CN115322258A

CN115322258A - Monoclonal antibody QKMA-1G41 and application thereof

Info

Publication number: CN115322258A
Application number: CN202210606383.1A
Authority: CN
Inventors: 王业富; 何瑜林; 董艳山
Original assignee: Wuhan Zhenfu Pharmaceutical Co ltd
Current assignee: Wuhan Zhenfu Pharmaceutical Co ltd
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-11-11
Anticipated expiration: 2042-05-31
Also published as: CN115322258B

Abstract

The invention provides a monoclonal antibody QKMA-1G41 and application thereof, belonging to the technical field of genetic engineering. The nucleotide sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:2, and the amino acid sequence of the heavy chain is shown as SEQ ID NO:3 is shown in the specification; the coding amino acid sequence of the light chain is shown as SEQ ID NO:4, respectively. The monoclonal antibody QKMA-1G41 can be used in histochemical method, enzyme linked immunosorbent assay and immunoblotting method for identifying and detecting bacillus subtilis fibrinolytic enzyme, fusion expression and preparation of targeted drugs.

Description

Monoclonal antibody QKMA-1G41 and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, and particularly relates to a monoclonal antibody QKMA-1G41 and application thereof.

Background

Thrombosis is caused by abnormal aggregation of a large amount of fibrin and platelets, is closely related to the occurrence of diseases such as myocardial infarction, cerebral infarction and stroke, and is a disease seriously threatening human health. During the fermentation process of natto, bacillus natto can produce natto kinase, an alkaline serine protease, which was discovered by Japan, et al, at the earliest in 1987, and both the bacillus natto and the natto kinase are effective thrombolytic enzymes in vivo and in vitro, which not only directly degrade fibrin, but also promote cells to release tissue plasminogen activator and degrade the fibrin, and the natto kinase is a main functional component of fermented foods such as natto, fermented soybean and the like, and is of great interest as a thrombolytic drug. Compared with clinical thrombolytic drugs (urokinase, streptokinase and the like), the nattokinase has the advantages of safety, low cost, easy oral administration, easy absorption, difficult bleeding tendency, less complications and the like, is an ideal green thrombolytic drug, and has wide development and application prospects. Nattokinase has various physiological functions such as platelet coagulation inhibition, blood pressure reduction, blood flow improvement and the like besides the function of dissolving thrombus, so the development of nattokinase-related products (such as nattokinase medicines, nattokinase health-care products, functional foods and the like) attracts wide attention in the world.

The bacillus subtilis fibrinolytic enzyme is fibrinolytic protease with the molecular weight of about 27KD, and can be used for treating thrombus diseases in human bodies by oral administration and injection administration. The Bacillus subtilis natto is used as a Bacillus subtilis natto. Bacillus subtilis QK02 can produce a protease highly homologous with nattokinase, namely Subtilisin QK, named plasmin QK. Compared with the existing clinical thrombolytic drugs, the plasmin QK has stronger thrombolytic specificity, higher thrombolytic activity and lower bleeding risk. The development of thrombolytic drugs has great application prospects. However, the development of thrombolytic drugs requires systemic and comprehensive pharmacokinetics and toxicokinetics. The premise of pharmacokinetic experiments is to establish a stable and reliable detection method. For the detection of protein drugs, firstly, an ELisa detection method based on monoclonal antibody antibodies is adopted. The bacillus subtilis plasmin QK monoclonal antibody can be applied to specific quantitative detection of the bacillus subtilis plasmin and can also be applied to identification tests of drug development. Meanwhile, the recombinant human lysozyme can be fused with bacillus subtilis fibrinolytic enzyme to develop a targeted thrombolytic drug. At present, the fish and the dragon in the natto market are mixed and are difficult to distinguish true from false. One of the main reasons is that there is no uniform and specific detection and identification method. Thrombolytic activity was assessed by the size of the fibrinolytic loop by means of a fibrin plate only. The method has large error and no specificity and specificity. The application of the monoclonal antibody can specially identify the bacillus subtilis plasmin and can reliably and quantitatively detect the protein content. Therefore, there is an urgent need in the art to develop a monoclonal antibody for the identification and detection of plasmin from Bacillus subtilis.

Disclosure of Invention

The invention aims to provide a monoclonal antibody QKMA-1G41 and application thereof, and the monoclonal antibody QKMA-1G41 can be used for histochemical method, enzyme linked immunosorbent assay and immunoblotting method for identification and detection of bacillus subtilis fibrinolytic enzyme, fusion expression and preparation of targeted drugs.

The technical scheme of the invention is realized as follows:

the invention provides a monoclonal antibody QKMA-1G41, wherein the nucleotide sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:2, respectively.

As a further improvement of the invention, the coding amino acid sequence of the heavy chain is shown in SEQ ID NO:3 is shown in the specification; the coding amino acid sequence of the light chain is shown as SEQ ID NO:4, respectively.

As a further development of the invention, the heavy chain comprises a heavy chain variable region VH and the light chain comprises a light chain variable region VL; wherein, the heavy chain variable region VH amino acid sequence is SEQ ID NO:3, light chain variable region VL amino acid sequence is SEQ ID NO: bits 1-113 of 4.

As a further improvement of the invention, the VH and VL antigenic determinant complementarity determining regions CDR consist of CDR1, CDR2, CDR3, wherein:

the amino acid sequence of the VH-CDR1 is 27 th to 34 th in SEQ ID NO. 3, and the sequence is GlyPheSerLeuSerTyrHis;

the amino acid sequence of VH-CDR2 is 51-57 th in SEQ ID NO. 3, and the sequence is MetTrpAsnAsnGlyAspThr;

the amino acid sequence of the VH-CDR3 is 96 th to 109 th in SEQ ID NO. 3, and the sequence is AlaArgGl mu glyThrThrValValThrValTrpAspTyr;

the amino acid sequence of the VL-CDR1 is 26 th to 31 th in SEQ ID NO. 4, and the sequence is GlnAsnAlAsnArg Phe;

the amino acid sequence of the VL-CDR2 is 49 th to 51 th in SEQ ID NO. 4, and the sequence is AsnThrAsn;

the amino acid sequence of VL-CDR3 is 88-96 th position in SEQ ID NO. 4, and the sequence is Le mu glnHisHisSerLeuTrpThr.

As a further improvement of the invention, the Bacillus subtilis plasmin is prepared by fermenting a strain Bacillus subtilis QK02, the strain is preserved in China center for type culture collection with the preservation number of CCTCC NO: M203078.

As a further improvement of the invention, the bacillus subtilis plasmin comprises plasmin QK and nattokinase.

As a further improvement of the invention, the antibody is a mouse IgG1 subtype monoclonal antibody.

The invention further protects the application of the monoclonal antibody QKMA-1G41 in the identification and detection of bacillus subtilis plasmin.

The invention further discloses application of the monoclonal antibody QKMA-1G41 in fusion expression of bacillus subtilis plasmin and preparation of targeted drugs.

The invention further protects the monoclonal antibody QKMA-1G41 hybridoma cell line which is preserved in China center for type culture collection with the preservation number of CCTCC NO: C2021307 and is classified and named as rat monoclonal hybridoma cell strain.

The invention has the following beneficial effects: the monoclonal antibody QKMA-1G41 can be used in histochemical method, enzyme linked immunosorbent assay and immunoblotting method for identifying and detecting bacillus subtilis fibrinolytic enzyme, fusion expression and preparation of targeted drugs.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The Bacillus subtilis QK02 strain is preserved in China center for type culture collection, the preservation place is Wuhan university in Wuhan, china, the preservation number is M203078, and the preservation date is 11/17/2003.

The monoclonal antibody QKMA-1G41 hybridoma cell line is preserved in China center for type culture collection, the preservation place is Wuhan university in Wuhan, china, the preservation number is CCTCC NO: C2021307, the preservation date is 2021, 12, 13 days, and the hybridoma cell line is named as a rat monoclonal hybridoma cell line in a classified manner.

Example 1: preparation and purification of monoclonal antibody for resisting bacillus subtilis plasmin QK

1. Antigen preparation

The bacillus subtilis plasmin QK antigen is produced and purified by the company, and the purity is more than 95 percent.

2. Animal immunization

6 Balb/C mice 6-9 weeks old, 4 Wistar rats 9 weeks old. The Freund complete adjuvant is used for the first main injection, and the Freund incomplete adjuvant is used for the later reinforcing injection, and the Freund incomplete adjuvant and the equal volume of antigen are fully and uniformly mixed and then injected. Back multiple injections. The main injection of 100 u g antigen/test mouse, strengthen injection of 50 u g antigen/test mouse.

3. Cell fusion and hybridoma cell cloning

One week prior to fusion, SP2/0 cells were revived and cultured normally to log phase. Rats to be fused were selected, sacrificed on the day of fusion by cervical dislocation, spleens were removed, splenocytes collected and counted by standard procedures. Myeloma cells and splenocytes were mixed in the ratio of 1:5, and subjected to cell fusion in the standard protocol, followed by culturing in HAT DMEM complete medium, 3 days after fusion to visualize hybridoma cells, 1/2HAT complete medium on day 7, and 1/2HT medium on day 8. Screening assays were started about 10 days after fusion. Cell fusion results: after fusion, HAT selective medium is used for culture, and a plurality of growing hybridoma cells are observed under a microscope, thereby proving that the fusion operation is successful. Cell supernatants were aspirated at 100. Mu.L/well for indirect ELISA detection. According to the ELISA result, positive wells were judged. And (4) picking and checking the positive holes detected by the whole plate by using a single-channel pipettor, and performing secondary recheck to further confirm the positive holes. Two rounds of subcloning were performed on the rescreened positive well cells.

Subcloning cells in the positive hole for the first time, adding an HT DMEM culture medium into the multiple holes for culture, observing under a microscope for about 7 days, detecting the hole with clone growth by indirect ELISA, and taking the hole with a high OD value as the positive hole; and (3) selecting the cells of the positive holes for secondary subcloning, detecting the stable and positive hybridoma cell strains as the cells for finally preparing the monoclonal antibody, and performing expanded culture.

4. Monoclonal antibody preparation and purification

The positive cells are subjected to amplification culture and injected into the abdominal cavity of an immunodeficient Balb/C mouse (sensitized by Freund's incomplete adjuvant), and abdominal swelling of the mouse is observed after 7-10 days, which indicates that ascites is generated. When the mouse has obvious ascites, the ascites is extracted in time. And purifying the ascites of the cells, wherein the purity of the purified antibody is more than 90%. And (4) performing protein A/G column affinity purification (IgG 1, igG2a, igG2b and IgG3 subtype antibody identification). Ascites were centrifuged and the pale yellow fluid aspirated to calculate volume. The ProteinA/G packing was loaded into a gravity purification column and washed three times with PBS, each time with 10 column volumes of PBS. The ascites fluid is filled into a column and mixed gently for 3h at 4 ℃. Discharging the liquid for later use. The ProteinA/G filler was washed three times with PBS, the antibody was eluted with a pre-cooled pH3.0 HCl-Glycine eluent, and the collected antibody was immediately neutralized with 10xPBS neutralizing solution. Antibody concentration was measured and high concentration collection tubes were pooled. The antibody was loaded at a higher concentration. Dialyzed against PBS and overnight at 4 ℃. After the concentration purity was checked, the concentration was adjusted to 2mg/mL.

Example 2: immunospecific detection of monoclonal antibody for antigen bacillus subtilis plasmin QK

1. Preparation of Polyacrylamide gels

1) Preparing a 15% separating gel solution: sequentially adding deionized water 2.3mL, 30% acrylamide 5mL, tris 2.5mL of pH8.8, 10% ammonium persulfate 100 μ L, 10% SDS 100 μ L, TEMED μ L;

2) Quickly adding a separating glue solution into the gap between the two glass plates, reserving a space for filling the concentrated glue, and carefully covering a layer of isopropanol on the separating glue solution;

3) After the separation gel is completely polymerized, draining the liquid on the gel as far as possible, and then completely sucking the residual liquid by using the edge of a paper towel;

4) Preparing a concentrated gel solution with the concentration of 5 percent: adding sequentially deionized water 2.7mL, 30% acrylamide 670. Mu. L, PH 8.8.8 Tris 500. Mu.L, 10% ammonium persulfate 40. Mu.L, 10% SDS 40. Mu. L, TEMED 6. Mu.L;

5) Quickly and directly pouring concentrated glue into the polymerized separation glue, and immediately inserting a clean comb into the concentrated glue solution;

6) After the gel was polymerized completely, the comb was carefully removed and the protein electrophoresis buffer was added to the electrophoresis tank.

2. Preparation of samples

1) Culturing and collecting cells to a 15mL centrifuge tube, centrifuging at 2000rpm for 5min, and removing supernatant;

2) Washing the culture medium twice, centrifuging at 2000rpm for 5min, and removing the supernatant;

3) After collection of the cells, 1mLPBS,0.1% NP40 was added, and the cells were lysed on ice for 10min;

4) Centrifuging at 10000rpm for 5min, collecting supernatant, adding equal volume of 2 × loading buffer, boiling in boiling water for 5min, subpackaging, and storing at-20 deg.C.

3. Electrophoresis

1) Mixing a sample to be detected (10 mu L) with 6 × loading buffer (10 mu L), slowly adding 15 mu L of the mixture into a sample tank by using a pipette gun, and pre-dyeing 10 mu L of a Maker;

2) Turning on a power supply, and adopting a voltage of 200V to carry out electrophoresis until the bromophenol blue loading buffer solution migrates to the bottom of the gel in the gel;

3) Cutting off power supply, taking out gel, soaking the gel in R250 Coomassie brilliant blue, dyeing, and boiling for 5min;

4) Taking out the dyed gel, soaking the gel in tap water for decolorization, boiling for about 20min, and observing the decolorized protein bands.

4. Rotary film

1) Taking the glue, cutting the glue into a proper size, and immersing the glue into a film buffer;

2) Soaking the PVDF membrane in methanol for 1min, transferring the PVDF membrane into a membrane transfer buffer, and soaking the filter paper into the membrane transfer buffer;

3) Leaching the graphite electrode by using a film transfer buffer, laying two pieces of filter paper, and dripping a little of the film transfer buffer;

4) Laying a film, and dripping a little of film buffer; spreading glue, and dripping a little film buffer (paying attention to not generate bubbles);

5) Finally, laying two pieces of filter paper, and dripping a little of membrane buffer;

5) Covering the electrode, and adjusting the voltage to the maximum value at 1.5mA/cm ² The gel volume is transferred to the membrane for 1.5h (the load voltage is not suitable to exceed 1V/cm) ² )。

5. Sealing of

1) The membrane was removed and washed three times with PBST, 5min each time (shaking on a horizontal shaker);

2) The membrane was removed and immersed in the blocking solution at 37 deg.C, 2h or 4 deg.C overnight (1% casein or 2% OVA in blocking solution).

6. Binding antibodies

2) The membrane was removed and soaked in primary antibody dilution diluted with 1% casein at 37 ℃,1h (primary antibody dilution 1:1000 );

3) The membrane was removed and washed three times with PBST, 5min each time (shaking on a horizontal shaker);

4) Taking out the membrane, soaking in secondary antibody diluted with 1% casein at 37 deg.C for 1 hr

5) The ratio of goat anti-mouse-HRP was 1:3000-1:5000, the ratio of goat anti-rabbit-HRP is 1.

7. Exposure to light

1) Diluting and mixing A, B luminescent liquid in equal proportion (each 500 mL), placing a film on a preservative film, uniformly dripping the AB mixed liquid on the film, covering the preservative film, and standing for 1min;

2) Opening the preservative film, sucking residual liquid on the surface by using filter paper, and fixing the preservative film in a cassette;

3) Placing the cassette in a darkroom, taking out the film, rapidly placing on the inner film of the cassette, closing the cassette, exposing for 1min according to the intensity of the visible fluorescence, and optionally pressing the sheet overnight if the strip is weak;

4) The cartridge was opened and the film was immediately removed and completely immersed in the developer for 1min.

Example 3: double-antibody sandwich Elisa detection of monoclonal antibody on antigen bacillus subtilis plasmin QK

1. Sandwich ELISA method

1) Coating antibody: rat anti-QKMA-9A 10 (another anti-Bacillus subtilis plasmin QK monoclonal antibody of the company) and QKMA-1G41 rabbit antibody were extracted with CBS buffer solution according to the ratio of 1:100 is diluted into working solution with the concentration of 100 mu L/hole, and the working solution is coated for 2h at 37 ℃;

2) And (3) sealing: 5% nonfat milk, 300. Mu.L/well, blocked at 37 ℃ for 1h;

3) Antigen dilution: taking C1145 natural protein (0.95 mg/mL), processing according to the information in the table, reacting for 2h at 37 ℃ at 100 mu L/hole;

4) Detecting an antibody: rat monoclonal antibodies QKMA-9A10 and QKMA-1G41 rabbit antibodies are taken, and the ratio of the two antibodies is 1:100 dilution, 100 mu L/hole, and reaction at 37 ℃ for 1h;

5) Secondary antibody: taking a coat Anti-Rat/Rat bit HRP, and carrying out reaction according to the weight ratio of 1: diluting with 5000, 100 μ L/hole, and reacting at 37 deg.C for 30min;

6) Color development: mixing TMB solution A and B solution in equal volume, reacting at room temperature for 20min with 100 μ L/hole;

7) And (4) terminating: taking 2M H2SO4 as stop solution, 50 mu L/hole, and reading the plate at 450 nm.

2. Sandwich ELISA Standard Curve assay

1) Coating antibody: taking a C1250-2# rabbit antibody, and using CBS buffer solution to perform reaction according to the weight ratio of 1: diluting 50% to obtain working solution, coating at 37 deg.C for 2 hr, and making into capsule with 100 μ L/hole;

2) And (3) sealing: 5% non fat milk, 300. Mu.L/well, blocked at 37 ℃ for 1h;

3) Antigen dilution: c1145 native protein (0.95 mg/mL) was sampled at 1:1000 initial dilution, subsequent continuous multiple dilution, 100 mu L/hole, reaction for 2h at 37 ℃;

4) Detecting an antibody: rat monoclonal antibodies 1G4-1 and 9A10 were taken, and the ratio was 1: diluting with 50, 100 mu L/hole, and reacting at 37 ℃ for 1h;

5) Secondary antibody: taking Goat Anti-Rat HRP, and mixing the materials according to the weight ratio of 1: diluting with 5000, 100 μ L/well, and reacting at 37 deg.C for 30min;

7) And (4) terminating: taking the stop solution 2M H ₂ SO ₄ 50 μ L/well loading, 450nm plate reading.

3. Test results

By establishing a standard curve, the monoclonal antibodies QKMA-1G41 and QKMA-9A10 are used as double-antibody sandwich detection, and the linear and fitting curve meets the standard.

Example 4: sequencing of monoclonal antibodies

1. Culturing the hybridoma cells. Recovering hybridoma cell strain, culturing, and expanding cell number to about 1 × 10 ⁷ Then, 1000rpm x 5min, and centrifuged to collect the cells.

2. And extracting cell RNA. Under the environment of an ultra-clean workbench, 1mL of Trizol reagent is added into centrifugal cells, the centrifugal cells are stood for 5min, 2mL of chloroform is added, the centrifugal cells are vigorously shaken for 15sec, the mixture is stood for 3min at room temperature and is multiplied by 15min at 12000rpm, an upper water sample layer is transferred to a new EP tube, 0.5mL of isopropanol is added, and the mixture is stood for 10min at room temperature. 12000rpm 10min. The supernatant was discarded, and then 1mL of 75% ethanol was added thereto for 7500rpmx 5min, and the precipitate was dried and 50. Mu.L of double distilled water was added. The purity was determined and quantified by agarose electrophoresis and stored at-70 ℃ for use.

3. Reverse transcription to prepare cDNA. mu.L of total cellular RNA, 6. Mu.L of RNase Free ddH2O, 0.5. Mu.L of oligo dT Primer, 0.5. Mu.L of PRIME Script RT Enzyme Mix I, 2. Mu.L of 5xPrime Script Buffer, mixed well, 15min at 37 ℃ and 5s at 85 ℃.

4. The cDNA is amplified. The above cDNAs were amplified separately using a library of designed mouse IgG VH VL primers. 5xPrime Star Buffer 10. Mu.L, dNTP 4. Mu.L, cDNA 1. Mu.L, forward primer 1. Mu.L, reverse primer 1. Mu.L, primeSTAR 0.5. Mu.L, water make up to 50. Mu.L. The PCR reaction was carried out under the following reaction conditions of 94 ℃ incubation for 5min, 94 ℃ denaturation 45s,63 ℃ annealing 45s,72 ℃ extension for 1min,30 cycles followed by 72 ℃ extension for 10min.

5. Agarose gel electrophoresis and gel recovery and sequencing. And (3) carrying out agarose gel electrophoresis on the PCR product, observing an electrophoresis result, and delivering the amplified product to sequencing. The nucleotide sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:2 is shown in the specification; the amino acid sequence of the heavy chain is shown as SEQ ID NO:3 is shown in the specification; the coding amino acid sequence of the light chain is shown as SEQ ID NO:4, respectively.

The identified hybridoma cell line is preserved in the national type culture collection at 12 months and 13 days in 2021 at the place of Wuhan university in Wuhan, china with the preservation number of CCTCC NO: C2021307, is named as a bacillus subtilis plasmin resistant monoclonal antibody QKMA-1G41 cell strain and is classified and named as a rat monoclonal hybridoma cell strain.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Wuhan Zhen Fu medicine GmbH

<120> monoclonal antibody QKMA-1G41 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1325

<212> DNA

<213> Bacillus subtilis

<400> 1

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ccaggaaaag gtctggagtg gatgggaata atgtggaata atggagacac ttcatataat 180

tcaactctca aatcccgact gagcatcagc agggacacct ccaagagcca aattttctta 240

aaaatgagca gtctgcaaat tgaagacaca gccacttact actgtgccag agaggggact 300

acagtggtga cagtttggga ttactggggc caaggagtca cggtcaccgt ctcctcggct 360

gaaacaacag ccccatctgt ctatccactg gctcctggaa ctgctctcaa aagtaactcc 420

atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtcac cgtgacctgg 480

aactctggag ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctggactc 540

tacactctca ccagctcagt gactgtaccc tccagcacct ggtccagcca ggccgtcacc 600

tgcaacgtag cccacccggc cagcagcacc aaggtggaca agaaaattgt gccaagggaa 660

tgcaatcctt gtggatgtac aggctcagaa gtatcatctg tcttcatctt ccccccaaag 720

accaaagatg tgctcaccat cactctgact cctaaggtca cgtgtgttgt ggtagacatt 780

agccagaatg atcccgaggt ccggttcagc tggtttatag atgacgtgga agtccacaca 840

gctcagactc atgccccgga gaagcagtcc aacagcactt tacgctcagt cagtgaactc 900

cccatcgtgc accgggactg gctcaatggc aagacgttca aatgcaaagt caacagtgga 960

gcattccctg cccccatcga gaaaagcatc tccaaacccg aaggcacacc acgaggtcca 1020

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ccacaggaaa actacaagaa cactccacct acgatggaca cagatgggag ttacttcctc 1200

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accaagctgg aaataaaacg ccggaccgag ctggaaataa aacgggcaga tgctgcacca 360

actgtatcca tcttcccacc atccatggaa cagttaacat ctggaggtgc cacagtcgtg 420

tgcttcgtga acaacttcta tcccagagac atcagtgtca agtggaagat tgatggcagt 480

gaacaacgag atggtgtcct ggacagtgtt actgatcagg acagcaaaga cagcacgtac 540

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Glu Val Lys Leu Val Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

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Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr

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His Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

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Gly Ile Met Trp Asn Asn Gly Asp Thr Ser Tyr Asn Ser Thr Leu Lys

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Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Ile Phe Leu

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Lys Met Ser Ser Leu Gln Ile Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

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Arg Glu Gly Thr Thr Val Val Thr Val Trp Asp Tyr Trp Gly Gln Gly

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Val Thr Val Thr Val Ser Ser Ala Glu Thr Thr Ala Pro Ser Val Tyr

115 120 125

Pro Leu Ala Pro Gly Thr Ala Leu Lys Ser Asn Ser Met Val Thr Leu

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

145 150 155 160

Asn Ser Gly Ala Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Gly Leu Tyr Thr Leu Thr Ser Ser Val Thr Val Pro Ser Ser

180 185 190

Thr Trp Ser Ser Gln Ala Val Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

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

210 215 220

Gly Cys Thr Gly Ser Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys

225 230 235 240

Thr Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val

245 250 255

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

260 265 270

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

275 280 285

Gln Ser Asn Ser Thr Leu Arg Ser Val Ser Glu Leu Pro Ile Val His

290 295 300

Arg Asp Trp Leu Asn Gly Lys Thr Phe Lys Cys Lys Val Asn Ser Gly

305 310 315 320

Ala Phe Pro Ala Pro Ile Glu Lys Ser Ile Ser Lys Pro Glu Gly Thr

325 330 335

Pro Arg Gly Pro Gln Val Tyr Thr Met Ala Pro Pro Lys Glu Glu Met

340 345 350

Thr Gln Ser Gln Val Ser Ile Thr Cys Met Val Lys Gly Phe Tyr Pro

355 360 365

Pro Asp Ile Tyr Thr Glu Trp Lys Met Asn Gly Gln Pro Gln Glu Asn

370 375 380

Tyr Lys Asn Thr Pro Pro Thr Met Asp Thr Asp Gly Ser Tyr Phe Leu

385 390 395 400

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

405 410 415

Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu

420 425 430

Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 4

<211> 221

<212> PRT

<213> Bacillus subtilis

<400> 4

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

1 5 10 15

Asp Arg Val Thr Ile Asn Cys Lys Ala Ser Gln Asn Val Asn Arg Phe

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Leu Asn Trp Tyr Gln Gln Lys Leu Gly Gly Ala Pro Lys Arg Leu Ile

35 40 45

Asp Asn Thr Asn Asn Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln His His Ser Leu Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Arg Thr Glu Leu Glu

100 105 110

Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser

115 120 125

Met Glu Gln Leu Thr Ser Gly Gly Ala Thr Val Val Cys Phe Val Asn

130 135 140

Asn Phe Tyr Pro Arg Asp Ile Ser Val Lys Trp Lys Ile Asp Gly Ser

145 150 155 160

Glu Gln Arg Asp Gly Val Leu Asp Ser Val Thr Asp Gln Asp Ser Lys

165 170 175

Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Ser Leu Thr Lys Val Glu

180 185 190

Tyr Glu Arg His Asn Leu Tyr Thr Cys Glu Val Val His Lys Thr Ser

195 200 205

Ser Ser Pro Val Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215 220

Claims

1. A monoclonal antibody QKMA-1G41, wherein the nucleotide sequence of the heavy chain is as shown in SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:2, respectively.

2. The monoclonal antibody QKMA-1G41 of claim 1, wherein the heavy chain encodes the amino acid sequence of SEQ ID NO:3 is shown in the specification; the coding amino acid sequence of the light chain is shown as SEQ ID NO:4, respectively.

3. The monoclonal antibody QKMA-1G41 of claim 1, wherein the heavy chain comprises a heavy chain variable region VH and the light chain comprises a light chain variable region VL; wherein, the heavy chain variable region VH amino acid sequence is SEQ ID NO:3, light chain variable region VL amino acid sequence is SEQ ID NO: bits 1-113 of 4.

4. The monoclonal antibody QKMA-1G41 as claimed in claim 3, wherein the VH and VL epitope complementarity determining regions CDR consist of CDR1, CDR2, CDR3, wherein:

5. The monoclonal antibody QKMA-1G41 of claim 1, wherein the Bacillus subtilis plasmin is prepared by fermenting Bacillus subtilis QK02 which is deposited in China center for type culture collection with the preservation number of CCTCC NO: M203078.

6. The monoclonal antibody QKMA-1G41 of claim 5, wherein said Bacillus subtilis plasmin comprises plasmin QK and nattokinase.

7. The monoclonal antibody QKMA-1G41 of claim 1, which is a mouse IgG1 subtype monoclonal antibody.

8. The use of the monoclonal antibody QKMA-1G41 as defined in any one of claims 1 to 7 for the identification and detection of B.subtilis plasmin.

9. The use of the monoclonal antibody QKMA-1G41 of any one of claims 1-7 in the fusion expression of Bacillus subtilis plasmin and the preparation of targeted drugs.

10. The monoclonal antibody QKMA-1G41 hybridoma cell line as claimed in any one of claims 1 to 7, which is deposited in China center for type culture Collection with the preservation number of CCTCC NO: C2021307.