CN108514635A - Recombinate trivalent adenovirus vaccine and its construction method - Google Patents

Abstract

The invention discloses a kind of recombination trivalent adenovirus vaccine and its construction methods.The recombination trivalent adenovirus vaccine is based on the human 3-type adenovirus that the areas E3 lack, corresponding coding segment in human 3-type adenovirus is replaced with the coding segment of the neutralizing epitope amino acid of the six adjacent body hypervariable regions of 7 type adenovirus of people, and the hexon full-length gene of 55 type adenovirus of someone is recombined into the areas E3 of original missing.The recombination trivalent adenovirus vaccine can generate anti-AD3, AD7, AD55 virucidin compared with high titre with inducing mouse; in vivo in mouse immune protest test; the vaccine can significantly protect mouse to be total to three-type-person's adenovirus by serious AD3, AD7, AD55 to be infected; it is substantially reduced even fully erased lung virus carrying capacity, illustrates that the vaccine candidate strain has trivalent vaccine protection feature.Since the trivalent vaccine Candidate Strain is single recombined adhenovirus, immunogenicity is strong, no In vivo recombination risk.

Description

Recombinate trivalent adenovirus vaccine and its construction method

Technical field

The present invention relates to molecular biology fields, more particularly, to a kind of recombination trivalent adenovirus vaccine and its structure side Method.

Background technology

Adenovirus (Adenovirus, Ad) earliest by Hilleman M.R. et al. in 1954 from operation excision it is acute Found in breathing problem infant body of gland and tonsillotome, it is separated so far go out 52 kinds of different serotypes human adenovirus, according to Different immunology, biology and biochemical characteristics can also be divided into 7 subgroups (A-G).Adenovirus often causes old man and children serious The major reason of acute respiratory infection and infantile diarrhea.(2009-2012) in recent years, according to Guangzhou respiratory tract infection Sick monitoring platform data are shown, (are less than 15 years old) in acute respiratory infection case in 4242 paediatrics, adenovirus infection is being exhaled Accounting 9% in the infection of road virus is inhaled, wherein accounting for 4.6% in the child less than two years old.3,7,14,55,11 type of B groups people and E groups 4 Type adenovirus is the important pathogen body of serious breathing problem such as acute respiratory infection and infant's fatal pneumonia, is infected Property it is strong, it is at home and abroad to have been reported that more.

The pathogenic popularity of adenovirus and the seriousness of harm make the prevention of adenovirus seem particularly significant.It is inoculated with adenopathy Malicious vaccine is to prevent one of adenovirus infection most efficient method.However current adenovirus vaccine production technology, it is mainly based upon Adenovirus inactivates or type is simply mixed in attenuated live vaccine, is easy to make vaccine genetic recombination occur in human body and generate new Virus then threatens human health, and genetic engineering subunit vaccine is due to the small peptide or virus single structure albumen of its expression It is difficult to effectively body be induced to generate the neutralizing antibody of enough high titres to protect people to resist viral infection again.Current people in the world 4 type of adenovirus vaccine someone, 7 type adenovirus vaccines, both vaccines are the oral enteric type vaccine of work, only in septic yanks Depot in use, application significantly reduces the incidence of U.S. army's heat generation respiratory tract infection.Since adenovirus is produced Raw neutralizing antibody can only neutralize the infection of homotype adenovirus, without cross-protection between different type adenovirus antibodies, because This, a kind of vaccine that can prevent a variety of high pathogenic adenovirus simultaneously of structure is that current clinical economics and research and production need most.

Invention content

Based on this, it is necessary to provide a kind of recombination trivalent adenovirus vaccine and its construction method.

A kind of construction method of recombination trivalent adenovirus vaccine, includes the following steps：

Step S1：The coding segment of the neutralizing epitope amino acid of 7 type adenovirus six adjacent body hypervariable regions of people is cloned into the areas E3 The six adjacent body hypervariable regions of the human 3-type adenovirus full-length genome carrier of missing obtain chimeric 7 types to replace corresponding coding segment The 3 type adenovirus vector of recombination of adenovirus epitope；

Step S2：It is recombined into 55 type of people in the former E3 zone positions of the 3 type adenovirus vector of recombination of chimeric 7 type adenovirus epitopes The hexon of adenovirus overall length coding segment to get.

In one of the embodiments, in the step S1, further include step S11：Compare 7 type adenovirus of people and 3 types The hexon amino acid sequence of adenovirus, and the 7 type adenovirus six adjacent body hypervariable regions of people are obtained according to the method for homologous modeling Neutralizing epitope amino acid sequence.

The neutralizing epitope amino acid of the 7 type adenovirus six adjacent body hypervariable regions of the people obtained in one of the embodiments, Sequence as shown in SEQ ID NO.11, corresponding coded slice in the human 3-type adenovirus full-length genome carrier being replaced The amino acid sequence of section coding is as shown in SEQ ID NO.9.

The sequence of coding segment corresponding with amino acid sequence shown in SEQ ID NO.11 in one of the embodiments, As shown in SEQ ID NO.12, the sequence such as SEQ ID of coding segment corresponding with amino acid sequence shown in SEQ ID NO.9 Shown in NO.10.

The coded sequence such as SEQ ID of the hexon of 55 type adenovirus of the overall length people in one of the embodiments, Shown in NO.40.

In one of the embodiments, in the step S1, further include following steps：

Step S12：By overlapping PCR method by the volume of the neutralizing epitope amino acid of 7 type adenovirus six adjacent body hypervariable regions of people Chip segment is cloned into human 3-type adenovirus six adjacent body hypervariable regions to replace corresponding coding segment, and after the complete recombination of acquisition Hexon gene；

Step S13：Hexon gene after the complete recombination is recombined into hexon shuttle vector, is obtained Recombinant shuttle plasmid carrier, the upstream homology arm containing hexon gene and downstream are homologous in the hexon shuttle vector Arm；

Step S14：Hexon gene in the recombinant shuttle plasmid carrier is recombined into the human 3-type of the areas the E3 missing To replace the hexon gene in original vector in adenovirus full-length genome carrier, 3 type of recombination of chimeric 7 type adenovirus epitopes is obtained Adenovirus vector.

The sequence of the primer used during the over-lap PCR of the step S12 in one of the embodiments, is respectively such as SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, shown in SEQ ID NO.29 and SEQ ID NO.30, acquisition The sequence of hexon gene after complete recombination is as shown in SEQ ID NO.41.

It is further comprising the steps of in one of the embodiments, in the step S2：

Step S21：Expand the hexon full-length gene of 55 type adenovirus of people；

Step S22：The hexon full-length gene of the 55 type adenovirus of people of amplification is recombined into the areas E3 shuttle vector, Obtain the areas recombination E3 shuttle vector；

Step S23：By the hexon full-length gene weight of the 55 type adenovirus of people in the areas recombination E3 shuttle vector Group enters 3 areas type adenovirus vector Yuan E3 of recombination of the chimeric 7 type adenovirus epitopes, obtains recombination trivalent adenovirus vaccine and carries Body；

Step S24：By the recombination trivalent Recombinant Adenoviral Vaccine Vector transfectional cell, obtained using rescue recombinant virus technology Recombinate trivalent adenovirus vaccine.

It is with shown in SEQ ID NO.38 and SEQ ID NO.39 in one of the embodiments, in the step S21 Sequence fragment be primer, using 55 type adenovirus of people as template amplification hexon full-length gene.

The weight that a kind of construction method using the recombination trivalent adenovirus vaccine described in any of the above-described embodiment is built Group trivalent adenovirus vaccine, the recombination trivalent adenovirus vaccine is based on the human 3-type adenovirus that the areas E3 lack, with 7 type of people The coding segment of the neutralizing epitope amino acid of the six adjacent body hypervariable regions of adenovirus replaces corresponding coded slice in human 3-type adenovirus Section, and recombinate the hexon full-length gene of 55 type adenovirus of someone in the areas E3 of original missing.

The present invention pass through the study found that mixed gland virus vaccine polyvalent have may recombinate and generate novel naturally in vivo The potential danger of virus, and the single six adjacent body hypervariable regions of recombined adhenovirus are limited for the position of modification, excessive change six Adjacent body structural proteins will influence adenovirus packaging.The recombination that the construction method of above-mentioned recombination trivalent adenovirus vaccine is built Trivalent adenovirus vaccine can generate anti-AD3, AD7, AD55 virucidin compared with high titre with inducing mouse, in vivo mouse In Immunization protection test, which can significantly protect mouse to be total to three-type-person's adenovirus by serious AD3, AD7, AD55 Infection, hence it is evident that reduce even fully erased lung virus carrying capacity, illustrate the vaccine candidate strain have trivalent vaccine protection feature. The neutralizing epitope small peptide non-immunogenicity of the traditional areas recombined adhenovirus E3 expression, and the present invention is aobvious by immunoblot experiment Show, the hexon of adenovirus AD55 is wrapped into the areas E3 of recombined adhenovirus, has immunogenicity.Due to trivalent vaccine candidate Strain is single recombined adhenovirus, and immunogenicity is strong, no In vivo recombination risk.

Description of the drawings

Fig. 1 is the construction strategy schematic diagram of recombined adhenovirus (MH1, MH2, MH5 and MH7).

Fig. 2 is the DNA replication dna growth curve of recombined adhenovirus MH1, MH2, MH5, MH7 and rAd Δ E3GFP.

Fig. 3 be recombined adhenovirus MH1, MH2, MH5, MH7 antiserum in intersecting between rAd Δs E3GFP, HAdV-7 With reaction titer determination, compareed using the mouse resisting anteserum of rAd Δs E3GFP and HAdV-7 as positive or negative.

The areas Tu4Wei E3 shuttle vector pSKcmv-egfp collection of illustrative plates.

Fig. 5 is the DNA replication dna growth song for recombinating trivalent adenovirus MH5 Δs E3-Ad55h and three type adenovirus HAdV-3 of people Line, MH5 Δ E3-Ad55h and HAdV-3 is in infection cell 12, harvest after 24,36,48,72 and 96 hours, using Q-PCR reagents Box detects gene copy number.

Fig. 6 is to recombinate trivalent adenovirus MH5 Δ E3-Ad55h antiserums to survey the neutralization titer of Adv3, Adv7, Adv55 etc. It is fixed.

Fig. 7 is to recombinate trivalent adenovirus MH5 Δs E3-Ad55h as the mouse immune plan of vaccine candidate strain and each Antibody titer variation after immune, wherein A. block arrows indicate that immunization time point, thin arrow indicate mouse blood sampling time point；B.0-8 It is immunized in week after front and back serum collection and potency is detected with ELISA.

Fig. 8 be the pre- immune recombination trivalent adenovirus MH5 Δs E3-Ad55h of BaLB/c mouse after attack malicious HAdV-3, HAdV-7 and HAdV-55 Protections, wherein A. experimental designs；B. malicious HAdV-3, mouse lung tissue are attacked after pre- immune MH5 Δs E3-Ad55h Virus load changes；C. malicious HAdV-7, the variation of mouse lung tissue virus load are attacked after pre- immune MH5 Δs E3-Ad55h；D. exempt from advance Malicious HAdV-55, the variation of mouse lung tissue virus load are attacked after epidemic disease MH5 Δs E3-Ad55h.

Fig. 9 be Western blot detect recombined adhenovirus MH5 Δ E3-Ad55h antiserums respectively with HAdV55, MH5 Δ The reaction of E3-Ad55h and MH5 hexons and HAdV55 epitope polypeptide R7 polyvalent antibodies and MH5 Δ E3-Ad55h hexons Antigen-antibody reaction, wherein A.HAdV-55 (1 swimming lane), MH5 Δs E3-Ad55h (2 swimming lane) and MH5 (3 swimming lane) are with SDS-PAGE Electrophoretic separation, then with western blot technologies respectively with anti-MH5 Δs E3-Ad55h seroreactions；B.HAdV-55 (1 swimming lane), MH5 Δs E3-Ad55h (2 swimming lane) and MH5 (3 swimming lane) are separated by electrophoresis with SDS-PAGE, then with western blot technologies respectively with Anti- ad55-R7 seroreactions.

Specific implementation mode

To facilitate the understanding of the present invention, below with reference to relevant drawings to invention is more fully described.In attached drawing Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes The embodiment of description.Keep the understanding to the disclosure more thorough on the contrary, purpose of providing these embodiments is Comprehensively.

Unless otherwise defined, all of technologies and scientific terms used here by the article and belong to the technical field of the present invention The normally understood meaning of technical staff is identical.Used term is intended merely to description tool in the description of the invention herein The purpose of the embodiment of body, it is not intended that in the limitation present invention.

The present invention is with the human 3-type adenovirus skeleton plasmid pBRAd3 Δs E3GFP (people of the areas the E3 missing containing green fluorescent protein 3 type adenovirus full-length genome plasmid vectors, full-length genome HAdv3-gz01, Construction and characterization of a replication–competent human adenovirus type3-based vector as a live-waccine candidate and a viral delivery vector,Qiwei Zhang, Xiaobo Su,Donald Seto,Bo jian Zheng,Xingui Tian,Huiying Sheng,Haitao Li, Youshao Wang,Rong Zhou,Vaccine,2009,27(8):Based on 1145-53), by comparing various adenovirus hominis Hexon amino acid sequence and method according to homologous modeling predict human 3-type and 7 type adenovirus six adjacent body hypervariable regions respectively The sequence of neutralizing epitope amino acid, then by 7 type adenovirus six adjacent body hypervariable regions of people (hypervariable regions, HVRs) The coded sequence of sequence HVR1, HVR2, HVR5 and HVR7 of prediction neutralizing epitope amino acid clone replace human 3-type gland respectively The correspondence six adjacent body hypervariable regions sequence of viral backbone plasmid pBRAd3 Δs E3GFP obtains the recombination for being fitted into 7 type adenovirus epitope of people Human 3-type adenovirus MH1, MH2, MH5, MH7, process refer to Fig. 1.In serum and test and the clear MH5 energy of results of animal Simultaneously generate the anti-human 3-type adenovirus of high-titer and anti-human 7 type adenovirus neutralizing antibody, using this bivalence recombinant adenovirus MH5 as Carrier is recombined into the coded sequence of 55 type of overall length adenovirus hominis (AdV5) hexon in the areas E3 of MH5, builds people 3,7,55 Type recombinates trivalent adenovirus vaccine Candidate Strain.

It is specific embodiment part below

1. being learnt by amino acid alignment and homologous modeling and forecasting, 7 type adenovirus (AD7) of human 3-type adenovirus (AD3) and people The sequence and its coded sequence of six adjacent body hypervariable regions HVR1, HVR2, HVR5 and HVR7 are as shown in table 1 below.

Table 1

2. the coded sequence of the neutralizing epitope amino acid of the six adjacent body hypervariable regions of 7 type adenovirus of people is substituted into human 3-type gland Viral vectors hexon corresponds to hypervariable region, and 7 type adenovirus epitope base sequence of people is introduced to 5 ' ends of primer by design primer, Carry out overlap PCR by saltation zone be spliced into complete hexon gene MHVR1, MHVR2, MHVR5 (SEQ ID NO.41) and MHVR7.Overlap PCR primers information such as the following table 2.

Table 2

Overlap PCR amplification detailed process is as follows：

Hexon base is amplified respectively using pBRAd Δs E3GFP as template with primer HexU and HVRnD, HVRnU and HexD The upper semisection and lower semisection of cause, PCR reaction systems see the table below 3.

Table 3

PCR response procedures are：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1.5min (1kb/min), 35 cycles；72 DEG C of extension 5min.

Gained PCR product is run into the purifying of agarose gel electrophoresis gel extraction, then corresponds to the progress of upstream and downstream segment Overlap PCR, which put up a bridge, to be expanded, and is first not added with upstream and downstream primer, PCR reaction systems see the table below 4.

Table 4

PCR response procedures are at this time：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1.5min (1kb/min), 10 cycles；72 DEG C of extension 5min.

Then hexon upstream and downstream primer HexU and HexD is added in above-mentioned PCR reaction systems again, reaction system is seen below Table 5.

Table 5

Response procedures are：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 2.5min (1kb/ Min), 25 cycles；72 DEG C of extension 10min.By bridging Overlap PCR product agarose gel electrophoresis, gel extraction is spare.

After 3. hexon gene PCR product is recovered, with ClaI+BamHI double digestions, while to hexon shuttle plasmid PBR22-L/R (contains the sequence upstream and downstream homology arm sequence Hexon as shown in SEQ ID NO.36 and SEQ ID NO.37 respectively The method of R and Hexon L, plasmid construction are shown in document Qiu, H., et al., Serotype-specific neutralizing antibody epitopes of human adenovirus type 3(HAdV-3)and HAdV-7reside in multiple hexon hypervariable regions.J Virol,2012.86(15):P.7964-75.) with same Enzymes double zyme cutting, respectively after gel extraction, connection conversion to Top10 Competent cells chooses single bacterium colony and carries out bacterium colony PCR Identification identifies that correctly clone send sequencing company sequencing identification, the shuttle plasmid built to be named as pBR22-L/R-mHexon (accordingly, and it is referred to as shuttle plasmid pBRL/R-MHVR1, pBRL/R-MHVR2, pBRL/R-MHVR5 and pBRL/R- MHVR7)。

4. with EcoRI+SalI double digestion shuttle plasmid pBRL/R-MHVR1, pBRL/R-MHVR2, pBRL/R-MHVR5 and PBRL/R-MHVR7, skeleton plasmid pBRAd3 Δs E3GFP are then with AvrII+PacI double digestions, and respectively after gel extraction, common-battery turns Into E. coli competent BJ5183 (stratagene companies), the LB plate screenings containing Amp are coated on, it is homologous in bacterium Recombination, obtains recombinant virus plasmid pBRAd Δs E3GFP-mHexon (pMH1, pMH2, pMH5, pMH7).PCR identifies monoclonal bacterium It falls, positive colony is extracted into plasmid after digestion identification and send the neutralizing epitope of 7 type adenovirus of sequencing company sequence verification people prediction Whether the coded sequence of amino acid is correctly cloned into the corresponding six adjacent body hypervariable regions of human 3-type adenovirus carrier.Through PCR, digestion and Positive colony after sequence verification is forwarded to 250mlLB fluid nutrient mediums and shakes bacterium, using taking out matter in QIAGEN companies endotoxin-free Grain extracts kit extracts plasmid.

5. will take out plasmid among the above discharges adenoviral gene group with AsisI linearization for enzyme restriction, and is transfected using lipo3000 Reagent transfects AD293 cells, and rescue recombinant virus obtains MH1, MH2, MH5, MH7.By dynamic to recombination adenoviral replication growth Force diagram measure it is found that the growth duplication characteristic of viral AD3E before recombined adhenovirus MH1, MH2, MH5, MH7 and transformation simultaneously Without change, Fig. 2 is seen.It is purified with CsCl density gradient centrifugations after mass propgation, measures OD260 and calculate virion number VPs/ml. Recombined adhenovirus MH1, MH2, MH5, MH7 are respectively with 1 × 10¹⁰SPF grades of Balb/c mouse are only immunized in VPs/, immune to take blood afterwards three times The anti-human 3-type adenovirus and anti-human 7 type adenovirus neutralization titer that its induction generates are measured clearly, see Fig. 3.Through comparing, recombinant adenovirus Malicious MH5 can generate the anti-human 3-type adenovirus of high-titer and anti-human 7 type adenovirus neutralizing antibody simultaneously, be made with this recombined adhenovirus MH5 To build trivalent vaccine alternate carrier.

It is as follows that recombinant virus saves detailed process：

Take recombinant adenovirus plasmid pBRAd Δs E3GFP-mHexon through AsiS I digestions, the extracting of phenol chloroform isoamyl alcohol, ethyl alcohol Precipitation recycling, is dissolved in aseptic deionized water, obtains recombination Ad3 Linearized genes group rAd Δ E3GFP-mHexon, ultraviolet spectrometry light Degree measurement OD260 is quantitative.

AD293 cells are after 0.25% pancreatin EDTA digestion, with containing 10%FBS, 100U/mL penicillin and 100mg/mL chains The DMEM culture mediums of mycin are diluted to cell density 1x10⁵Cells/mL takes 0.5ml to be uniformly layered in 24 porocyte culture plates, It is incubated overnight, waits for that cell growth is used as to transfect to 60-80%.

Take Ad3 Linearized genes group rAd Δs E3GFP-mHexon about 750ng per hole according to transfection reagent Lipofectamine^TM LTX and PLUS^TMThe operation of Reagent specifications is transfected, and the DMEM containing 2%FBS is changed after 6h Culture medium continues to cultivate.Due to carrying green fluorescence protein gene on adenovirus vector pBRAd Δs E3GFP, thus can directly lead to Cross fluorescence inverted microscope observation cell transfecting efficiency.Observe the change of AD293 cell fluorescences expression daily after transfecting 3~7d Change, if it is possible to successfully save out virus, then can be observed to occur fluorescence aggregation in cell and have typical cells lesion (CPE). If still failing to see apparent cell fluorescence clustering phenomena or CPE after 10d, cell and supernatant are collected, after freeze thawing 3 times 12000r/min centrifuges 2min, takes the fresh AD293 cells of supernatant superinfection and two generation of blind passage, during which continues whether to observe cell Occur expressing green fluorescence because infecting recombined adhenovirus.Transfection one is repeated if it can not see the fluorescence that intracellular accumulation increases It is secondary.

6. design primer AD55HexonF (SEQ ID NO.38：5’-GCTACCGGTCGCCACCATGGCCACCCCATCGA TGCTGCC-3 ') and AD55HexonR (SEQ ID NO.39：5 '-GCAGAATTCTTACGTGGTAGCGTTACCGGC-3 '), with Adenovirus hominis Ad55 (Strain Ad55-Shanxi-Y16) expands hexon full-length gene order (SEQ ID NO.40, method See below), with AgeI and EcoR I double digestions PCR products and the areas E3 shuttle vector pSKcmv-egfp (collection of illustrative plates is shown in Fig. 4), glue recycling Carrier and hexon gene are connected afterwards, replaces the GFP reporter genes of the former areas E3 shuttle vector pSKcmv-egfp, are obtained new Shuttle plasmid pSKcmv-AD55H.It is mono- with EcoRV and NotI double digestion shuttle plasmids pSKcmv-AD55H and RsrII again The above-mentioned candidate 3 of digestion, 7 type bivalent vaccine carrier pMH5, while by its electrotransformation to competence B5183, by homologous recombination, It is positive to screen PCR, correct monoclonal pMH5- Δs E3-Ad55H is identified in digestion identification and sequencing.Extract endotoxin-free matter Grain, and postgenome is discharged with AsiSI digestions, it is transfected into AD293 cells using lipo3000 transfection reagents, rescue recombination gland The same MH5- Δs E3-Ad55H of viral methods.The growth facsimile log indifference of recombined adhenovirus MH5- Δs E3-Ad55H and AD3E It is different, show improved vaccine candidate strain still duplication fertility having the same, sees Fig. 5.After mass propgation, CsCl is close Degree gradient centrifugation purification obtains a large amount of recombined adhenovirus MH5- Δ E3-Ad55H, measures virion number and TCID50.

The detailed mistake of hexon full-length gene order is expanded with adenovirus hominis Ad55 (Strain Ad55-Shanxi-Y16) Journey：Hexon full-length gene order is expanded with primer AD55HexonF and AD55HexonR, template is adenovirus hominis Ad55 (Ad55-Shanxi-Y16).Amplification program is as follows：98 DEG C of pre-degeneration 2min；98 DEG C of denaturation 10s, 55 DEG C of annealing 20s, 68 DEG C are prolonged Stretch 30s (1kb/10s), 35 cycles；68 DEG C of extension 5min.PCR reactions use TAKARA companies long segment high fidelity enzymeGXL DNA Polymerase。

7. candidate trivalent vaccine MH5- Δs E3-Ad55H attacks malicious protective effect.

Balb/c mouse are divided into 6 groups, every group 20, MH5- Δ E3-Ad55H blank groups are immunized in complete blank group, are immunized MH5- Δs E3-Ad55H attacks malicious AD3 groups, and immune MH5- Δs E3-Ad55H attacks malicious AD7 groups, and immune MH5- Δs E3-Ad55H attacks poison AD55 groups.Immune group mouse is immunized in four times, and immune amount is 1 × 10 every time¹⁰VPs/ only, attacks malicious group and attacks malicious virus quantity and be 10⁵TCID50.Experimental result shows that immune group mouse can generate high-titer anti-AD3, AD7, and compared in the anti-AD55 of high titre And antibody, see Fig. 6 and Fig. 7.Challenge viral dosage shows that trivalent vaccine MH5- Δs E3-Ad55H can make mouse generate protection antibody AD3, AD7 and AD55 infection are resisted, sees Fig. 8.

8. running PAGE electrophoresis to purify recombinant virus MH5- Δs E3-Ad55H, anti-AD7 and more grams of anti-AD55 mouse are used respectively Grand antibody is primary antibody, and sheep anti-mouse igg antibody is secondary antibody, detects recombinant virus surface antigenicity, the results show that in anti-AD7 and table Position is illustrated in recombinant virus hexon, and AD55 hexons also have small part to be wrapped into recombined adhenovirus MH5- Δs E3- Its new structural proteins is formed among the capsid protein of Ad55H, thus it is speculated that possible Ad55 and AD3, AD7 are B group adenovirus, and six Adjacent body homology is high so that the similar hexon of structure is assembled into same virus, sees Fig. 9.

Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, it is all considered to be the range of this specification record.

Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Sequence table

<110>The First Affiliated Hospital of Guangzhou Medical University

GIRD Pharmaceutical Technology Co., Ltd.

Guangdong Province South Mountain Medicine innovation research institute

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<213>Artificial sequence (Artificial Sequence)

<400> 22

tttatcagca taaattactg tgtcagttcc agaagttgaa tctattctaa taggcagc 58

<210> 23

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 23

cccatcgatg atgccccaat gggca 25

<210> 24

<211> 54

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 24

tctggaactg acacagtaat ttatgctgat aaaacatatc agccagagcc tcaa 54

<210> 25

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 25

ccgggatcca cctcaaaagt catgtccagc 30

<210> 26

<211> 55

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 26

atcgtagtta gcaacaatat ttttgccgtc gaaaaattcc atatcaatat ctggt 55

<210> 27

<211> 52

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 27

attttctgtg tacattacaa tatctggatc gtagttagca acaatatttt tg 52

<210> 28

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 28

cccatcgatg atgccccaat gggca 25

<210> 29

<211> 52

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 29

ccagatattg taatgtacac agaaaatgta aatttggaaa ctccagacag tc 52

<210> 30

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 30

ccgggatcca cctcaaaagt catgtccagc 30

<210> 31

<211> 46

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 31

agaacctgca tctgttttaa ctttaacgcc ttgatacctg tgccct 46

<210> 32

<211> 55

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 32

aactgtggtg tcatctttgt cccacttttc agaacctgca tctgttttaa cttta 55

<210> 33

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 33

cccatcgatg atgccccaat gggca 25

<210> 34

<211> 55

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 34

cttttcaccc tgtttctact gtggtgtcaa gttgatacag ctaatgaaat agcca 55

<210> 35

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 35

ccgggatcca cctcaaaagt catgtccagc 30

<210> 36

<211> 3435

<212> DNA

<213>3 type adenovirus (Adenovirus type 3)

<400> 36

catggatgag cccaccctgc tttatcttct tttcgaagtc ttcgacgtgg tcagagtgca 60

ccagccacac cgcggcgtca tcgaggccgt ctacctgcgc acaccgttct cggccggcaa 120

cgccaccaca taagaagcct cttgcttctt gcaagcagca gctgcagcca tgacgtgcgg 180

gtccggaaac ggctccagcg agcaagagct caaagccatc gtccgagacc tgggctgcgg 240

accctatttc ctgggaacct ttgacaagcg tttcccgggg tttatggccc ccgacaagct 300

cgcctgcgcc atagtcaaca ctgccggacg cgagacgggg ggagagcact ggctggcttt 360

tggttggaac ccgcgctaca acacctgcta cctttttgat ccttttgggt tctcggatga 420

gcggcttaaa cagatttacc agtttgagta cgaggggctc ctgcgtcgca gtgcccttgc 480

taccaaagac cgctgcatca ccctggaaaa gtccacccaa agcgtgcagg gtccgcgctc 540

agccgcctgt ggactttttt gctgtatgtt ccttcatgcc tttgtgcact ggcccgaccg 600

ccccatggac ggaaacccca ccatgaagtt gctgactggg gtgtccaaca gcatgctcca 660

atcaccccaa gtccagccca ccttgcgccg caaccaggag gtgctatacc gcttcctaaa 720

cacccactca tcttactttc gttctcaccg cgcgcgcatc gaaagggcca ccgcgtttga 780

ccgtatggat atgcaataag tcatgtaaaa ccgtgttcaa taaacagcac tttattttta 840

catgcactga ggctctggtt ttgctcattc gtttcatcat ttcacttaga agtcgaatgg 900

gttctggcgg gagtcagagt gacccgcggg cagggatacg ttgcggaact gtaacctgtt 960

ctgccacttg aactcgggga ttactagctt gggaactgga atttcgggaa aggtgtcttg 1020

ccacaacttt ctggtcagtt gcatagcgcc aagcaggtca ggagcagaga tcttgaaatc 1080

acagttgggg ccggcattct ggacacggga gttgcggtac actgggttgc aacactggaa 1140

caccatcaac gctgggtgtc ttacgcttgc caacacggtt gggtcactga tggtagtcac 1200

atccaagtct tcagcattgg ccatcccaaa gggggtcatc ttacatgtct gcctgcccat 1260

cacgggagcg cagcctggct tgtggttgca atcacaatga atggggatca gcatcatcct 1320

ggcttggttg ggagttatcc ctgggtacac agccttcatg aaggcttcgt actgcttaaa 1380

agcttcctgg gccttacttc cctcggtgta gaacatccca caggacttac tggaaaattg 1440

attagtagta cagttggcat cattcacaca gcagcgggca tcgttgttgg ccaactgaac 1500

cacatttctg ccccagcggt tttgggtgat cttggctctg tctggattct ccttcatagc 1560

gcgctgcccg ttctcgctcg ccacatccat ctcaataatg tggtccttct ggatcatgat 1620

agtgccatgc aggcatttca ccttgccttc ataatcggtg catccatgag cccacagagc 1680

gcacccggtg cactcccaat tattgtgggc gatctcggaa taataatgta ccaatccctg 1740

catgaatctt cccatcattg ttgtcaaggt cttcatgctg gtaaatgtca gcgggatgcc 1800

acggtgctcc tcgttcacat actggtggca gatacgcttg tattgctcgt gctgctctgg 1860

catcagcttg aaagaggttc tcagatcatt atccagcctg tacctttcca ttagcacagc 1920

catcacttcc atgcccttct cccaggcaga taccaggggc agactcaaag gattcctaac 1980

agcaataaaa gtagctcctt tagctatagg gtcattcttg tcgatcttct caacacttct 2040

tttgccatcc ttctcaatga tgcgcaccgg ggggtagctg aagcccacgg ccaccaactg 2100

agcctgttct ctttcttctt cactatcctg actgatgtct tgcagaggga catgcttggt 2160

cttcctgggc ttcttcttgg gagggatcgt gggaggactg ttgctccgct ccggagacag 2220

ggatgactgc gacgtttcgc ttaccaatac cacctggctc tcggtagaag aaccggaccc 2280

cacgcgacgg taggtgttcc tcttcggggg cagaggtgga ggcgactgag atgggctgcc 2340

tggccttgga ggcggatggc tggcagagcc cattccgcgt tcgggggtgt gctcccggtg 2400

gcggtcgctt gactgatttc ctccgcggct ggccattgtg ttctcctagg cagagaaaca 2460

acagacatgg aaactcagcc atcactgcca acatcgctac acctcgcccc cagcagcgac 2520

gaggaggaga gcttaaccac cccaccaccc agtcccgcta ccaccacctc tacccttgat 2580

gatgaggagg aggtcaacgc agcccaggag atgcaggcgc aggataatgt gaaaacggaa 2640

gagattgagg cagatgtcga gcaggacccg ggctatgtga cgccggcgga gcacgaggag 2700

gagctgaaac gctttctaga cagagaggat gacgaccgcc cagagcatca agcagatggc 2760

gtttaccagg aggctgggat cagggatcat gtcgccgact acctcaccgg ccttggtggg 2820

gaggacgtgc tcctcaaaca tctagcaagg cagtcgatca tagttaaaga cgcattgctc 2880

aatctcactg aagtgcccat cagtgtggaa gagctcagcc gcgcctacga gctgaacctc 2940

ttttcgcctc aggtaccccc caagcggcag ccaaacggca cctgcgaggc caaccctcga 3000

ctcaacttct atccagcttt tactatcccc gaagtgttgg ccacctacca catctttttt 3060

aagaaccaaa agattccagt ctcctgccgc gccaaccgca cccgcgccga tgccctgctc 3120

aacttgggtc cgggagctcg cttacctgat atagcttcct tggaagaggt tccaaagatc 3180

tttgagggtc tgggaagtga tgagacacgg gccgcaaatg ctctgcaaca gggagagaat 3240

gacatggatg aacatcacag cgctctggtg gaactggagg gtgacaatgc ccggattgca 3300

gtgctcaagc gcagtatcgt tgtcacccat tttgcctacc ccgctgttaa cctgcccccc 3360

aaagttatgg gcgctgtcat ggaccatctg ctcatcaaac gagcaagacc cctttcagaa 3420

aaccagaaca tgcag 3435

<210> 37

<211> 1685

<212> DNA

<213>3 type adenovirus (Adenovirus type 3)

<400> 37

tatccagcct gaggtcaaag taagacctat caaggaagtg gcgccaggtt tgggagtaca 60

aaccttcgac atcaagattc ccaccgagtc catggaagtg cagaccgaac ctgcaaaacc 120

cacaaccacc tcaattgagg tgcaaacgga accctggacg cccgcgcccg ttgtcgcccc 180

cagcaccact cgaagatcac gacgaaagta cggcccagca agtcttctaa tgcccaacta 240

tgctctgcac ccatccatca ttcccactcc gggttacaga ggcactcgct actatcgaaa 300

ccggagcaac acctctcgcc gccgcaaacc acctgcaagt cgcactcgca gtcgccgccg 360

ccgcaacact gccagcaaag tgactcccgc cgccctggtg cggagagtgt accgcgatgg 420

tcgcgctgaa cctctgacgc tgccgcgcgc gcgctaccat ccaagcatca ccacttaatg 480

actgttgacg ctgcctcctt gcagatatgg ccctcacttg ccgccttcgc gtccccatta 540

ctggctaccg aggaagaaac tcgcgccgta gaaggatgtt ggggcgaggg atgcgccgcc 600

acagacgaag gcgcgctatc agcagacgat tagggggtgg ctttttgcca gctcttatac 660

ccatcatcgc cgcagcgatc ggggcgatac caggcatagc ttccgtggcg gttcaggcct 720

cgcagcgcca ctaacattgg aaaaaactta taaataaaaa atagaatgga ctctgacgct 780

cctggtcctg tgactatgtt tttgtagaga tggaagacat caatttttca tccctggctc 840

cgcgacacgg cacgaggccg tacatgggca cctggagcga catcggcacg agccaactga 900

acgggggcgc cttcaattgg agcagtatct ggagcgggct taaaaatttt ggctcgaccg 960

taaaaaccta tgggaacaga gcttggaaca gcagcacagg gcaggctctg agaaataagc 1020

ttaaggaaca aaacttccaa cagaaggtgg tcgatgggat cgcctctggt attaacggcg 1080

tagtggattt ggccaaccag gctgtacaaa aacagataaa cagccgcctg gacccgccgc 1140

ccgcaacccc tggtgaaatg gaagtggagg aagaacttcc tccgctggaa aagcggggcg 1200

acaagcgtcc gcgacccgag ctggagcaga cgctggtgac gcgcgcagac gagccccctt 1260

catacgagga ggcagtaaag ctcggaatgc ccactaccag gcctgtagct cacatggcta 1320

ccggggtaat gaaaccttct cagacacatc gacccgccac cttggacttg cctcctcccc 1380

ctgcttctgc ggcacctgtt cccaaacctg tcgctaccag aaagcccacc gccgtacagc 1440

ccgtcgccgt agccagaccg cgtcctgggg gcacaccgcg cccgaaagca aactggcaaa 1500

gtactctgaa cagcatcgtg ggtctgggcg tgcagagtgt aaagcgccgt cgctgctatt 1560

aattaaatat ggagtagcgc ttaacttgct tgtctgtgtg tatgtatcat caccacgccg 1620

ccgcagcaga ggagaaagga agaggtcgcg cgccgaggct gagttgcttt caagatggcc 1680

acccc 1685

<210> 38

<211> 39

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 38

gctaccggtc gccaccatgg ccaccccatc gatgctgcc 39

<210> 39

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 39

gcagaattct tacgtggtag cgttaccggc 30

<210> 40

<211> 2841

<212> DNA

<213>55 type adenovirus (Adenovirus type 55)

<400> 40

atggccaccc catcgatgct gccccagtgg gcatacatgc acatcgccgg acaggatgct 60

tcggagtacc tgagtccggg tctggtgcag ttcgcccgcg ccacagacac ctacttcaat 120

ctgggaaata agtttagaaa tcccaccgta gcgccgaccc acgatgtgac caccgatcgt 180

agccagcggc tcatgttgcg cttcgtgccc gttgaccggg aggacaatac atactcttac 240

aaagtgcggt acacactggc cgtgggcgac aacagagtgc tggatatggc cagcacgttc 300

tttgacatta ggggcgtgtt ggacagaggt cccagtttca aaccctattc tggtacggct 360

tacaactctc tggctcctaa aggcgctcca aatacatctc agtggattgc agaaggtgta 420

aaaaatggtg aggagcgcgt aacagaagag gaaaacaata ctactactta cacttttggc 480

aatgctcccg taaaagctga agctgaaatt acaaaagaag gactcccaat aggtttgaaa 540

gtttcagatg aagaaagtaa accgatttat gctgataaaa catatcagcc agaacctcag 600

ctgggagatg aaacttggac tgaccttgat ggaaagaccg aaaagtatgg gggcagggct 660

ctcaaaccag atactaaaat gaaaccatgc tacgggtcct ttgccaaacc tactactgtg 720

aaaggcggtc aggcaaaacc aaaaacaacg gagcagccaa atcagaaagt cgaatatgat 780

attgacatgg agttttttga tgcggcatca cagaaaacaa acttaagtcc taaaattgtc 840

atgtatgcag aaaatgtaaa tttggaaact ccagacactc atgtagtgta caaacctgaa 900

tcagaagaca caagttccga agctaatttg ggacaacagt ctatgcccaa cagacccaac 960

tacattggct tcagagataa ctttattgga cttatgtact ataacagtac tggtaacatg 1020

ggggtgctgg ctggtcaagc gtctcagtta aatgcagtgg ttgacttgca ggacagaaac 1080

acagaacttt cttaccaact cttgcttgac tctctgggcg acagaaccag atactttagc 1140

atgtggaatc aggctgtgga cagttatgat cctgatgtac gtgttattga aaatcatggt 1200

gtggaagatg aacttccaaa ctattgtttt ccactgaatg gcataggggt tccaacaacc 1260

agttacaaat caatagtttc aaatggagac aatgcaccta attggaagga acctgaagta 1320

aatggaacaa gtgagatcag acagggtaat ttgtctgcca tggaaattaa ccttcaagcc 1380

aatctatggc gaagtttcct ttattccaat gtggctctgt atctcccaga ctcgtacaaa 1440

tacaccccgt ccaatgtcac tcttccagaa aacaaaaaca cctacgacta catgaacggg 1500

cgggtggtgc cgccatctct agtagacacc tatgtgaaca ttggcgccag gtggtctctg 1560

gatgctatgg acaatgtcaa cccattcaac caccaccgta acgctggctt gcgttaccga 1620

tccatgcttc tgggtaacgg acgttatgtg cctttccaca tacaagtgcc tcaaaaattc 1680

ttcgctgtca aaaacctgct gcttctccca ggctcctaca cttatgagtg gaacttcaga 1740

aaggatgtga acatggtgct acagagttcc cttggtaacg acctacgggt agatggcgcc 1800

agcatcagtt tcacgagcat caacctctat gctacctttt tccccatggc tcacaacacc 1860

gcttccaccc ttgaagccat gctgcggaat gacaccaatg atcagtcatt caacgactac 1920

ctatctgcag ctaacatgct ctatcccatt cctgccaatg caaccaatat tcccatttcc 1980

attccttctc gcaactgggc ggctttcaga ggctggtcat ttaccagact caaaaccaaa 2040

gaaactccct ctttggggtc tggatttgac ccctactttg tctattctgg ttctattccc 2100

tacctggatg gtaccttcta cctgaaccac acttttaaga aggtttccat catgtttgac 2160

tcttcagtga gctggcctgg aaatgacagg ttactatctc ccaacgaatt tgaaataaag 2220

cgcactgtgg atggcgaagg ctacaacgta gcccaatgca acatgaccaa agactggttc 2280

ttggtacaga tgctcgccaa ctacaacatc ggctatcagg gcttctacat tccagaagga 2340

tacaaagatc gcatgtattc atttttcaga aacttccagc ccatgagcag gcaggtggtt 2400

gatgaggtca attacaaaga cttcaaggcc gtcgccatac cctaccaaca caacaactct 2460

ggctttgtgg gttacatggc tccgaccatg cgccaaggtc aaccctatcc cgctaactat 2520

ccctatccac tcattggaac aactgccgta aatagtgtta cgcagaaaaa gttcttgtgt 2580

gacagaacca tgtggcgcat accgttctcg agcaacttca tgtctatggg ggcccttaca 2640

gacttgggac agaacatgct ttatgccaac tcagctcatg ctctggacat gacctttgag 2700

gtggatccca tggatgagcc caccctgctt tatcttctct tcgaagtttt cgacgtggtc 2760

agagtgcatc agccacatcg cggcatcatc gagacagtct acctgcgtac accgttctcg 2820

gccggtaacg ctaccacgta a 2841

<210> 41

<211> 2832

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 41

atggccaccc catcgatgat gccccaatgg gcatacatgc acatcgccgg acaggatgct 60

tcggagtacc tgagtccggg tctggtgcag ttcgcccgtg caacagacac ctacttcagt 120

atggggaaca aatttagaaa ccccacagtg gcgcccaccc acgatgtgac caccgatcgt 180

agccagcgcc tgatgctgcg cttcgtgccc gttgaccggg aagacaatac ctactcttac 240

aaagttcgct acacgctggc tgtaggcgac aacagagtgc ttgacatggc cagcacattc 300

tttgacattc ggggggtgct tgatagaggt cctagcttca agccatattc cggcacagct 360

tacaattcac tcgctcctaa gggcgcgccc aatacatctc agtggatagt tacaacgaat 420

cgagacaatg cagtaactac caccacaaac acatttggca ttgcttccat gaagggagac 480

aatattacta aagaaggttt gcaaattggg aaagacatta ccactactga aggagaagaa 540

aagcccattt atgccgataa aacatatcag ccagagcctc aagttggaga agaatcatgg 600

actgatactg atggaacaaa tgaaaagttt ggtggaagag cccttaaacc agctaccaac 660

atgaagccat gctacgggtc ttttgcaaga cctacaaaca taaaaggggg ccaagctaaa 720

aacagaaaag taaaaccaac aaccgaagga ggggttgaaa ctgaggaacc agatattgat 780

atggaatttt tcgatggtag agaagctgct gacgcttttt cgcctgaaat tgtgctttac 840

acggaaaatg taaatttgga aactccagac agtcatgtgg tatataaacc aggaacgtct 900

gataactctc atgcaaattt gggtcaacaa gccatgccta acagacccaa ttacattgga 960

ttcagggata actttgtagg cctaatgtac tacaacagta ctggaaatat gggagttttg 1020

gctggccaag catcacaact gaatgcagtg gttgacttgc aggacagaaa tactgaactg 1080

tcatatcagc ttttgcttga ttctctggga gacagaacca gatacttcag catgtggaat 1140

caggctgtgg acagttacga tcccgatgtt cgcattattg aaaatcatgg catcgaggat 1200

gaactgccta attactgttt tcctctggat ggcataggac cagggcacag gtatcaaggc 1260

attaaagtta aaaccgatga cgctaatgga tgggaaaaag atgctaatgt tgatacagct 1320

aatgaaatag ccataggaaa caacctggct atggaaatta atatccaagc taacctttgg 1380

agaagttttc tgtactccaa tgtggctttg taccttccag atgtttacaa gtacacgcca 1440

cctaacatta ctttgcccac taacaccaac acctatgagt acatgaacgg gcgagtggta 1500

tccccatctc tggttgattc atacatcaac atcggcgcca ggtggtctct tgacccaatg 1560

gacaatgtga atccattcaa ccaccaccgc aatgctggtc tgcgctacag gtccatgctt 1620

ctgggaaatg gtcgttatgt gcctttccac atacaagtgc ctcaaaaatt ctttgctgtc 1680

aaaaacctac ttcttctacc tggctcctac acctacgagt ggaacttcag aaaggatgtg 1740

aacatggtcc tgcaaagttc ccttggaaat gacctcagaa cagatggtgc taccataagt 1800

ttcaccagca tcaatctcta tgccaccttc ttccccatgg ctcacaacac agcttccacc 1860

cttgaagcca tgctgcgcaa cgataccaat gatcagtcat ttaacgacta cctctctgca 1920

gctaacatgc tttaccccat tcctgccaat gcaaccaaca ttccaatttc catcccatct 1980

cgcaactggg cagccttcag gggctggtcc ttcaccaggc tcaaaaccaa ggagactcca 2040

tctcttggat cagggttcga tccctacttc gtatattctg gatctattcc ctacctggat 2100

ggcaccttct accttaacca cactttcaag aaggtctcca tcatgtttga ctcctcagtc 2160

agctggcctg gcaatgacag gctgttgagc ccaaatgagt ttgaaatcaa gcgcactgtg 2220

gacggggaag gatacaacgt ggcacaatgc aacatgacca aagactggtt cctagttcag 2280

atgcttgcca actacaacat tggctaccag ggcttttaca tccctgaggg atacaaggat 2340

cgcatgtact cttttttcag aaacttccag cctatgagca ggcaggtggt tgatgaggtt 2400

aattacactg actacaaagc cgtcacctta ccataccaac acaacaactc tggctttgta 2460

gggtaccttg cacctactat gagacaaggg gaaccttacc cagccaatta tccatacccg 2520

ctcatcggaa ctactgccgt taagagtgtt acccagaaaa agttcctgtg cgacaggacc 2580

atgtggcgca ttcccttctc cagcaacttc atgtccatgg gggcccttac cgacctggga 2640

cagaacatgc tctatgccaa ctcagcccat gcgctggaca tgacttttga ggtggatccc 2700

atggatgagc ccaccctgct ttatcttctt ttcgaagtct tcgacgtggt cagagtgcac 2760

cagccacacc gcggcgtcat cgaggccgtc tacctgcgca caccgttctc ggccggcaac 2820

gccaccacat aa 2832

Claims (10)

1. a kind of construction method of recombination trivalent adenovirus vaccine, which is characterized in that include the following steps：

Step S1：The coding segment of the neutralizing epitope amino acid of 7 type adenovirus six adjacent body hypervariable regions of people is cloned into the areas E3 missing Human 3-type adenovirus full-length genome carrier six adjacent body hypervariable regions to replace corresponding coding segment, obtain chimeric 7 type adenopathies The 3 type adenovirus vector of recombination of malicious epitope；

Step S2：It is recombined into 55 type adenopathy of people in the former E3 zone positions of the 3 type adenovirus vector of recombination of chimeric 7 type adenovirus epitopes Poison hexon overall length coding segment to get.

2. the construction method of recombination trivalent adenovirus vaccine as described in claim 1, which is characterized in that in the step S1 In, further include step S11：The hexon amino acid sequence of people 7 type adenovirus and 3 type adenovirus is compared, and according to homologous modeling Method obtain 7 type adenovirus six adjacent body hypervariable regions of the people neutralizing epitope amino acid sequence.

3. the construction method of recombination trivalent adenovirus vaccine as claimed in claim 2, which is characterized in that the people 7 of acquisition The sequence of the neutralizing epitope amino acid of type adenovirus six adjacent body hypervariable regions is as shown in SEQ ID NO.11, the people that is replaced The amino acid sequence of corresponding coding fragment coding is as shown in SEQ ID NO.9 in 3 type adenovirus full-length genome carriers.

4. the construction method of recombination trivalent adenovirus vaccine as claimed in claim 3, which is characterized in that with SEQ ID NO.11 Shown in amino acid sequence it is corresponding coding segment sequence as shown in SEQ ID NO.12, with ammonia shown in SEQ ID NO.9 The sequence of the corresponding coding segment of base acid sequence is as shown in SEQ ID NO.10.

5. the construction method of recombination trivalent adenovirus vaccine as described in claim 1, which is characterized in that 55 type of overall length people The coded sequence of the hexon of adenovirus is as shown in SEQ ID NO.40.

6. such as the construction method of recombination trivalent adenovirus vaccine according to any one of claims 1 to 5, which is characterized in that Further include following steps in the step S1：

Step S12：By overlapping PCR method by the coded slice of the neutralizing epitope amino acid of 7 type adenovirus six adjacent body hypervariable regions of people Section is cloned into human 3-type adenovirus six adjacent body hypervariable regions to replace corresponding coding segment, and six after the complete recombination of acquisition Adjacent body gene；

Step S13：Hexon gene after the complete recombination is recombined into hexon shuttle vector, is recombinated Shuttle vector, upstream homology arm and downstream homology arm containing hexon gene in the hexon shuttle vector；

Step S14：Hexon gene in the recombinant shuttle plasmid carrier is recombined into the human 3-type adenopathy of the areas the E3 missing To replace the hexon gene in original vector in malicious full-length genome carrier, the 3 type adenopathy of recombination of chimeric 7 type adenovirus epitopes is obtained Poisonous carrier.

7. the construction method of recombination trivalent adenovirus vaccine as claimed in claim 6, which is characterized in that in the step S12 Over-lap PCR during the sequence of primer that uses respectively as SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, Shown in SEQ ID NO.29 and SEQ ID NO.30, the sequence such as SEQ ID of the hexon gene after the complete recombination of acquisition Shown in NO.41.

8. such as the construction method of recombination trivalent adenovirus vaccine according to any one of claims 1 to 5, which is characterized in that It is further comprising the steps of in the step S2：

Step S21：Expand the hexon full-length gene of 55 type adenovirus of people；

Step S22：The hexon full-length gene of the 55 type adenovirus of people of amplification is recombined into the areas E3 shuttle vector, is obtained Recombinate the areas E3 shuttle vector；

Step S23：The hexon full-length gene of 55 type adenovirus of people in the areas recombination E3 shuttle vector is recombined into 3 areas type adenovirus vector Yuan E3 of recombination of the chimeric 7 type adenovirus epitopes, obtain recombination trivalent Recombinant Adenoviral Vaccine Vector；

Step S24：By the recombination trivalent Recombinant Adenoviral Vaccine Vector transfectional cell, recombinated using rescue recombinant virus technology Trivalent adenovirus vaccine.

9. the construction method of recombination trivalent adenovirus vaccine as claimed in claim 8, which is characterized in that in the step S21 In, it is to expand by template of 55 type adenovirus of people using sequence fragment shown in SEQ ID NO.38 and SEQ ID NO.39 as primer Increase hexon full-length gene.

10. a kind of construction method using such as recombination trivalent adenovirus vaccine according to any one of claims 1 to 9 is built The recombination trivalent adenovirus vaccine arrived, which is characterized in that the human 3-type adenopathy that the recombination trivalent adenovirus vaccine is lacked with the areas E3 Based on poison, human 3-type adenovirus is replaced with the coding segment of the neutralizing epitope amino acid of the six adjacent body hypervariable regions of 7 type adenovirus of people In corresponding coding segment, and recombinate the hexon full-length gene of 55 type adenovirus of someone in the areas E3 of original missing.