CN108178799B

CN108178799B - A kind of nano antibody of anti-CA 125 sugar antigen and its application

Info

Publication number: CN108178799B
Application number: CN201810056379.6A
Authority: CN
Inventors: 宋海鹏; 于建立; 刘原源
Original assignee: Shenzhen Creation Nanometer Antibody Technology Co Ltd
Current assignee: Shenzhen Creation Nanometer Antibody Technology Co Ltd
Priority date: 2018-01-20
Filing date: 2018-01-20
Publication date: 2019-10-25
Anticipated expiration: 2038-01-20
Also published as: CN110590952B; CN110590952A; CN110642951A; CN108178799A; CN110642951B

Abstract

The invention discloses a kind of nano antibodies of anti-CA 125 sugar antigen, the nano antibody has unique 3 complementary determining regions CDR1, CDR2, CDR3, and the invention also discloses application of the nano antibody in preparation tumor therapeutic agent and tumour diagnostic reagent.Anti-CA 125 nano antibody provided by the invention has the identification and binding ability of high special to CA125, and has significant ADCC effect to ovarian cancer cell, and the accurately image of tumour can be realized in Mice Body.

Description

A kind of nano antibody of anti-CA 125 sugar antigen and its application

Technical field

The invention discloses a kind of antibody, more specifically, the invention discloses a kind of nano antibodies.

Background technique

CA125 Ovarian Cancer Associated Antigen is found in 1981, is the associated antigen of ovarian epithelial class cancer.By embryonic period, embryonic phase Epithelial cells are not secreted under normal circumstances or are seldom secreted, but when malignant change occurs for ovary, even if not having clinically There is performance or when being pathologically difficult to, CA125 value can also be increased, thus is preferable ovarian cancer diagnosis and screening indexes, and Transfer and prognosis with oophoroma have substantial connection.It is initially to be mediated by Bast etc. by ovary cell line OVCA433 immunogene Mouse monoclonal antibody OC125 response then identifies and confirms its presence.CA125 antigen is the sugar that molecular mass reaches 200ku Albumen has film mating type and two kinds of state properties of sequestered concurrently, be so far, study most comprehensive ovarian cancer serum marker it One.90% advanced ovarian cancer patients serum's CA125 concentration is in raising in various degree.

Although the methods of the operation of ovarian cancer patients first choice or chemotherapy, prognosis is not still very ideal.Tumor recurrence, especially It is intraperitoneal recurrence and chemotherapy resistance, often an important factor for influence prognosis.Currently, the therapeutic scheme that oophoroma is new, special It is not that biological therapy technology is quickly grown, and certain applications are in vitro and In vivo study and clinical test.Oophoroma biology Treatment includes the diversified forms such as cell factor, monoclonal antibody, transgenosis therapy.In all multipaths, Ovarian Cancer Associated Antigen CA125 monoclonal antibody therapy is more noticeable.The technology of CA125 mab treatment oophoroma includes that antigen is anti-at present Nanocrystal composition mediates human body immune response and radioactive element and anti-tumor drug targeting to induce two classes.Antigen antibody complex Mediated immunity is to achieve the purpose that remove lesion, self-regeneration regulation by the antitumor reaction of exactor body endogenous.It is applied to Most representative antibody is anti-CA 125/anti- T cell surface molecular bispecific antibody (BsAb) and mouse list in the technology Clonal antibody B43.13.Anti-CA 125/anti- T cell surface molecular bispecific antibody passes through anti-with ovarian cancer cell surface C A125 Original combines, and then identifies inducing T cell with anti-T cell surface molecular epitope, and the cell toxicant for generating T cell for tumor tissues is made With lesion is killed.In addition to this, which can also excite body itself to generate and maintain the active immunity shape of long period State.At present although anti-CA 125/anti- T cell surface molecular BsAb shows excellent therapeutic effect, but still has more problem urgently It is to be solved, for example detailed vivo pharmacokinetic feature is still not clear；Prejudge its vivo applications dosage and activation T lymph Cytotoxicity degree is more difficult after reaction；The horizontal lower and toxicity of preparation process humanization is more apparent；BsAb, tumour and It is both needed to further increase in terms of the specificity and affinity of T lymphocyte；Antibody Fc fragment function still needs to improve；Antibody tumor group It is not high to knit permeability, thus brings in-vivo tumour and positions not strong, the non-higher technical problem of purpose clearance rate.Mouse Dan Ke Grand antibody B43.13, which then passes through, forms immune complex in conjunction with CA125 antigen, starts classical idiotypic immunity response.It is acted on Mechanism is that source of people anti-mouse antibody activates antiidiotype chain reaction, and then causes the polyclonal antibody humoral immunity for CA125 Response.This immune response therapy tolerance is good, has no adverse reaction or the pause medicining condition such as non-compliant, but can not remain people Body cannot also overcome tumor immune escape mechanism to the sensitive response status of tumour cell.Radioactive element and anti-tumor drug Targeting inductive technology is another CA125 mab treatment technology.After cytoreductive surgery in ovarian cancer, residual tumor Cell is still in intraperitoneal plantation.Under general scenario, this kind of repopulating cell or cell cluster clinic are not easy to find, chemotherapy resistance rate is high, But it is quite fragile under radio-immunity destruction.Based on the feature, the development of oophoroma radioimmunotherapy is swift and violent.Current puts It penetrates the mouse monoclonal antibody that immunotherapy mainly selects the epitope containing anti-CA 125, after isotope labelling, targeting positioning and puts It treats.Multitest confirms that after radioactive element label, CA125 monoclonal antibody and the compatibility of its antigen do not weaken.However, The direct radio-labeled law limitation of traditional monoclonal antibody is more, radiotoxicity accumulation and entity especially in reticuloendothelial system Tumor curative effect is not good enough.Reticuloendothelial system accumulation greatly weakens isotope marked antibodies targeting positioning rate.It is put as solid tumor It is not good enough to penetrate immunotherapeutic, generally lays the blame on and absorbs concentration lower than most suitable radiotherapy concentration for tumor tissues, plasma clearance is low and marrow Toxicity etc..

Based on the CA125 prominent characteristic shown in terms of clinical diagnosis and in the huge applications of therapeutic field of tumor Prospect develops the specific binding antibody for CA125, and improving clinical diagnosis and therapeutic efficiency becomes the urgent of the prior art Demand.But some disadvantages based on conventional antibodies, such as affinity is not high, Immune discrimination inefficiency, for some hidden The higher antigen of degree is difficult to reach ideal combination and neutralization.

1993, Hamers-Casterman etc. was the study found that camellid (camel, dromedary camel and yamma) Have found a kind of only have heavy chain homodimer (H in vivo₂) antibody, be mainly IgG₂And IgG₃Type, such antibody is due to lacking Weary light chain, then by this antibody be known as only heavy chain antibody (Heavy chain only like Antibody, HCAbs), antigen-binding site is made of a structural domain, referred to as the area VHH, therefore such antibody is also referred to as single domain Antibody or single domain antibody (sdAb).Since such antibody is the variable region sequences after removing constant region, molecular weight only has 15kD, About 10 nanometers of diameter, therefore also referred to as nano antibody (Nbs).In addition, also observing this kind of single domain in shark Antibody, referred to as VNAR.The antibody of this only heavy chain was intended only as a kind of human B cell's proliferative disease (heavy chain disease) originally Pathological forms are recognized by people.The type antibody may be mutation and missing due to genomic level and lead to heavy chain CH1 Structural domain cannot express, so that the heavy chain given expression to lacks CH1, to lack the binding ability with light chain, therefore form one kind Heavy chain homodimer.

For the scFv of four conventional chain antibodies, nano antibody scFv phase corresponding in terms of affinity When, but in solubility, stability, the resistance to aggregation, refolding, expression productivity and DNA operation, library construction and 3- Surmount scFv in terms of the easiness of D structure determination.

Nano antibody has the smallest functional antigen binding fragment of the HCAbs in adult camel body, has height Stability and high affinity with antigen binding can interact with albumen crack and enzyme active sites, and the effect of being allowed to be similar to Inhibitor.Therefore, nano antibody can provide new thinking to design small molecule enzyme inhibitor from peptide aids drug.Due to only The manufacture of heavy chain, nano antibody is easy compared with monoclonal antibody.The peculiar property of nano antibody is such as in extreme temperature and pH environment In stability, can be manufactured at low cost with big yield.Therefore, nano antibody has very big valence in the treatment and diagnosis of disease Value also has very big development prospect in the antibody target diagnosing and treating of tumour.

It is an object of the invention to provide the superior functions that one kind can give full play to nano antibody, both have excellent spy Specific Antigen binding ability, and conventional antibodies entity tumor poor permeability can be overcome, the inherent shortcomings such as targeting effect is low it is anti- The nano antibody of CA125, and further provide for its tumour especially treatment of ovarian cancer drug and diagnostic preparation preparation in Using.

Summary of the invention

Based on foregoing invention purpose, present invention firstly provides a kind of nano antibody of anti-CA 125, the nano antibody Variable region has 3 complementary determining regions CDR1, CDR2, CDR3, wherein

(1) CDR1 sequence amino acid sequence described in SEQ ID NO.1 forms, and CDR2 sequence is as described in SEQ ID NO.2 Amino acid sequence composition, CDR3 sequence amino acid sequence described in SEQ ID NO.3 form, or

(2) CDR1 sequence is made of following amino acid sequences: the amino acid sequence described in SEQ ID NO.7 forms, CDR2 Sequence amino acid sequence described in SEQ ID NO.8 forms, and CDR3 sequence amino acid sequence described in SEQ ID NO.9 forms, Or

(3) CDR1 sequence amino acid sequence described in SEQ ID NO.12 forms, and CDR2 sequence is by SEQ ID NO.13 institute Amino acid sequence composition is stated, CDR3 sequence amino acid sequence described in SEQ ID NO.14 forms.

In a preferred technical solution, the variable region sequences of the nano antibody

(1) amino acid sequence described in SEQ ID NO.4 forms, or

(2) amino acid sequence described in SEQ ID NO.10 forms, or

(3) amino acid sequence described in SEQ ID NO.15 forms.

In one more preferably technical solution, the nano antibody also has constant region, the nano antibody Constant-region sequences amino acid sequence described in SEQ ID NO.5 forms.

Second, the present invention also provides a kind of nucleotide coding sequence for encoding above-mentioned nano antibody sequence, the codings Sequence is as shown in SEQ ID NO.6, perhaps as shown in SEQ ID NO.11 or as shown in SEQ ID NO.16.

Third, the present invention provides a kind of expression vectors containing above-mentioned nucleotide coding sequence.

In a preferred technical solution, the carrier is pMES4.

4th, the present invention provides a kind of host cell containing above-mentioned expression vector, the cell is Escherichia coli BL₂₁ (DE₃)。

5th, the present invention also provides application of the above-mentioned nano antibody in preparation tumor therapeutic agent.

In a preferred technical solution, the tumour is ovarian neoplasm.

Finally, the present invention provides above-mentioned nano antibodies to prepare the application in tumour diagnostic reagent.

The nano antibody of anti-CA 125 provided by the invention, due to making described anti-with the region sequence of unique CDR1,2 and 3 Body has special identification and binding ability to CA125 antigen.And it is not reacted with other non-specific cross-reaction albumen. Nano antibody provided by the invention has significant ADCC effect, is capable of splitting for the tumour cell OVCAR-3 of inducing expression CA125 Solution, and do not have this effect then for the A431 cell for not expressing CA125, it is shown that answering in tumor therapeutic agent preparation Use prospect.Moreover, anti-CA 125 nano antibody provided by the invention can realize the accurately image of tumour in Mice Body, display Application prospect in diagnostic reagent preparation and animal imaging.

Detailed description of the invention

Fig. 1 .SDS-PAGE and Western blot detection affinity purification CA125 identifies map；

Fig. 2 .SDS-PAGE and Western blot detection molecules sieve purifying CA125 identify map；

Fig. 3 .pMES4 expression vector structural schematic diagram；

The total serum IgE electroresis appraisal map that Fig. 4 is extracted；

Fig. 5 first round PCR amplification antibody variable gene electroresis appraisal map；

Fig. 6 second takes turns PCR amplification antibody variable gene electroresis appraisal map；

Fig. 7 .pMES4 carrier double enzyme digestion reaction product electroresis appraisal map；

Fig. 8 bacterium colony PCR identifies transformant electroresis appraisal map；

Fig. 9 nano antibody expresses SDS-PAGE and identifies map；

Figure 10 fusion expression vector constructs schematic diagram；

Figure 11 nano antibody purifies SDS-PAGE map；

Figure 12 nano antibody ADCC effect curve figure.

Specific embodiment

The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.But examples are merely exemplary for these, does not constitute any restrictions to protection scope of the present invention.

The preparation of 1. anti-CA 125 nano antibody of embodiment

The preparation of 1.1CA125 antigen

1.1.1 saturated ammonium sulphate method purifies the CA125 in ovarian cancer patients ascites: by 2L ascites in magnetic stirring apparatus Top ice bath stirring side slowly adds 228g ammonium sulfate, makes final concentration of the 20% of ammonium sulfate, and 4 DEG C stand overnight, 10000rpm from The heart 10 minutes, retain supernatant.Supernatant is slowly added into 524g ammonium sulfate on magnetic stirring apparatus top ice bath stirring side, makes sulfuric acid Final concentration of the 60% of ammonium, 4 DEG C stand overnight, and 10000rpm is centrifuged 10 minutes, retain precipitating.It is blown with about 400mL PBS solution It beats and mixes precipitating, 10000rpm is centrifuged 10 minutes, retains supernatant.Supernatant is surpassed draw 4000rpm with 100kD to be concentrated into 200mL is packed as 50mL/ pipe.

1.1.2 the CA125 in immunoaffinity chromatography purifying ovarian cancer patients ascites

The Affi-Gel Hz HydrazideGel for having replaced buffer is added in a 10mL centrifuge tube and removal aoxidizes The Anti-h CA125McAb of agent, is mixed by inversion, with sealed membrane tube sealing mouth, after centrifuge tube insertion buoy is fixed, at 30 DEG C Horizontal shaker in mix 10-24 hours.By centrifuge tube be stored at room temperature until Hydrazide Gel be settled down to tube bottom, use 1mL Micropipettor removal upper solution after, 5mL PBS solution is added, is mixed by inversion, is stored at room temperature until Hydrazide Gel It is settled down to tube bottom, removes upper solution with the micropipettor of 1mL, repetition is washed 3-5 times with 5mL PBS solution.It will be crosslinked Affi-Gel Hz Hydrazide Gel and 40mL ascites concentrate are added in a 50mL centrifuge tube, at 30 DEG C, 240rpm water Affine combination is stayed overnight in yawing bed.Affi-Gel Hz Hydrazide Gel and ascites mixed liquor are crossed into gravity column, collection penetrates Liquid.10 column volumes (elution foreign protein) is eluted with the PBS solution of the NaCl containing 0.5M, with 0.1M citric acid (pH 3.0) elution 5 A column volume (elution destination protein), collects eluent.Affi-Gel Hz Hydrazide Gel PBS solution is balanced 5 Column volume.The eluent of collection 100kD is surpassed into draw 4000rpm concentration and replaces it with PBS solution, until final volume is 1mL, It is saved in -20 DEG C.By 0.22 μm of membrane filtration of the eluent of collection, with equilibrated Affi-Gel Hz Hydrazide Gel in a 50mL centrifuge tube, in 30 DEG C, 240rpm horizontal shaker affine combination stay overnight, through reproducibility SDS-PAGE, The CA125 of Western blot and mass spectral analysis, affinity purification (is the portion of CA125 at size 180kD, 55kD and 25kD Point protein band) in containing part human serum albumins (protein band that size is 70kD), (Fig. 1: M is 180 wide spectrum egg of rainbow White molecular weight marker；1-3 is the CA125 after affinity purification).

1.1.3 molecule sieve separation CA125 and albumin are utilized

After separating affinity purification using HiPrep 16/60Sephacryl S-300High Resolution (GE) The eluent of collection 10kD super filter tube 4000rpm concentrate eluant to 1mL is passed through reproducibility SDS- by CA125 and albumin PAGE and Western blot analyzes purification effect, and (Fig. 2: M is 180 wide spectrum protein markers of rainbow；1 is without molecular sieve The CA125 of purifying；2-9 is the CA125 eluent of Fractional Collections).It can be seen that containing big in the albumen of the collection at 3-5 sections and 7-9 sections Partial human serum albumins, therefore the albumen that 2 sections and 6 sections are collected merges concentration to get the higher CA125 egg of purity is arrived It is white.

The building and screening of 1.2 anti-CA 125 nano antibody phage display libraries

1.2.1 alpaca is immune: one sheep of healthy adult alpaca is chosen, by the CA125 of purifying and Freund's adjuvant by 1:1's Ratio mixes, and alpaca is immunized by the way of dorsal sc multi-point injection by 6-7 μ g/Kg, is immunized four times altogether, immunization interval 2 Week.Alpaca peripheral blood is acquired later, for constructing phage display library.

1.2.2. the separation of hunchbacked source lymphocyte: according to the art conventional program from the hunchbacked source anticoagulated whole blood of acquisition Analysis lymphocyte, every 2.5 × 10⁷1mL RNA separation agent is added in a living cells, and 1mL is taken to carry out RNA extraction, and remaining -80 DEG C It saves.

1.2.3 Total RNAs extraction: extracting total serum IgE according to the art conventional program, adjusts concentration with RNase-free water To 1 μ g/ μ L (see Fig. 4).

1.2.4. reverse transcription synthesizes cDNA: according to the Reverse Transcriptase kit specification (transcripor of Roche company First stand cDNA synthesis KIT) using the RNA of 2.3 steps acquisition as template progress reverse transcription cDNA.

1.2.5 antibody variable gene expands: the cDNA that reverse transcription is obtained carries out PCR reaction as template.Amplification is altogether Two-wheeled is carried out, the primer sequence of first round PCR is as follows:

CALL001:GTCCTGGCTGCTCTTCTACAAGG

CALL002:GGTACGTGCTGTTGAACTGTTCC

PCR reaction condition and program are as follows: 95 DEG C 5 minutes；95 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds, 30 circulation；72 DEG C of 7 minutes bands using Ago-Gel QIAquick Gel Extraction Kit glue recycling 700bp or so, finally extremely with water adjustment nucleic acid concentration (Fig. 5: M marks 5ng/ μ l for Trans 2K DNA molecular amount；1 is first round PCR product).

The primer sequence of second wheel PCR is as follows:

VHH-Back:GATGTGCAGCTGCAGGAGTCTGGRGGAGG

VHH-For:CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

PCR reaction condition and program are as follows: 95 DEG C 5 minutes；95 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds, 15 circulation；72 Using PCR product QIAquick Gel Extraction Kit purified pcr product, (Fig. 6: M marked for Trans 2K DNA molecular amount DEG C 7 minutes；1 is second Take turns PCR product).

1.2.6 vector construction: by pMES4 (being purchased from Biovector, structural schematic diagram is shown in Fig. 3) and second of PCR product PstI, BstEII double digestion are carried out respectively, are taken second of PCR product after 1.5 μ g digestions after carrier and 450ng digestion, are added 15 μ L T₄DNA ligase supplements buffer and water to 150 μ L total volumes, and 16 DEG C connect overnight and recycle connection product.Use PCR Product QIAquick Gel Extraction Kit carries out product recycling, 20 μ L water elutions.1% Normal Agarose Gel detects pMES4 carrier double digestion knot (Fig. 7: M marks fruit for Trans 2K DNA molecular amount；1 is the non-digested plasmid of pMES4 carrier；2 is after pMES4 carrier double digestions Product).

1.2.7 electrotransformation and storage capacity measurement: the connection product of 10 μ L after purification is taken, is added to containing 50 μ L Escherichia coli It is placed in electroporation (the ECM630 electroporation of U.S. BTX) in the pre-cooling electricity revolving cup of TG1 competent cell and carries out electrotransformation, takes out electricity Revolving cup recovers and cultivates transformant.18 clones are selected at random, and carrying out bacterium colony PCR identification, (Fig. 8: M is Trans 2K DNA points Son amount label；1-19 is the monoclonal PCR identification product selected at random).According to PCR positive rate calculate storage capacity (storage capacity=gram Grand number × extension rate × PCR identifies positive rate × 10).

Primer sequence is as follows:

MP57:TTATGCTTCCGGCTCGTATG

GIII:CCACAGACAGCCCTCATAG

1.2.8 Phage amplification: the bacterium solution of recovery is taken to be seeded in YT-AG culture medium, 37 DEG C of 200rpm cultures to culture Object OD₆₀₀=0.5.It takes out 10ml bacterium solution and is added 4 × 10¹⁰VCSM13,37 DEG C it is static infection 30 minutes.4000rpm, room temperature centrifugation 10 minutes, remove supernatant.Thallus, 37 DEG C of 200rpm trainings are resuspended with 2 × YT-AK (containing ampicillin and kanamycins) culture medium It supports overnight.In centrifuging and taking supernatant 40ml pipe, 10ml PEG/NaCl (20%/2.5M) solution is added and is sufficiently mixed, in centrifugation abandoning Clearly, precipitating washs centrifugation with 1ml ice PBS, and the PEG/NaCl for taking 250 μ l of supernatant to be pre-chilled is mixed well and washed resuspension.

Measurement phage titre: TG1 is cultivated to OD₆₀₀=0.4, with LB culture medium gradient dilution bacteriophage, take multiple proportions dilute The bacteriophage TG1 culture mixed culture released, next day observes Plaques assay situation in culture plate, to plaque number in 30-300 Dilution gradient plate count and calculate phage titre (pfu) according to the following formula.

Phage titre (pfu/ml)=extension rate × plaque number × 100

1.2.9 nano antibody screens: by ELISA method with CA125 antigen selection positive colony.With CA125 antigen packet By elisa plate, 5%BSA closing, PBST washing.100 μ l phage supernatants are added in every hole, and 37 DEG C are placed 1 hour.Supernatant is abandoned, The secondary antibody of the anti-M13 of mouse of HRP label is added, 37 DEG C are placed 1 hour.Supernatant is abandoned, TMB solution is added, is incubated at room temperature 5 hours, 2M sulfuric acid terminate liquid is added in every hole, is read with microplate reader 450nm.

1.2.10 expression and purifying of the nano antibody in Escherichia coli: selecting the clone of the phage E LSIA result positive, It extracts plasmid and converts to bacterial strain BL₂₁Competent cell induces nano antibody protein expression with IPTG, and collecting supernatant, (pericentral siphon mentions Take object), and periplasmic extract is dialysed to PBS, it is purified using Ni-NTA resin, is eluted using various concentration imidazoles And collection, the sample of collection is subjected to the analysis of reduced form protein electrophoresis, finally carries out dialysis nano antibody in PBS.

Immune, cell separation, the building of phage library, the screening of nano antibody by alpaca filter out 3 plants altogether and resist The nano antibody of CA125.Sequencing result is analyzed with Vector NTI software, login IMGT (http:// www.imgt.org/IMGT_vquest), antibody light chain and heavy chain gene are analyzed, to determine the framework region of variable region (Framework Regions, FR) and complementary determining region (Complementary Determining Regions, CDR).

Nano antibody VHH-CA125-4H10 heavy chain nucleotide sequence is variable region amino acid sequence shown in SEQ ID NO.6 It is classified as shown in SEQ ID NO.4, wherein 1-20 amino acids sequence is FR1,21-28 amino acids sequence is CDR1, the 29-45 amino acids sequence is FR2, and 46-53 amino acids sequence is CDR2, and 54-91 amino acids sequence is FR3, the 92-108 amino acids sequence is CDR3, and 109-113 amino acids sequence is FR4.

Nano antibody VHH-CA125-2H7 heavy chain nucleotide sequence is SEQ ID NO.11, and variable region amino acid sequence is Shown in SEQ ID NO.10, wherein 1-20 amino acids sequence is FR1,21-28 amino acids sequence is CDR1,29- 45 amino acids sequences are FR2, and 46-53 amino acids sequence is CDR2, and 54-91 amino acids sequence is FR3,92- 108 amino acids sequences are CDR3, and 109-113 amino acids sequence is FR4.

Nano antibody VHH-CA125-4C1 heavy chain nucleotide sequence is SEQ ID NO.16, and variable region amino acid sequence is SEQ ID NO.15, wherein 1-20 amino acids sequence be FR1,21-28 amino acids sequence be CDR1,29-45 Amino acid sequence is FR2, and 46-52 amino acids sequence is CDR2, and 53-90 amino acids sequence is FR3,91-108 Amino acid sequence is CDR3, and 109-113 amino acids sequence is FR4.

Expression, the purifying of 1.3 nano antibodies filtered out

1.3.1 nano antibody original strain TG1 amplification and nano antibody recombinant plasmid transformed Escherichia coli BL₂₁(DE₃): it will Original strain TG1 glycerol stock containing nano antibody nucleic acid is inoculated in the fresh LB-A culture medium of 5mL according to 1:1000 ratio, 37 DEG C, 200rpm is incubated overnight.Next day extracts plasmid using Plasmid mini kit (OMEGA) to specifications.It is verified Above-mentioned 1 μ l of plasmid conversion is mixed gently in 100 μ l competent cells afterwards, is placed 30 minutes on ice, 42 DEG C of water-bath thermal shocks 90 seconds, ice bath was 3 minutes cooling.To centrifuge tube be added 600 μ l LB culture mediums, 37 DEG C shaken cultivation 60 minutes.100 μ l of supernatant is taken, It is coated on LB-A plate with triangle spreader, 37 DEG C of inversion overnight incubations.

1.3.2 the inducing expression and extraction of nano antibody: the above-mentioned monoclonal colonies of picking are in LB-A culture medium, 37 DEG C of vibrations Swing overnight incubation.Next day, take the bacterium solution that the fresh LB-A culture medium of 100ml is added according to 1:100 ratio, 37 DEG C of shaken cultivations 3 are small Up to bacterium solution OD₆₀₀=0.8 or so, final concentration of 1mM IPTG, 30 DEG C of overnight inductions are added.Third day, 8000rpm, centrifugation 10 Minute collects thallus, and precipitating is resuspended in the pre-cooling TES buffer that 1.5mL is added.After ice bath 2 minutes, gentle agitation 30 seconds, this is repeated Circulation 6 times.Add 3.0ml TES/4 (TES is diluted with water 4 times), after gentle agitation 30 seconds, ice bath stands 2 minutes, same to repeat It vibrates and stands step totally 6 times.9000rpm, 4 DEG C are centrifuged 10 minutes, collect the supernatant (periplasmic extract) of about 4.5mL, will be upper It is clear to carry out protein electrophoresis analysis.

1.3.3 the purifying and identification of nano antibody: after IMAC Sepharose (GE company) is resuspended, 2ml is taken to be added to In gravity column, 30 minutes are stood, makes sepharose natural subsidence in gravity column bottom, outflow saves buffer.2 times of columns are added The nickel sulfate solution (0.1M) of volume flows out nickel sulfate solution according to the flow velocity of about 8 seconds/drop；The balance of 10 times of column volumes is added Buffer balances and washs sepharose, and flow velocity remains unchanged；After sample is diluted using 2 times of equilibration buffer, gravity is added In column, adjusting flow velocity is 6 seconds/drop, and collection penetrates liquid；10 times of column volume washing buffer washing sepharose are added, maintain stream Speed is constant, collects cleaning solution；The elution buffer of 3 times of column volumes is added, flow velocity maintains 6 seconds/drop, and collection contains destination protein Eluent；Finally sequentially add the equilibration buffer of 10 times of column volumes, the pure water of 10 times of column volumes and 10 times of column volumes 20% ethanol washing sepharose, and retain 20% ethyl alcohol of 4ml finally to save pillar.The sample of above-mentioned collection respectively into (Fig. 9: M is 180 wide spectrum protein markers of rainbow for row SDS-PAGE detection；1-3 be Escherichia coli inducing expressions after purification Nano antibody VHH-CA125-4H10, VHH-CA125-2H7 and VHH-CA125-4C1).

The affine determination of activity of embodiment 2. anti-CA 125 nano antibody and antigen

2.1 chip antigens coupling: by the sodium-acetate buffer (pH 5.5, pH 5.0, pH 4.5, pH of the different pH of antigen 4.0) it is configured to the working solution of 20 μ g/mL, while preparing the NaOH actified solution of 50mM, it is mutual using Biacore T100 albumen Template method in function analysis system instrument is to the electrostatic knot between antigen and chip (GE company) surface of condition of different pH Conjunction is analyzed, and reaches 5 times of RL as standard using the increased amount of signal, the pH system of the suitable most partial neutral of selection is simultaneously as needed Adjust condition of the antigen concentration as coupling when.Chip is coupled according to the template method carried in instrument: wherein 1 channel Blank conjugation pattern, 2 channel selecting Target conjugation patterns are selected, target is set as designed theoretical coupling amount.It was coupled Journey is probably 60 minutes time-consuming.

2.2 analyte concentrations setting conditional FP tree and regeneration condition optimization: hand sampling mode is taken, 1,2 channels are selected 2-1 mode sample introduction, flow velocity are set as 30 μ L/ minutes.Sampling condition is 120 seconds, 30 μ L/ minutes.Regeneration condition is 30 seconds, 30 μ L/ minutes.Continue sky first and walks running buffer until all baselines are stable.Prepare the biggish nano antibody of concentration span Solution is configured with running buffer, it is proposed that 200 μ g/mL, 150 μ g/mL, 100 μ g/mL, 50 μ g/mL, 20 μ g/mL, 10 μ of setting G/mL, 2 μ g/mL.Prepare actified solution, the actified solution of selection four pH gradients of glutamate acid system: 1.5,2.0,2.5, 3.0.200 μ g/mL analyte sample of hand sampling is observed 2 channels, is regenerated from the regeneration buffer of most partial neutral pH, directly Line of response after to the regeneration of 2 channels returns to and baseline sustained height.200 μ g/mL analyte samples of hand sampling again, observation The signal intensity in the channel 2-1 simultaneously records binding capacity, is carried out again with the actified solution for finally making line of response return to baseline in previous step After life, again receive 200 μ g/mL analyte sample of hand sampling, observe the channel 2-1 signal intensity and record binding capacity with just now Combination numerical quantity comparison, if deviation less than 5%, that is, think this pH actified solution be optimal actified solution, if again into The binding capacity of sample is relatively low, then continues to be tested with the regeneration buffer of lower pH.With the best actified solution of selection, as every Chip surface regenerative agent after secondary sample introduction.The analyte concentration sample that sample introduction is arranged above respectively, and to the knot of each concentration Resultant is analyzed, the final concentration gradient for determining that affinity test is required.

2.3 affinity test: by the sample concentration gradient optimized, actified solution, the template method carried using instrument (being provided with sampling condition is 60 seconds, 30 μ L/ minutes；Dissociation time: 600 seconds；Regeneration condition: 30 seconds, 30 μ L/ minutes) to receiving Affinity between meter Kang Ti and antigen is tested.The signal condition in the channel 2-1 is observed at any time.Affinity test process is general It is 200 minutes time-consuming.In specific experiment, the nanometer body on chip is captured into signal value appropriate, is then run with system slow Fliud flushing HBS-EP (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05%P20) is injected into core with 30 L/ minutes flow velocitys of μ On piece obtains the dynamic process of nano antibody and antigen interactions.3 nano antibodies are tested using this method respectively and are resisted The ability of former association and dissociation.

2.4 interpretations of result: the combination dissociation curve of suitable several concentration gradients is selected to use the mode of 1:1binding All curves are fitted, the important parameters such as affinity numerical value and binding constant and dissociation constant are finally obtained.The three of screening Strain nano antibody affinity reaches 10^-10。

Table 1: nano antibody affinity data

Sample number into spectrum	Binding constant	Dissociation constant	Affinity
				VHH-CA125-4H10	2.381×10⁺⁵	6.775×10^-5	2.85E-10
VHH-CA125-2H7	5.374×10⁺⁴	3.857×10^-5	7.18E-10
				VHH-CA125-4C1	1.593×10⁺⁵	1.012×10^-4	6.35E-10

The ADCC activity measurement of 3. anti-CA 125 nano antibody of embodiment induction

Using primer, using VHH-pMES4 as template, PCR amplification nano antibody gene.Primer sequence is as follows:

F:CCGAAATTCGAGTCTGGAGGAGG

R:GGAAGATCTCTGGGTCCCCTGGCCC

Using EcoR I and Bgl II restriction enzyme (NEB) by PCR product and fusion expression vector pFUSE-hIgG1- Fc carries out double digestion respectively, and (carrier schematic diagram has the encoding gene of antibody constant region, the coding base referring to Figure 10, the carrier Because the amino acid sequence given expression to is shown in SEQ ID NO.5).Using T4 ligase (NEB) by after double digestion carrier with receive The connection of rice antibody gene overnight, utilizes the big extraction reagent kit of endotoxin-free (Tiangeng) to extract plasmid after converting DH5 α competence.Transfection 293 cell of people, using the method for protein A affinity chromatography, purified fusion has constant-region sequences from the culture supernatant of 293 cells (the result is shown in Figure 1 1:M is 180 wide spectrum protein markers of rainbow to anti-CA 125 nano antibody；1-3 be Fc amalgamation and expression after purification Antibody VHH-CA125-4H10-Fc, VHH-CA125-2H7-Fc and VHH-CA125-4C1-Fc).In 96 hole cell microwell plates Interior culture OVCAR-3 and A431 cell (3 × 10⁴/ hole) 48 hours, LAK cell (lymphokine then is added according to special ratios The killing cell of activation, lymphokine activated killer cells), anti-CA 125 merge nano antibody VHH- CA125-4H10-Fc, VHH-CA125-2H7-Fc, VHH-CA125-4C1-Fc or IgG antibody control, LAK cell and target are thin The ratio of born of the same parents is 1,5,10,15,20:1, and antibody concentration is 2 μ g/ml.In 5%CO₂Lower 37 DEG C of environment are incubated for 6 hours, suck LAK cell and dead tumour cell carry out cell viability measurement using MTS method.Cytotoxicity (%)=[experiment target cell OD₄₉₀The OD of/control target cell₄₉₀]×100

The results show that anti-CA 125 merges nano antibody VHH-CA125-4H10-Fc, VHH-CA125-2H7-Fc, VHH- CA125-4C1-Fc can significantly induce the ADCC activity of LAK cell, and tumor cell lysis rate is between 55-75%, in not table Up to this cracking phenomenon is not observed in the A431 cell of CA125 (result is referring to Figure 12).

4. anti-CA 125 nano antibody of embodiment positions the in-vivo imaging of xenograft tumor mouse

Prepare OVCAR-3 cell solution (5 × 10⁶OVCAR-3 cell is dissolved in 0.2ml PBS), it is subcutaneously injected small in SCID Mouse metal is given respectively by the method for tail vein injection after tumour growth 20 days in the back of mouse¹⁸⁸Re label resists CA125 nano antibody VHH-CA125-4H10, VHH-CA125-2H7, VHH-CA125-4C1 or control antibodies IgG (30 μ g/ 0.1ml), imaging positioning is carried out to the transplantation tumor of mouse in small animal living body imaging system.

The results show that anti-CA 125 nano antibody provided by the invention can be realized in Mice Body tumour it is accurate at Picture can be used for the accurate in-vivo diagnostic of related neoplasms in the future.

Sequence table

<110>Shenzhen Guo Chuan nano antibody Technology Co., Ltd.

<120>a kind of nano antibody of anti-CA 125 sugar antigen and its application

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 8

<212> PRT

<213> Lama pacos

<400> 1

Glu Phe Thr Leu Glu His Tyr Ala

1 5

<210> 2

<211> 8

<212> PRT

<213> Lama pacos

<400> 2

Ile Ser Ser Ser Gln Glu Asn Thr

1 5

<210> 3

<211> 17

<212> PRT

<213> Lama pacos

<400> 3

Ala Ala Arg Arg Phe Gly Leu Cys Val Ile Ser Pro Gly Tyr Phe Glu

1 5 10 15

Val

<210> 4

<211> 113

<212> PRT

<213> Lama pacos

<400> 4

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Glu Phe Thr Leu Glu His Tyr Ala Ile Gly Trp Phe

20 25 30

Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Gly Cys Ile Ser Ser

35 40 45

Ser Gln Glu Asn Thr Tyr Val Glu Asp Ser Ala Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Asp Asn Val Lys Asn Thr Val Tyr Leu Gln Met Asn Ser

65 70 75 80

Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Arg Phe

85 90 95

Gly Leu Cys Val Ile Ser Pro Gly Tyr Phe Glu Val Trp Gly Gln Gly

100 105 110

Thr

<210> 5

<211> 231

<212> PRT

<213> Homo sapiens

<400> 5

Ala His His Ser Glu Asp Pro Ser Ser Lys Cys Pro Lys Cys Pro Gly

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Thr Val Phe Ile Phe Pro Pro Lys Pro

20 25 30

Lys Asp Val Leu Ser Ile Thr Arg Lys Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Gly Lys Glu Asp Pro Glu Ile Glu Phe Ser Trp Ser Val

50 55 60

Asp Asp Thr Glu Val His Thr Ala Glu Thr Lys Pro Lys Glu Glu Gln

65 70 75 80

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln

85 90 95

Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys Gly Gln Thr

115 120 125

Arg Glu Pro Gln Val Tyr Ala Leu Ala Pro His Arg Glu Glu Leu Ala

130 135 140

Lys Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly Phe Phe Pro Ala

145 150 155 160

Asp Ile Asn Val Glu Trp Gln Arg Asn Gly Gln Pro Glu Ser Glu Gly

165 170 175

Thr Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp Gly Thr Tyr Phe

180 185 190

Leu Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp Gln Gln Gly Glu

195 200 205

Val Phe Thr Cys Val Val Met His Glu Ala Leu His Asn His Ser Thr

210 215 220

Gln Lys Ser Ile Ser Gln Ser

225 230

<210> 6

<211> 1050

<212> DNA

<213> Homo sapiens

<400> 6

gagtctggag gaggcttggt gcagcctggg gggtctctga gactctcctg tgcagcctct 60

gaattcactt tggaacatta tgctataggc tggttccgcc aggccccagg gaaggagcgt 120

gagggggtcg gatgtataag tagtagtcaa gaaaacacat atgttgaaga ctccgcgaag 180

ggccgattca ccatctccag agataatgtc aagaacacgg tatatctgca gatgaacagt 240

ctgaaacctg aggacacagc cgtttattac tgtgcagcac gaagattcgg gctatgtgta 300

atatcccctg ggtatttcga agtttggggc cagggcaccc aggtcaccgt ctcctcggcg 360

caccacagcg aagaccccag ctccaagtgt cccaaatgcc caggccctga gctccttgga 420

gggcccacgg tcttcatctt ccccccgaaa cccaaggacg tcctctccat cacccgaaaa 480

cctgaggtca cgtgcgttgt ggtggacgtg ggtaaggaag accctgagat cgagttcagc 540

tggtccgtgg atgacacaga ggtacacacg gctgagacaa agccaaagga ggaacagttc 600

aacagcacgt accgcgtggt cagcgtcctg cccatccagc accaggactg gctgacgggg 660

aaggaattca agtgcaaggt caacaacaaa gctctcccag cccccatcga gaggaccatc 720

tccaaggcca aagggcagac ccgggagccg caggtgtacg ccctggcccc acaccgggaa 780

gagctggcca aggacaccgt gagcgtaacc tgcctggtca aaggcttctt cccagctgac 840

atcaacgttg agtggcagag gaacgggcag ccggagtcag agggcaccta cgccaccacg 900

ctgccccagc tggacaacga cgggacctac ttcctctaca gcaaactctc cgtgggaaag 960

aacacgtggc agcagggaga agtcttcacc tgtgtggtga tgcacgaggc tctacacaat 1020

cactccaccc agaaatccat ctcccagtct 1050

<210> 7

<211> 8

<212> PRT

<213> Lama pacos

<400> 7

Asp Ala Pro His Ser Pro Tyr Thr

1 5

<210> 8

<211> 8

<212> PRT

<213> Lama pacos

<400> 8

Ile Thr Trp Ser Gly Gly Thr Thr

1 5

<210> 9

<211> 17

<212> PRT

<213> Lama pacos

<400> 9

Ala Val Gly Leu Gly Gly Gly Ala Tyr Arg Glu Asp His Gln Tyr Asp

1 5 10 15

Tyr

<210> 10

<211> 113

<212> PRT

<213> Lama pacos

<400> 10

Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Glu Arg Ser Asp Ala Pro His Ser Pro Tyr Thr Met Gly Trp Phe

20 25 30

Arg Gln Ala Pro Gly Lys Asp Arg Glu Phe Val Ala Thr Ile Thr Trp

35 40 45

Ser Gly Gly Thr Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Gly Ile Ala Lys Asn Thr Ala Tyr Leu Gln Met Asn Thr

65 70 75 80

Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Gly Leu Gly

85 90 95

Gly Gly Ala Tyr Arg Glu Asp His Gln Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr

<210> 11

<211> 1050

<212> DNA

<213> Homo sapiens

<400> 11

gagtctggag gaggattggt gcaggctggg ggctctctga gactctcctg tgaacgttcg 60

gacgcaccgc acagtcccta taccatgggc tggttccgcc aggctccagg gaaggaccgt 120

gaatttgtag caacaattac ttggagtggt ggtaccacac tctatgcaga ctccgtgaag 180

ggccgattca ccatctccag aggcattgcc aagaacacgg catatcttca aatgaacact 240

ctgaaacctg aggacacggc cgtttattac tgtgcagttg gactgggggg cggtgcctat 300

cgagaagatc atcagtatga ctactggggc caggggaccc aggtcaccgt ctcctcggcg 360

caccacagcg aagaccccag ctccaagtgt cccaaatgcc caggccctga gctccttgga 420

gggcccacgg tcttcatctt ccccccgaaa cccaaggacg tcctctccat cacccgaaaa 480

cctgaggtca cgtgcgttgt ggtggacgtg ggtaaggaag accctgagat cgagttcagc 540

tggtccgtgg atgacacaga ggtacacacg gctgagacaa agccaaagga ggaacagttc 600

aacagcacgt accgcgtggt cagcgtcctg cccatccagc accaggactg gctgacgggg 660

aaggaattca agtgcaaggt caacaacaaa gctctcccag cccccatcga gaggaccatc 720

tccaaggcca aagggcagac ccgggagccg caggtgtacg ccctggcccc acaccgggaa 780

gagctggcca aggacaccgt gagcgtaacc tgcctggtca aaggcttctt cccagctgac 840

atcaacgttg agtggcagag gaacgggcag ccggagtcag agggcaccta cgccaccacg 900

ctgccccagc tggacaacga cgggacctac ttcctctaca gcaaactctc cgtgggaaag 960

aacacgtggc agcagggaga agtcttcacc tgtgtggtga tgcacgaggc tctacacaat 1020

cactccaccc agaaatccat ctcccagtct 1050

<210> 12

<211> 8

<212> PRT

<213> Lama pacos

<400> 12

Gly Asn Thr Phe Asn Ile Asn Ala

1 5

<210> 13

<211> 7

<212> PRT

<213> Lama pacos

<400> 13

Ile Thr Ser Gly Gly Ser Thr

1 5

<210> 14

<211> 18

<212> PRT

<213> Lama pacos

<400> 14

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

1 5 10 15

Asp Tyr

<210> 15

<211> 113

<212> PRT

<213> Lama pacos

<400> 15

Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Gly Asn Thr Phe Asn Ile Asn Ala Met Gly Trp Tyr

20 25 30

Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Thr Ser

35 40 45

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

50 55 60

Ser Arg Asp Asn Ala Lys Asn Thr Val Phe Leu Gln Met Asn Ser Leu

65 70 75 80

Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Val Gln Thr Arg

85 90 95

Leu Pro Asn Ser Pro Asp Arg Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr

<210> 16

<211> 1050

<212> DNA

<213> Homo sapiens

<400> 16

gagtctgggg gaggcttggt gcaggctggg gggtctctga gactctcctg tgcagcctct 60

ggaaacactt tcaatatcaa tgccatgggc tggtaccgcc aggctccagg gaagcagcgc 120

gagttggtcg ctacaattac tagtggtggt agcacaaact atgcagactc cgtgaagggc 180

cgattcacca tctcgagaga caacgccaag aacacggtgt ttctgcaaat gaacagcctg 240

aaaccagagg acacagccgt ctattactgt aatgcggtcc agacgcgact tccgaacagc 300

ccagaccgtt atgagtatga ctactggggc caggggaccc aggtcaccgt ctcctcggcg 360

caccacagcg aagaccccag ctccaagtgt cccaaatgcc caggccctga gctccttgga 420

gggcccacgg tcttcatctt ccccccgaaa cccaaggacg tcctctccat cacccgaaaa 480

cctgaggtca cgtgcgttgt ggtggacgtg ggtaaggaag accctgagat cgagttcagc 540

tggtccgtgg atgacacaga ggtacacacg gctgagacaa agccaaagga ggaacagttc 600

aacagcacgt accgcgtggt cagcgtcctg cccatccagc accaggactg gctgacgggg 660

aaggaattca agtgcaaggt caacaacaaa gctctcccag cccccatcga gaggaccatc 720

tccaaggcca aagggcagac ccgggagccg caggtgtacg ccctggcccc acaccgggaa 780

gagctggcca aggacaccgt gagcgtaacc tgcctggtca aaggcttctt cccagctgac 840

atcaacgttg agtggcagag gaacgggcag ccggagtcag agggcaccta cgccaccacg 900

ctgccccagc tggacaacga cgggacctac ttcctctaca gcaaactctc cgtgggaaag 960

aacacgtggc agcagggaga agtcttcacc tgtgtggtga tgcacgaggc tctacacaat 1020

cactccaccc agaaatccat ctcccagtct 1050

Claims

1. a kind of nano antibody of anti-CA 125 sugar antigen, which is characterized in that the variable region of the nano antibody is mutual with 3 It mends and determines area CDR1, CDR2, CDR3, wherein CDR1 sequence amino acid sequence described in SEQ ID NO.12 forms, CDR2 sequence Column amino acid sequence described in SEQ ID NO.13 forms, and CDR3 sequence amino acid sequence described in SEQ ID NO.14 forms.

2. nano antibody according to claim 1, which is characterized in that the variable region sequences of the nano antibody are by SEQ ID The composition of amino acid sequence described in NO.15.

3. containing the antibody of nano antibody variable region as claimed in claim 2, which is characterized in that the antibody also has constant Constant-region sequences amino acid sequence described in SEQ ID NO.5 in area, the nano antibody forms.

4. a kind of polynucleotides of antibody described in coding claim 3, the coded sequence is as shown in SEQ ID NO.16.

5. a kind of expression vector containing polynucleotides as claimed in claim 4.

6. carrier according to claim 5, which is characterized in that the carrier is pMES4.

7. a kind of host cell containing expression vector described in claim 6, the cell is e. coli bl21 (DE3).

8. application of the antibody as claimed in claim 3 in preparation tumor therapeutic agent.

9. application according to claim 8, which is characterized in that the tumour is ovarian neoplasm.

10. nano antibody of any of claims 1 or 2 is preparing the application in tumour diagnostic reagent.