CN108728462A

CN108728462A - H3N2 type canine influenza virus shuttle intracellular antibodies TAT-2C

Info

Publication number: CN108728462A
Application number: CN201810538741.3A
Authority: CN
Inventors: 田园; 岳玉环; 张国利; 吴广谋; 祝令伟; 余晓颖; 孙红; 刘雨玲; 马洪圆
Original assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Current assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2018-11-02
Anticipated expiration: 2038-05-30
Also published as: CN108728462B

Abstract

The invention discloses a kind of H3N2 types canine influenza virus shuttle intracellular antibody, H3N2 viruses are influenza A, and most important neutralizing antibody comes from surface glycoprotein hemagglutinin（HA）, therefore HA becomes previous main research target；However influenza virus has variability, and the immunological cross-reaction between Different Variation branch is weaker, the antibody of M1 can by with M1 protein bindings, inhibit its activity, the duplication, transcription and release of influenza virus interfered, to play antiviral effect；We select M1 albumen to prepare corresponding antibodies to obtain stable potency, be coupled it with the TAT protein PTD that large biological molecule can be transduceed into cell thus, expressed fusion protein TAT PTD-M1 ScFv, the shuttle antibody for preparing anti-canine influenza virus provides new approach for the treatment of dog influenza.

Description

H3N2 type canine influenza virus shuttle intracellular antibodies TAT-2C

Technical field

The invention belongs to bioengineering and disease prevention and cure fields, are protected for H3N2 type canine influenza virus more particularly to preparing Keep antigen shuttle intracellular antibody TAT-2C and this antibody to the antivirus action of H3N2 type canine influenza virus.

Background technology

For a long time, it has been recognized that the influenza virus of canine not various hypotypes of easy infection, therefore dog under natural conditions It is excluded always except the susceptible range of influenza virus, but virologist in 2004 isolates dog out of Florida dog race body Influenza virus（Canine influenze virus, CIV）, and after confirming infection of the CIV in each kind of dog, it is this to recognize It knows the talent and is changed.Hereafter studies have shown that this once matched the influenza virus to raise fear in dog industry and pet-breeder in the U.S., The actually variant of equine influenza virus H3N8.Canine influenza virus H3N8 is lethal as a kind of emerging canine pathogenic microorganism Rate may be up to 8%.This H3N8 dogs influenza is after equine influenza virus makes a variation, to form the ability propagated between horse to dog. As a kind of epidemic disease of kainogenesis, almost all of dog all lacks immunity to this virus.South Korea researcher report in 2008 The dog influenza of H3N2 hypotypes occurs, and the genetic fragment of several different avian influenza virus is contained in this viral genome, with The avian influenza virus homology of Southeast Asia prevalence reaches 95.5%-98.9%, and experiment proves that H3N2 subtype influenza virus has been provided with The ability that dog is propagated, researcher think that this virus is likely to become second popular of canine influenza virus i.e..2006 China is found that the sick dog with flu symptom, and 4 plants of influenza As are separated to from sick dog nose swab, and evolutionary analysis shows 4 The H3N2 CIV germlines branch that 8 genes of a strain are isolated from dog in 2007 with South Korea is close, and artificial liver support proves Separation strains can infect dog and can directly be propagated between dog, show that fowl source H3N2 CIV have been enter into China.2009-2012 is in It is separated to CIV in the multiple province ,city and area's disease dogs of state, is found in artificial liver support, being isolated from the influenza virus of dog can not only infect Dog, moreover it is possible to infecting mouse and pig.Show that H3N2 CIV have adapted to mammal completely, be successfully realized across host propagation, Exist simultaneously the risk that the span host of new mammal propagates.

Dog influenza is a kind of emerging infectious disease of dog, and different cultivars, all ages and classes dog can all infect, for canine influenza virus Research foreign countries be only limitted to vaccine research, and the domestic research for canine influenza virus is still in the blank phase, still without corresponding Prevent and treat the appearance of drug.Dog has special status as companion animals in present human lives.Due to dog and people And the intimate contact of wild animal, provide more chances to intermediate propagate of influenza virus.Therefore, once there is dog influenza Be very popular and break out and will will produce serious consequence, it is necessary to accelerate the research and development of CIV related vaccines and medicine.

In the case of viral constantly generation drug resistance, novel antibodies drug becomes reply canine influenza virus and causes potential stream Feel pandemic effective means.With the development of technique for gene engineering, the development of genetic engineering antibody is very fast, single-chain antibody It is small with its specificity height, molecular weight, it is simple in structure, it is low compared with parental antibody immunogenicity, maximum it can mitigate in clinical application The unique advantage of allergy caused by foreign protei has attracted the sight of numerous scientific research persons, technology of preparing to tend to be ripe, Especially display technique of bacteriophage more improves the screening efficiency of antibody and antibody gene.Single-chain antibody is to disease of viral infection Treatment will play an important role.

H3N2 viruses are influenza A, and most important neutralizing antibody comes from surface glycoprotein hemagglutinin（HA）, therefore HA becomes previous main research target.However influenza virus has variability, and the immune friendship between Different Variation branch Fork reaction is weaker.In view of the influence of virus variation, the antibody based on conservative antigen component can produce different subtype influenza virus Raw antivirus action.Influenza matrix albumen M1 is the main structural proteins of influenza virus, is located on the inside of virus envelope, passes through With host cell target protein in conjunction with and participate in and the processes such as duplication, transcription, release of regulation and control virus.M1 protein sequences are conservative, because This for M1 antibody can by with M1 protein bindings, inhibit its activity, interfere the duplication, transcription and release of influenza virus, To play antiviral effect.We select M1 albumen to prepare corresponding antibodies to obtain stable potency, not by virus thus Make a variation the antibody preparation influenced.

M1 albumen is located on the inside of virus envelope, and antibody needs to enter the effect of infection cell competence exertion.Protein transduction domain （Protein transduction domain,PTD）It is small peptide fragment of the energy mediating proteins across cell membrane, big point can be carried Son effectively enters cell by biomembrane.The Protein transport that PTD is mediated, can independent of receptor, channel, energy and encytosis Lipid bilayer to directly act on all types cell completes transmembrane movement, and its cross-film function does not have species spy It is anisotropic.Since PTD is identified and is identified, there are hundreds of compounds and protein to be entered by Successful transductions different thin Born of the same parents, and show corresponding bioactivity.In the PTD having found, human immunodeficiency virus-1（HIV-1）TAT protein PTD is that most study, function exact PTD, Tat PTD can be by the polypeptide being attached thereto, protein and DNA with a kind of concentration The mode of dependence efficiently and rapidly imports into the cell, and the normal configuration of cell and function are unaffected.Although protein transduction At present still under study for action, but it will can directly have medicative large biological molecule and be sent into cells play biology effect mechanism It answers this characteristic to provide new thinking for the biological therapy of disease, thus is received significant attention in medical research field.1997 Year, the discoveries such as Vives, Tat PTD are 11 amino acid positioned at 47~57（YGRKKRRQRRR）, it is one and is rich in The polypeptide fragment of basic amino acid.The biological transduction properties for seeing TAT PTD, by after the segment and M1 ScFv amalgamation and expressions its M1 ScFv can be brought to virus infected cell into, targeting prevents it from playing biological function in the M1 albumen of intracellular, inhibits stream The assembly and release of Influenza Virus, to play antiviral effect.

In conclusion it is target antigen to select canine influenza virus conserved sequence M1 albumen, sieved using dog phage antibody library The high-affinity single-chain antibody for selecting anti-M1 albumen, it is even with the TAT protein PTD that large biological molecule can be transduceed into cell Connection, expressed fusion protein TAT PTD-M1 ScFv prepare the shuttle antibody of anti-canine influenza virus, are carried for the treatment of dog influenza For new approach.

Invention content

The object of the present invention is to provide a kind of H3N2 type dogs streams for H3N2 type canine influenza virus conserved sequence M1 albumen Influenza Virus shuttle intracellular antibody TAT-2C, this antibody can enter cytosis in target, can be used for controlling for H3N2 type dogs influenza disease It treats.

H3N2 types canine influenza virus shuttle intracellular antibody TAT-2C, its nucleotide sequence such as sequence table SEQ ID NO.1；

H3N2 type canine influenza virus shuttle intracellular antibody TAT-2C, its amino acid sequence is as shown in sequence table SEQ ID NO. 3；

Applications of the H3N2 type canine influenza virus shuttle intracellular antibody TAT-2C in terms of preparing treatment H3N2 type dog influenza medicines；

The diagnostic kit of H3N2 type canine influenza virus, it includes albumen shown in SEQID NO. 3.

It is A type influenzas the present invention provides a kind of H3N2 types canine influenza virus shuttle intracellular antibody TAT-2C, H3N2 virus Virus, most important neutralizing antibody come from surface glycoprotein hemagglutinin（HA）, therefore HA becomes previous main research target；So And influenza virus has variability, and the immunological cross-reaction between Different Variation branch is weaker, the antibody of M1 can lead to Cross with M1 protein bindings, inhibit its activity, the duplication, transcription and release of influenza virus are interfered, to play antiviral effect； Thus we select M1 albumen prepare corresponding antibodies with obtain stable potency, by its with large biological molecule can be transduceed into cell TAT protein PTD coupling, expressed fusion protein TAT PTD-M1 ScFv prepare the shuttle antibody of anti-canine influenza virus, are The treatment of dog influenza provides new approach.

Description of the drawings

Fig. 1 is M1 protein expressing plasmid pET-SUMO-M1 PCR qualification results；M：DL2000 DNA Marker；1：M1 tables Up to carrier pET-SUMO-M1 PCR results；

Fig. 2 is expression of results of the PET-SUMO-M1 protein expression engineering bacterias after induction；M：Protein Marker；1：It is negative right According to；2：Induce thalline ultrasound precipitation；3：Induce thalline ultrasound supernatant；4：Induce full bacterium；

Fig. 3 is M1 albumen after purification；M:Marker;1:SUMO digestions and after purification sample；

Fig. 4 is the PCR qualification results of 10 plants of positive strains；

Fig. 5 is M1-scFv after purification；M:Marker;1:10%-55% saturation degree ammonium sulfate precipitation samples;2:Flow through 3:Purifying Sample afterwards；

Fig. 6 is the amplification of M1-ScFv genes；M:Marker；1-2:TAT-ScFv PCR products;

Fig. 7 is the induced expression result of recombinant expression plasmid pET-28a-TAT-M1 ScFv；M：Molecular weight of albumen Marker；1: Negative control；2-3:Recombinant expression plasmid pET-28a-TAT-M1ScFv inductions；

Fig. 8 is the scFv of purifying；M:Marker；

1:TAT-scFv expresses bacterium induced ultrasonic supernatant；

2:Cu2+200 imidazoles elution samples; 3:The TAT-scFv of purifying.

Specific implementation mode

Embodiment 1：Structure, expression and the purifying of M1 albumen of M1 recombinant expression plasmids pET-SUMO-M1

Primer M1P1, M1P2 are designed and synthesized, using dog H3N2 cDNA as template PCR amplifications M1 protein gene, and is cloned into PET- In SUMO carriers, build plasmid pET-SUMO-M1, be then transferred to T-shot competent cells, the agar plate of the resistance containing kan into Row preliminary screening.Picking individual colonies are cultivated in LB liquid medium；Plasmid, PCR identifications, production are extracted with plasmid QIAquick Gel Extraction Kit Object is analyzed through 1% agarose gel electrophoresis, obtains the band of 750 bp or so, size is consistent with the target gene of insertion, and carries out The measurement of sequence, it was demonstrated that target fragment is correctly inserted into carrier, successfully constructs recombinant plasmid pET-SUMO-M1（See Fig. 1）.

By recombinant plasmid pET-SUMO-M1 conversion expression bacterium e. coli bl21s（DE3）, after IPTG induced expressions, SDS- PAGE results are shown：Recombinant protein SUMO-M1 has an apparent expression band, size to be consistent with theoretical value at the places 40KD or so, ultrasound Destination protein is mainly in ultrasonic supernatant afterwards, it was demonstrated that destination protein is expressed with soluble form（See Fig. 2）.

The purifying of M1 albumen takes its supernatant after the cracking of induced expression thalline ultrasound, is precipitated with 20-45% AS, precipitates with PB (pH 7.0) crosses ion-exchange chromatography after being resuspended（SP FF）, with the PB linear elutions of the Nacl containing 0.5M, collect destination protein and wash De- peak, crosses Cu²⁺Column, buffer system are PB+0.5M Nacl, are eluted respectively with 50mM, 150mM imidazoles, destination protein is in 150 mM In imidazoles eluting peak.It is 50 mM that 150 mM imidazole elutions, which are diluted to imidazole concentration, and SUMO protease, 30 degree of digestions are added 2h.Digestion products cross Cu²⁺Column, 20mM imidazoles elutes destination protein, then is concentrated with SP FF, obtains M1 egg of the purity 90% or more In vain, meet the needs of phage antibody library screening（See Fig. 3）.

Embodiment 2：The amplification of phage antibody library

1, structure dog phage antibody library structure

Dog separation of lymphocytes:6 peripheral blood lymphocytes separation of beasle dog, extract total serum IgE, reverse transcription synthesizes cDNA；Expand Increase VH genes, upstream and downstream are separately added into Nco I, I restriction enzyme sites of Xho；VL genes are expanded, upstream and downstream are separately added into Sal I, Not I Restriction enzyme site；VH digestions are connected into PIT2 carriers；VL digestions are connected into PIT2 carriers.

2, the carrier electrotransformation e. coli tg1 built is coated with TYE tablets（Containing 100 Portugals μ g/mL Amp+1% of final concentration Grape sugar）, 37 DEG C are incubated overnight, and random picking monoclonal carries out bacterium solution PCR identifications from tablet, calculate recombination fraction, and estimate library Hold（Storage capacity=clone's number × extension rate × recombination fraction）And the dog bacteriophage scFv antibody libraries preserved（Storage capacity is 2.5 × 10⁷）It connects In the 250mL 2 × TY culture mediums for entering preheating（Containing 100 μ g/mL Amp+1% glucose of final concentration）, 37 DEG C of shaken cultivations are extremely OD600 is 0.4（About 2h）.50mL bacterium solutions are taken to be added 2.0 × 10¹¹Helper phage KM13,37 DEG C of water-bath 30min.By bacterium solution With 4 DEG C, 3300g centrifuges 20min, precipitation 100mL2 × TY culture mediums (100 μ g/mLAmp+50 μ g/mL Kana+ containing final concentration 0.1% glucose) it is resuspended, 30 DEG C of shaking overnight incubations of 250rpm.4 DEG C of bacterium solution, 3300g 30min will be centrifuged overnight, and collect supernatant 20mL PEG/NaCl solution is added in about 80mL（Final concentration of 20% PEG-6000,2.5mol/L NaCl）, mixing is placed on Lh on ice.4 DEG C of 3300g centrifuge 30min, and precipitation is resuspended with 4m1 PBS, is mixed well.4 DEG C of 11600g centrifuge 10min, take Supernatant, 4 DEG C of preservations are carried out at the same time phage titre for screening of phage antibody library and measure.

Embodiment 3：The screening of anti-M1-scFv

The M1 albumen of purifying is antigen coat in 96 hole elisa Plates, and 4 DEG C overnight.Next day abandons supernatant, with 2% Milk-PBS 37 DEG C closing 2h, the phage antibody library of preparation is added（Titre is 1.0 × 10¹³pfu）, it acutely shakes be incubated 60min at room temperature, it is quiet Set 60min.After discard liquid, washed 10 times with the PBS containing 0.1%Twenn-20, will be per residual liquid in hole gently after washing It pats dry, 50 μ L eluents is added per hole（Pancreatin-the PBS of 5mg/mL）, 10min is acutely shaken at room temperature, and wash-out bacteriophage is collected 4 DEG C of preservations.

With the Phage Infection E.coli TG1 under elution, and it is coated on TYE tablets（Containing 100 μ g/mLAmp's and 1% Glucose）37 DEG C are incubated overnight.Phage library is expanded using helper phage KM13, bacteriophage is recycled by PEG/NaCl.Weight Multiple above procedure 3 times, totally 4 wheel screening.

Phage Infection after screeningE.ColiHB2151 after induced expression, is identified using ELISA, sets microplate reader measurement OD values（Wavelength is 490nm）, each sample does diplopore measurement, takes OD average values.Positive colony bacterial strain determines that standard is：OD values are 3 times or more of negative control.

Two Specific PCR primers of synthesis expand scFv full genome segments：

P3: 5’—CAG GAA ACA GCT ATG AC—3’

P4: 5’ —CTA TGC GGC CCC ATT CA—3’

Amplify the segment of 900bp or so, it was demonstrated that obtain complete single-chain antibody (Fig. 4).

Embodiment 4：The expression and purifying of M1-scFv

ELISA positive strains are transferred in 5mL 2 × TY culture mediums（Glucose containing 100 μ g/mLAmp and 1%）, 37 DEG C of cultures Overnight.Next day, 200 μ L overnight cultures of switching are in 2 × TY culture mediums（Glucose containing 100 μ g/mLAmp and 0.1%）, 37 DEG C It cultivates to OD600 to 0.9（About 4 h）, final concentration of 1 mmol/L IPTG are added, 30 DEG C of shaken cultivations induce overnight.Next day Induction bacterium solution 4200rpm is centrifuged into 20 min, takes supernatant, with 10%-55% ammonium sulfate precipitations, 30 mmol/L PB of precipitation （pH7.2）It is resuspended, the dialysed overnight in PBS, dialysis sample carries out rProtein-A FF affinity chromatographys, and elution samples are saturating with PBS Overnight, for 12%SDS-PAGE analysis shows that destination protein size is about 31000 Da, scFv purity after purification meets progress for analysis The requirement of antivirus test.（Fig. 5）

Embodiment 5：Structure, expression and the purifying of TAT-M1 ScFv

Design 2 primers of synthesis：P5, P6 introduce EcoR I, III restriction enzyme sites of Hind respectively, extract M1-ScFv bacterial strain plasmids （TAT-4F, TAT-2C bacterial strain plasmid）, PCR is carried out as template（Fig. 6）.

P5: 5` GTGAATTCATGAAATACCTATTGCCT 3`

P6: 5` GCAAGCTTCTATGCGGCCCCATTCAG 3`

The amplified production that above-mentioned PCR reactions are recycled using gel electrophoresis, uses EcoR I and III double digestion PCR amplifications of Hind to return respectively Receive product and carrier pET28a-TAT-GFP, T4 ligase connection PCR product and carrier segments, transformed competence colibacillus Escherichia coli DH5 α, PCR identify that positive colony, extraction PCR identify correct recombinant plasmid sequencing, the results showed that：TAT-M1 ScFv segments with Correct reading frame is cloned into expression vector pET-28a.

The PET28a-TAT-M1 ScFv Calcium Chloride Methods of structure are transformed into BL21 (DE3), picking single bacterium colony is inoculated into Shaken cultivation adds IPTG induced expressions to 0.5 or more bacterium solution OD600 ≈ in LB liquid medium.Induction thalline is collected, is carried out 12%SDS-PAGE is detected, not induce engineering bacteria as negative control, the results show that occurring an apparent table at about 30KDa Up to band, it is completely the same to be expected size with fusion protein（Fig. 7）.

The purifying of TAT-M1 ScFv takes its supernatant after the cracking of induced expression thalline ultrasound, crosses metal-chelating Cu²⁺Column delays Flushing system is PBS (pH7.2), is eluted respectively with 20mM, 200mM imidazoles, destination protein is in 200 mM imidazoles eluting peaks.200 MM imidazole elutions cross affinity column（rProteinA FF）, elution samples are dialysed with PBS, that is, obtain the TAT-M1 of purifying ScFv（Fig. 8）.

Embodiment 6：The bioactivity of TAT-M1 ScFv detects

Postdigestive mdck cell is spread into 96 porocyte culture plates (3 × 10⁴A cells/well), wait for that cell grows up to single layer suction and abandons training Base is supported, cell is washed 3 times with DMEM, is separately added into the H3N2 viruses of different dilutions：50 TCID50,100 TCID50,150 TCID50,200 TCID50,250 TCID50,300 TCID50,350 TCID50,400 TCID50, negative control hole are added PBS, 37 DEG C of incubation 3.5h, abandons extracellular fluid, PBS washes cell 2 times, is separately added into TAT-2C, TAT-4F (10.8 μ g/ of purifying Hole), positive control adds PBS, 37 DEG C of effect 1.5h, suction to abandon extracellular fluid, and DMEM (containing 2%FBS), 37 DEG C of trainings overnight are added per hole It supports.Next day does hemagglutination test with overnight culture supernatant.

Hemagglutination test：Culture supernatant is added in reaction plate, 50 holes μ L/, each sample does multiple holes, then is added into every hole 0.85% chicken erythrocyte suspension, 50 holes μ L/, are placed at room temperature for 30min, reaction plate are uprightly observed result.

As a result H3N2 virus activities can be neutralized by showing shuttle intracellular antibody, in TAT-2C, TAT-4F and the effect of H3N2 viruses Valence is respectively 250TCID50,200TCID500（It is shown in Table 1）.

1 hemagglutination test result table of table

* the TCID50 of H3N2 is 10^-4.5/0.1mL。

Sequence table

<110>Military medical research institute of Academy of Military Sciences military affairs veterinary institute

<120>H3N2 type canine influenza virus shuttle intracellular antibodies TAT-2C

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 906

<212> DNA

<213>Dog (Canis lupus familiaris)

<400> 1

tatggtcgta aaaaacgtcg tcagcgtcgt cgtgaattca tgaaatacct attgcctacg 60

gcagccgctg gattgttatt actcgcggcc cagccggcca tggccgaggt gcagctgttg 120

gagtctgggg gaggcttggt acagcctggg gggtccctga gactctcctg tgcagcctct 180

ggattcacct ttagcagcta tgccatgagc tgggtccgcc aggctccagg gaaggggctg 240

gagtgggtct cagatattag taagtctggt tctaagacat cgtacgcaga ctccgtgaag 300

ggccggttca ccatctccag agacaattcc aagaacacgc tgtatctgca aatgaacagc 360

ctgagagccg aggacacggc cgtatattac tgtgcggaaa tgccttctgt ttttgactac 420

tggggccagg gaaccctggt caccgtctcg agcggtggag gcggttcagg cggaggtggc 480

agcggcggtg gcgggtcgac ggacatccag atgacccagt ctccatcctc cctgtctgca 540

tctgtaggag acagagtcac catcacttgc cgggcaagtc agagcattag cagctattta 600

aattggtatc agcagaaacc agggaaagcc cctaagctcc tgatctatga ggcatccaag 660

ttgcaaagtg gggtcccatc aaggttcagt ggcagtggat ctgggacaga tttcactctc 720

accatcagca gtctgcaacc tgaagatttt gcaacttact actgtcaaca gctgaatcat 780

cggcctcaga cgttcggcca agggaccaag gtggaaatca aacgggcggc cgcacatcat 840

catcaccatc acggggccgc agaacaaaaa ctcatctcag aagaggatct gaatggggcc 900

gcatag 906

<210> 2

<211> 906

<212> DNA

<213>Dog (Canis lupus familiaris)

<400> 2

tatggtcgta aaaaacgtcg tcagcgtcgt cgtgaattca tgaaatacct attgcctacg 60

gcagccgctg gattgttatt actcgcggcc cagccggcca tggccgaggt gcagctgttg 120

gagtctgggg gaggcttggt acagcctggg gggtccctga gactctcctg tgcagcctct 180

ggattcacct ttagcagcta tgccatgagc tgggtccgcc aggctccagg gaaggggctg 240

gagtgggtct caggtattaa tagtacgggt aagctgacaa agtacgcaga ctccgtgaag 300

ggccggttca ccatctccag agacaattcc aagaacacgc tgtatctgca aatgaacagc 360

ctgagagccg aggacacggc cgtatattac tgtgcgaaaa ggaggcttct gtttgactac 420

tggggccagg gaaccctggt caccgtctcg agcggtggag gcggttcagg cggaggtggc 480

agcggcggtg gcgggtcgac ggacatccag atgacccagt ctccatcctc cctgtctgca 540

tctgtaggag acagagtcac catcacttgc cgggcaagtc agagcactag cagctattta 600

aattggtatc agcagaaacc agggaaagcc cctaagctcc tgatctataa ggcatcctac 660

ttgcaaagtg gggtcccatc aaggttcagt ggcagtggat ctgggacaga tttcactctc 720

accatcagca gtctgcaacc tgaagatttt gcaacttact actgtcaaca gcggtataat 780

tctcctgcta cgttcggcca aagggaccaa agtggaaatc aaacggcggc cgcacatcat 840

catcaccatc acggggccgc agaacaaaaa ctcatctcag aagaggatct gaatggggcc 900

gcatag 906

<210> 3

<211> 301

<212> PRT

<213>Dog (Canis lupus familiaris)

<400> 3

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Phe Met Lys Tyr

1 5 10 15

Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro

20 25 30

Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

35 40 45

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

50 55 60

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

65 70 75 80

Glu Trp Val Ser Asp Ile Ser Lys Ser Gly Ser Lys Thr Ser Tyr Ala

85 90 95

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

100 105 110

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

115 120 125

Tyr Tyr Cys Ala Glu Met Pro Ser Val Phe Asp Tyr Trp Gly Gln Gly

130 135 140

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser

165 170 175

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

180 185 190

Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

195 200 205

Lys Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys Leu Gln Ser Gly

210 215 220

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

225 230 235 240

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

245 250 255

Gln Leu Asn His Arg Pro Gln Thr Phe Gly Gln Gly Thr Lys Val Glu

260 265 270

Ile Lys Arg Ala Ala Ala His His His His His His Gly Ala Ala Glu

275 280 285

Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala

290 295 300

<210> 4

<211> 301

<212> PRT

<213>Dog (Canis lupus familiaris)

<400> 4

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Phe Met Lys Tyr

1 5 10 15

Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro

20 25 30

Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

35 40 45

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

50 55 60

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

65 70 75 80

Glu Trp Val Ser Gly Ile Asn Ser Thr Gly Lys Leu Thr Lys Tyr Ala

85 90 95

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

100 105 110

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

115 120 125

Tyr Tyr Cys Ala Lys Arg Arg Leu Leu Phe Asp Tyr Trp Gly Gln Gly

130 135 140

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser

165 170 175

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

180 185 190

Ser Gln Ser Thr Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

195 200 205

Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Tyr Leu Gln Ser Gly

210 215 220

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

225 230 235 240

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

245 250 255

Gln Arg Tyr Asn Ser Pro Ala Thr Phe Gly Gln Arg Asp Gln Ser Gly

260 265 270

Asn Gln Thr Ala Ala Ala His His His His His His Gly Ala Ala Glu

275 280 285

Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala

290 295 300

Claims

1.H3N2 types canine influenza virus shuttle intracellular antibody TAT-2C, its nucleotide sequence such as sequence table SEQ ID NO.1.

2.H3N2 type canine influenza virus shuttle intracellular antibody TAT-2C, its amino acid sequence such as 3 institutes of sequence table SEQ ID NO. Show.

3. H3N2 types canine influenza virus shuttle intracellular antibody TAT-2C described in claim 1 is preparing treatment H3N2 type dog influenzas Application in terms of medicine.

The diagnostic kit of 4.H3N2 type canine influenza virus, it includes albumen shown in SEQID NO. 3.