CN107602672A

CN107602672A - The adenovirus cilia protein peptide of recombination expression, adenovirus subunit vaccine and preparation method thereof

Info

Publication number: CN107602672A
Application number: CN201710764336.9A
Authority: CN
Inventors: 田新贵; 周荣; 廖嘉仪; 周志超; 李潇
Original assignee: First Affiliated Hospital of Guangzhou Medical University
Current assignee: First Affiliated Hospital of Guangzhou Medical University
Priority date: 2017-08-30
Filing date: 2017-08-30
Publication date: 2018-01-19
Anticipated expiration: 2037-08-30
Also published as: CN107602672B

Abstract

The invention discloses a kind of adenovirus cilia protein peptide of recombination expression, adenovirus subunit vaccine and preparation method thereof, the adenovirus cilia protein peptide of the recombination expression has such as SEQ ID No:Amino acid sequence shown in 2, the vaccine include above-mentioned adenovirus cilia protein peptide.High, the immune mouse of the adenovirus cilia protein peptide expression quantity of the present invention can be with induced high titers neutralizing antibody, and immunogenicity is strong, can be used as adenovirus subunit vaccine candidate, and production is convenient with purifying, cost is low；Cross-neutralization antibody can be induced, multivalence adenovirus vaccine candidate can be used as；A kind of multivalence adenovirus subunit vaccine candidate can be also used as with other types knob compatibilities；Can be as the immunizing antigen and detection antigen for preparing wide spectrum neutralization activity monoclonal antibody or Human monoclonal antibody.

Description

Adenovirus cilia protein peptide, adenovirus subunit vaccine and its preparation of recombination expression Method

Technical field

The invention belongs to gene engineering technology field, more particularly it relates to which a kind of adenovirus of recombination expression is fine Hairless protein peptide, adenovirus subunit vaccine, multivalence adenovirus subunit vaccine and preparation method thereof.

Background technology

Adenovirus hominis (human Adenovirus, HAdV) can infect a variety of mucosal tissues, as intestines and stomach, respiratory tract, Urogenital tract and canthus conjunctiva etc., cause ARI (ARI), acute gastroenteritis, ephritis, cornea conjunctivitis, The even serious lethal infection of the mucosal infections of the self limitings such as cystitis.Adenovirus is popular extensively, propagates soon, in recent years more There are outburst and popular, serious threat to social public health security during individual countries and regions.All age group crowd can be by gland Virus infection, wherein Susceptible population is mostly patient (such as bone-marrow transplantation, human immunodeficiency of infant and immune function depression Poison infection), it can often cause serious even lethal infection；Adenovirus is to cause the important pathogen of heat generation breathing problem in army Body.HAdV infection outbreaks mostly occur in the closing such as kindergarten, new military camp or crowded place, on a large scale the weight caused by it in the groove Disease pneumonia case fatality rate is higher, or even the severe HAdV pneumonia of untreated or the death rate of disseminated disease can exceed that 50%. Pneumonia is particularly acute caused by adenovirus, can not only cause heart failure, respiratory failure, can also be caused and is involved outside lung, such as Encephalitis, hepatic lesion, myocarditis or cardiac damage etc., death is even resulted in, and generally prognosis is poor.

So far the adenovirus hominis having found has 7 group of 50 various serotype, more than 69 kinds genotype.Adenovirus is toxicity of causing a disease One of main pathogens of pneumonia, B groups, C groups, E groups adenovirus can cause the 3 of respiratory tract infection, wherein people B groups adenovirus Type, 7 types, 14 types, 55 types and the type adenovirus of E groups 4 be cause ARI (ARI) and infant's fatal pneumonia it is important Pathogen, infectiousness is strong, often results in eruption and prevalence, at home and abroad to have been reported that more.The domestic popular main type of strain is 3 types and 7 Type, highly pathogenicity strain HAdV-B55 existing more eruption and prevalences in the army in the multiple provinces in the whole nation and the masses in recent years, One of main pathogens for causing pneumonia as the country.

In the adenovirus for causing infant and acute respiratory tract infections in adults ARI, the type of adenovirus hominis B groups 3,7 types, 14 types and 55 types are most important several types.Wherein 3 type adenovirus mainly result in infant's severe infections, do not cause typically adult tight Double infection contaminates.The adenovirus infection of relatively other types, the type adenovirus (HAdV-B7) of B groups people 7, the type of people 55 (HAdV-B55) infection Caused symptoms of pneumonia is generally more serious.It is domestic to have had HAdV-B7 severe infections and the report of eruption and prevalence always since the sixties Road.The report of respiratory tract infection and serious pneumonia outbreak of epidemic caused by emerging HAdV-B55 adenovirus is more and more in recent years It is more, in China in the existing more eruption and prevalences of the military camp and colleges and universities that the crowd is dense, have become important prevalence virus Strain, causes people more to pay attention to.Child and acute respiratory tract infections in adults are in state caused by the type of adenovirus hominis 14 (HAdV-B14) It is inside and outside to have outburst Case report, and severe pneumonia is frequently resulted in, the pathogenic HAdV- of the height particularly occurred in recent years The popular serious threat public health of 14p1 strains.HAdV-B55 is a kind of adenovirus of genome recombination, and its genome includes fibre Hair gene derives from the type adenovirus of people 11 essentially from 14 type adenovirus, only six adjacent body hypervariable regions, and this restructuring variation causes It can not be neutralized by HAdV-B14 antiserums.Restructuring is the major way that adenovirus is evolved, and recombinates that there may be biological characteristics The New-type adenovirus of change, there is stronger pathogenic or stronger communicable new strain, it will serious threat human health.

The pathogenic popularity of adenovirus and the seriousness of harm cause the preventing and treating of adenovirus to seem particularly significant, alive at present Also without for adenovirus, specifically effective medicine, its treatment mainly take symptomatic treatment, be inoculated with adenovirus in the range of boundary Subunit vaccine is one of prevention adenovirus infection most efficient method.Therefore, it is necessary to it is sub- single to develop a kind of multivalence adenovirus Position vaccine, common adenovirus type infection can be prevented, and to be adapted to general population particularly infant to use.

4 types, the 7 non-attenuated live vaccines of type enteric solubility being only used at present in septic yanks.Conventional polyvaccine Preparation method is the separation other clinical strain of different shaped, respectively culture purified, and polyvaccine, but this is made after proper ratio compatibility Kind method, which prepares multivalence adenovirus subunit vaccine, its defect, first, is easily recombinated between different type adenoviral gene groups, There may be the stronger New-type adenovirus of virulence；Secondly, the duplication propagation efficiency of different type adenovirus is different, causes development more Compatibility is difficult during valency vaccine.The current type of adenovirus hominis subunit vaccine someone 4,7 type adenovirus subunit vaccines in the world, this Two kinds of vaccines are oral enteric type vaccine living, are only used in the depot of septic yanks, and its application significantly reduces The incidence of disease of U.S. army's heat generation respiratory tract infection.But this vaccine can only prevent the type of people 4,7 type adenovirus infections, to other wide General popular type such as 3 types, 14 types and 55 types do not have prevention effect, and this vaccine is also not suitable for making in general population or children With, it is still necessary to explore development of new adenovirus subunit vaccine.

Adenovirus hominis is no coating icosahedral symmetry structure, and major capsid protein includes hexon, penton, penton , can the specific neutralizing antibody of induction type in vivo including penton pedestal (penton base) and dynein (fiber) Response.Dynein is most important receptor binding protein, and the virus of its head domain (knob) and host cell surface is special Specific receptor combines the absorption for completing virus.Restructuring hexon polypeptide is immunized due to that can not form correct 3 aggressiveness conformation Mouse or can not can only induce very weak Neutralizing antibody response.There is document report to show that the adenovirus knob such as the type of people 5,2 types can be with The Trimeric structures of solubility are self-assembled into, Neutralizing antibody response can be induced after immune mouse.Compared with eukaryotic expression system, greatly Enterobacteria expression system earlier, be broadly applied to biomedicine field, and be generally acknowledged efficient, inexpensive, safe The good expression system of property.The vaccine yield of Escherichia coli is high, cost is low, and it is (special towards the whole world to be especially suitable for exploitation Most developing countries) new generation vaccine.So if can be in E. coli adenovirus subunit epidemic disease Seedling, will be more valuable.

At present, by clinic application, its cost is low, preparation side for the recombinant protein vaccine of Bacillus coli expression such as hepatitis C vaccine Just, can be expressed in document report knob polypeptides before in Escherichia coli, including the type of C groups 5,2 types, the type of B groups 3, and Other groups of part types.Only 5 types and 2 types have had been reported that neutralization, and having been reported that proves that dynein is to neutralize antigen, but does not have There is the report as the application of adenovirus subunit vaccine.Cross-neutralization between B group adenovirus difference types Knob is not reported Road.

The content of the invention

It is fine the invention provides a kind of adenovirus of recombination expression the defects of in order to overcome above-mentioned prior art based on this Hairless protein peptide, adenovirus subunit vaccine, multivalence adenovirus subunit vaccine and preparation method thereof.

In order to realize foregoing invention purpose, this invention takes following technical scheme：

A kind of adenovirus cilia protein peptide of recombination expression, the adenovirus cilia protein peptide of the recombination expression have such as SEQ ID No:Amino acid sequence shown in 2.

In wherein some embodiments, the adenovirus cilia protein peptide of the recombination expression is by such as SEQ ID No:Shown in 1 Base sequence coded by.

Present invention also offers the preparation method of the adenovirus cilia protein peptide of above-mentioned recombination expression, comprise the following steps：

(1), using the type adenoviral gene group of people 55 as template, SEQ ID No:13~14 enter performing PCR for primer pair expands To 55 type knob genes, it is connected to after BamHI and HindIII double digestion PCR fragments in prokaryotic expression carrier PQE30；

(2) Escherichia coli Top10 Competents, are converted, picking single bacterium colony carries out gene sequencing, taken and theoretical sequence one Cause the bacterium colony conservation without mutation and extract plasmid；

(3) it, will identify that correct expression plasmid translation table reaches bacterium BL21 competence, and choose single bacterium colony and be seeded to the LB containing Amp Culture medium shakes training, adds IPTG to induce, and thalline is collected by centrifugation；

(4) thalline, lysozyme or ultrasonic treatment bacterium, is resuspended with PBS, takes supernatant after high speed centrifugation, uses nickel Post is purified, and gradient imidazoles is washed and eluted, and eluent removes imidazoles, produces the adenovirus cilia protein of recombination expression Peptide.

Present invention also offers a kind of adenovirus subunit vaccine, the adenovirus that the vaccine includes above-mentioned recombination expression is fine Hairless protein peptide.

In wherein some embodiments, the vaccine also includes phosphaljel adjuvant, the adenovirus of the recombination expression Dynein peptide is matched with the phosphaljel adjuvant with same volume.

Present invention also offers a kind of multivalence adenovirus subunit vaccine, the vaccine includes the adenopathy of above-mentioned recombination expression Malicious dynein peptide.

Present invention also offers a kind of multivalence adenovirus subunit vaccine, the vaccine includes the adenopathy of above-mentioned recombination expression Malicious dynein peptide and SEQ ID No:The adenovirus cilia protein peptide of the recombination expression of amino acid sequence shown in 4.

In wherein some embodiments, the multivalence adenovirus subunit vaccine also includes phosphaljel adjuvant, described The adenovirus cilia protein peptide of recombination expression is matched with the phosphaljel adjuvant with same volume.

Present invention also offers a kind of multivalence adenovirus subunit vaccine, the vaccine includes the adenopathy of above-mentioned recombination expression Malicious dynein peptide, SEQ ID No:The adenovirus cilia protein peptide and SEQ ID of the recombination expression of amino acid sequence shown in 4 No:The adenovirus cilia protein peptide of the recombination expression of amino acid sequence shown in 6.

Present invention also offers a kind of monoclonal antibody, the monoclonal antibody is the adenovirus using above-mentioned recombination expression Dynein peptide prepares as immunogene or detection antigen.

Compared with prior art, the invention has the advantages that：

1st, the present invention will be connected in PQE30 after 55 type knob gene double digestions, then is converted BL21 (DE3) and expressed, Bacterium is collected, lysozyme lysis, is purified with nickel post；The knob polypeptides exhibits of purifying are homotrimer structure, expression quantity High, immune mouse can be with induced high titers neutralizing antibody, and immunogenicity is strong, can be used as adenovirus subunit vaccine candidate, raw Production is convenient with purifying, cost is low；

2nd, the type adenovirus knob of people 55 of the invention can be induced in extremely strong anti-human 3-type, 14 types, 55 type adenovirus intersection With effect antibody, multivalence adenovirus subunit vaccine candidate can be used as；Using 55 type adenovirus and other several type knob Also a kind of multivalence adenovirus subunit vaccine candidate can be used as after mixing；

3rd, restructuring knob polypeptides of the invention can be as preparing exempting from for wide spectrum neutralization activity monoclonal antibody or Human monoclonal antibody Epidemic disease antigen and detection antigen.

Brief description of the drawings

Drawings below is used to illustrate specific embodiments of the present invention, is not used in and defines this hair that claim is defined Bright scope.

Fig. 1 is the SDS-PAGE of adenovirus cilia protein knob purified products in the embodiment of the present invention 1 through Coomassie brilliant blue The collection of illustrative plates of dyeing, wherein, -1 is does not boil sample, and -2 be to boil sample；

Fig. 2 is that restructuring knob protein immunizations mice serum is micro- to the cell of respective type adenovirus in the embodiment of the present invention 2 Amount neutralizes experimental result；

Fig. 3 is that knob protein immunizations mice serum (10000 times of dilutions) ELISA detection knots are recombinated in the embodiment of the present invention 2 Fruit.

Embodiment

Unless otherwise defined, the technical field of all technologies used in the present invention and scientific terminology with belonging to the present invention The implication that is generally understood that of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality The purpose of example is applied, is not used in the limitation present invention.Term "and/or" used in the present invention includes the listed of one or more correlations The arbitrary and all combination of project.

For the ease of understanding the present invention, the present invention is described more fully below with reference to relevant drawings.In accompanying 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.On the contrary, the purpose for providing these embodiments is to make the understanding to the disclosure more thorough Comprehensively.

Reagent or raw material used in following examples, unless otherwise specified, derive from commercially available.

Ad3 described in this specification embodiment is HAdv3-gz01 Strain, full-length genome Genbank：DQ099432. The whole genome sequence of Ad7 Strain (Ad7-GZ08) described in this specification embodiment GenBank：GQ478341.1；Ad14 Strain (Ad14-GZ01) whole genome sequence GenBank：JQ824845.1；Ad55 Strain (Ad55-Shanxi-Y16) Hexon gene sequence GenBank：KF911353.1；Ad4 Strain (Ad4-GZ01) whole genome sequence GenBank： KF006344.1；Replication defect type Ad5 Strain (AdEASY) derives from pAdEasy-1, its No. GenBank：AY370909.2. Knob genes are consistent with these Strain in this specification embodiment, and amino acid sequence is without mutation；Experiment is neutralized in the present embodiment Deng also using these strains.

Embodiment 1 recombinates the expression of adenovirus hominis knob polypeptides

1st, to human 3-type, 7 types, 14 types, 4 types, 5 type adenovirus knob genes carry out the optimization of Escherichia coli preferred codons with Bacillus coli expression is adapted to, is respectively synthesized gene, and be cloned into prokaryotic expression carrier PQE30 (Qiagen companies)；

2nd, using the type adenoviral gene group of people 55 as template, performing PCR, amplification 55 are entered with primer pair A55Kn-RH/A55Kn-UB Type knob genes, above-mentioned prokaryotic expression carrier PQE30 is connected to after BamHI and HindIII double digestion PCR fragments, and (Qiagen is public Department), Escherichia coli Top10 Competents are converted, picking single bacterium colony carries out gene sequencing.Take no mutation consistent with theoretical sequence Bacterium colony conservation and extract plasmid.

A55Kn-RH:AGCTAATTAAGCttagtcgtcttctctgatgtagta(SEQ ID No：13)

A55Kn-UB:ACCATCACGGATCCaacaacatttgcattgatgac(SEQ ID No：14)；

3rd, it will identify that correct expression plasmid translation table reaches bacterium BL21 (DE3) competence, and choose single bacterium colony inoculation 5ml LB 37 DEG C of (Amp for adding 100ug/ml) culture medium shakes training overnight, is all seeded to 200ml LB (Amp for adding 100ug/ml) cultures 37 DEG C of base shakes training 3-4hr, adds 0.5mM IPTG to induce 4-5 hours or low temperature (18 DEG C) overnight induction at 37 DEG C, bacterium is collected by centrifugation Body.Thalline, lysozyme (2mg is resuspended with PBS (1mM EDTA, 10% glycerine, 1%Triton X100,10mM imidazoles) Enzyme/g thalline) or ultrasonic wave (3s/3s, 60 times) cracking bacterium, supernatant is taken after 12000g high speed centrifugations, is purified with nickel post, Washed and eluted with 10mM, 20mM, 200Mm, 400mM gradient imidazoles, eluent centrifuges displacement PBS with 10kDa super filter tubes Buffer solution (1mM EDTA, 10% glycerine) removes imidazoles, and purifying protein is frozen in -20 DEG C.

The albumen of purifying shows as homotrimer structure.Ad3, Ad4, Ad5, Ad7, Ad14Knob recombinant protein are used 0.5mM IPTG induce 5h in 37 DEG C, express, purifying；Ad55knob recombinant proteins be 0.5mM IPTG in 18 DEG C of overnight inductions, Expression, purifying；Ad4, Ad5, Ad7, Ad14knob expressing quantity are high, solvable and be in tripolymer；Ad3knob albumen is solvable, but The trimerization scale of construction is less；Ad55knob albumen soluble-expressions, in tripolymer, but expression quantity is relatively low (such as Fig. 1).

Recombinating adenovirus hominis knob polypeptides, there is following base composition and amino acid to form respectively：

1、Ad55-Knob：Base sequence such as SEQ ID No:Shown in 1, amino acid sequence such as SEQ ID No:Shown in 2；

2、Ad7-Knob：Base sequence such as SEQ ID No:Shown in 3, amino acid sequence such as SEQ ID No:Shown in 4；

3、Ad4-Knob：Base sequence such as SEQ ID No:Shown in 5, amino acid sequence such as SEQ ID No:Shown in 6；

4、Ad3-Knob：Base sequence such as SEQ ID No:Shown in 7, amino acid sequence such as SEQ ID No:Shown in 8；

5、Ad14-Knob：Base sequence such as SEQ ID No:Shown in 9, amino acid sequence such as SEQ ID No:Shown in 10；

6、Ad5-Knob：Base sequence such as SEQ ID No:Shown in 11, amino acid sequence such as SEQ ID No:Shown in 12.

The Study On Immunogenicity of the recombinant protein of embodiment 2

To analyze the immunogenicity of the recombinant protein of embodiment 1, using recombinant protein plus Aluminium phosphate adjuvant (ADJU-PHOS, Brenntag Biosector) mouse is immunized after emulsification, it is immunized four times altogether, 40ug albumen is immunized in every mouse every time, and last is exempted from Epidemic disease gathers serum after 7 days, carry out in ELISA detections and cell micro and test.

As a result as shown in Figures 2 and 3, the results showed that the immune mouse of various restructuring knob can induce high titre antibody should Answer, to the neutralizing antibody titers about 500-4000 (Fig. 2) of respective type adenovirus, ELISA detections are carried out with recombinant protein antigen Its titre can reach 10⁶-10⁷(Fig. 3).

Cross-neutralization of anti-adenovirus cilia protein head (knob) serum of the mouse of embodiment 3 to different type adenovirus Analysis

The anti-dynein head serum after four times immune for gathering embodiment 2 carries out cross-neutralization detection, as a result As shown in table 1.

Cross-neutralization of the anti-adenovirus cilia protein head serum of the mouse of table 1 to different type adenovirus

-:It is not detected by neutralization

The result of table 1 is found：Some types have cross-neutralization, and this intersection to a variety of type adenovirus in same group Neutralization is very strong.Ad55 types Knob can be as a kind of prevention people 14 type, 3 types, the multivalence adenovirus epidemic disease of 55 type adenovirus Seedling candidate.

Mouse experiment is immunized after 4 different types of embodiment restructuring knob mixed in equal amounts

According to the result of cross reaction between type, multivalence adenovirus candidate vaccine, Yi Zhongshi are prepared using various combination mode 55 types, 7 type adenovirus restructuring knob add Aluminium phosphate adjuvant that mouse, each every mouse immune is immunized after homogenous quantities mix in vitro Total protein concentration 60ug (every kind of 30ug)；Another kind be 55 types, 4 types, 7 type adenovirus restructuring knob in vitro homogenous quantities mixing after plus Mouse is immunized in Aluminium phosphate adjuvant, each every mouse immune total protein concentration 90ug (every kind of 30ug)；Intramuscular injection, it is immunized altogether twice After collect antiserum, carry out cell in vitro microneutralization experiment, neutralization titer of the detection antiserum to each type adenovirus.

As a result immune mouse can be induced in anti-3 type, 7 types, 14 types, 55 type adenovirus after showing 55 types, 7 type knob mixing And antibody, neutralization titer 1000-4000, a kind of 4 valency adenovirus subunit vaccines can be used as to use, various neutralization titer Difference is not notable (table 2).

Neutralization of the mouse resisting anteserum to each type adenovirus is immunized after the type of 2 people of table 55,7 type adenovirus knob mixing

Mouse, which is immunized, after 55 types, 4 types, 7 type knob mixing can induce in anti-3 type, 4 types, 7 types, 14 types, 55 type adenovirus And antibody, neutralization titer 1000-4000, a kind of 5 valency adenovirus candidate vaccines can be used as to use, various neutralization titer is poor Different not notable (table 3).

Neutralization of the mouse resisting anteserum to each type adenovirus is immunized after the type of 3 people of table 55,4 types, 7 type adenovirus knob mixing to make With

Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality Apply 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, the scope that this specification is recorded all is considered to be.

Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art Say, 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 Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Sequence table

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Met Leu Leu Thr Ile Thr Leu Asn Gly Thr Asp Asp Thr Thr Ser Ala

145 150 155 160

Tyr Ser Ile Ser Phe Ser Tyr Thr Trp Thr Asn Gly Ser Tyr Ile Gly

165 170 175

Ala Thr Phe Gly Ala Asn Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu

180 185 190

<210> 7

<211> 588

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 7

aatagcattg cactgaaaaa taacaccctg tggaccggtc cgaaaccgga agccaattgt 60

attattgaat atggcaaaga aaatccggat agtaaactga ccttaattct ggttaaaaat 120

ggcggcattg ttaatggcta tgtgaccctg atgggcgcct cagattatgt taataccctg 180

tttaaaaaca aaaatgtgag cattaatgtg gaactgtatt ttgatgcaac cggtcatatt 240

ctgccggatc tgtcaagtct gaaaaccgat ctgcagctga aatacaaaca gaccacccat 300

tttagcgcac gcggctttat gccgtctacc accgcctatc cgtttgtgct gccgaatgca 360

ggcaccgata acgaaaatta tatttttggt cagtgttatt acaaggcaag cgatggcgca 420

ctgtttccgc tggaagtgac cgtgaccctg aataagcgtc tgccggatag tcgtaccagc 480

tatgtgatta cctttctgtg gagcctgaat gcaggcttag ccccggaaac cacccaggcc 540

accttaatta cctctccgtt tacctttagc tatattaccg aagatgat 588

<210> 8

<211> 196

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 8

Asn Ser Ile Ala Leu Lys Asn Asn Thr Leu Trp Thr Gly Pro Lys Pro

1 5 10 15

Glu Ala Asn Cys Ile Ile Glu Tyr Gly Lys Glu Asn Pro Asp Ser Lys

20 25 30

Leu Thr Leu Ile Leu Val Lys Asn Gly Gly Ile Val Asn Gly Tyr Val

35 40 45

Thr Leu Met Gly Ala Ser Asp Tyr Val Asn Thr Leu Phe Lys Asn Lys

50 55 60

Asn Val Ser Ile Asn Val Glu Leu Tyr Phe Asp Ala Thr Gly His Ile

65 70 75 80

Leu Pro Asp Leu Ser Ser Leu Lys Thr Asp Leu Gln Leu Lys Tyr Lys

85 90 95

Gln Thr Thr His Phe Ser Ala Arg Gly Phe Met Pro Ser Thr Thr Ala

100 105 110

Tyr Pro Phe Val Leu Pro Asn Ala Gly Thr Asp Asn Glu Asn Tyr Ile

115 120 125

Phe Gly Gln Cys Tyr Tyr Lys Ala Ser Asp Gly Ala Leu Phe Pro Leu

130 135 140

Glu Val Thr Val Thr Leu Asn Lys Arg Leu Pro Asp Ser Arg Thr Ser

145 150 155 160

Tyr Val Ile Thr Phe Leu Trp Ser Leu Asn Ala Gly Leu Ala Pro Glu

165 170 175

Thr Thr Gln Ala Thr Leu Ile Thr Ser Pro Phe Thr Phe Ser Tyr Ile

180 185 190

Thr Glu Asp Asp

195

<210> 9

<211> 603

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 9

aataacattt gtattgatga taatattaat accctgtgga ccggtgtgaa tccgaccgaa 60

gccaattgtc agatgatgga tagtagcgaa agcaatgatt gtaaactgat tctgacctta 120

gttaaaaccg gcgcactggt gaccgccttt gtgtatgtga ttggcgtttc aaataacttt 180

aatatgttaa ccacctatcg taatattaat tttaccgcag aactgttctt cgatagcgca 240

ggcaatctgc tgacctctct gtctagtctg aaaaccccgc tgaatcataa aagtggtcag 300

aatatggcaa ccggtgccat taccaatgcc aagagcttta tgccgagcac caccgcctat 360

ccgtttaaca acaattcacg cgaaaattat atatacggca cctgtcatta taccgcctca 420

gatcataccg cctttccgat tgatattagt gttatgctga atcagcgtgc cattcgtgca 480

gataccagct attgtattcg cattacctgg agttggaata ccggcgatgc cccggaaggc 540

cagacctctg caaccaccct ggtgaccagt ccgtttacct tttattatat tcgcgaagat 600

gat 603

<210> 10

<211> 201

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 10

Asn Asn Ile Cys Ile Asp Asp Asn Ile Asn Thr Leu Trp Thr Gly Val

1 5 10 15

Asn Pro Thr Glu Ala Asn Cys Gln Met Met Asp Ser Ser Glu Ser Asn

20 25 30

Asp Cys Lys Leu Ile Leu Thr Leu Val Lys Thr Gly Ala Leu Val Thr

35 40 45

Ala Phe Val Tyr Val Ile Gly Val Ser Asn Asn Phe Asn Met Leu Thr

50 55 60

Thr Tyr Arg Asn Ile Asn Phe Thr Ala Glu Leu Phe Phe Asp Ser Ala

65 70 75 80

Gly Asn Leu Leu Thr Ser Leu Ser Ser Leu Lys Thr Pro Leu Asn His

85 90 95

Lys Ser Gly Gln Asn Met Ala Thr Gly Ala Ile Thr Asn Ala Lys Ser

100 105 110

Phe Met Pro Ser Thr Thr Ala Tyr Pro Phe Asn Asn Asn Ser Arg Glu

115 120 125

Asn Tyr Ile Tyr Gly Thr Cys His Tyr Thr Ala Ser Asp His Thr Ala

130 135 140

Phe Pro Ile Asp Ile Ser Val Met Leu Asn Gln Arg Ala Ile Arg Ala

145 150 155 160

Asp Thr Ser Tyr Cys Ile Arg Ile Thr Trp Ser Trp Asn Thr Gly Asp

165 170 175

Ala Pro Glu Gly Gln Thr Ser Ala Thr Thr Leu Val Thr Ser Pro Phe

180 185 190

Thr Phe Tyr Tyr Ile Arg Glu Asp Asp

195 200

<210> 11

<211> 585

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 11

ggcgccatta ccgtgggcaa caaaaacaat gataaactga ccctgtggac caccccggcc 60

ccgagtccga attgtcgctt aaatgcagaa aaagatgcca aactgacctt agtgctgacc 120

aaatgtggta gtcagattct ggccaccgtg agcgtgttag cagtgaaagg ctcactggcc 180

ccgattagcg gcaccgttca gagcgcacat ctgattattc gctttgatga aaatggcgtg 240

ctgttaaaca atagctttct cgatccggaa tattggaatt ttcgtaacgg agacctaacc 300

gaaggcaccg catataccaa tgcagttggc tttatgccga atctgtcagc ctatccgaaa 360

tcacatggga aaaccgccaa aagtaatatt gtgtctcagg tgtatctgaa tggcgataaa 420

accaaaccgg tgaccttaac cattaccctg aatggcaccc aggaaaccgg cgataccacc 480

ccgtctgcct atagtatgtc attttcttgg gattggagcg gtcataatta tattaacgag 540

atttttgcaa cctctagcta tacctttagt tatattgcac aggaa 585

<210> 12

<211> 195

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 12

Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp

1 5 10 15

Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu Lys Asp

20 25 30

Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala

35 40 45

Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile Ser Gly

50 55 60

Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val

65 70 75 80

Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn

85 90 95

Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met

100 105 110

Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser

115 120 125

Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Val

130 135 140

Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr

145 150 155 160

Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn

165 170 175

Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile

180 185 190

Ala Gln Glu

195

<210> 13

<211> 36

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 13

agctaattaa gcttagtcgt cttctctgat gtagta 36

<210> 14

<211> 35

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 14

accatcacgg atccaacaac atttgcattg atgac 35

Claims

A kind of 1. adenovirus cilia protein peptide of recombination expression, it is characterised in that the adenovirus cilia protein of the recombination expression Peptide has such as SEQ ID No:Amino acid sequence shown in 2.
2. the adenovirus cilia protein peptide of recombination expression according to claim 1, it is characterised in that the recombination expression Adenovirus cilia protein peptide is by such as SEQ ID No:Coded by base sequence shown in 1.
3. the preparation method of the adenovirus cilia protein peptide of the recombination expression described in claim 1 or 2, it is characterised in that including Following steps：

(1), using the type adenoviral gene group of people 55 as template, SEQ ID No:13~14 enter performing PCR for primer pair expands to obtain 55 Type knob genes, it is connected in prokaryotic expression carrier PQE30 after BamHI and HindIII double digestion PCR fragments；

(2) Escherichia coli Top10 Competents, are converted, picking single bacterium colony carries out gene sequencing, takes nothing consistent with theoretical sequence The bacterium colony conservation of mutation simultaneously extracts plasmid；

(3) it, will identify that correct expression plasmid translation table reaches bacterium BL21 competence, and choose single bacterium colony and be seeded to the LB cultures containing Amp Base shakes training, adds IPTG to induce, and thalline is collected by centrifugation；

(4) thalline, lysozyme or ultrasonic treatment bacterium, is resuspended with PBS, takes supernatant after high speed centrifugation, is entered with nickel post Row purifying, gradient imidazoles are washed and eluted, and eluent removes imidazoles, produce the adenovirus cilia protein peptide of recombination expression.
4. a kind of adenovirus subunit vaccine, it is characterised in that the vaccine includes the recombination expression described in claim 1 or 2 Adenovirus cilia protein peptide.
5. adenovirus subunit vaccine according to claim 4, it is characterised in that the vaccine also includes phosphaljel Adjuvant, the adenovirus cilia protein peptide of the recombination expression are matched with the phosphaljel adjuvant with same volume.
6. a kind of multivalence adenovirus subunit vaccine, it is characterised in that the vaccine includes the restructuring described in claim 1 or 2 The adenovirus cilia protein peptide of expression.
7. multivalence adenovirus subunit vaccine according to claim 6, it is characterised in that the vaccine also includes aluminum phosphate Gel Adjuvant, the adenovirus cilia protein peptide of the recombination expression are matched with the phosphaljel adjuvant with same volume.
8. a kind of multivalence adenovirus subunit vaccine, it is characterised in that the vaccine includes the restructuring described in claim 1 or 2 The adenovirus cilia protein peptide and SEQ ID No of expression:The adenovirus cilium egg of the recombination expression of amino acid sequence shown in 4 White peptide.
9. a kind of multivalence adenovirus subunit vaccine, it is characterised in that the vaccine includes the restructuring described in claim 1 or 2 Adenovirus cilia protein peptide, the SEQ ID No of expression:The adenovirus cilia protein peptide of the recombination expression of amino acid sequence shown in 4, And SEQ ID No:The adenovirus cilia protein peptide of the recombination expression of amino acid sequence shown in 6.
10. a kind of monoclonal antibody, it is characterised in that the monoclonal antibody is using restructuring as claimed in claim 1 or 2 The adenovirus cilia protein peptide of expression prepares as immunogene or detection antigen.