CN117866086B

CN117866086B - Monoclonal antibody binding to human caveolin-1 and its application

Info

Publication number: CN117866086B
Application number: CN202410052268.3A
Authority: CN
Inventors: 谢暖; 吴海; 刘政泽; 李志友; 代海艳; 黄谧
Original assignee: Wuhan Abclonal Inc
Current assignee: Wuhan Abclonal Inc
Priority date: 2024-01-12
Filing date: 2024-01-12
Publication date: 2025-02-25
Anticipated expiration: 2044-01-12
Also published as: CN117866086A

Abstract

The application relates to the technical field of human microcapsule protein-1, in particular to a monoclonal antibody combined with human microcapsule protein-1 and application thereof. Examples high affinity rabbit monoclonal antibodies against human microcapsule-1 were successfully developed by single B cell screening and culture techniques, with high affinity and specificity to human microcapsule-1, and can be used to prepare immunoblotting and immunocombination kits for detection of human microcapsule-1. The monoclonal antibody also has important clinical value for diagnosis and treatment of human microcapsule protein-1 and related diseases.

Description

Monoclonal antibody combined with human microcapsule protein-1 and application thereof

Technical Field

The application relates to the technical field of human microcapsule protein-1, in particular to a monoclonal antibody combined with human microcapsule protein-1 and application thereof.

Background

Microcapsule protein-1 (riveolin-1) is a transmembrane scaffold protein, a major structural component of cytoplasmic membrane microcapsules, involved in a variety of complex cellular physiological processes, and interrelated with a variety of membrane structures, including the endoplasmic reticulum and golgi apparatus. Caveolin-1 is synthesized and inserted into endoplasmic reticulum, transported to Golgi apparatus and cell surface to form fovea, and Caveolin-1 can be recovered from cytoplasmic membrane in reverse order. Human Caveolin-1 (Homo sapiens Caveolin-1) consists of 178 amino acids, has an oligomerization region and a CSD region (Caveolin scaffold region), and the conserved region between amino acids 60-70 is necessary for the Caveolin-1 to leave the endoplasmic reticulum, and amino acids between 71-80 regulate the Caveolin-1 oligomer to the Golgi apparatus, phosphorylate at serine 80, convert Caveolin-1 into soluble secreted proteins, and directly affect and regulate signal molecule activity via CSD.

Caveolin-1 is used as a scaffold protein, can increase or inhibit the activity of cytoplasmic membrane microcapsule related proteins, inhibit cell migration or promote tumor formation, regulate apoptosis signal paths, influence the growth proliferation condition in the cell cycle, negatively regulate and control angiogenesis or positively regulate angiogenesis under different tissues and different conditions, and has certain anti-inflammatory activity. Thus, caveolin-1 is widely focused as one of the members of the family of cellar proteins closely related to tumors, its function and its relation to cancer are the current research hotspots, and although the role and role played by scientists on Caveolin-1 at different cancers and different stages of cancer are one, it is considered to be important for malignant phenotype transformation of cancer and is a target protein for cancer treatment.

Therefore, the development of the human Caveolin-1 antibody has very important significance for detecting the human Caveolin-1 protein.

Disclosure of Invention

The embodiment of the application provides a rabbit monoclonal antibody of high-affinity human microcapsule protein-1, so as to solve one of the technical problems to a certain extent. Examples high affinity rabbit monoclonal antibodies against human microcapsule-1 were successfully developed by single B cell screening and culture techniques, with high affinity and specificity to human microcapsule-1, and therefore, the monoclonal antibodies can be used to prepare kits for detecting human microcapsule-1 levels. The monoclonal antibody provided by the application has important clinical value for diagnosis and treatment of human microcapsule protein-1 and related diseases.

Therefore, the application at least discloses the following technical scheme:

In a first aspect, embodiments disclose a monoclonal antibody that specifically binds to human microcapsule-1, comprising:

3 Complementarity Determining Regions (CDRs) of a light chain variable region (VL) defined according to the Kabat numbering system, a VL CDR1 as shown in SEQ ID NO.5, a VL CDR2 as shown in SEQ ID NO.6, and a VL CDR3 as shown in SEQ ID NO. 7;

and/or

3 Complementarity Determining Regions (CDRs) of a heavy chain variable region (VH) defined according to the Kabat numbering system, a VH CDR1 as shown in SEQ ID NO. 8, a VH CDR2 as shown in SEQ ID NO. 9, and a VH CDR3 as shown in SEQ ID NO. 10.

In a second aspect, embodiments disclose a monoclonal antibody that specifically binds human microcapsule protein-1, comprising a light chain variable region (VL) having a sequence as shown in SEQ ID NO. 2 and a heavy chain variable region (VH) having a sequence as shown in SEQ ID NO. 7.

In a third aspect, the examples disclose a monoclonal antibody that specifically binds human microcapsule-1, comprising a light chain having a sequence as shown in SEQ ID NO.1 and a heavy chain having a sequence as shown in SEQ ID NO. 6.

In a fourth aspect, embodiments disclose an immunoblotting detection kit for detecting human microglobulin-1, the kit comprising a monoclonal antibody according to any of the first to third aspects.

In a fifth aspect, embodiments disclose an immunohistochemical detection kit for detecting human microglobulin-1, the kit comprising a monoclonal antibody as described in any one of the first to third aspects.

In a sixth aspect, the embodiments disclose the use of a monoclonal antibody according to any of the first to third aspects for the preparation of a reagent or kit for detecting human microglobulin-1.

Drawings

FIG. 1 shows the detection of the His tag of Caveolin-1 protein by SDS-PAGE gel as provided in the examples, lane 1 and lane 2are respectively 0.4mg/mL and 0.2mg/mL BSA, lane 3 is marker, lane 4 is supernatant ① (abbreviated as "upper 1"), lane 5 is flow-through (abbreviated as "FT") during purification, lane 6 is 2-fold dilution of Caveolin-1 protein collected (abbreviated as "X2"), and lane 7 is 10-fold dilution of Caveolin-1 protein collected (abbreviated as "X10").

FIG. 2 is a schematic diagram of the structure of an expression vector containing a heavy chain constant region of a rabbit monoclonal antibody constructed in the example, in which pBR322 origin and f1origin are replication promoters in E.coli (E.Coli), AMPCILLIN is a plasmid resistance gene, CMV immearlypromotor is a promoter in eukaryote, SV40 PAterminator is a tailing signal, and HEAVY CHAIN constant is a nucleotide sequence of the heavy chain constant region of a rabbit monoclonal antibody.

FIG. 3 is a schematic diagram of the structure of an expression vector containing a rabbit monoclonal antibody light chain constant region constructed in the example, in which pBR322 origin and f1origin are replication promoters in E.coli (E.Coli), AMPCILLIN is a plasmid resistance gene, CMV immearlypromotor is a promoter in eukaryote, SV40 PAterminator is a tailing signal, and LIGHT CHAIN constant is a nucleotide sequence of a rabbit monoclonal antibody light chain constant region.

FIG. 4 shows the detection results of Caveolin-1 in a cell sample HeLa by using the immunoblotting kit constructed by the rabbit monoclonal antibody provided in the example.

FIG. 5 shows the detection results of Caveolin-1 in Mouse lung tissue and rabbit lung tissue by using the immunoblotting kit constructed by the rabbit monoclonal antibody provided in the example, "Mouse lung" is a Mouse lung cell sample with high expression of Caveolin-1 from 6-8 weeks c57bl/6 mice, and "Rat lung" is a Mouse lung cell sample with high expression of Caveolin-1 from SD rats.

FIG. 6 is a chart showing the detection of the staining localization of Caveolin-1 in human lung samples by using the immunohistochemical kit constructed by the rabbit monoclonal antibody provided by the embodiment of the application.

FIG. 7 is a chart showing the staining localization of Caveolin-1 in human colon samples using the immunohistochemical kit constructed by the rabbit monoclonal antibody according to the embodiment of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents which are not specifically described in the present application are conventional reagents and are commercially available, and the methods which are not specifically described in the present application are conventional experimental methods and are known from the prior art.

Interpretation of the terms

In the present application, the term "antibody" is to be interpreted in the broadest sense, having a variety of antibody structures, including, but not limited to, Y-type antibodies, so-called full length antibodies, antigen binding portions of Y-type antibodies, and genetic or chemical modifications thereof. Wherein "antigen binding portion" refers to one or more portions or fragments of a Y-type antibody that retains the ability of the antibody to specifically bind to human microcapsule protein-1.

In the present application, the term "monoclonal antibody" (mAb) includes a population of highly homogeneous antibodies having substantially identical antigenic determinants. That is, the individual antibodies are essentially identical in the population, except for the small number of mutations that may occur naturally. Monoclonal antibodies may exhibit a single binding specificity and affinity for a particular epitope on an antigen. Each monoclonal antibody may be directed against the same or substantially the same epitope on the antigen, as compared to a polyclonal antibody which typically comprises antibodies directed against different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring preparation by any particular method. The antibodies can be prepared by a variety of methods including, but not limited to, hybridoma methods, recombinant DNA methods, phage antibody libraries, B cell screening and culturing, and the like.

In the present application, the terms "rabbit antibody", "anti-human microglobulin-1 rabbit monoclonal antibody", "human microglobulin-1 rabbit monoclonal antibody" or the modifier "rabbit" in similar terms means that the Complementarity Determining Regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. In one example, a rabbit monoclonal antibody to human microcapsule-1 may comprise the CDRs and Framework Regions (FR) of an antibody from a rabbit immunoglobulin sequence. In one embodiment, the rabbit antibody or rabbit monoclonal antibody against human microcapsule-1 may comprise CDRs from an antibody of rabbit immunoglobulin sequence. In one example, the rabbit monoclonal antibody against human microglobulin-1 may be an antibody whose CDR regions are derived from rabbit immunoglobulin sequences, while the FR is derived from germline immunoglobulin sequences of other mammals (e.g., mice or humans). The term "rabbit monoclonal antibody against human microglobulin-1" may also comprise antibodies having amino acid residues encoded by non-rabbit immunoglobulin sequences, e.g. mutations introduced by random or point-specific mutations in vitro, or by somatic mutations in vivo.

In the present application, the term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains, of which the two chains with the larger molecular weight are called heavy chains (HEAVY CHAIN, H) and the two chains with the smaller molecular weight are called light chains (LIGHT CHAIN, L). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The heavy and light chains vary widely in about 110 amino acid sequences near the N-terminus, with the other portions of the amino acid sequences being relatively constant. Thus, the regions of the light and heavy chains that vary widely in amino acid sequence near the N-terminus are referred to as variable regions (V) that account for 1/4 and 1/2 of the heavy and light chains, respectively, and the regions near the C-terminus that are relatively stable in amino acid sequence are referred to as constant regions (constantregion, C) that account for 3/4 and 1/2 of the heavy and light chains, respectively.

The V region of the heavy chain and the V region of the light chain are referred to as VH and VL, respectively. Each of VH and VL contains a region of highly variable 3 amino acid composition and arrangement sequence, known as the hypervariable region (hypervariable region, HVR) or complementarity determining region (complementarity determining region, CDR), including HVRl (CDRl), HVR2 (CDR 2) and HVR3 (CDR 3), wherein HVR3 (CDR 3) varies to a greater extent. Typically, VH comprises 3 complementarity determining regions VH CDR1, VH CDR2 and VH CDR3, and VL comprises 3 complementarity determining regions VL CDR1, VL CDR2 and VL CDR3. The 3 complementarity determining regions of VH and VL, individually or collectively, constitute the antigen-binding site of an antibody, and determine the specificity of an antibody, which is the site at which an antibody recognizes and binds an antigen. In the V region, the amino acid composition and arrangement order of the regions outside the CDRs are relatively conserved, called Framework Regions (FR). VH or VL has four framework regions, denoted FR1, FR2, FR3 and FR4, respectively. By substitution or alteration of the framework regions, multiple antibodies of the same specificity can be provided while ensuring that the site of antibody recognition and antigen binding is unchanged.

The C chains of the heavy and light chains are referred to as CH and CL, respectively. CL lengths of different classes (kappa or lambda) of Ig are substantially identical, but CH lengths of different classes of Ig are different, e.g., igG, igA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CHl, CH2, CH3, and CH4.

In the present application, the term "framework region" or "framework region" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.

Antibodies to

The embodiment of the application discloses an antibody, which specifically binds to human microcapsule protein-1.

In some embodiments, the antibody comprises Complementarity Determining Regions (CDRs) of a light chain variable region (VL) defined according to the 3 Kabat numbering system, VL CDR1, VL CDR2 and VL CDR3.VL CDR1 is shown as SEQ ID NO. 5, VL CDR2 is shown as SEQ ID NO. 6, and VL CDR3 is shown as SEQ ID NO. 7.

In some embodiments, the antibody comprises Complementarity Determining Regions (CDRs) of a heavy chain variable region (HL) defined according to the 3 Kabat numbering system, VH CDR1, VH CDR2, and VH CDR3.VL CDR1 is shown as SEQ ID NO. 8, VL CDR2 is shown as SEQ ID NO. 9, and VL CDR3 is shown as SEQ ID NO. 10.

In some embodiments, the antibody comprises Complementarity Determining Regions (CDRs) of a light chain variable region (VL) defined according to 3 Kabat numbering systems, VL CDR1, VL CDR2, and VL CDR3, and Complementarity Determining Regions (CDRs) of a heavy chain variable region (HL) defined by 3 Kabat numbering systems, VH CDR1, VH CDR2, and VH CDR3.VL CDR1 is shown as SEQ ID NO. 5, VL CDR2 is shown as SEQ ID NO.6, and VL CDR3 is shown as SEQ ID NO. 7. VL CDR1 is shown as SEQ ID NO. 8, VL CDR2 is shown as SEQ ID NO. 9, and VL CDR3 is shown as SEQ ID NO. 10.

In certain embodiments, the anti-human microcapsule protein-1 antibody may have a Y-type molecular structure. In one embodiment, the anti-human microcapsule-1 antibody may comprise a pair of heavy chains and a pair of light chains. The heavy chain may include one heavy chain variable region and one or more heavy chain constant regions. Mammalian antibodies generally comprise five types of heavy chains, gamma, delta, alpha, mu and epsilon, and the corresponding compositions of antibodies are referred to as IgG, igD, igA, igM and IgE five antibodies. The light chain may be a smaller polypeptide subunit relative to the heavy chain. The light chain may include a light chain variable region and a light chain constant region. VL is typically the N-terminal part of the light chain, exhibiting higher variability in amino acid sequence. VL between different antibodies has a specific amino acid sequence. In one embodiment, both the heavy chain variable region VH and the light chain variable region VL are useful for recognizing and binding human microcapsule protein-1.

In some embodiments, CDRs located in the VL and VH regions can be separated from each other by FR. FR is a conserved region in the sequence structure. FR can generally act as a scaffold to allow CDRs to form a three-dimensional structure that can specifically bind to an antigen (e.g., human microcapsule protein-1). The three-dimensional structure of FR may be conserved among different antibodies.

Antibody preparation

The monoclonal antibodies provided by the embodiments of the present application can be prepared by a variety of methods including, but not limited to, hybridoma methods, recombinant DNA methods, phage antibody libraries, single B cell screening and culture, and the like.

In some embodiments, the monoclonal antibody is prepared by immunizing New Zealand white rabbits by taking recombinant expressed human microcapsule protein-1 as an immunogen, sorting antigen-specific B lymphocytes from spleen cells of the white rabbits, screening positive B lymphocytes capable of secreting the specific antibody after antibody function verification, amplifying antibody heavy chain and light chain genes of the positive B lymphocytes, and then carrying out recombinant expression to obtain the rabbit monoclonal antibody resisting human MSH 2.

In some embodiments, the method of preparing human microcapsule protein-1 comprises:

(1) Construction of expression plasmid

PCR amplification was performed using a plasmid (Thermo FISHER SCIENTIFIC CDNA/ORF Library-Plate Map (s)) containing the human Caveolin-1CDS sequence as a template, with primers of Caveolin-1-Primer-F (shown as SEQ ID NO. 11) and Caveolin-1-Primer-F (shown as SEQ ID NO. 12) for expression of recombinant human Caveolin-1 (shown as SEQ ID NO. 13) consisting of amino acids 1 to 80 from the N-terminus of the human Caveolin-1 protein.

Wherein the PCR reaction system comprises 1. Mu.L of the template, 1. Mu.L of the forward primer (10 mM), 1. Mu.L of the reverse primer (10 mM), 25. Mu.L of 2X Gloria Hi-FI PCR MASTER Mix with GC Buffer (Wohan Eboltag Biotechnology Co., ltd.) and 22. Mu.LN.FH ₂ O. The PCR amplification procedure was performed at 98℃for 30s, followed by 30 cycles of 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally, the reaction solution was kept at 72℃for 5min, and the reaction solution was kept at 4 ℃. Purifying the amplified PCR product, loading the purified PCR product on an expression vector in a homologous recombination mode, wherein the prokaryotic expression vector is pGEX-4T-AB1 (commercial transformation vector), and preserving plasmids after verification by sequencing (Jin Kairui Biotechnology Co., ltd.) to obtain the expression plasmid for expressing human Caveolin-1.

(2) Recombinant expression of proteins

The expression plasmid for expression of human Caveolin-1 was transformed into E.coli Rosetta strain and incubated overnight at 37℃on LB agar plates (containing 75. Mu.g/mL ampicillin) to obtain several single colony transformants.

The single colony transformant was inoculated into 2mL of LB medium (75. Mu.g/mL ampicillin) and cultured at 37℃and 220rpm until the OD600nm of the bacterial solution was 0.4 to 0.6. Then, transferring 2mL of culture of each strain into 400mL of LB expression medium, culturing again at 37 ℃ and 220rpm until the OD600nm of the bacterial liquid reaches about 0.45-0.55, adding 0.8mM IPTG, continuously inducing at 37 ℃ for 3-4 h, centrifuging at 4000rpm for 10min, and taking the precipitate to obtain the bacterial body.

(3) Purification of recombinant proteins

The cells were resuspended in 30mL of a lysis solution (50 mM Tris-300mM NaCl) and fixed in ice boxes. Selecting an amplitude transformer, placing the amplitude transformer into a bacteria breaking cabin, wherein the power is 350W, the bacteria breaking time is 3s, the interval time is 3s, the time is counted down for 5min, then placing the amplitude transformer into an ice-water mixture for cooling for 5min, and repeating the steps for breaking bacteria for 5min. After the completion of the sterilization, the mixture was centrifuged at 9000rpm for 10min to obtain a supernatant ① and a precipitate.

Recombinant Caveolin-1 was extracted from supernatant ① by affinity chromatography using Ni affinity chromatography resin. As a result of purification, as shown in FIG. 1, recombinant Caveolin-1 having a purity of 80% comprising a Caveolin-1 protein fragment and a glutathione-transferase (GST) tag was obtained.

In some embodiments, the method of making a monoclonal antibody to human microcapsule-1 comprises:

(1) Immunization of animals

The recombinant human Caveolin-1 protein prepared in the example is used as immunogen, and 200 mug of immunogen is immunized by each New Zealand white rabbit. Prior to the first immunization, the immunogen was mixed with an equivalent amount of complete Freund's adjuvant (purchased from Sigma) to make an emulsion, and injected subcutaneously in the abdomen and back of rabbits at multiple points. After the first immunization, 100. Mu.g of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma Co.) every 3 weeks to prepare an emulsifier, which was injected subcutaneously in the abdomen and back of rabbits in multiple spots to boost the immunization twice. After three immunizations, rabbit serum samples were collected, titers against human Caveolin-1 protein were determined by ELISA, titers were determined by ELISA after serum dilution at 1:243000, rabbits with OD450nm exceeding 0.2 were boosted by 200. Mu.g of immunogen subcutaneously by multiple injections, and spleens were obtained 3 days later.

(2) Isolation of spleen cells

Washing the spleen of the rabbit obtained in the above way with basic culture medium under aseptic condition, shearing off excessive connective tissue and fat, and grinding in cell screen. Collecting grinding fluid (namely, membrane inner cells), centrifuging at 400g for 5min at room temperature, removing supernatant, reserving cells, adding 13mL of RBC red blood cell lysate at room temperature (purchased from BioGems company), blowing off for 1min, performing red blood cell lysis, adding 37mL of basic culture medium, mixing, and stopping red blood cell lysis. Centrifugation was performed at 400g for 5min at room temperature, the supernatant was removed, 40mL of room temperature basal medium was added, and the pellet was gently blown off with a pipette to resuspend the cells. Centrifuging at 400g for 5min at room temperature again, removing supernatant, reserving cells, adding 20mL of normal temperature basal medium, gently blowing off cell clusters by using a pipettor to resuspend the cells, filtering the resuspended cells again by using a cell screen to remove clustered cells, and counting the cells.

(3) B lymphocyte sorting and culture

B lymphocyte sorting and culturing, see patent 'method for efficiently separating single antigen-specific B lymphocyte from spleen cell (publication No. CN 110016462A)' and patent 'an in vitro B lymphocyte culturing system and application (publication No. CN 111518765A)'.

(4) Cloning of genes encoding Rabbit monoclonal antibodies

The cultured B cell supernatants were used to identify positive clones by antigen coated ELISA. Cells of positive clones were collected and lysed, and RNA was extracted using Quick-RNATM MicroPrep kit (available from ZYMO) and reverse transcribed into cDNA. The cDNA is used as a template, a PCR method is adopted to amplify the naturally paired rabbit monoclonal antibody chain variable region (VL) and heavy chain variable region (VH) genes from the cDNA of the corresponding positive clone, and a plurality of clones are selected for sequencing.

The PCR reaction system was as follows, 4. Mu.L of cDNA, 1. Mu.L of 10mM forward primer, 1. Mu.L of 10mM reverse primer, 12.5. Mu.L of 2X Gloria HiFi (supplied by Wohan Eboltag Biotechnology Co., ltd.) and 6.5. Mu.LN.F H ₂ O. The PCR amplification procedure comprises pre-denaturing at 98deg.C for 30s, performing 40 cycles at 98deg.C for 10s,64 deg.C for 30s, and 72 deg.C for 30s, maintaining at 72 deg.C for 5min, and storing the obtained reaction solution at 4deg.C

The light chain variable region Primer pair is VL-Primer-F, SEQ ID NO. 14, VL-Primer-R, SEQ ID NO. 15.

The heavy chain variable region Primer pair is VH-Primer-F, SEQ ID NO. 16, VH-Primer-R, SEQ ID NO. 17.

(5) Construction of heavy and light chain expression vectors

The heavy chain genes and the light chain genes of the plurality of rabbit monoclonal antibodies selected in the steps are respectively loaded on the expression vectors, and the used mammal expression vector pBR322 is shown in figures 2 and 3. Wherein, the coding gene of the selected light chain is shown as SEQ ID NO.18, and the coding gene of the selected light chain variable region is shown as SEQ ID NO. 19. The coding gene of the selected heavy chain is shown as SEQ ID NO.20, and the coding gene of the selected heavy chain variable region is shown as SEQ ID NO. 21.

(6) Monoclonal antibody preparation and purification

Mammalian cell (e.g., CHO) expression vectors containing the heavy chain constant region (FIG. 2) and the light chain constant region (FIG. 3) of the rabbit monoclonal antibodies were routinely linearized with NheI, xbaI and XbaI, bamHI restriction enzymes, respectively.

Purifying the amplified PCR product, constructing heavy chain variable region gene and light chain variable region gene into corresponding mammal expression vectors by adopting a homologous recombination mode, carrying out sequencing verification, transfecting the expression vectors containing the corresponding rabbit monoclonal antibody light chain gene and heavy chain gene into 293F cells together, carrying out transfection for 72-96 hours, continuously culturing the obtained transformant, collecting culture solution, purifying and obtaining rabbit monoclonal antibody identifying human Caveolin-1 from the centrifuged supernatant by using protein A affinity gel resin centrifugation, verifying the purity of the antibody by using 12% SDS-PAGE gel electrophoresis, carrying out amino acid sequencing on the purified rabbit monoclonal antibody of the human Caveolin-1 protein, and preserving at a low temperature of-20 ℃ for standby.

As a result, the obtained rabbit monoclonal antibody has a light chain amino acid sequence shown in SEQ ID NO.1, a heavy chain amino acid sequence shown in SEQ ID NO.2, a light chain variable region amino acid sequence shown in SEQ ID NO.3, a heavy chain variable region amino acid sequence shown in SEQ ID NO.4, a light chain complementarity determining region VL CDR1 amino acid sequence shown in SEQ ID NO.5, a light chain complementarity determining region VL CDR2 amino acid sequence shown in SEQ ID NO.6, a light chain complementarity determining region VL CDR3 amino acid sequence shown in SEQ ID NO.7, a heavy chain complementarity determining region VH CDR1 amino acid sequence shown in SEQ ID NO.8, a heavy chain complementarity determining region VH CDR2 amino acid sequence shown in SEQ ID NO.9, and a heavy chain complementarity determining region VH CDR3 amino acid sequence shown in SEQ ID NO. 10. And the rabbit monoclonal antibody has kappa chain as the light chain constant region and IgG1 type as the heavy chain constant region.

In some examples, the specificity of the rabbit monoclonal antibody of the human Caveolin-1 protein prepared in the above examples was examined using HeLa cells (cell line of the cell bank of the national academy of sciences) highly expressing Caveolin-1. HeLa cells (cell line of cell bank of China academy of sciences) which express Caveolin-1 are taken, the recognition specificity of the monoclonal antibody is detected by an immunoblotting method, and 7% polyacrylamide gel electrophoresis is carried out. Gel protein bands were transferred to PVDF membranes in an electrotransfer system in a conventional manner. The membranes were incubated in TBST blocking solution containing 3% nonfat milk powder for 1h at room temperature, and the Caveolin-1 rabbit monoclonal antibody provided in the examples (1:10000 dilution) was added and incubated overnight at 4 ℃. After washing the membrane with TBST, goat anti-rabbit secondary antibody (purchased from JacksonImmunoResearch, cat. No. 111-035-045) diluted 1:10000 was added and incubated for 1 hour at room temperature. And (3) washing the film again by TBST, adding ECL color development liquid, developing, and exposing for 90 seconds. As a result, as shown in FIG. 4, the overall length of the Caveolin-1 is 178aa, the theoretical molecular weight is 22kd, and the rabbit monoclonal antibody of the human Caveolin-1 protein obtained by screening has better specificity and detects a specific target band.

In some examples, the recognition specificity of the monoclonal antibody was detected by immunoblotting using murine lung cell samples (6-8 week c57bl/6 mice and SD rats) that highly expressed Caveolin-1, and 7% polyacrylamide gel electrophoresis was performed. Gel protein bands were transferred to PVDF membranes in an electrotransfer system in a conventional manner. The membranes were incubated in TBST blocking solution containing 3% nonfat milk powder for 1h at room temperature, and the Caveolin-1 rabbit monoclonal antibody provided in the examples (1:10000 dilution) was added and incubated overnight at 4 ℃. After washing the membrane with TBST, goat anti-rabbit secondary antibody (purchased from JacksonImmunoResearch, cat. No. 111-035-045) diluted 1:10000 was added and incubated for 1 hour at room temperature. And (3) washing the film again by TBST, adding ECL color development liquid, developing, and exposing for 10s. As a result, as shown in FIG. 5, the full length of the Caveolin-1 is 178aa, the theoretical molecular weight is 20kD, the specificity of the rabbit monoclonal antibody of the human Caveolin-1 protein obtained by screening is good, and specific target bands (two bands are two sheared bodies of the Caveolin-1 respectively, and the size of the posttranslational modification after phosphorylation and lipidation is 24kD and 21 kD) are detected.

In some detection cases, the rabbit monoclonal antibody of the prepared human Caveolin-1 protein is used as a primary antibody, and the Caveolin-1 in the human lung sample and the human colon sample is qualitatively detected by adopting an immunohistochemical staining method. The specific process comprises the following steps:

(1) Chip selection:

Immunohistochemical chip, cat# TMA000-42, BAOOST.

(2) IHC staining and analysis

The slice baking method comprises the steps of respectively placing paraffin slices (a human lung sample and a human colon sample) on a slice frame in the same direction, placing the slices in a 56 ℃ incubator for 30min, simultaneously placing a dewaxing liquid 1 cylinder into the 56 ℃ incubator, dewaxing until water, placing the paraffin slices together with the slice frame into the dewaxing liquid 1 cylinder, taking out the paraffin slices from the incubator and placing the paraffin slices at normal temperature for 5min, taking out the paraffin slices and immersing the paraffin slices into a normal-temperature dewaxing liquid 2 cylinder, and placing the paraffin slices into the dewaxing liquid 2, the dewaxing liquid 3, the absolute ethyl alcohol 1, the absolute ethyl alcohol 2 and the absolute ethyl alcohol 3 in sequence, wherein each dewaxing liquid reagent cylinder is 5min, each absolute ethyl alcohol reagent cylinder is 3min, and washing the paraffin slices with running water for 3min.

Antigen retrieval, high pressure thermal retrieval with 0.01M sodium citrate retrieval solution (pH 6.0).

Endogenous peroxidase inactivation, namely immersing and washing the slices with PBS buffer solution for 3 times each for 1min, removing the buffer solution on the slices, immersing the slices into 3% hydrogen peroxide solution completely, and incubating for 10min at room temperature.

Sealing, namely immersing and washing the slide glass for 3 times with PBS buffer solution for 3min each time, removing the buffer solution on the slide glass, delinting a tissue region to be detected on the slide glass with an immunohistochemical water pen, dripping sealing liquid-PBS sealing liquid into the delineating region, horizontally placing the slide glass in an incubation wet box with water at the bottom, incubating for 30min at normal temperature, and starting timing from the dripping of the sealing liquid.

The first antibody is incubated, namely, removing blocking solution, dripping the first antibody diluted by PBS buffer solution (the dilution ratio of the antibody is 1:150) on a tissue slice, horizontally placing the tissue slice in an incubation wet box, incubating for 60 minutes at normal temperature, removing antibody working solution, quickly rinsing the PBS buffer solution for 1 time, soaking and washing for 3 times by the PBS buffer solution for 3 minutes each time, and repeatedly lifting up and down for a plurality of times during the soaking and washing (the dilution ratio of the first antibody is 1:150).

The secondary antibody incubation comprises dripping a working solution (Dako REAL EnVision Detection System, peroxidase/DAB, rabbit/Mouse, HRP; dako) of the instant secondary antibody on a tissue slice, horizontally placing in an incubation wet box, incubating at normal temperature for 25min, removing reagent on the slice, rapidly rinsing with buffer PBS for 1 time, soaking and washing with buffer PBS for 3 times and 3min each time, repeatedly lifting up and down for multiple times during soaking and washing

The color development is carried out, namely, a color development liquid working solution is dripped on the tissue slice, and the color change condition is closely observed under a microscope to obtain proper dyeing strength;

counterstaining, namely immersing the slightly drained slice into Mayer's hematoxylin to counterstain the slice for 1min, and washing the slice with running water for 3min after counterstaining is finished;

Bluing, namely immersing the slightly drained slice into a saturated aqueous solution of lithium carbonate to blu for 3s, and cleaning the slice with running water for 3min;

dewatering, namely soaking the cleaned slices in absolute ethyl alcohol for 1 time, lifting up and down for a plurality of times during the soaking period, and taking out after timing for 10 seconds;

And (3) sealing the slice, namely dripping a proper amount of neutral gum into the center of the slice, covering a cover slip, wherein the proper amount of gum is required, and the tissue is required to be completely covered after the cover slip is added, and the slice cannot be scanned by overflowing the gum.

The immunohistochemical staining results were classified into positive and negative. Positive expression must be positive at the cell and tissue specific antigenic sites. Immunohistochemical staining was performed using the anti-human Caveolin-1 rabbit monoclonal antibody prepared in the above example as a primary antibody, and the results are shown in FIG. 6 and FIG. 7. The staining is clear and has no nonspecific staining, and the background is clean. By combining with an immunoblotting result, the anti-human Caveolin-1 rabbit monoclonal antibody specifically recognizes human Caveolin-1 protein, has no nonspecific staining, and effectively avoids false positive results.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims

1. A monoclonal antibody that specifically binds to human microcystin-1, comprising:

three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VL CDR1 as set forth in SEQ ID NO:5, VL CDR2 as set forth in SEQ ID NO:6, and VL CDR3 as set forth in SEQ ID NO:7; and

Three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VH CDR1 as shown in SEQ ID NO:8, VH CDR2 as shown in SEQ ID NO:9, and VH CDR3 as shown in SEQ ID NO:10.

2. A monoclonal antibody that specifically binds to human microcystin-1, the monoclonal antibody comprising:

The light chain variable region (VL) of the sequence shown in SEQ ID NO: 3; and

The heavy chain variable region (VH) has the sequence shown in SEQ ID NO:4.

3. A monoclonal antibody that specifically binds to human microcystin-1, the monoclonal antibody comprising:

The light chain of the sequence shown in SEQ ID NO: 1; and

The heavy chain has the sequence shown in SEQ ID NO:2.

4. A nucleic acid molecule carrying a gene encoding the monoclonal antibody according to any one of claims 1 to 3.

5. A host cell, which carries the nucleic acid molecule according to claim 4 and is capable of autonomously expressing the monoclonal antibody according to any one of claims 1 to 3.

6. A kit for detecting human microcystin-1, comprising: the monoclonal antibody according to any one of claims 1 to 3.

7. The kit according to claim 6, wherein the kit is selected from the group consisting of an immunohistochemistry kit, an immunoblotting kit, and an immunoprecipitation kit.

8. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent or kit for detecting human microcystin-1.