CN109651510A - Anti- Eno1 antibody and application thereof - Google Patents

Anti- Eno1 antibody and application thereof Download PDF

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CN109651510A
CN109651510A CN201811471173.6A CN201811471173A CN109651510A CN 109651510 A CN109651510 A CN 109651510A CN 201811471173 A CN201811471173 A CN 201811471173A CN 109651510 A CN109651510 A CN 109651510A
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antibody
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CN109651510B (en
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陈思敏
邹最
郭诗雨
田复波
邹泽强
安毛毛
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Shanghai Changzheng Hospital
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Shanghai Jiapi Pharmaceutical Technology Co Ltd
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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Abstract

The invention discloses anti-Eno1 antibody and application thereof, are related to antibody technique field.A kind of specific binding fungi Eno1 protein antibodies or its segment, the antibody or its segment have specific binding affinity to fungi Eno1 albumen；The antibody includes heavy chain variable region (VH) and light chain variable region (VL)；Wherein, the heavy chain variable region (VH) is combined comprising CDR below: VH-CDR1, VH-CDR2 and VH-CDR3；The light chain variable region (VL) is combined comprising CDR below: VL-CDR1, VL-CDR2 and VL-CDR3.Antibody provided by the invention has with pathomycete Eno1 protein binding and inhibits the ability of fungi attack to host cell, can combine individually or with existing antimycotic chemicals for treating invasive infections with fungi.

Description

Anti- Eno1 antibody and application thereof
Technical field
The present invention relates to antibody technique fields, and in particular to a kind of anti-Eno1 antibody.
Background technique
Fungi enolase1 (enolase, Eno1) is also known as 2- phosphoric acid-D- glycerol salt hydrolysis enzyme, by 440 amino acid groups At the basic function of enolase is to participate in glycolysis reaction, can be catalyzed 2-phosphoglyceric acid (2-PGE) and phosphoenolpyruvate third Ketone mutually converts.Recent study discovery, invasive fungi enolase are also distributed in invasion other than being distributed in cytoplasm Fungal cell's wall surface.Eno1 molecule can be by activating fibrinolytic system to infect host in conjunction with plasminogen.Fibrinolysin Original is the precursor of fibrin lyase, is present in most vertebrate bodies, is the key component of fibrinolytic system.Fibrinolysin Original can cut N-terminal portion sequence under the action of fibrinolysin original activator protein (plasminogen activator), conversion For the fibrinolysin (plasmin) with serine protease, it is clostridiopetidase A that it, which can convert collagen proenzyme, then therewith Fibrin degradation and other extracellular matrixs such as laminin and fibronectin together.The enol on invasive fungi surface After changing enzyme combination fibrinolysin original, its activation can be accelerated to be fibrinolysin and cause a series of downstream reactions, invaded in this way Property fungi can be degraded its tissue barrier using host's fibrinolytic system, infected or diffusion mobility with carrying out tissue.Inhibit fungi Cell surface Eno1 molecular activity can effectively inhibit invasion of the fungi to host.It has been reported that immune candida albicans The rabbit anteserum of Eno1 albumen is able to suppress adherency of the candida albicans to people's intestinal epithelial cell, prompts Eno1 albumen in candida albicans It played an important role in adherency host epithelial cells.Mouse immune candida albicans Eno1 albumen infects candida albicans again When, mouse kidney, brain tissue, lung, spleen carry bacterium amount and substantially reduce.The adaptive immune response that this phenomenon may be mediated with Th1 It is related.In conclusion fungi Eno1 albumen is highly conserved into the cell in pathomycete, biological function it is important (with fungi virulence, Stress reaction etc. is closely related)；The interior positioning of fungal cell is different from mammalian cell, and (mammalian cell Eno1 is located at cell In slurry；Fungal cell Eno1 is located at cell wall), it will not be right based on fungal cell's wall surface Eno1 protein design antibody drug Human body cell Eno1 albumen impacts, and will not be replaced that (small molecule compound can pass through cell by small molecule compound drug Film influences human body cell Eno1 protein function)；Fungi Eno1 protein structure can be in infected patient Immune inducing in vivo protection antibody It generates.So Eno1 albumen is the ideal action target spot of anti-fungal infection antibody drug.
In recent years, with the development of therapeutic treatment technology, such as hematopoietic stem cell transplantation, solid organ transplantation, high intensity Immunosuppressor, broad-spectrum antibiotic, various tube indwelling technologies be widely used and intensive care patient, aged patient, AIDS Patient's increases so that invasive infections with fungi disease incidence increases year by year.Invasive infections with fungi is in China, the U.S. and European institute Interior infection occupies 4-6.Since invasive fungi has high degree of adaptability, it is transformed into mycelia in vivo, forms biofilm Afterwards also can height drug resistance, in addition the diagnostic method of the research and development of novel antifungal drugs and related early stage are made slow progress, so that invasion Property fungal infection case fatality rate is up to 40%, causes to seriously threaten to human health.The case fatality rate of candidemia is up to 40%, invades The case fatality rate that attacking property aspergillin infection fails timely diagnosis and treatment is up to 90% or more, has become the infection for seriously threatening human health Property disease.
Current clinically alternative antifungal drug is very limited, using it is more be still triazole antifungal agent object Such as Fluconazole, Itraconazole, voriconazole, with prolonged application clinically, antifungal agent resistance phenomenon is on the rise；Some The congenital drug resistance such as fungi such as candida krusei, aspergillus fumigatus；Fungi has high-adaptability feature, forms biofilm in vivo, turns Become after mycelia also can height drug resistance, or even there is the prevalence of " super drug resistance " fungi, drug resistance and also become invasive fungi One of the main reason for treatment of infection fails.So studying efficient novel antifungal drugs is clinically urgently to be resolved ask Topic.There has been no industrialization for the monoclonal antibody of targeting fungi Eno1 albumen exploitation at present.
Summary of the invention
It is an object of the invention to: overcome the deficiencies of the prior art and provide a kind of specific binding fungi Eno1 albumen Antibody or its function fragment, and its purposes is provided based on the antibody or its function fragment.
To realize above-mentioned target, the present invention provides the following technical scheme that
A kind of specific binding fungi Eno1 protein antibodies or its segment, the antibody or its segment are to fungi Eno1 albumen With specific binding affinity；
The antibody includes heavy chain variable region (VH) and light chain variable region (VL)；
Wherein, the heavy chain variable region (VH) is combined comprising CDR below: VH-CDR1, VH-CDR2 and VH-CDR3；
The light chain variable region (VL) is combined comprising CDR below: VL-CDR1, VL-CDR2 and VL-CDR3；
The heavy chain variable region are as follows:
VH-CDR1 amino acid: GFNIKDYY,
VH-CDR2 amino acid: IDPENGNN,
VH-CDR3 amino acid: ARYEGNYVSY,
The light chain variable region are as follows:
VL-CDR1 amino acid: QSIVHSNGYTY,
VL-CDR2 amino acid: KVS,
VL-CDR3 amino acid: FQSSHVPYT.
Further, the heavy chain variable region (VH) includes sequence selected from the following: with amino acid shown in SEQ ID NO:1 Sequence has the amino acid sequence of at least 75% identity；Preferably, at least 75% identity be for example, at least 80%, it is excellent Choosing at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, The identity of any percentage of 98% or even 99% identity etc. >=75%.
And/or the light chain variable region (VL) includes sequence selected from the following: with amino acid shown in SEQ ID NO:2 Sequence has the amino acid sequence of at least 75% identity.Preferably, at least 75% identity be for example, at least 80%, it is excellent Choosing at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, The identity of any percentage of 98% or even 99% identity etc. >=75%.
Further, the antibody or its segment include heavy chain variable region selected from the following (VH) and light chain variable region (VL) Combination:
The amino acid sequence as shown in SEQ ID NO:1 has at least with amino acid sequence shown in SEQ ID NO:1 The heavy chain variable region of the amino acid sequence of 75% identity；
Have the light chain of at least amino acid sequence of 75% identity can with amino acid sequence shown in SEQ ID NO:2 Become area.
Further, the antibody be monoclonal antibody, single-chain antibody, single domain antibody, complete or partial humanized antibody or Any one in person's chimeric antibody；Preferably, the antibody is IgA, IgD, IgE, IgG or IgM, more preferably IgG1.
The segment is selected from scFv, Fab, F (ab ') of the antibody2Or Fv segment.
On the other hand, the present invention also provides a kind of nucleic acid molecules, codings such as the heavy chain in afore mentioned antibodies or its segment CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain or light chain.
On the other hand, the present invention also provides a kind of conjugate, the conjugate includes antibody above-mentioned or its segment.
On the other hand, the present invention also provides a kind of carriers, and it includes nucleic acid molecules above-mentioned.The carrier can be true Nuclear expression carrier, prokaryotic expression carrier, artificial chromosome or phage vector etc..
On the other hand, the present invention also provides a kind of pharmaceutical compositions, and it includes antibody above-mentioned or its segment, Huo Zhehe Acid molecule perhaps conjugate or carrier；And optional pharmaceutically acceptable auxiliary material.
On the other hand, the present invention also provides antibody above-mentioned or its segment perhaps nucleic acid molecules above-mentioned or aforementioned Conjugate perhaps carrier above-mentioned or pharmaceutical composition above-mentioned are preparing the medicine for preventing or treating fungal infection Purposes in object.
On the other hand, the present invention also provides a kind of methods for diagnosing fungal infection, above-mentioned anti-the method includes making Body or its segment perhaps nucleic acid molecules above-mentioned perhaps conjugate above-mentioned perhaps carrier above-mentioned or drug above-mentioned Composition is in contact with the sample from subject.
The fungal infection is skin and soft tissue infection, deep fungal infection or invasive infections with fungi.
Preferably, the fungi is the disease fungus that can cause mammal such as human infection, preferably Mycotoruloides, Cryptococcus and mould category fungi, such as candida albicans.
Preferably, other antifungal drugs such as triazole antifungal agent object (such as Fluconazole, Yi Qukang be can be combined with Azoles, voriconazole, posaconazole, Chinese mugwort Saperconazole etc.), echinocandin antifungal agent object (such as Caspofungin, anidulafungin, Mikafen etc.), polyene antifungal medicine (such as anphotericin etc.), Allylamines antifungal drug (such as Terbinafine etc.) Or miazines antifungal drug (such as 5-flurocytosine etc.) makes composition of medicine.
The present invention due to using the technology described above, compared with prior art, as an example, has the following advantages that and accumulates Pole effect: the antibody has specific binding affinity to fungi Eno1 albumen, therefore has and pathomycete Eno1 albumen In conjunction with and inhibit the ability of fungi attack to host cell, can combine individually or with existing antimycotic chemicals for treating Invasive infections with fungi.
Invasive infections with fungi is mostly immuuoeorapromised host, and the present invention is as monoclonal antibody drug, from immune The angle of adjusting treats fungal infection, has unique advantage.Eno1 is predominantly located on fungal cell wall simultaneously, and mammal is thin Born of the same parents do not have cell wall structure, and cell membrane surface does not have homologous protein, and Eno1 monoclonal antibody drug is predominantly targeting in fungi On cell wall antigen, possibility of missing the target is extremely low, and potential toxic side effect is small.
Detailed description of the invention
Fig. 1 is the recombination table of the candida albicans Eno1 full length protein sequence of fusion His label provided in an embodiment of the present invention It reaches.
Fig. 2 is the purifying of recombination candida albicans Eno1 albumen provided in an embodiment of the present invention.
Fig. 3 is the result point of the combination provided in an embodiment of the present invention that antibody and Eno1 albumen are detected by ELISA method Analysis figure.
Fig. 4 is that Eno1 antibody provided in an embodiment of the present invention inhibits candida albicans Eno1 protein active experimental result.
Fig. 5 is candidemia animal model replication provided in an embodiment of the present invention and mab treatment effect detection As a result.
Specific embodiment
Anti- Eno1 antibody disclosed by the invention and application thereof is made below in conjunction with the drawings and specific embodiments further detailed Explanation.It should be noted that the combination of technical characteristic described in following embodiments or technical characteristic is not construed as Isolated, they can be combined with each other to reach superior technique effect.In the drawings of the following embodiments, each attached drawing institute The identical label occurred represents identical feature or component, can be apply to different embodiments.Therefore, once a certain Xiang Yi It is defined in a attached drawing, then in subsequent attached drawing does not need that it is further discussed.
It should be noted that may not make in detail for technology, method and apparatus known to person of ordinary skill in the relevant It discusses, but in the appropriate case, the technology, method and apparatus should be considered as authorizing part of specification.Show herein Out and in all examples of discussion, any occurrence should be construed as merely illustratively, not as limitation.Therefore, The other examples of exemplary embodiment can have different values.
Embodiment 1: the recombinant expression of the candida albicans Eno1 PROTEIN C terminal sequence of fusion His label
Using candida albicans Eno1 PROTEIN C end amino acid as purpose sequence, artificial synthesized corresponding base sequence, and utilize Restriction enzyme site NdeI and XhoI are cloned into the Pet-21a plasmid of the label containing His.Recombinant plasmid transformed is thin to competence In born of the same parents BL21 (DE3) pLysS, Yu Ci picking single colonie is seeded in the LB liquid medium containing 100 ampicillins μ g/ml, 37 DEG C of shaken cultivations are stayed overnight.It is incubated overnight bacterium solution and is seeded in the LB liquid medium containing 100 ampicillins μ g/ml by 1: 100, 200rpm is in 37 DEG C of shaken cultivations to OD600About 0.6-0.8 IPTG to final concentration of 0.5mM is added into bacterium solution, in 37 DEG C Induce 4.5h.Bacterium solution after inducing is taken, 8,000rpm centrifugation 3min collect thallus, -80 DEG C of preservations.Merge the white beads of His label The recombinant expression of bacterium Eno1 full length protein sequence is shown in Figure 1.
Eno1 full length protein amino acid sequence:
Express the base sequence of candida albicans Eno1 full length protein:
Embodiment 2: the purifying of recombination candida albicans Eno1 albumen
The Escherichia coli of inducing expression recombination candida albicans Eno1 albumen are crushed with Ultrasonic Cell Disruptor, 180W works between 3s Have a rest 3s, time 7-9min；13,000rpm centrifugation 30min, collect supernatant, with 0.22 μm of L filter filtration sterilization；At room temperature will Ni column, in mixing 1h on rotary mixer, Ni column is loaded into filled column with supernatant；With the BD liquid elution of 5 times of bed volumes (contain imidazole concentration 30mM) and Ni column non-specific binding albumen, it is non-discolouring to be washed till albumen developing solution, then with 5 times of bed volumes BB liquid (contain imidazole concentration 300mM) elution destination protein；Eluent containing destination protein is concentrated using the concentration tube of 10KD Displacement solvent is PBS buffer solution.Electrophoretogram is shown in Figure 2.
Embodiment 3: recombination candida albicans Eno1 protein immunization Balb/c mouse
With reference to Antibodies a Laboratory Manual, Second Edition (Edward A.Greenfield 2012) 8 week old Balb/c mouse, were immunized for the total 42 days processes in interval with 14 days.By immunogene candida albicans Eno1 albumen It is emulsified in completely or in incomplete Freund's adjuvant, and it is injected in mouse the nape of the neck, root of the tail portion, abdomen stock in a manner of unilateral At ditch 3 in subcutaneous tissue and cavum peritoneale.It takes and exempts from after detecting antibody titer with ELISA method in immune 35th day tail vein blood Epidemic disease mouse boosting cell is merged with myeloma cell.
Embodiment 4: screening, identification and the antibody sequence measurement of anti-Eno1 protein monoclonal antibody hybridoma cell strain
Take recombination candida albicans Eno1 protein immunization Balb/c Mouse spleen cells and myeloma cell P3X63Ag8.653 It is merged using PEG or electro' asion method, fused hybridoma is inoculated in 96 orifice plates, be added after 24 hours The culture medium of culture medium containing HAT and HT screens hybridoma；After cultivating 10-14 days in 96 orifice plates, cell conditioned medium is taken to carry out ELISA experiment, screening can secrete the hybridoma mother clone of anti-Eno1 antibody.
The hybridoma mother for secreting anti-Eno1 antibody clone is added to 96 orifice plates for being covered with feeder cells using limiting dilution assay In, the microscopically observation after 2-3 days simultaneously marks monoclonal cell, can be secreted after the 7th day by ELISA experiment screening anti- The monoclonal hybridoma of Eno1 monoclonal antibody.
After the monoclonal hybridoma for secreting anti-Eno1 monoclonal antibody is expanded culture, according to RNAfast200 reagent Box (Shanghai Fei Jie Bioisystech Co., Ltd) specification step extracts cell total rna；Utilize 5 × PrimeScript RT Master Mix (Takara) is by hybridoma total serum IgE reverse transcription at cDNA；Use degenerate primer (Anke Krebber.1997) and Extaq PCR reagent (Takara) expands antibody's light chain variable region IgVL (κ) and heavy chain variable region VHSequence Column；Pcr amplification product is purified using PCR clean-up Gel extraction kit (Macherey-Nagel company)； PCR product will be expanded according to pClone007SimpleVector Kit kit (Qing Ke Biotechnology Co., Ltd) specification It is connected to carrier T and converts competent escherichia coli cell, carry out DNA sequencing after bacterial strain amplification, extracting plasmid and obtain monoclonal Antibody variable sequences.
The combination of embodiment 5:ELISA method detection antibody and Eno1 albumen
Fungi Eno1 albumen is diluted to 1 μ g/ml, every 100 μ l of hole with PBS buffer solution and is coated in 96 orifice plate (Microwell 96F 167008, Thermo) 4 DEG C be incubated overnight；Next day takes out 96 orifice plates, with PBST (containing 0.5%PBS) board-washing, infiltrates every time Residual moisture is thoroughly dried after 1min.It is separately added into the PBST containing 5%BSA of 200 μ l in sample well, is placed in 37 DEG C of closing 1h；So PBST board-washing is used afterwards, and dries moisture in hole.4 DEG C of 100 μ l of sample to be tested overnight incubations are added into 96 orifice plates respectively.It takes out 100 μ l of secondary antibody is added with hole every after PBST board-washing after 96 orifice plates, is placed in 37 DEG C of incubation 1h.It is washed 5 times with PBST again, every hole is added 100 μ l Substrate Solution (Invitrogen), in 37 DEG C of incubation 10min；Every hole is added 50 μ l of 2N sulfuric acid and terminates instead Absorbance is detected after answering at microplate reader (Multiskcin FC, Thermo) 450nm wavelength.
Testing result is shown in Figure 3.
Embodiment 6:Eno1 antibody inhibits the experiment of candida albicans Eno1 protein active
1ug Eno1 albumen is mixed with 9ug, 37 degree of incubators is put into and is incubated for 10 minutes.It is separately added into kit reagent (Sigma company)；2 μ l, Enolase Converter of Enolase Substrate Mix, 2 μ l, Enolase Developer 2 μ l Peroxidase Substrate, 2 μ l supplies reaction system to 50 μ l with Enolase Assay Buffer.After mixing Sample be put into 37 degree of incubation 2h, with multi-function microplate reader read 570nm absorption photometric value.
Experimental result is shown in Figure 4.
Embodiment 7: candidemia animal model replication and mab treatment effect detection will cultivate at 12 hours It is resuspended in PBS buffer solution, and adjust bacterial concentration after PBS buffer solution is washed 3 times in logarithmic growth phase candida albicans.Using The mode of tail vein injection infects C57BL/6 mouse.Model mice is randomly divided into model group, antimycotic small molecule according to weight Medication therapy groups, monoclonal antibody drug treatment group, antimycotic small-molecule drug+monoclonal antibody drug treatment group；Infection is read After pearl bacterium 2 hours, groups of animals is given relative medicine by tail vein and is treated, and model group gives the PBS buffer solution of equal volume. Execution mouse takes kidney, weighing, is homogenized, is coated on after experimental animal perhaps observes and records life span or infection 48h daily Tissue is detected on SDA solid medium carries bacterium amount.
Testing result is shown in Figure 5.
Other embodiments
In the above description, the disclosure is not intended to for its own to be limited to these aspects.But at this Within the scope of the protection of goal of disclosure, each component can selectively and operatively be merged with arbitrary number.In addition, As the term of " comprising " and " having " should default being interpreted as including property or open, rather than exclusive or envelope Closing property, unless it is explicitly defined as opposite meaning.All technologies, science and technology or otherwise term all meet this field skill The meaning that art personnel are understood, unless it is defined as opposite meaning.The public term found in dictionary should be in correlation It is not idealized very much or is impractically explained very much under the background of technical documentation, unless present disclosure is clearly defined as that Sample.Any change, the modification that the those of ordinary skill in field of the present invention does according to the disclosure above content, belong to claims Protection scope.
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Pro Leu Tyr Lys His Ile Ala Asn Ile Ser Asn Ala Lys Lys Gly Lys
130 135 140
Phe Val Leu Pro Val Pro Phe Gln Asn Val Leu Asn Gly Gly Ser His
145 150 155 160
Ala Gly Gly Ala Leu Ala Phe Gln Glu Phe Met Ile Ala Pro Thr Gly
165 170 175
Val Ser Thr Phe Ser Glu Ala Leu Arg Ile Gly Ser Glu Val Tyr His
180 185 190
Asn Leu Lys Ser Leu Thr Lys Lys Lys Tyr Gly Gln Ser Ala Gly Asn
195 200 205
Val Gly Asp Glu Gly Gly Val Ala Pro Asp Ile Lys Thr Pro Lys Glu
210 215 220
Ala Leu Asp Leu Ile Met Asp Ala Ile Asp Lys Ala Gly Tyr Lys Gly
225 230 235 240
Lys Val Gly Ile Ala Met Asp Val Ala Ser Ser Glu Phe Tyr Lys Asp
245 250 255
Gly Lys Tyr Asp Leu Asp Phe Lys Asn Pro Glu Ser Asp Pro Ser Lys
260 265 270
Trp Leu Ser Gly Pro Gln Leu Ala Asp Leu Tyr Glu Gln Leu Ile Ser
275 280 285
Glu Tyr Pro Ile Val Ser Ile Glu Asp Pro Phe Ala Glu Asp Asp Trp
290 295 300
Asp Ala Trp Val His Phe Phe Glu Arg Val Gly Asp Lys Ile Gln Ile
305 310 315 320
Val Gly Asp Asp Leu Thr Val Thr Asn Pro Thr Arg Ile Lys Thr Ala
325 330 335
Ile Glu Lys Lys Ala Ala Asn Ala Leu Leu Leu Lys Val Asn Gln Ile
340 345 350
Gly Thr Leu Thr Glu Ser Ile Gln Ala Ala Asn Asp Ser Tyr Ala Ala
355 360 365
Gly Trp Gly Val Met Val Ser His Arg Ser Gly Glu Thr Glu Asp Thr
370 375 380
Phe Ile Ala Asp Leu Ser Val Gly Leu Arg Ser Gly Gln Ile Lys Thr
385 390 395 400
Gly Ala Pro Ala Arg Ser Glu Arg Leu Ala Lys Leu Asn Gln Ile Leu
405 410 415
Arg Ile Glu Glu Glu Leu Gly Ser Glu Ala Ile Tyr Ala Gly Lys Asp
420 425 430
Phe Gln Lys Ala Ser Gln Leu
435
<210> 4
<211> 1332
<212> PRT
<213>artificial synthesized (the candida albicans Eno1 albumen of fusion His label)
<400> 4
Gly Gly Ala Thr Cys Cys Ala Thr Gly Thr Cys Thr Thr Ala Cys Gly
1 5 10 15
Cys Cys Ala Cys Thr Ala Ala Ala Ala Thr Cys Cys Ala Cys Gly Cys
20 25 30
Cys Ala Gly Ala Thr Ala Cys Gly Thr Cys Thr Ala Cys Gly Ala Cys
35 40 45
Thr Cys Cys Ala Gly Ala Gly Gly Thr Ala Ala Cys Cys Cys Ala Ala
50 55 60
Cys Cys Gly Thr Thr Gly Ala Ala Gly Thr Thr Gly Ala Thr Thr Thr
65 70 75 80
Cys Ala Cys Cys Ala Cys Cys Gly Ala Cys Ala Ala Ala Gly Gly Thr
85 90 95
Thr Thr Ala Thr Thr Cys Ala Gly Ala Thr Cys Ala Ala Thr Thr Gly
100 105 110
Thr Cys Cys Cys Ala Thr Cys Thr Gly Gly Thr Gly Cys Cys Thr Cys
115 120 125
Thr Ala Cys Thr Gly Gly Thr Gly Thr Cys Cys Ala Cys Gly Ala Ala
130 135 140
Gly Cys Thr Thr Thr Gly Gly Ala Ala Thr Thr Gly Ala Gly Ala Gly
145 150 155 160
Ala Thr Gly Gly Thr Gly Ala Cys Ala Ala Ala Thr Cys Cys Ala Ala
165 170 175
Ala Thr Gly Gly Thr Thr Ala Gly Gly Thr Ala Ala Ala Gly Gly Thr
180 185 190
Gly Thr Thr Thr Thr Gly Ala Ala Ala Gly Cys Cys Gly Thr Thr Gly
195 200 205
Cys Cys Ala Ala Thr Gly Thr Thr Ala Ala Thr Gly Ala Cys Ala Thr
210 215 220
Cys Ala Thr Thr Gly Cys Cys Cys Cys Ala Gly Cys Thr Thr Thr Ala
225 230 235 240
Ala Thr Ala Ala Ala Ala Gly Cys Cys Ala Ala Gly Ala Thr Cys Gly
245 250 255
Ala Thr Gly Thr Thr Gly Thr Cys Gly Ala Cys Cys Ala Ala Gly Cys
260 265 270
Thr Ala Ala Gly Ala Thr Thr Gly Ala Thr Gly Ala Ala Thr Thr Cys
275 280 285
Thr Thr Gly Thr Thr Gly Thr Cys Cys Thr Thr Gly Gly Ala Cys Gly
290 295 300
Gly Thr Ala Cys Thr Cys Cys Ala Ala Ala Cys Ala Ala Ala Thr Cys
305 310 315 320
Cys Ala Ala Ala Thr Thr Gly Gly Gly Thr Gly Cys Cys Ala Ala Thr
325 330 335
Gly Cys Thr Ala Thr Cys Thr Thr Gly Gly Gly Thr Gly Thr Thr Thr
340 345 350
Cys Thr Thr Thr Gly Gly Cys Thr Gly Cys Thr Gly Cys Cys Ala Ala
355 360 365
Thr Gly Cys Thr Gly Cys Cys Gly Cys Thr Gly Cys Thr Gly Cys Thr
370 375 380
Cys Ala Ala Gly Gly Cys Ala Thr Thr Cys Cys Ala Thr Thr Gly Thr
385 390 395 400
Ala Cys Ala Ala Ala Cys Ala Cys Ala Thr Thr Gly Cys Cys Ala Ala
405 410 415
Cys Ala Thr Thr Thr Cys Cys Ala Ala Thr Gly Cys Cys Ala Ala Gly
420 425 430
Ala Ala Ala Gly Gly Thr Ala Ala Ala Thr Thr Cys Gly Thr Thr Thr
435 440 445
Thr Gly Cys Cys Ala Gly Thr Thr Cys Cys Ala Thr Thr Cys Cys Ala
450 455 460
Ala Ala Ala Cys Gly Thr Thr Thr Thr Gly Ala Ala Cys Gly Gly Thr
465 470 475 480
Gly Gly Thr Thr Cys Cys Cys Ala Thr Gly Cys Thr Gly Gly Thr Gly
485 490 495
Gly Thr Gly Cys Thr Thr Thr Ala Gly Cys Thr Thr Thr Cys Cys Ala
500 505 510
Ala Gly Ala Ala Thr Thr Thr Ala Thr Gly Ala Thr Thr Gly Cys Cys
515 520 525
Cys Cys Ala Ala Cys Thr Gly Gly Thr Gly Thr Cys Thr Cys Cys Ala
530 535 540
Cys Thr Thr Thr Cys Thr Cys Thr Gly Ala Ala Gly Cys Thr Thr Thr
545 550 555 560
Gly Ala Gly Ala Ala Thr Thr Gly Gly Thr Thr Cys Ala Gly Ala Ala
565 570 575
Gly Thr Thr Thr Ala Cys Cys Ala Cys Ala Ala Cys Thr Thr Gly Ala
580 585 590
Ala Ala Thr Cys Thr Thr Thr Gly Ala Cys Cys Ala Ala Gly Ala Ala
595 600 605
Gly Ala Ala Ala Thr Ala Cys Gly Gly Thr Cys Ala Ala Thr Cys Cys
610 615 620
Gly Cys Thr Gly Gly Thr Ala Ala Cys Gly Thr Cys Gly Gly Thr Gly
625 630 635 640
Ala Cys Gly Ala Ala Gly Gly Thr Gly Gly Thr Gly Thr Thr Gly Cys
645 650 655
Thr Cys Cys Ala Gly Ala Thr Ala Thr Cys Ala Ala Ala Ala Cys Thr
660 665 670
Cys Cys Ala Ala Ala Gly Gly Ala Ala Gly Cys Thr Thr Thr Gly Gly
675 680 685
Ala Cys Thr Thr Gly Ala Thr Cys Ala Thr Gly Gly Ala Thr Gly Cys
690 695 700
Cys Ala Thr Thr Gly Ala Cys Ala Ala Ala Gly Cys Cys Gly Gly Thr
705 710 715 720
Thr Ala Cys Ala Ala Ala Gly Gly Thr Ala Ala Gly Gly Thr Thr Gly
725 730 735
Gly Thr Ala Thr Thr Gly Cys Cys Ala Thr Gly Gly Ala Thr Gly Thr
740 745 750
Thr Gly Cys Thr Thr Cys Ala Thr Cys Thr Gly Ala Ala Thr Thr Cys
755 760 765
Thr Ala Cys Ala Ala Gly Gly Ala Cys Gly Gly Thr Ala Ala Ala Thr
770 775 780
Ala Cys Gly Ala Cys Thr Thr Gly Gly Ala Cys Thr Thr Thr Ala Ala
785 790 795 800
Ala Ala Ala Cys Cys Cys Ala Gly Ala Ala Thr Cys Cys Gly Ala Cys
805 810 815
Cys Cys Ala Thr Cys Thr Ala Ala Ala Thr Gly Gly Thr Thr Gly Thr
820 825 830
Cys Thr Gly Gly Cys Cys Cys Ala Cys Ala Ala Thr Thr Gly Gly Cys
835 840 845
Thr Gly Ala Cys Thr Thr Ala Thr Ala Thr Gly Ala Ala Cys Ala Ala
850 855 860
Thr Thr Gly Ala Thr Thr Thr Cys Cys Gly Ala Ala Thr Ala Cys Cys
865 870 875 880
Cys Ala Ala Thr Thr Gly Thr Thr Thr Cys Thr Ala Thr Thr Gly Ala
885 890 895
Ala Gly Ala Thr Cys Cys Ala Thr Thr Cys Gly Cys Thr Gly Ala Ala
900 905 910
Gly Ala Thr Gly Ala Cys Thr Gly Gly Gly Ala Thr Gly Cys Thr Thr
915 920 925
Gly Gly Gly Thr Cys Cys Ala Cys Thr Thr Cys Thr Thr Thr Gly Ala
930 935 940
Ala Ala Gly Ala Gly Thr Thr Gly Gly Thr Gly Ala Cys Ala Ala Gly
945 950 955 960
Ala Thr Cys Cys Ala Ala Ala Thr Thr Gly Thr Cys Gly Gly Thr Gly
965 970 975
Ala Thr Gly Ala Thr Thr Thr Gly Ala Cys Thr Gly Thr Cys Ala Cys
980 985 990
Thr Ala Ala Cys Cys Cys Thr Ala Cys Cys Ala Gly Ala Ala Thr Cys
995 1000 1005
Ala Ala Gly Ala Cys Thr Gly Cys Cys Ala Thr Thr Gly Ala Ala Ala
1010 1015 1020
Ala Gly Ala Ala Ala Gly Cys Cys Gly Cys Thr Ala Ala Thr Gly Cys
1025 1030 1035 1040
Thr Thr Thr Gly Thr Thr Gly Thr Thr Gly Ala Ala Gly Gly Thr Thr
1045 1050 1055
Ala Ala Cys Cys Ala Ala Ala Thr Thr Gly Gly Thr Ala Cys Thr Thr
1060 1065 1070
Thr Gly Ala Cys Thr Gly Ala Ala Thr Cys Thr Ala Thr Ala Cys Ala
1075 1080 1085
Ala Gly Cys Thr Gly Cys Thr Ala Ala Cys Gly Ala Thr Thr Cys Thr
1090 1095 1100
Thr Ala Cys Gly Cys Thr Gly Cys Thr Gly Gly Thr Thr Gly Gly Gly
1105 1110 1115 1120
Gly Thr Gly Thr Cys Ala Thr Gly Gly Thr Thr Thr Cys Cys Cys Ala
1125 1130 1135
Cys Ala Gly Ala Thr Cys Cys Gly Gly Thr Gly Ala Ala Ala Cys Cys
1140 1145 1150
Gly Ala Ala Gly Ala Thr Ala Cys Thr Thr Thr Cys Ala Thr Thr Gly
1155 1160 1165
Cys Thr Gly Ala Cys Thr Thr Gly Thr Cys Ala Gly Thr Thr Gly Gly
1170 1175 1180
Thr Thr Thr Ala Ala Gly Ala Thr Cys Thr Gly Gly Thr Cys Ala Ala
1185 1190 1195 1200
Ala Thr Cys Ala Ala Gly Ala Cys Thr Gly Gly Thr Gly Cys Thr Cys
1205 1210 1215
Cys Ala Gly Cys Thr Ala Gly Ala Thr Cys Thr Gly Ala Ala Ala Gly
1220 1225 1230
Ala Thr Thr Gly Gly Cys Cys Ala Ala Ala Thr Thr Gly Ala Ala Cys
1235 1240 1245
Cys Ala Ala Ala Thr Cys Thr Thr Gly Ala Gly Ala Ala Thr Cys Gly
1250 1255 1260
Ala Ala Gly Ala Ala Gly Ala Ala Thr Thr Ala Gly Gly Thr Thr Cys
1265 1270 1275 1280
Thr Gly Ala Ala Gly Cys Thr Ala Thr Cys Thr Ala Cys Gly Cys Thr
1285 1290 1295
Gly Gly Thr Ala Ala Ala Gly Ala Thr Thr Thr Cys Cys Ala Ala Ala
1300 1305 1310
Ala Gly Gly Cys Thr Thr Cys Thr Cys Ala Ala Thr Thr Gly Cys Thr
1315 1320 1325
Cys Gly Ala Gly
1330

Claims (10)

1. a kind of specific binding fungi Eno1 protein antibodies or its segment, the antibody or its segment have fungi Eno1 albumen There is specific binding affinity；
The antibody includes heavy chain variable region (VH) and light chain variable region (VL)；
Wherein, the heavy chain variable region (VH) is combined comprising CDR below: VH-CDR1, VH-CDR2 and VH-CDR3；
The light chain variable region (VL) is combined comprising CDR below: VL-CDR1, VL-CDR2 and VL-CDR3；
The heavy chain variable region are as follows:
EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMNWVKQRPEQGLEWIGWIDPENGNNIYDPKFQGKASITA DKSSNTAYLQLSSLTSEDTAVYYCARYEGNYVSYWAQGTLVTVSA (SEQ ID NO:1),
VH-CDR1 amino acid: GFNIKDYY,
VH-CDR2 amino acid: IDPENGNN,
VH-CDR3 amino acid: ARYEGNYVSY,
The light chain variable region are as follows:
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGYTYLEWYLQKPGQSPKLLMYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYYCFQSSHVPYTFGGGTKLEIK (SEQ ID NO:2),
VL-CDR1 amino acid: QSIVHSNGYTY,
VL-CDR2 amino acid: KVS,
VL-CDR3 amino acid: FQSSHVPYT.
2. antibody according to claim 1 or its segment, it is characterised in that: the heavy chain variable region (VH) include selected from Under sequence: with amino acid sequence shown in SEQ ID NO:1 have at least 75% identity amino acid sequence；
And/or the light chain variable region (VL) includes sequence selected from the following: with amino acid sequence shown in SEQ ID NO:2 Amino acid sequence at least 75% identity.
3. antibody according to claim 1 or its segment, it is characterised in that: the antibody or its segment are comprising selected from following Heavy chain variable region (VH) and light chain variable region (VL) combination:
The amino acid sequence as shown in SEQ ID NO:1 has at least 75% with amino acid sequence shown in SEQ ID NO:1 The heavy chain variable region of the amino acid sequence of identity；
With the light chain variable with amino acid sequence shown in SEQ ID NO:2 at least amino acid sequence of 75% identity Area.
4. antibody according to any one of claim 1 to 3 or its segment, it is characterised in that: the antibody is monoclonal Any one in antibody, single-chain antibody, single domain antibody, complete or partial humanized antibody or chimeric antibody；
The segment is selected from scFv, Fab, F (ab ') of the antibody2Or Fv segment.
5. a kind of nucleic acid molecules, as described in any one of claims 1 to 4 antibody or the heavy chain CDR in its segment, gently are encoded Chain CDR, heavy chain variable region, light chain variable region, heavy chain or light chain.
6. a kind of conjugate, the conjugate includes antibody described in any one of Claims 1-4 or its segment.
7. a kind of carrier, it includes nucleic acid molecules as claimed in claim 5；
The carrier can be carrier for expression of eukaryon, prokaryotic expression carrier, artificial chromosome and phage vector etc..
8. a kind of pharmaceutical composition, it includes antibody according to any one of claims 1 to 4 or its segments, or comprising Nucleic acid molecules as claimed in claim 5 perhaps comprising conjugate as claimed in claim 6 or include such as claim 7 The carrier；
And optional pharmaceutically acceptable auxiliary material.
9. antibody described in any one of Claims 1-4 or its segment perhaps nucleic acid molecules described in claim 5 or Perhaps carrier as claimed in claim 7 or pharmaceutical composition according to any one of claims 8 exist conjugate as claimed in claim 6 Prepare the purposes in the drug for preventing or treating fungal infection.
10. it is a kind of diagnose fungal infection method, the method includes make antibody described in any one of Claims 1-4 or Its segment perhaps nucleic acid molecules described in claim 5 perhaps conjugate as claimed in claim 6 or claim 7 institute The carrier or pharmaceutical composition according to any one of claims 8 stated are in contact with the sample from subject.
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