CN115304677A

CN115304677A - Monoclonal antibody QKMA-9A10 for resisting bacillus subtilis plasmin and application thereof

Info

Publication number: CN115304677A
Application number: CN202210693831.6A
Authority: CN
Inventors: 王业富; 董艳山; 肖帅
Original assignee: Hubei Zhen Fu Pharmaceutical Co ltd
Current assignee: Hubei Zhen Fu Pharmaceutical Co ltd
Priority date: 2022-06-19
Filing date: 2022-06-19
Publication date: 2022-11-08

Abstract

The invention provides a monoclonal antibody QKMA-9A10 for resisting bacillus subtilis fibrinolytic enzyme and application thereof, belonging to the technical field of genetic engineering. The antibody comprises a heavy chain and a light chain, wherein the coding amino acid sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the coding amino acid sequence of the light chain is shown as SEQ ID NO:2, respectively. The monoclonal antibody QKMA-9A10 of the anti-bacillus subtilis fibrinolytic enzyme can be used in 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.

Description

Monoclonal antibody QKMA-9A10 for resisting bacillus subtilis plasmin and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a monoclonal antibody QKMA-9A10 for resisting bacillus subtilis plasmin and application thereof.

Background

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 the nattokinase produced by fermenting 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 test proves that 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. In addition, nattokinase, as a bacillus subtilis fibrinolytic enzyme, can be orally taken to play a thrombolytic effect. Natto, a functional food originated from Japan, contains nattokinase. China is also a large consuming country of natto related products. However, the current natto market is mixed with fish and dragon, and the true and false are hard to distinguish. 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-9A10 for resisting bacillus subtilis plasmin and application thereof, and the monoclonal antibody QKMA-9A10 for resisting the bacillus subtilis plasmin can be used for histochemical method, enzyme-linked immunosorbent assay and immunoblotting method in identification and detection of the bacillus subtilis plasmin and fusion expression and preparation of targeted drugs.

The technical scheme of the invention is realized as follows:

the invention provides a monoclonal antibody QKMA-9A10 for resisting bacillus subtilis plasmin, which comprises a heavy chain and a light chain, wherein the coding amino acid sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the coding amino acid sequence of the light chain is shown as SEQ ID NO:2, 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:1, and the light chain variable region VL amino acid sequence is SEQ ID NO: bits 1-106 of 2.

As a further improvement of the invention, the VH and VL epitope 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 GlyPheSerLeuThrThrTyrHis;

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 glyPheLysSerAspHisValMetAspAla;

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

the amino acid sequence of the VL-CDR2 is the 50 th to the 52 th positions in SEQ ID NO. 4, and the sequence is HisThrAsn;

the amino acid sequence of VL-CDR3 is 89-96 th in SEQ ID NO. 4, and the sequence is Le mu glnHisAsnSerLeuTrpThr.

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

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

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

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

The invention further protects the application of the monoclonal antibody QKMA-9A10 for resisting the bacillus subtilis plasmin in the identification and detection of the bacillus subtilis plasmin.

The invention further discloses an application of the monoclonal antibody QKMA-9A10 for resisting the bacillus subtilis plasmin in fusion expression of the bacillus subtilis plasmin and preparation of targeted drugs.

The invention further protects the monoclonal antibody QKMA-9A10 hybridoma cell line for resisting the bacillus subtilis plasmin, which is preserved in China center for type culture collection with the preservation number of CCTCC NO: C2021306, is classified and named as a rat monoclonal hybridoma cell strain, and has the preservation date of 2021 year, 12 months and 13 days.

The invention has the following beneficial effects: the monoclonal antibody QKMA-9A10 for resisting the bacillus subtilis fibrinolytic enzyme can be used for histochemical method, enzyme linked immunosorbent assay and immunoblotting method in identification and detection of the 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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within 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 months and 17 days in 2003.

The monoclonal antibody QKMA-9A10 hybridoma cell line for resisting bacillus subtilis plasmin is preserved in China center for type culture collection, the preservation place is Wuhan university in China, the preservation number is CCTCC NO: C2021306, the hybridoma cell line is classified and named as 'rat monoclonal hybridoma cell strain QKMA-9A 10', and the preservation date is 2021, 12 months and 13 days.

The invention provides a monoclonal antibody QKMA-9A10 for resisting bacillus subtilis plasmin, which comprises a heavy chain and a light chain, wherein the coding amino acid sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the coding amino acid sequence of the light chain is shown as SEQ ID NO:2, 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:1, and the light chain variable region VL amino acid sequence is SEQ ID NO: bits 1-106 in 2.

The VH and VL antigenic determinant complementarity determining region CDR consists of CDR1, CDR2 and CDR3, wherein:

the amino acid sequence of VH-CDR2 is 51-57 th bit 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 glyPheLysSerRaspHisValMetAspAla;

the amino acid sequence of VL-CDR3 is 89-96 th of SEQ ID NO. 4, and the sequence is Le mu glnHisAsnSerLeuTrpThr.

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, is coated overnight at 4 ℃, and has purity of more than 95 percent.

2. Animal immunization

6 Balb/C mice 5-8 weeks old and 4 Wistar rats 8 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, and the boost 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. Screening assays were started about 10 days after fusion. Cell fusion results: after fusion, HAT selective medium is used for culture, and observation is carried out under a microscope, a plurality of growing hybridoma cells are observed, and the success of the fusion operation is proved. 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. (since the positive well cell line obtained by the first subcloning is not stable yet and may contain multiple hybridoma cells, it is generally accepted that the hybridoma cells are a single cell line after the second subcloning and are determined to be positive). 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 was packed in a column and gently mixed for 3 hours at 4 ℃. Discharging the liquid for later use. The ProteinA/G pad was washed three times with PBS, the antibody was eluted with pre-cooled pH3.0 HCL-Glycine eluent, and the collected antibody was immediately neutralized with 10 × PBS neutralization solution. And detecting the concentration of the antibody, and combining the high-concentration collection tubes. The antibody was loaded at a higher concentration at elution. Dialyzed against PBS and overnight at 4 ℃. After the concentration purity was checked, the concentration was adjusted to 2mg/mL.

Example 2: immunospecificity detection of monoclonal antibody to antigen bacillus subtilis plasmin QK

1. Preparation of Polyacrylamide gels

1) Preparing a 15% separating gel solution: sequentially adding 2.3mL of deionized water, 5mL of 30% acrylamide, 2.5mL of Tris (pH8.8), 100 μ L of 10% ammonium persulfate, 100 μ L of 10% SDS, and 6 μ L of TEMED (after TEMED is added, the separation gel immediately starts to polymerize, so that the separation gel is immediately and uniformly mixed);

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 deionized water 2.7mL, acrylamide 30% 670. Mu.L, tris 500. Mu.L of pH8.8, ammonium persulfate 10% 40. Mu.L, SDS 10% 40. Mu.L, and TEMED 6. Mu.L in this order (after TEMED is added, the separation gel immediately starts to polymerize, so it should be mixed uniformly quickly);

5) Pouring concentrated gel directly on the polymerized separating gel, and immediately inserting a clean comb (carefully avoiding air bubbles) into the concentrated gel solution;

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

2. Preparation of samples

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

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

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

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

3. Electrophoresis

1) Mixing a sample to be detected (10. Mu.L) with a 2 × (or 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-staining 10. Mu.L of a marker;

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 the power supply, taking out the gel, soaking the gel in R250 Coomassie brilliant blue for dyeing, and boiling for 5 minutes;

4) Taking out the dyed gel, soaking the gel in tap water for decolorization, boiling for about 20 minutes, 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 1 minute, transferring the PVDF membrane into a membrane transfer buffer, and soaking filter paper into the membrane transfer buffer (the PVDF membrane and the filter paper are cut into the same size as the glue);

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

4) Laying a film, and dripping a little film buffer; spreading glue, and dripping a little of film buffer;

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

5) Cover power-onThe voltage is adjusted to the maximum value at 1.5mA/cm ² The gel volume is transferred to the membrane for 1.5 hours (the load voltage is not more than 1V/cm) ² )。

5. Sealing of

1) The membrane was removed, washed three times with PBST and shaken on a shaker at 150rpm for 5 minutes each time;

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

6. Binding antibodies

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

3) Taking out the membrane, washing the membrane with PBST for three times, and oscillating the membrane on a 150rpm shaking table for 5 minutes each time;

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

5) Goat anti-mouse-HRP 1:3000-1:5000, goat anti-rabbit-HRP 1.

7. Exposure method

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

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 dark room, taking out the film, quickly placing the film on the inner film of the cassette, closing the cassette, and exposing according to the intensity of the visible fluorescence (the exposure time is 1 minute, and if the strip is weak, the sheeting can be selected overnight);

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

Example 3: the monoclonal antibody is used for detecting double-antibody sandwich Elisa of 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-9A10 rabbit antibodies were extracted with CBS buffer solution according to the ratio of 1:100 is diluted into working solution, 100 mu L/hole, and the working solution is coated for 2 hours at 37 ℃;

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

3) Antigen dilution: taking C1145 natural protein (0.95 mg/mL), reacting at 37 ℃ for 2 hours in 100 mu L/hole;

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

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 mu L/hole, and reacting at 37 ℃ for 30 minutes;

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

7) And (4) terminating: taking stop solution 2 MH ₂ SO ₄ 50 μ L/well, 450nm plate read.

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 into working solution at 50 μ L/hole, and coating at 37 deg.C for 2 hr;

3) Antigen dilution: the C1145 native protein (0.95 mg/mL) was sampled at a rate of 1:1000 initial dilution, subsequent continuous multiple dilution, 100 mu L/hole, reaction at 37 ℃ for 2 hours;

4) And (3) 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 1 hour;

5) Secondary antibody: taking coat Anti-Rat HRP, and mixing the materials according to the proportion of 1: diluting with 5000, 100 mu L/hole, and reacting at 37 ℃ for 30 minutes;

7) And (4) terminating: taking stop solution 2M H ₂ SO ₄ 50 μ L/well, and 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 At 1000rpm for 5 min, the cells were harvested by centrifugation.

2. And extracting cell RNA. Adding 1mL of Trizol reagent into the centrifugal cells under an ultra-clean workbench environment, standing for 5 minutes, adding 2mL of chloroform, violently shaking for 15 seconds, standing for 3 minutes at room temperature, multiplying by 12000rpm for 15 minutes, transferring an upper water sample layer to a new EP tube, adding 0.5mL of isopropanol, and standing for 10 minutes at room temperature. 12000rpm 10 min. The supernatant was discarded, and 1mL of 75% ethanol was added thereto at 7500rpm for 5 minutes, and the precipitate was dried, and 50. Mu.L of double distilled water was added thereto. The purity was identified and quantified by agarose electrophoresis and stored at-70 ℃ for future use.

3. Reverse transcription to prepare cDNA. Cellular Total RNA 1. Mu.L, RNase Free ddH ₂ O6. Mu.L, oligo dT Primer 0.5. Mu.L, PRIME Script RT Enzyme Mix I0.5. Mu.L, 5xPrime Script Buffer 2. Mu.L, mixed well, 15 minutes at 37 ℃ and 5 seconds at 85 ℃.

4. The cDNA is amplified. The cDNA was amplified 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 by incubating at 94 ℃ for 5 minutes, denaturing at 94 ℃ for 45 seconds, annealing at 63 ℃ for 45 seconds, extending at 72 ℃ for 1 minute, and extending at 72 ℃ for 10 minutes after 30 cycles.

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:3 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:4 is shown in the specification; the amino acid sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the coding amino acid sequence of the light chain is shown as SEQ ID NO:2, 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: C2021306, and is named as the cell line of the anti-bacillus subtilis plasmin monoclonal antibody QKMA-9A 10.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Sequence listing

<110> Hubei Zhenfu medicine Co., ltd

<120> monoclonal antibody QKMA-9A10 for resisting bacillus subtilis plasmin and application

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

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

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

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

65 70 75 80

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

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Arg Glu Gly Phe Lys Ser Asp Ser His Val Met Asp Ala Trp Gly Gln

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Gly Ala Ser Val Thr Val Ser Ser Ala Glu Thr Thr Ala Pro Ser Val

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

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

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

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

180 185 190

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

195 200 205

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

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

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

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

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

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

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

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

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

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

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

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ccaggaaaag gtctggagtg gatgggagta atgtggaata atggagacac atcatataat 180

tcagctctca aatcccgact gagcgtcagc agggacacct ccaagagcca agttttctta 240

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aagtccgatt cccatgttat ggatgcctgg ggtcaaggag cttcagtcac tgtctcctca 360

gctgaaacaa cagccccatc tgtctatcca ctggctcctg gaactgctct caaaagtaac 420

tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt caccgtgacc 480

tggaactctg gagccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgga 540

ctctacactc tcaccagctc agtgactgta ccctccagca cctggtccag ccaggccgtc 600

acctgcaacg tagcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgccaagg 660

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acagctcaga ctcatgcccc ggagaagcag tccaacagca ctttacgctc agtcagtgaa 900

ctccccatcg tgcaccggga ctggctcaat ggcaagacgt tcaaatgcaa agtcaacagt 960

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<400> 4

gacatccaga tgacccagtc tccttcattc ctgtctgcat ctgtgggaga cagagtcact 60

atcaactgca aagcgagtca gaatattaac aagtacttaa gctggtatca gcagaagctt 120

ggagaagctc ccaaacgcct gatatatcat acaaacaatt tgcaaacagg catcccatca 180

aggttcagtg gcggtggatc tggtacagat ttcacactca ccatcagcag cctgcagcct 240

gaggattttg ccacatattt ctgcttgcaa cataatagtt tgtggacgtt cggtggaggg 300

accaagctgg aattgaaacg ggcagatgct gcaccaactg tatccatctt cccaccatcc 360

atggaacagt taacatctgg aggtgccaca gtcgtgtgct tcgtgaacaa cttctatccc 420

agagacatca gtgtcaagtg gaagattgat ggcagtgaac aacgagatgg tgtcctggac 480

agtgttactg atcaggacag caaagacagc acgtacagca tgagcagcac cctctcgttg 540

accaaggttg aatatgaaag gcataacctc tatacctgtg aggttgttca taagacatca 600

tcctcacccg tcgtcaagag cttcaacagg aatgagtgtt a 641

Claims

1. A monoclonal antibody QKMA-9A10 resisting bacillus subtilis plasmin, comprising a heavy chain and a light chain, wherein the coding amino acid sequence of the heavy chain is shown as SEQ ID NO:1 is shown in the specification; the coding amino acid sequence of the light chain is shown as SEQ ID NO:2, respectively.

2. The monoclonal antibody QKMA-9A10 against Bacillus subtilis plasmin, according to claim 1, wherein said 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:1, and the light chain variable region VL amino acid sequence is SEQ ID NO: bits 1-106 in 2.

3. The monoclonal antibody QKMA-9A10 against Bacillus subtilis plasmin of claim 2, wherein the VH and VL epitope Complementarity Determining Regions (CDRs) consist of CDR1, CDR2, CDR3, wherein:

4. The monoclonal antibody QKMA-9A10 against Bacillus subtilis plasmin according to claim 1, wherein the nucleotide sequence of the heavy chain thereof is as shown in SEQ ID NO:3 is shown in the specification; the nucleotide sequence of the light chain is shown as SEQ ID NO:4, respectively.

5. The monoclonal antibody QKMA-9A10 against Bacillus subtilis plasmin according to claim 1, wherein the antibody is a mouse IgG1 subtype monoclonal antibody.

6. The monoclonal antibody QKMA-9A10 against Bacillus subtilis plasmin according to claim 1, wherein the Bacillus subtilis plasmin is prepared by fermenting Bacillus subtilis QK02, and the Bacillus subtilis plasmin is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M203078.

7. The monoclonal antibody QKMA-9A10 against Bacillus subtilis plasmin according to claim 1 wherein said Bacillus subtilis plasmin comprises plasmin QK and nattokinase.

8. The use of the monoclonal antibody QKMA-9A10 against Bacillus subtilis plasmin according to any one of claims 1-7 in the identification and detection of Bacillus subtilis plasmin.

9. The use of the monoclonal antibody QKMA-9A10 against Bacillus subtilis plasmin of any one of claims 1-7 in fusion expression of Bacillus subtilis plasmin and preparation of targeted drugs.

10. The monoclonal antibody QKMA-9A10 hybridoma cell line against Bacillus subtilis plasmin according to any one of claims 1-7, wherein said hybridoma cell line is deposited in China center for type culture Collection with the preservation number of CCTCC NO: C2021306.