgggt?tcttatacca?gcaattacat?ttatcatact 3720

aacagtccta?aagttcattg?ctaatatttt?tcacacaagt?aatcaagaga?ataggctaaa 3780

atcagtactt?agaaagataa?aggaagagtt?tgaaaaaaca?aaaggctcaa?tggtatgtga 3840

tgtctgcaag?tatgagtgtg?aaacctataa?agaattaaag?gcacacgggg?tatcatgccc 3900

ccaatctcaa?tgtccttact?gttttactca?ttgtgaaccc?acagaagcag?cattccaagc 3960

tcattacaag?gtatgccaag?ttactcacag?attcagggat?gatctaaagg?tcgacatggc 4020

aactatggag?gaattacaga?gggaaatcaa?tgcccatgag?ggtcaattag?tgatagccag 4080

gcagaaggtg?agggatgcag?aaaaacagta?tgaaaaggat?ccagatgagt?tgaacaagag 4140

aacattaact?gaccgagagg?gcgttgcagt?atctatccag?gcaaaaattg?atgagttaaa 4200

aaggcaactg?gcagatagga?ttgcaactgg?gaaaaacctt?gggaaggaac?aagatccaac 4260

aggggtggag?cctggagacc?atctgaaaga?gaggtcaatg?ctcagttatg?gtaatgtgct 4320

ggatttaaac?catttggata?ttgatgaacc?tacaggacag?acagcagact?ggctgagcat 4380

catcgtctat?cttacatcct?ttgtcgtccc?gatacttctg?aaagctctgt?atatgttgac 4440

aacaaggggg?aggcaaacta?ccaaggataa?taaagggacc?cggattcgat?ttaaggatga 4500

tagctcgttc?gaggatgtta?acggtatccg?gaaaccaaaa?catctttacg?tgtccttgcc 4560

aaatgcacag?tcaagcatga?aggcagaaga?gattacacct?ggtagatata?gaacagcagt 4620

ctgtgggctc?taccctgcac?agattaaggc?acggcagatg?atcagtccag?ttatgagtgt 4680

aattggtttt?ctagcattag?caaaggactg?gagtgatcgt?atcgaacaat?ggttaattga 4740

accttgcaag?cttcttccag?atacagcagc?agttcatcat?catcatcatc?atgcggccgc 4800

cactgtgctg?gatgatccga?gctcggtacc?aagcttaagt?ttaaaccgct?gatcagcctc 4860

gactgtgcct?tctagttgcc?agccatctgt?tgtttgcccc?tcccccgtgc?cttccttgac 4920

cctggaaggt?gccactccca?ctgtcctttc?ctaataaaat?gaggaaattg?catcgcattg 4980

tctgagtagg?tgtcattcta?ttctgggggg?tggggtgggg?caggacagca?agggggagga 5040

ttgggaagac?aatagcaggc?atgctgggga?tgcggtgggc?tctatggctt?ctgaggcgga 5100

aagaaccagc?agatctgcag?atctgaattc?atctatgtcg?ggtgcggaga?aagaggtaat 5160

gaaatggcat?tatgggtatt?atgggtctgc?attaatgaat?cggccaacgc?gcggggagag 5220

gcggtttgcg?tattgggcgc?tcttccgctt?cctcgctcac?tgactcgctg?cgctcggtcg 5280

ttcggctgcg?gcgagcggta?tcagctcact?caaaggcggt?aatacggtta?tccacagaat 5340

caggggataa?cgcaggaaag?aacatgtgag?caaaaggcca?gcaaaaggcc?aggaaccgta 5400

aaaaggccgc?gttgctggcg?tttttccata?ggctccgccc?ccctgacgag?catcacaaaa 5460

atcgacgctc?aagtcagagg?tggcgaaacc?cgacaggact?ataaagatac?caggcgtttc 5520

cccctggaag?ctccctcgtg?cgctctcctg?ttccgaccct?gccgcttacc?ggatacctgt 5580

ccgcctttct?cccttcggga?agcgtggcgc?tttctcaatg?ctcacgctgt?aggtatctca 5640

gttcggtgta?ggtcgttcgc?tccaagctgg?gctgtgtgca?cgaacccccc?gttcagcccg 5700

accgctgcgc?cttatccggt?aactatcgtc?ttgagtccaa?cccggtaaga?cacgacttat 5760

cgccactggc?agcagccact?ggtaacagga?ttagcagagc?gaggtatgta?ggcggtgcta 5820

cagagttctt?gaagtggtgg?cctaactacg?gctacactag?aaggacagta?tttggtatct 5880

gcgctctgct?gaagccagtt?accttcggaa?aaagagttgg?tagctcttga?tccggcaaac 5940

aaaccaccgc?tggtagcggt?ggtttttttg?tttgcaagca?gcagattacg?cgcagaaaaa 6000

aaggatctca?agaagatcct?ttgatctttt?ctacggggtc?tgacgctcag?tggaacgaaa 6060

actcacgtta?agggattttg?gtcatgagat?tatcaaaaag?gatcttcacc?tagatccttt 6120

tgatcctccg?gcgttcagcc?tgtgccacag?ccgacaggat?ggtgaccacc?atttgcccca 6180

tatcaccgtc?ggtactgatc?ccgtcgtcaa?taaaccgaac?cgctacaccc?tgagcatcaa 6240

actcttttat?cagttggatc?atgtcggcgg?tgtcgcggcc?aagacggtcg?agcttcttca 6300

ccagaatgac?atcaccttcc?tccaccttca?tcctcagcaa?atccagccct?tcccgatctg 6360

ttgaactgcc?ggatgccttg?tcggtaaaga?tgcggttagc?ttttacccct?gcatctttga 6420

gcgctgaggt?ctgcctcgtg?aagaaggtgt?tgctgactca?taccaggcct?gaatcgcccc 6480

atcatccagc?cagaaagtga?gggagccacg?gttgatgaga?gctttgttgt?aggtggacca 6540

gttggtgatt?ttgaactttt?gctttgccac?ggaacggtct?gcgttgtcgg?gaagatgcgt 6600

gatctgatcc?ttcaactcag?caaaagttcg?atttattcaa?caaagccgcc?gtcccgtcaa 6660

gtcagcgtaa?tgctctgcca?gtgttacaac?caattaacca?attctgatta?gaaaaactca 6720

tcgagcatca?aatgaaactg?caatttattc?atatcaggat?tatcaatacc?atatttttga 6780

aaaagccgtt?tctgtaatga?aggagaaaac?tcaccgaggc?agttccatag?gatggcaaga 6840

tcctggtatc?ggtctgcgat?tccgactcgt?ccaacatcaa?tacaacctat?taatttcccc 6900

tcgtcaaaaa?taaggttatc?aagtgagaaa?tcaccatgag?tgacgactga?atccggtgag 6960

aatggcaaaa?gcttatgcat?ttctttccag?acttgttcaa?caggccagcc?attacgctcg 7020

tcatcaaaat?cactcgcatc?aaccaaaccg?ttattcattc?gtgattgcgc?ctgagcgaga 7080

cgaaatacgc?gatcgctgtt?aaaaggacaa?ttacaaacag?gaatcgaatg?caaccggcgc 7140

aggaacactg?ccagcgcatc?aacaatattt?tcacctgaat?caggatattc?ttctaatacc 7200

tggaatgctg?ttttcccggg?gatcgcagtg?gtgagtaacc?atgcatcatc?aggagtacgg 7260

ataaaatgct?tgatggtcgg?aagaggcata?aattccgtca?gccagtttag?tctgaccatc 7320

tcatctgtaa?catcattggc?aacgctacct?ttgccatgtt?tcagaaacaa?ctctggcgca 7380

tcgggcttcc?catacaatcg?atagattgtc?gcacctgatt?gcccgacatt?atcgcgagcc 7440

catttatacc?catataaatc?agcatccatg?ttggaattta?atcgcggcct?cgagcaagac 7500

gtttcccgtt?gaatatggct?cataacaccc?cttgtattac?tgtttatgta?agcagacagt 7560

tttattgttc?atgatgatat?atttttatct?tgtgcaatgt?aacatcagag?attttgagac 7620

acaacgtggc?tttgttgaat?aaatcgaact?tttgctgagt?tgaaggatca?gatcacgcat 7680

cttcccgaca?acgcagaccg?ttccgtggca?aagcaaaagt?tcaaaatcac?caactggtcc 7740

acctacaaca?aagctctcat?caaccgtggc?tccctcactt?tctggctgga?tgatggggcg 7800

attcaggcct?ggtatgagtc?agcaacacct?tcttcacgag?gcagacctca?gcgctagatt 7860

attgaagcat?ttatcagggt?tattgtctca?tgagcggata?catatttgaa?tgtatttaga 7920

aaaataaaca?aataggggtt?ccgcgcacat?ttccccgaaa?agtgccacct?gacgt 7975

Claims (1)

1. be used to the recombinant adenovirus high-expression vector that promotes that Hantaan virus fusion rotein G1S0.7 effectively offers; Its preparation method is: the recombinant adenovirus transfer vector G1S0.7-pCAG to containing mosaic gene G1S0.7 reconstructs; Ubiquitin antigen presentation molecular gene is connected on this carrier, and its complete genome sequence is shown in SEQ ID NO:1.

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