A monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein and its preparation method and application

Abstract

The present invention discloses a monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein and a preparation method and application thereof, and belongs to the field of antibody technology. The monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein of the present invention has the characteristics of high affinity and strong specificity. When the monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein of the present invention or its antigen binding portion is used to detect enterotoxigenic Escherichia coli in a biological sample, it has the advantages of high sensitivity, strong specificity, and reliable detection results, which is helpful for the prediction or diagnosis of enterotoxigenic Escherichia coli infection diseases and has a very high clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to a monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein, and a preparation method and application thereof. Background Art

[0002] Enterotoxigenic Escherichia coli Escherichia coli ETEC (ETEC) is an important pathogen that causes diarrhea in humans and young animals (newborn piglets, calves, lambs and weaned piglets). When newborn animals are infected with ETEC, they often die from severe watery diarrhea and rapid dehydration, with high morbidity and mortality rates.

[0003] After enterotoxigenic E. coli infects young animals, it binds to the receptors of the small intestinal epithelial cells through the adhesive pili on the surface of the bacteria, settles and reproduces, and then produces two types of enterotoxins: heat-sensitive enterotoxin and heat-resistant enterotoxin. Adhesive pili and enterotoxin are the main pathogenic factors of ETEC. Enterotoxin can cause water and electrolyte metabolism disorders, leading to diarrhea and even death. The pili related to the adhesion of enterotoxigenic E. coli in humans and livestock include K88, K99, F41, 987P, CFⅠ, CFⅡ, CFⅢ, CFⅣ, etc.

[0004] Therefore, monoclonal antibodies against enterotoxigenic Escherichia coli K99 pilus proteins can provide technical support for the detection of diseases such as diarrhea caused by enterotoxigenic Escherichia coli. However, the art lacks highly sensitive and specific monoclonal antibodies against enterotoxigenic Escherichia coli K99. Summary of the invention

[0005] In order to solve the above technical problems, the inventors used a bovine enterotoxigenic Escherichia coli as an antigen for animal immunization and screened to obtain a hybridoma cell line, whose product, 6D3 monoclonal antibody, has high affinity and specificity for bovine enterotoxigenic Escherichia coli and does not cross-react with other bacterial antigens, thereby completing the present invention.

[0006] The first aspect of the present invention provides a monoclonal antibody or an antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein, the monoclonal antibody or the antigen-binding portion thereof comprising:

[0007] A heavy chain variable region, wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID No. 7 to SEQ ID No. 9, respectively; and

[0008] The amino acid sequences of the light chain variable region, CDR-L1, CDR-L2, and CDR-L3, are shown in SEQ ID No. 12 to SEQ ID No. 14, respectively.

[0009] In some embodiments of the present invention, the monoclonal antibody or antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein comprises:

[0010] A heavy chain variable region, the amino acid sequence of which is shown in SEQ ID No. 6; and

[0011] The amino acid sequence of the light chain variable region is shown in SEQ ID No. 11.

[0012] In the present invention, the monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein has an Igκ light chain constant region subtype and an IgG1 heavy chain constant region subtype.

[0013] In the present invention, the antigen binding portion refers to the antigen binding fragment of an antibody and an antibody analog, which generally includes at least part of the antigen binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. In some embodiments of the present invention, the antigen binding portion is selected from any one of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment and a scFv antibody. Among them,

[0014] The "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain.

[0015] A "Fab' fragment" contains a light chain and a portion of a heavy chain including a VH domain, a CH1 domain and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 fragment.

[0016] A "F(ab')2 fragment" contains two light chains and a portion of two heavy chains including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0017] An "Fv fragment" contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0018] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, wherein these domains are contained in a single polypeptide chain.

[0019] In the present invention, the K99 pilus protein is also referred to as K99 antigen. Different enterotoxigenic Escherichia coli carry different types of pilus proteins, and K99 pilus is one of the main pilus carried by such pathogenic bacteria. The monoclonal antibody of the present invention can be used to detect K99 pilus protein or K99 antigen, thereby achieving the diagnosis or prediction of enterotoxigenic Escherichia coli infection diseases.

[0020] In some embodiments of the present invention, the monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein is a recombinant antibody. Further, the monoclonal antibody or its antigen-binding portion comprises:

[0021] A heavy chain, the amino acid sequence of which is shown in SEQ ID No. 19; and

[0022] The light chain has an amino acid sequence as shown in SEQ ID No. 25.

[0023] The second aspect of the present invention provides a gene encoding a monoclonal antibody or an antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein as described in any one of the first aspect of the present invention.

[0024] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No. 5; the nucleotide sequence encoding the light chain variable region is shown as SEQ ID No. 10.

[0025] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain is shown as SEQ ID No. 20; the nucleotide sequence encoding the light chain variable region is shown as SEQ ID No. 26.

[0026] The third aspect of the present invention provides a recombinant vector comprising the gene encoding the monoclonal antibody or antigen binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein described in any one of the first aspect of the present invention as described in the second aspect of the present invention.

[0027] The recombinant vector may refer to a cloning vector or an expression vector, which may be obtained by connecting the encoding nucleic acid with a commercially available vector (such as a plasmid or a viral vector). The recombinant vector in the present invention is not particularly limited, and commonly used plasmids may be used, such as pSeTag2, PEE14, pMH3, pcDNA3.1, pcDNA3.4, pTT5, etc.

[0028] In some embodiments of the present invention, the nucleic acid encoding the full-length heavy chain variable region and the nucleic acid encoding the full-length light chain variable region are respectively connected to different expression vectors to obtain two recombinant vectors.

[0029] In some specific embodiments of the present invention, the coding nucleotide sequences of mouse heavy chain signal peptide, full-length heavy chain variable region, and mouse IgG1 constant region are cloned into an expression vector to form a recombinant vector expressing the heavy chain variable region; the coding nucleotide sequences of mouse Kappa chain signal peptide, full-length light chain variable region, and mouse Kappa chain constant region are cloned into an expression vector to form a recombinant vector expressing the light chain variable region.

[0030] In some preferred embodiments of the present invention, the mouse heavy chain signal peptide amino acid sequence is shown in SEQ ID No. 15, and its nucleotide sequence is shown in SEQ ID No. 16; the mouse IgG1 constant region amino acid sequence is shown in SEQ ID No. 17, and its nucleotide sequence is shown in SEQ ID No. 18. Thus, the obtained heavy chain amino acid sequence is shown in SEQ ID No. 19, and its nucleotide sequence is shown in SEQ ID No. 20.

[0031] In some preferred embodiments of the present invention, the mouse Kappa chain signal peptide amino acid sequence is shown in SEQ ID No. 21, and its nucleotide sequence is shown in SEQ ID No. 22; the mouse Kappa chain constant region amino acid sequence is shown in SEQ ID No. 23, and its nucleotide sequence is shown in SEQ ID No. 24. Thus, the obtained light chain amino acid sequence is shown in SEQ ID No. 25, and its nucleotide sequence is shown in SEQ ID No. 26.

[0032] The fourth aspect of the present invention provides a recombinant cell, comprising the recombinant vector described in the third aspect of the present invention.

[0033] The recombinant cell carries the aforementioned gene, recombinant vector or transformant or monoclonal antibody or antigen binding fragment against enterotoxigenic Escherichia coli K99 pilus protein. The recombinant cell is obtained by transfecting or transforming the recombinant vector.

[0034] According to an embodiment of the present invention, the recombinant cell can efficiently express the above-mentioned monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein under suitable conditions.

[0035] It should be noted that the recombinant cell of the present invention is not particularly limited and can be a prokaryotic cell, a eukaryotic cell or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cell can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, insect cells such as Spodoptera frugiperda, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In certain embodiments, the recombinant cell of the present invention is preferably a mammalian cell, including BHK cells, CHO cells, 293F cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0036] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of monoclonal antibodies against enterotoxigenic Escherichia coli K99 pilus proteins described in this application. It is easy for those skilled in the art to understand that conditions suitable for the expression of monoclonal antibodies against enterotoxigenic Escherichia coli K99 pilus proteins include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of monoclonal antibodies against enterotoxigenic Escherichia coli K99 pilus proteins according to the specific environment of the laboratory.

[0037] The fifth aspect of the present invention provides a method for preparing a monoclonal antibody or an antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein, comprising the following steps:

[0038] The gene described in the second aspect of the present invention is cloned into an expression vector to obtain a recombinant vector, and the recombinant vector is then transfected or transformed into a host cell to obtain a recombinant cell, and the recombinant cell is induced to express.

[0039] In some embodiments of the present invention, the preparation method comprises the following steps:

[0040] The recombinant vector described in the third aspect of the present invention is transfected or transformed into a host cell to obtain a recombinant cell, and the recombinant cell is induced to express.

[0041] In some embodiments of the present invention, the preparation method includes the step of inducing the expression of the recombinant cell described in the fourth aspect of the present invention.

[0042] The sixth aspect of the present invention provides the use of any monoclonal antibody or antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein described in the first aspect of the present invention in the preparation of a kit for detecting enterotoxigenic Escherichia coli in a biological sample.

[0043] In the present invention, the detection of enterotoxigenic Escherichia coli is further used for prediction or diagnosis of diseases infected by enterotoxigenic Escherichia coli. Diseases infected by enterotoxigenic Escherichia coli include but are not limited to diarrhea and the like.

[0044] In some embodiments of the present invention, the enterotoxigenic E. coli is detected by immunoassay using the monoclonal antibody against enterotoxigenic E. coli K99 pilus protein or its antigen binding portion, wherein the immunoassay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, in situ hybridization, immunofluorescence, Western blot, and flow cytometry.

[0045] The seventh aspect of the present invention provides a kit for predicting or diagnosing enterotoxigenic Escherichia coli infection diseases, comprising any monoclonal antibody or antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein described in the first aspect of the present invention.

[0046] Beneficial Effects of the Invention

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein of the present invention has high affinity, and the indirect ELISA detection result shows that the EC50 of the monoclonal antibody against bovine enterotoxigenic Escherichia coli K99 is 12.37 ng / mL.

[0049] The monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein of the present invention has strong specificity, strongly reacts with bovine enterotoxigenic Escherichia coli K99 antigen, and does not cross-react with antigens of Salmonella, Shigella, Enterobacter cloacae, Enterobacter sakazakii, Staphylococcus and Pseudomonas aeruginosa.

[0050] When the monoclonal antibody against enterotoxigenic Escherichia coli K99 pilus protein or its antigen-binding portion of the present invention is used to detect bovine enterotoxigenic Escherichia coli in biological samples, it has the advantages of high sensitivity, strong specificity and reliable detection results, which is helpful for the prediction or diagnosis of bovine enterotoxigenic Escherichia coli infection diseases and has very high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The type and subclass identification of the 6D3 mAb are shown.

[0052] Figure 2 The reactivity of the recombinant antibodies to different bacterial antigens is shown.

[0053] Figure 3The results of the detection of the EC50 values ​​of the recombinant antibodies are shown. DETAILED DESCRIPTION

[0054] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0055] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0056] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0058] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0059] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0060] Example 1 Preparation of hybridoma cell lines

[0061] 1. Antigen Preparation

[0062] Bovine enterotoxigenic Escherichia coli (ETEC) k99 (China Inspection and Metrology Co., Ltd., VIP(S)24609) was inoculated on solid LB bacterial culture medium by the plate line method, inverted in a 37°C incubator for 18-24 hours, and when colonies appeared on the culture medium, a single clone was picked and inoculated into liquid LB culture medium for amplification. When the liquid culture medium became obviously turbid, it was centrifuged at 3000rpm for 20min, the supernatant was discarded, 1% formaldehyde was added for fixation overnight, centrifuged at 3000rpm for 20min, the supernatant was discarded, and the culture was resuspended with physiological saline for counting and stored in a 4°C refrigerator.

[0063] 2. Mouse Immunization

[0064] Five 6-8 week old female Balb / c mice were selected and the formaldehyde-inactivated bacterial solution (1×10 8 Mice were immunized by intraperitoneal injection of 0.5 mL / mouse. Mice were immunized again on days 7, 14, 21, and 28, and spleen cells were collected on day 31 for cell fusion.

[0065] 3. Cell Fusion

[0066] After grinding and counting the mouse spleen cells, mix them with mouse myeloma sp2 / 0 cells in a ratio of 3:1 in a 50mL centrifuge tube. After centrifugation (1500r / min, 3min), aspirate the supernatant, tap the bottom of the tube to evenly distribute the cells, place the centrifuge tube in a water bath preheated at 37°C, and slowly add 1mL of cell fusion agent (50% PEG-1500) within 1min. After standing for 1min, gradually add 40mL of DMEM medium that has been preheated to 37°C to dilute the PEG and make it ineffective. After centrifugation (1500r / min, 3min), discard the supernatant. After resuspending in HAT medium, spread 100,000 spleen cells / well and 200μL per well on a 96-well cell culture plate with feeder cells added in advance. Place in a 5% (V / V), 37°C CO 2 Culture in an incubator.

[0067] 4. Screening of positive wells and cell cloning

[0068] When the cells in the wells grow to cover a quarter of the bottom area of ​​the wells, the culture medium supernatant is aspirated by indirect ELISA to screen the positive cell wells. The positive cell strains that do not cross-react with the antigens of Salmonella, Shigella, Enterobacter cloacae, Enterobacter sakazakii, Staphylococcus and Pseudomonas aeruginosa are selected and cloned and cultured five times by limiting dilution method until the positive rate reaches 100%.

[0069] The monoclonal antibody produced by the obtained hybridoma cell line 6D3 has high affinity and specificity for the bovine enterotoxigenic Escherichia coli K99 antigen and does not cross-react with other bacterial antigens. After expansion and cultivation, the cell line is used to prepare ascites and frozen in liquid nitrogen.

[0070] Example 2 Large-scale preparation of monoclonal antibodies

[0071] Take 10-12 week old female Balb / c mice and inject 0.3 mL of Freund's incomplete adjuvant intraperitoneally. 7 days later, inject 0.3 mL of 10 6 The cell suspension of hybridoma cells was injected. The abdominal cavity of the mouse was obviously swollen 7-10 days after injection. The mouse was killed by cervical distension and ascites was collected. The supernatant was collected by centrifugation at 12000rpm / min for 3min, which was the ascites-type monoclonal antibody (6D3 monoclonal antibody). 6D3 monoclonal antibody was purified by protein A column chromatography and stored at -80℃.

[0072] Example 3 Detection of monoclonal antibody types and subclasses

[0073] The monoclonal antibody was identified using a mouse monoclonal antibody subclass identification kit. Bovine enterotoxigenic Escherichia coli K99 was diluted to 1×10 6 / mL, 100μL per well was added to the ELISA plate, repeated for 16 wells, and incubated at 37℃ for 2h. After shaking off the coating solution, wash with PBST 3 times. Take 50μL of 6D3 monoclonal antibody diluted 2000 times and add it to each well of the ELISA plate, incubate at 37℃ for 1h, wash with PBST 3 times, take 50μL of each of the 8 enzyme-labeled antibodies for detecting antibody Ig types and subclasses in the kit and add them to each well, set 3 replicates for each enzyme-labeled antibody, incubate at 37℃ for 30min, wash with PBST 3 times, add TMB substrate colorimetric solution, add 50μL of 2M H 2 SO 4 After color development is stopped, the OD450nm value is read using an ELISA reader. The antibody Ig type and subclass corresponding to the positive well is the antibody type and subclass of the monoclonal antibody.

[0074] After testing, it was determined that the antibody type of 6D3 monoclonal antibody was IgG type, of which the light chain constant region subtype was Igκ type and the heavy chain constant region subtype was IgG1 type (such as Figure 1 as shown).

[0075] Example 4 Cloning of heavy chain and light chain variable region genes of 6D3 monoclonal antibody

[0076] 1. Hybridoma Cell Culture and Total RNA Extraction

[0077] The cells were cultured in DMEM complete medium containing 20% ​​FBS at 37°C and 5% CO 2 Hybridoma cells were cultured in an incubator until the cell number reached 1×10 7 Total RNA was extracted from cells using a total RNA extraction kit (purchased from Tiangen).

[0078] 2. RNA Reverse Transcription

[0079] RNA reverse transcription was completed using TIANGEN genome cDNA first-strand synthesis premix reagent. The reaction system is as follows:

[0080] Table 1 RNA reverse transcription reaction system

[0081]

[0082] Table 2 Reverse transcription reaction conditions

[0083]

[0084] 3. Gene Amplification

[0085] Design upstream and downstream primers for antibody light and heavy chains.

[0086] Heavy chain variable region upstream primer HF:

[0087] CAGGTTCAGCTGCAGCAGTC (SEQ ID No. 1)

[0088] Heavy chain variable region downstream primer HR:

[0089] GCAGAGACAGTGACCAGAG (SEQ ID No. 2)

[0090] Light chain variable region upstream primer LF:

[0091] GACATTGTGATGACCCAGTCTCAC (SEQ ID No. 3)

[0092] Light chain variable region downstream primer LR:

[0093] TTTTATTTCCAGCTTGGTCCCCC (SEQ ID No. 4)

[0094] The amplified cDNA was used as a template, and the heavy chain and light chain were amplified using the synthetic heavy chain variable region primers and light chain variable region primers, respectively. The PCR program was as follows: 98°C pre-denaturation for 8 min, 28 cycles with the following parameters: 98°C denaturation for 30 sec, 55°C annealing for 30 sec, 72°C extension for 30 sec; and then 72°C full extension for 7 min.

[0095] The PCR reaction system for the heavy chain variable region was as follows: cDNA 1 μL, heavy chain variable region upstream primer (10 μM) 1 μL, heavy chain variable region downstream primer (10 μM) 1 μL, 5× PrimeSTAR Buffer 10 μL, dNTP Mixture 4 μL, Primestar DNA Polymerase 0.5 μL, ddH 2 O 32.5 μL.

[0096] The PCR reaction system for the light chain variable region was as follows: cDNA 1 μL, light chain variable region upstream primer (10 μM) 1 μL, light chain variable region downstream primer (10 μM) 1 μL, 5× PrimeSTAR Buffer 10 μL, dNTP Mixture 4 μL, Primestar DNA Polymerase 0.5 μL, ddH 2 O 32.5 μL.

[0097] 4. Cloning and screening of PCR amplification products

[0098] The PCR products were subjected to 1.5% agarose gel electrophoresis, and the antibody light and heavy chain variable region fragments were recovered using a PCR product recovery kit (Tiangen). The fragments were inserted into the pEASY-Blunt Cloning Vector vector using the transgen pEASY-Blunt Cloning Kit blunt-end ligation kit and transformed into DH5ɑ competent cells (Amp resistance), and the recombinant positive clones were screened and sequenced.

[0099] The nucleotide sequence encoding the heavy chain variable region is shown below:

[0100] CAGGTTCAGCTCCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAAGCTTCTGGCTACACATTCAGTACCTCTTGGATGAACTGGGTGAAACAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACGGATTTATCCTGGAGATGGAGATACTAATTAC AATGGGAAGTTCAAGGACAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTGCAGGAGCCTACCTTCAACCCCATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID No. 5)

[0101] The amino acid sequence of the heavy chain variable region is shown below:

[0102] QVQLQQSGPELVKPGASVKISCKASGYTFSTSWMNWVKQRPGQGLEWIGRIYPGDGDTNYNGKFKDKATLTADKSSNTAYMQLSSLTSVDSAVYFCAGAYLQPHAMDYWGQGTSVTVSS (SEQ ID No. 6)

[0103] The heavy chain variable region sequence was analyzed (Kabat) to obtain its CDR region. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the light chain variable region are:

[0104] CDR-H1: TSWMN (SEQ ID No. 7)

[0105] CDR-H2: RIYPGDGDTNYNGKFKD (SEQ ID No. 8)

[0106] CDR-H3:AYLQPHAMDY (SEQ ID No. 9)

[0107] The nucleotide sequence encoding the light chain variable region is shown below:

[0108] GATATCCAGATGACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTTTTTACACTGGTATCAGCAGAAACCAGATGGAACTTTTCAACTCCTGATCTACTACACATCAAGATTA CACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTACCAACCTGGAGCTAGAAGATATTGCCACTTACTTTTGCCAACAGGGTGATACGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID No. 10)

[0109] The amino acid sequence of the light chain variable region is shown below:

[0110] DIQMTQTTSSLSASLGDRVTISCRASQDISNFLHWYQQKPDGTFQLLIYYTSRLHSGVPSRFSGSGSGTDYSLTITNLELEDIATYFCQQGDTLPWTFGGGTKLEIK (SEQ ID No. 11)

[0111] The light chain variable region sequence was analyzed (Kabat) to obtain its CDR region. The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are:

[0112] CDR-L1: RASQDISNFLH (SEQ ID No. 12)

[0113] CDR-L2: YTSRLHS (SEQ ID No. 13)

[0114] CDR-L3: QQGDTLPWT (SEQ ID No. 14)

[0115] 5. Variable region amino acid sequence and homology analysis

[0116] The heavy chain and light chain gene sequences were compared and analyzed in the NCBI database. The analysis results showed that the monoclonal antibody light chain variable region gene sequence had the highest homology with the mouse immunoglobulin kappa chain variable region (Sequence ID: PP321885.1), with a homology of 314 / 321 and a homology percentage of 98%. The monoclonal antibody light chain variable region amino acid sequence had the highest homology with the mouse immunoglobulin kappa chain variable region amino acid sequence (Sequence ID: AAT76279.1), with a homology of 100 / 107 and a homology percentage of 93%.

[0117] The heavy chain variable region gene sequence has the highest homology with the mouse immunoglobulin heavy chain variable region (Sequence ID: AF004331.1), with a homology of 339 / 361 and a homology percentage of 94%. The monoclonal antibody heavy chain variable region amino acid sequence has the highest homology with the mouse immunoglobulin heavy chain variable region amino acid sequence (Sequence ID: AML31199.1), with a homology of 106 / 119 and a homology percentage of 89%.

[0118] Example 5 Preparation of recombinant antibodies

[0119] The coding nucleotide sequences of mouse heavy chain signal peptide, full-length heavy chain variable region, and mouse IgG1 constant region were synthesized and cloned into the expression vector PTT5.

[0120] The mouse heavy chain signal peptide amino acid sequence is as follows:

[0121] MTRLTVLALLAGLLASSRA (SEQ ID No. 15)

[0122] The encoding nucleotide sequence is as follows:

[0123] ATGACCCGGCTGACAGTCCTGGCCCTGCTGGCTGGTCTGCTGGCATCTTCTCGTGCG (SEQ ID No. 16)

[0124] The amino acid sequence of the mouse IgG1 constant region is as follows:

[0125] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTA QTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID No. 17)

[0126] The encoding nucleotide sequence is as follows:

[0127] GCCAAAACGACACCCCCATCTGTCTATCCACTGGCGCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACACTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGGAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGAACACGAATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA(SEQ IDNo. 18)

[0128] The heavy chain amino acid sequence obtained is as follows:

[0129] MTRLTVLALLAGLLASSRAQVQLQQSGPELVKPGASVKISCKASGYTFSTSWMNWVKQRPGQGLEWIGRIYPGDGDTNYNGKFKDKATLTADKSSNTAYMQLSSLTSVDSAVYFCA GAYLQPHAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDK KIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEK TISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID No. 19)

[0130] The encoding nucleotide sequence is as follows:

[0131]

[0132] The coding nucleotide sequences of mouse Kappa chain signal peptide, full length light chain variable region, and mouse Kappa chain constant region were selected, synthesized, and cloned into the expression vector PTT5.

[0133] The amino acid sequence of mouse Kappa chain signal peptide is as follows:

[0134] METDTLLLWVLLLWVPGSTGD (SEQ ID No. 21)

[0135] The encoding nucleotide sequence is as follows:

[0136] ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCTGGTTCTACTGGTGAC (SEQID No. 22)

[0137] The amino acids of the mouse kappa chain constant region are as follows:

[0138] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID No. 23)

[0139] The encoding nucleotide sequence is as follows:

[0140] CGGGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCCGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAAC AGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID No. 24)

[0141] The light chain amino acid sequence obtained is as follows:

[0142] METDTLLLWVLLLWVPGSTGDDIQMTQTTSSLSASLGDRVTISCRASQDISNFLHWYQQKPDGTFQLLIYYTSRLHSGVPSRFSGSGSGTDYSLTITNLELEDIATYFCQQGDTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID No. 25)

[0143] Its encoded nucleotide sequence is as follows:

[0144] (SEQ ID No. 26)

[0145] One day before transfection, 2.0 × 10 6 293F cells were inoculated at a density of 10 / mL. The cell density on the second day of culture can reach 4.0×10 6 After cell counting on the second day of culture, the cell viability was >95% and the viable cell density was ≥4.0×10 6 cells / mL, it can be used directly; if the cell density is lower than 4.0×10 6 Cells were collected by centrifugation (1000 rpm, 5 min) and centrifuged at 4.0 × 10 6Resuspend in culture medium at a density of 100 μg / mL, prepare 1 mg / mL PEI solution as transfection reagent in advance, prepare DNA-PEI mixed solution at a ratio of 1:3, add the mixed solution to the culture medium, and culture for 5-7 days, or end the culture when the activity is lower than 60%.

[0146] The cell culture medium was centrifuged at 8000 rpm for 20 min, the supernatant was collected and filtered with a 45 μm filter, and then purified with a protein A column to obtain the recombinant antibody, which was filtered and sterilized and stored at -20°C.

[0147] Example 6 Specificity detection of recombinant antibodies

[0148] The test objects are bovine enterotoxigenic Escherichia coli K99, Salmonella, Shigella, Enterobacter cloacae, Enterobacter sakazakii, Staphylococcus aureus and Pseudomonas aeruginosa. The above antigens are diluted to 1×10 6 / mL, 100 μL was added to each well of the ELISA plate, the coating solution was used as the blank control, and the specificity of the recombinant antibody was detected by indirect ELISA. The primary antibody was 6D3 monoclonal antibody diluted 2000 times, and the enzyme-labeled secondary antibody GAM-HRP was diluted 10000 times.

[0149] The above tests showed that the recombinant antibody reacted strongly with the antigen of bovine enterotoxigenic Escherichia coli K99, but did not cross-react with antigens of other bacteria (such as Figure 2 Therefore, the recombinant antibody has strong species specificity and can be used to detect human and animal diseases caused by enterotoxigenic Escherichia coli.

[0150] Example 7 EC50 test of recombinant antibodies

[0151] The EC50 value of the recombinant antibody was determined by indirect ELISA. Bovine enterotoxigenic Escherichia coli K99 was diluted to 1×10 6 / mL, 100μL per well was added to the ELISA plate, the coating solution was used as the blank control, overnight at 4℃, washed 3 times with PBST, and blocked with skim milk powder at 37℃ for 1h. The recombinant antibody was diluted 3-fold from 10μg / mL to 11 gradients, 50μL per well, and the last well was PBS, incubated at 37℃ for 1h. After washing 3 times with PBST, the secondary antibody GAM-HRP diluted 10000 times was added, 50μL per well, and incubated at 37℃ for 30min. After washing 3 times with PBST, TMB substrate colorimetric solution was added for color development, and 50μL 2M H 2 SO 4 After stopping the color development, the OD450nm value was read using an enzyme reader. After testing, the EC50 value of the recombinant antibody was determined to be 12.37 ng / mL ( Figure 3 as shown).

[0152] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A monoclonal antibody or an antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein, characterized in that: The monoclonal antibody or antigen-binding portion thereof comprises: A heavy chain variable region, wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID No. 7 to SEQ ID No. 9, respectively; and The amino acid sequences of the light chain variable region, CDR-L1, CDR-L2, and CDR-L3, are shown in SEQ ID No. 12 to SEQ ID No. 14, respectively.

2. The monoclonal antibody or antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein according to claim 1, characterized in that: The monoclonal antibody or antigen-binding portion thereof comprises: A heavy chain variable region, the amino acid sequence of which is shown in SEQ ID No. 6; and The amino acid sequence of the light chain variable region is shown in SEQ ID No.

11.

3. The monoclonal antibody or antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein according to claim 1 or 2, characterized in that: The antigen binding portion is selected from any one of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment and a scFv antibody.

4. A gene encoding a monoclonal antibody or an antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein according to any one of claims 1 to 3.

5. The gene according to claim 4, characterized in that The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 5; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No.

10.

6. A recombinant vector, characterized in that: Comprising the gene according to claim 4 or 5.

7. A recombinant cell, characterized in that Comprising the recombinant vector according to claim 6.

8. A method for preparing a monoclonal antibody or an antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein, characterized in that: The following steps are involved: The gene according to claim 4 or 5 is cloned into an expression vector to obtain a recombinant vector, and the recombinant vector is then transfected or transformed into a host cell to obtain a recombinant cell, and the recombinant cell is induced to express.

9. Use of the monoclonal antibody or antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein according to any one of claims 1 to 3 in the preparation of a kit for detecting enterotoxigenic Escherichia coli in a biological sample.

10. A kit for diagnosing enterotoxigenic Escherichia coli infection, characterized in that: The invention comprises a monoclonal antibody or an antigen-binding portion thereof against enterotoxigenic Escherichia coli K99 pilus protein as described in any one of claims 1 to 3.

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