An anti-human CD11b engineered antibody and its application

Abstract

The present invention provides an anti-human CD11b engineered antibody and application, the antibody includes a heavy chain variable region and a light chain variable region; the CDR1, CDR2, and CDR3 of the heavy chain variable region are amino acid sequences shown as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; the CDR1, CDR2, and CDR3 of the light chain variable region are amino acid sequences shown as SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. The anti-human CD11b engineered antibody of the present invention provides an anti-human CD11b monoclonal antibody light chain variable region and a heavy chain variable region, and the anti-CD11b engineered antibody produced by the recombinant expression of the two can specifically bind to human CD11b molecules, has high sensitivity and high specificity, and can be used for the detection of human CD11b molecules; at the same time, it can be used to develop detection reagents for auxiliary diagnosis of diseases related to abnormal expression of CD11b molecules.

Description

Technical Field

[0001] The present invention relates to an antibody for detection and diagnosis, and in particular to an anti-human CD11b engineered antibody and its application. Background Art

[0002] CD11 antigen-like family member B (CD11b) is a 170 kDa membrane glycoprotein, also known as integrin αM (ITGAM), which is mainly expressed on the surface of bone marrow-derived cells, including granulocytes, monocytes and macrophages. CD11b binds to a variety of ligands, including members of the intercellular adhesion molecule family, complement protein iC3b and fibrinogen, mediating granulocyte adhesion, migration and tissue recruitment, thereby participating in the body's immune regulation. In the field of biomedical testing, the development of CD11b antibodies has important application value and provides a new strategy for disease diagnosis, treatment monitoring and immune status assessment.

[0003] Detection of CD11b is of great significance for the evaluation of immune regulation function, such as the detection of CD11b molecules on the surface of granulocytes. Granulocytes are a heterogeneous group of white blood cells, including neutrophils, eosinophils, basophils and mast cells. Neutrophils are the most abundant granulocytes in the human body and are the first line of defense against infection. When the body is stimulated by necrotic tissue or pathogens, the toll-like receptor-related signaling pathway stimulates CD11b. CD11b mediates neutrophils to migrate to the lesion site through a series of actions such as bundling, rolling, crawling, cell blockade / firm adhesion / migration across endothelial cells, and exerts the neutrophil effector to fight against the effect of infectious factors. CD11b is a classic rolling / adhesion marker for evaluating the migration state of neutrophils, and is also an important marker for neutrophils to move from a resting state to an activated state. CD11b also plays a key role in the phagocytosis of opsonized particles, such as ic3b-coated bacteria, apoptotic cells and immune complexes (ic), which is crucial for limiting unnecessary inflammatory immune responses. iC3b ligation with CD11b also leads to the production of anti-inflammatory cytokines such as IL-10 and TGF-β.

[0004] Therefore, cloning the light and heavy chain variable region genes from CD11b hybridoma cells and constructing engineered antibodies that specifically recognize CD11b have very important practical application value for the detection of patient granulocyte immune function. Summary of the invention

[0005] In view of this, the present invention aims to overcome the defects in the prior art and proposes an anti-human CD11b engineered antibody and its application.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] The present invention provides an anti-human CD11b engineered antibody, which comprises a heavy chain variable region and a light chain variable region;

[0008] The CDR1, CDR2 and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0009] The CDR1, CDR2 and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0010] Furthermore, FR1, FR2, FR3, and FR4 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively;

[0011] The FR1, FR2, FR3 and FR4 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0012] The anti-human CD11b engineered antibody is of IgG1 subtype.

[0013] Furthermore, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.16.

[0014] The present invention also provides a nucleic acid molecule, which comprises a nucleotide sequence encoding the anti-human CD11b engineered antibody.

[0015] The present invention also provides an expression vector, which contains the nucleic acid molecule.

[0016] The present invention also provides a cell, which contains the nucleic acid molecule or the expression vector.

[0017] The present invention also provides a use of the anti-human CD11b engineered antibody in preparing a detection reagent for CD11b molecules.

[0018] Furthermore, the anti-human CD11b engineered antibody is used in the preparation of a detection reagent for CD11b molecules on the surface of granulocytes.

[0019] The present invention also provides a use of the anti-human CD11b engineered antibody in the preparation of a detection reagent for auxiliary diagnosis of diseases associated with abnormal expression of CD11b molecules.

[0020] The present invention also provides an immunoassay kit, which comprises the anti-human CD11b engineered antibody.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The anti-human CD11b engineered antibody described in the present invention provides a light chain variable region and a heavy chain variable region of an anti-human CD11b monoclonal antibody. The anti-CD11b engineered antibody produced by the recombinant expression of the two can specifically bind to human CD11b molecules with high sensitivity and high specificity, and can be used for the detection of human CD11b molecules; at the same time, it can be used to develop detection reagents for auxiliary diagnosis of diseases related to abnormal expression of CD11b molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The electrophoresis diagram of the purified product protein described in Example 3 of the present invention;

[0024] Figure 2 This is an activity detection diagram of the commercially available CD11b antibody (2.0 μg) described in Example 4 of the present invention: wherein, Figure A shows that CD45 distinguishes granulocytes from lymphocytes, and Figure B shows that the positive rate of granulocytes is 99.9%;

[0025] Figure 3 This is an activity detection diagram of the commercially available CD11b antibody (1.0 μg) described in Example 4 of the present invention: wherein, Figure A shows that CD45 distinguishes granulocytes from lymphocytes, and Figure B shows that the positive rate of granulocytes is 98.4%;

[0026] Figure 4 This is an activity detection diagram of the commercially available CD11b antibody (0.5 μg) described in Example 4 of the present invention: wherein, Figure A shows that CD45 distinguishes granulocytes from lymphocytes, and Figure B shows that the positive rate of granulocytes is 99.7%;

[0027] Figure 5 This is an activity detection diagram of the CD11b engineered antibody (2.0 μg) described in Example 4 of the present invention: wherein, Figure A shows that CD45 distinguishes granulocytes from lymphocytes, and Figure B shows that the positive rate of granulocytes is 99.8%;

[0028] Figure 6 This is an activity detection diagram of the CD11b engineered antibody (1.0 μg) described in Example 4 of the present invention: wherein, Figure A shows that CD45 distinguishes granulocytes from lymphocytes, and Figure B shows that the positive rate of granulocytes is 99.0%;

[0029] Figure 7This is an activity detection diagram of the CD11b engineered antibody (0.5 μg) described in Example 4 of the present invention, wherein Figure A shows that CD45 distinguishes granulocytes from lymphocytes, and Figure B shows that the positive rate of granulocytes is 99.0%. DETAILED DESCRIPTION

[0030] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0031] The present invention will be described in detail below with reference to the embodiments.

[0032] Example 1 Cloning of the light and heavy chain variable region genes of mouse anti-human CD11b antibodies

[0033] 1. RNA extraction: Using Trizol one-step method, 1) take about 1×10 hybridoma cells 6 1) Add 1mL Trizol, mix by pipetting, and let stand at room temperature for 10 min; 2) Add 0.2mL chloroform, shake vigorously for 15 sec, and let stand at room temperature for 2-3 min; 3) Centrifuge at 12000rpm, 4℃ for 15 min; 4) Take the supernatant, add 0.5mL isopropanol and let stand at room temperature for 15 min; 5) Centrifuge at 12000rpm, 4℃ for 15 min; 6) Discard the supernatant, add 1mL 75% ethanol to rinse, centrifuge at 7500rpm, 4℃ for 5min; 7) Discard the supernatant, dry the precipitate, and dissolve it in 30μL DEPC water;

[0034] 2. Reverse transcription to cDNA (40 μL): Use TransScript All-in-one First-Strand cDNA Synthesis SuperMix for qPCR kit for reverse transcription reaction, take 1.0 μg of total RNA, TransScript All-in-one SuperMix for qPCR 4 μL, gDNA remover 4 μL, DEPC water to 20 μL, react at 42°C for 15 min, incubate at 85°C for 5 min, and store at 20°C;

[0035] 3. PCR amplification of the light and heavy chain variable region genes of CD11b antibody

[0036] Light chain variable region gene PCR amplification reaction system (50 μL): Design universal degenerate primers: upstream primer 5'-GACATT GTG CTC ACC CAG WCT SMH-3' (SEQ ID NO.17), downstream primer 5'-CCG TTAGAT CTC CARBTT KGT SCS-3' (SEQ ID NO.18); cDNA was used as template and amplified with high-fidelity pfu DNA polymerase; PCR cycle program was 94℃ 5 min; 94℃ 30 sec, 55℃ 30 sec, 72℃ 30 sec, for a total of 30 cycles; finally, extension at 72℃ for 10 min;

[0037] Heavy chain variable region gene PCR amplification reaction system (50 μL): upstream primer 5'-CAG GTS MARCTGCAGSAG TCW GG-3' (SEQ ID NO.19); downstream primer 5'-TGA GGA GAC KGT GAC HGT GGT SCC-3' (SEQ ID NO.20); cDNA was used as template and high-fidelity pfu DNA polymerase was used for amplification; the PCR cycle program was 94℃ for 5min; 94℃, 30 sec, 55℃, 30 sec, 72℃, 30 sec, for a total of 35 cycles; and the final extension was 72℃ for 10 min.

[0038] 4. Construction of sequencing vector: pClone007 Blunt Simple Vector Kit was purchased from Qingke Biotechnology; the PCR products of the light and heavy chain variable region genes were recovered, connected to the pClone007 Blunt Simple Vector vector, and transformed with calcium chloride according to the conventional method. The positive clones were screened at a concentration of 100μg / mL ampicillin and sent for sequencing. The gene completely conformed to the characteristics of several conservative framework amino acids of antibodies in the protein database, and the sequence was the antibody gene sequence. They were named pClone007-VH and pClone007-VL respectively.

[0039] Example 2 Construction of engineered antibody expression vectors pTT5-H and pTT5-L

[0040] Based on the sequences of the light and heavy chain variable regions and the sequence of the vector pTT5 containing the mouse light and heavy chain constant regions, primers for amplifying the heavy chain variable region (PDH-F and PDH-R), primers for amplifying the light chain variable region (PDL-F and PDL-R), primers for amplifying the heavy chain vector (ZTH-F and ZTH-R), and primers for amplifying the light chain vector (ZTL-F and ZTL-R) were designed and synthesized. <h2 style=";text-align:left;direction:ltr">

[0041] <h2 style=";text-align:left;direction:ltr"> PDH-F:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0042] <h2 style=";text-align:left;direction:ltr"> 5'-TTCCAGGTTCCACTGGTGACCAGGTGAAGCTGCAGGAGTCTG-3'(SEQ ID NO.21)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0043] <h2 style=";text-align:left;direction:ltr"> PDH-R:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0044] <h2 style=";text-align:left;direction:ltr"> 5'-GATGGGGGTGTCGTTTTGGCTGAGGAGACAGTGAGAGTGGGTGC-3'(SEQ ID NO.22)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0045] <h2 style=";text-align:left;direction:ltr"> PDL-F:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0046] <h2 style=";text-align:left;direction:ltr"> 5'-TTCCAGGTTCCACTGGTGACGATGTTGTGATGACCCAAACTCC-3'(SEQ ID NO.23)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0047] <h2 style=";text-align:left;direction:ltr"> PDL-R:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0048] <h2 style=";text-align:left;direction:ltr"> 5'-ACAGTTGGTGCAGCATCAGCCCCGTTTTATTTCCAGCCTGGT-3'(SEQ ID NO.24)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0049] <h2 style=";text-align:left;direction:ltr"> ZTH-F:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0050] <h2 style=";text-align:left;direction:ltr"> 5'-GCCAAAACGACACCCCCA-3'(SEQ ID NO.25)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0051] <h2 style=";text-align:left;direction:ltr"> ZTH-R:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0052] <h2 style=";text-align:left;direction:ltr"> 5'-GTCACCAGTGGAACCTGGAACC-3'(SEQ ID NO.26)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0053] <h2 style=";text-align:left;direction:ltr"> ZTL-F:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0054] <h2 style=";text-align:left;direction:ltr"> 5'-GCTGATGCTGCACCAACTGTAT-3'(SEQ ID NO.27)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0055] <h2 style=";text-align:left;direction:ltr"> ZTL-R:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0056] <h2 style=";text-align:left;direction:ltr"> 5'-GTCACCAGTGGAACCTGGAACC-3'(SEQ ID NO.28)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0057] Light chain variable region gene PCR amplification reaction system (50μL): upstream primer PDL-F; downstream primer PDL-R; pClone007-VL as template, high-fidelity pfu DNA polymerase amplification; PCR cycle program: 94℃ 5 min; 94℃ 30 sec, 58℃ 60sec, 72℃ 30 sec, 30 cycles in total; finally extension at 72℃ for 10 min.

[0058] Heavy chain variable region gene PCR amplification reaction system (50μL): upstream primer PDH-F; downstream primer PDH-R; pClone007-VH as template, high-fidelity pfu DNA polymerase amplification; PCR cycle program: 94℃ 5 min; 94℃ 30 sec, 58℃ 60sec, 72℃ 30 sec, 30 cycles in total; finally 72℃ extension for 10 min.

[0059] Amplification of the light chain linearized vector: upstream primer ZTL-F; downstream primer ZTL-R; pTT5-CD34-L (synthetic sequence, containing the mouse light chain constant region nucleotide sequence) as a template, high-fidelity pfu DNA polymerase amplification; PCR cycle program is 94℃ 5 min; 94℃ 30 sec, 58℃ 60sec, 72℃ 4 min, a total of 30 cycles; finally 72℃ extension for 10 min.

[0060] Amplification of the heavy chain linearized vector: upstream primer ZTH-F; downstream primer ZTH-R; using pcDNA-CD34-H (synthetic sequence, containing the nucleotide sequence of the mouse heavy chain constant region) as a template, high-fidelity pfu DNA polymerase amplification; the PCR cycle program was 94℃ 5 min; 94℃ 30 sec, 58℃ 60sec, 72℃ 4 min, for a total of 30 cycles; and finally extending at 72℃ for 10 min.

[0061] The PCR products were recovered separately, and the recovered light chain variable region PCR products and heavy chain variable region PCR products were recombined with the recovered linearized vector products using recombinase. After recombination, the DH5α cloning strain was transformed, and a single colony was picked for expansion and culture. The samples were sent for sequencing, and the clones with correct sequencing were used to transfect 293 cells.

[0062] Example 3 Expression and purification of anti-human CD11b engineered antibodies

[0063] 1. Expression of engineered antibody CD11b antibody: Take the prepared 293 cells out of the incubator, prepare two 15mL sterile centrifuge tubes, add 5mL SPM culture medium and 100μg sterile plasmid DNA (including light chain expression vector and heavy chain expression vector) to one of them, and gently blow and mix; take another centrifuge tube, add 5mL SPM and 500μL transfection reagent, and gently blow and mix; transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently blow and mix; let stand at room temperature for 10 minutes to prepare the plasmid-vector complex; take out the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and put them back into the CO2 constant temperature shaker for shaking culture; add auxiliary materials on the first day, and harvest the cell supernatant on the 6th day;

[0064] 2. Purification of anti-human CD11b engineered antibody: The cell supernatant collected in the previous step was purified using a Protein A affinity column. After equilibration of the column with PBS, the sample was loaded and eluted with glycine at a pH of 3.0. The eluted pure antibody was replaced with a G25 column solution, and the solution was replaced with PBS. Samples were taken for electrophoresis testing. The electrophoresis pattern is shown below. Figure 1 Then aliquot and store at -20°C.

[0065] Example 4 Activity determination of anti-human CD11b engineered antibody

[0066] Normal human peripheral blood test: add 100 μL normal human anticoagulated peripheral blood to each tube, add different amounts of engineered antibody CD11b and commercially available antibody CD11b (Beijing Kuangbo Biotechnology Co., Ltd., Catalog No. A6241), incubate at room temperature in the dark for 1 hour, then add 0.5 μg APC-labeled secondary antibody, incubate at room temperature in the dark for 30 min; add 2 mL hemolysin, react at room temperature in the dark for 10 min, centrifuge at 1000 rpm for 5 min, and discard the supernatant; add matching CD45-PerCP, incubate at room temperature in the dark for 30 min; add 2 mL cold PBS buffer, centrifuge at 1000 rpm for 5 min, and discard the supernatant; add 300 μL PBS buffer containing 1% paraformaldehyde, and detect by flow cytometry. The results are as follows Figure 2-7 As shown, the engineered antibody CD11b of the present invention and the commercially available antibody CD11b have the same effect on granulocyte population and percentage.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-human CD11b engineered antibody, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; The CDR1, CDR2 and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; The CDR1, CDR2 and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

2. The anti-human CD11b engineered antibody according to claim 1, characterized in that: The FR1, FR2, FR3, and FR4 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively; The FR1, FR2, FR3 and FR4 of the light chain variable region are the amino acid sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

3. The anti-human CD11b engineered antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

16.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule comprises a nucleotide sequence encoding the anti-human CD11b engineered antibody according to any one of claims 1 to 3.

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule according to claim 4.

6. A cell, characterized in that: The cell comprises the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. Use of the anti-human CD11b engineered antibody according to any one of claims 1 to 3 in the preparation of a detection reagent for CD11b molecules.

8. The use according to claim 7, characterized in that: The anti-human CD11b engineered antibody is used in preparing a detection reagent for CD11b molecules on the surface of granulocytes.

9. An immunoassay kit, characterized in that: The kit comprises the anti-human CD11b engineered antibody according to any one of claims 1 to 3.

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