CN114702580B - Monoclonal antibody specifically binding human serum albumin and application thereof - Google Patents
Monoclonal antibody specifically binding human serum albumin and application thereof Download PDFInfo
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
- G01N2333/765—Serum albumin, e.g. HSA
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- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Abstract
The invention discloses a monoclonal antibody specifically binding to human serum albumin and application thereof, comprising an antibody with a heavy chain variable region with an amino acid sequence SEQ ID NO.1 and a light chain variable region with an amino acid sequence SEQ ID NO. 3. The human serum albumin monoclonal antibody can be used for detecting human serum albumin, accurately detects the concentration of HSA, and has important significance for liver risk diagnosis, renal syndrome and diabetes diagnosis.
Description
Technical Field
The invention belongs to the fields of biotechnology and molecular immunology, and particularly relates to a monoclonal antibody specifically combined with human serum albumin and application thereof.
Background
Antibodies (abs) are important effector molecules that mediate fluid immunity, and protect the body during viral infection by preventing pathogenic microorganisms from adhering to target cell receptors primarily through specific binding to the corresponding antigen. The discovery of the antibody not only makes great contribution in the fields of diagnosis, treatment, analysis and detection of harmful substances and the like of human diseases, but also is an important component of biological medicine, and the second biological medicine product wave is led after the recombinant protein.
Human Serum Albumin (HSA) is a soluble monomeric protein that constitutes half of the total amount of protein in the blood. Albumin is used as a basic carrier for carrying and delivering fatty acids, steroids, hormone molecules, etc., and its stable inert nature is an important factor in maintaining blood pressure. Serum albumin is a globular, non-glycosylated, serum protein having a molecular weight of 65 kilodaltons and having 585 amino acids. This protein (albumin precursor) is then processed by the transformation of the golgi apparatus to remove the guide polypeptide and is secreted extracellularly. Serum albumin has 35 cysteines, and in blood albumin is a monomer with 17 disulfide bonds. When the polypeptide is not secreted, the albumin product in the yeast cell is in a mismatched state, will lose 90% of its antigenicity (as compared to the natural state albumin in plasma), and form insoluble albumin aggregates, without the biological activity of albumin. Human serum albumin is now used clinically, and is extracted from human plasma.
Albumin is the main component in blood, and has a half-life of 20 days or more, and its content in human body is 40-50 g/L blood. Albumin is also commonly used as a stabilizer for drugs in pharmaceutical formulation compositions, especially in the manufacture of biopharmaceuticals and vaccines. In conclusion, after the human serum albumin and the therapeutic protein gene are fused in saccharomycetes or vertebrate cells by utilizing the genetic engineering technology to express the recombinant fusion protein, the fusion protein has great advantages of resisting enzymolysis in organisms, greatly improving the service life of the therapeutic protein in vivo and in vitro, namely, improving the stability and longer half-life of the therapeutic protein in serum and during storage, achieving the long-acting of protein drugs, greatly reducing the administration frequency and obtaining better therapeutic effects in clinical treatment of serious diseases.
When microalbumin is detected in human urine, it reflects that the kidneys may have abnormal leakage of protein. The urine microalbumin detection index can be used as an early diagnosis index of chronic diseases such as diabetes, nephropathy and the like, and has important clinical significance for quantitative detection of albumin in urine. HSA is also an important prognostic marker in hypoalbuminemia and many other pathological conditions caused by malnutrition and inflammation. The concentration of HSA in body fluids is also considered a marker of body health, and early assessment of disease can be made based on this. HSA is produced mainly by hepatocytes, and many studies have demonstrated that liver disease causes a decrease in HSA concentration, and detection of HSA content can be a central factor in liver risk diagnosis.
Therefore, the method for specifically and sensitively detecting the urine microalbumin also has important application value, and the key point of the method is that the sensitivity and the specificity of the anti-albumin monoclonal antibody, namely the human serum albumin monoclonal antibody, are high and low in detection sensitivity and specificity of the anti-albumin, and clinically examine whether patients potentially suffer from chronic diseases such as diabetes, nephropathy and the like, so that the method has important clinical significance for early detection of illness and early treatment.
Disclosure of Invention
The invention provides a monoclonal antibody specifically combined with human serum albumin, which is used for preparing a reagent for detecting the human serum albumin, and comprises a heavy chain variable region with an amino acid sequence SEQ ID NO.1 and an antibody with a light chain variable region with an amino acid sequence SEQ ID NO. 3.
The invention also provides a functional fragment of the antibody, wherein the functional fragment is selected from Fab, F (ab) 2, fab' and Fv.
The invention also provides a nucleic acid sequence which codes for the above antibody or antibody functional fragment. The nucleic acid sequence comprises a nucleic acid sequence shown in SEQ ID NO.2 for encoding a heavy chain variable region and a nucleic acid sequence shown in SEQ ID NO.4 for encoding a light chain variable region.
The invention also provides an epitope peptide combined by the antibody, which is an amino acid sequence shown as SEQ ID NO. 5.
The invention also provides an expression vector comprising a nucleic acid sequence as shown in SEQ ID NO.2 encoding a heavy chain variable region and a nucleic acid sequence as shown in SEQ ID NO.4 encoding a light chain variable region.
The invention also provides a host cell transformed with the above expression vector.
The present invention also provides a method for producing an antibody or a functional fragment of an antibody of interest, comprising the steps of culturing the above-mentioned host cell, and collecting the antibody or the functional fragment of the antibody of interest from the culture obtained by the above-mentioned steps.
The invention also provides a modified anti-human serum albumin antibody or a functional fragment of the antibody, which is characterized in that: comprising the above antibody or functional fragment of the antibody and at least one moiety selected from the group consisting of oligosaccharides, non-protein moieties, therapeutic agents, prophylactic agents and diagnostic agents.
The invention also provides a pharmaceutical composition, which is characterized in that: comprising the above antibody or functional fragment of the antibody, the above modified anti-human serum albumin antibody or functional fragment of the antibody, and a pharmaceutically acceptable carrier.
The invention also provides a kit for diagnosis or treatment, comprising the above antibody or functional fragment, or the above modified anti-human serum albumin antibody or functional fragment of the antibody, or the above pharmaceutical composition, and instructions for using the antibody or functional fragment, the modified antibody or functional fragment, or the pharmaceutical composition for diagnosis or treatment.
The antigen epitope sequence of the human serum albumin combined by the monoclonal antibody HSA mAb provided by the invention is shown as SEQ ID NO. 5.
The nucleic acid sequences encoding the monoclonal antibodies of the invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleic acid sequence of the monoclonal antibody of the present invention are derived from the nucleic acid sequence of the present invention and are equivalent to the sequence of the present invention as long as the monoclonal antibody is encoded and human serum albumin is resisted. The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleic acid sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleic acid sequence of the protein consisting of the amino acid sequences shown in sequence 2 and/or sequence 4 of the coding sequence table of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The beneficial effects are that:
the invention provides a monoclonal antibody HSA mAb capable of specifically binding human serum albumin, which can be used for preparing a human serum albumin detection reagent; the accurate detection of HSA concentration has important significance for liver risk diagnosis, kidney syndrome and diabetes diagnosis.
The monoclonal antibody of the invention has the advantages compared with the commercial products that the monoclonal antibody of the invention has the advantages that: 1. the antibody provided by the invention has the advantages that the specific antibody sequence is already identified, and the biological background is clear; 2. the antibody provided by the invention has definite antigen epitope already identified, and the sequence difference between the epitope and common bovine serum albumin is large, and the antibody is not combined with bovine serum albumin through Western Blot test (shown in figure 2).
Drawings
Fig. 1: SDS-PAGE detection after the expression and purification of the monoclonal antibody HSA mAb (the sample and the loading amount of 3 lanes from left to right are sequentially shown as a marker, 8 mu l, an HSA mAb,5 mu l and a pig IgG,5 mu l);
fig. 2: after the expression and purification of the monoclonal antibody HSA mAb, the binding activity of the monoclonal antibody HSA mAb on the human serum albumin is detected (a marker, an 8 mu l sample, a 5 mu g of human serum albumin and a 5 mu g of bovine serum albumin are sequentially carried out from left to right);
fig. 3: monoclonal antibody hsa mab immunoprecipitation of phage display library to detect hsa mab epitope (differential analysis of hsa mab group versus PBS control group);
fig. 4: the monoclonal antibody HSA mAb and phage display library immunoprecipitates were subjected to 5 rounds of biopanning, with varying degrees of epitope enrichment.
Detailed Description
To facilitate an understanding of the examples provided herein, certain frequently occurring terms are defined herein.
As used herein, the term "amino acid" refers to any organic compound containing an amino group (-NH 2) and a carboxyl group (-COOH), possibly in the form of a free radical or alternatively as part of a peptide bond after condensation. "twenty naturally occurring amino acids" are understood in the art and refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), valine (val or V), which are building blocks of natural proteins.
The term "antibody" as used herein refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, fab ', (Fab') 2, fv, and single chain antibody (SCA or scFv) fragments, which are capable of binding to an epitope of an antigen. These functional fragments can be made using methods well known in the art and described further below, which retain the ability to selectively bind to an antigen (e.g., a polypeptide antigen) of an antibody from which they are derived. Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and can be made by digestion of whole antibody molecules with papain, resulting in fragments consisting of the complete light chain and a portion of the heavy chain. Fab' fragments of antibody molecules can be obtained by treating a whole antibody molecule with pepsin followed by reduction, resulting in a molecule consisting of an intact light chain and a portion of the heavy chain. Two Fab' fragments were obtained per antibody molecule treated in this way. The (Fab') 2 fragment of the antibody can be obtained by treating the whole antibody molecule with pepsin without subsequent reduction. The (Fab ') 2 fragment is a dimer of two Fab' fragments bound together by two disulfide bonds. Fv fragments are defined as genetically engineered fragments comprising a light chain variable region and a heavy chain variable region and representing both chains.
Single chain antibodies ("SCAs" or scFvs) are genetically engineered single chain molecules containing a light chain variable region and a heavy chain variable region, linked by a suitable flexible polypeptide spacer, and may comprise other amino acid sequences at the amino-and/or carboxy-terminus. For example, a single chain antibody may comprise a tether segment for ligation to a coding polynucleotide. Functional single chain antibodies typically contain a sufficient portion of the light chain variable region and a sufficient region of the heavy chain variable region so as to preserve the properties of full length antibodies for binding to a specific target molecule or epitope.
The term "functional fragment" as used herein refers to a molecule other than and including a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of functional fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') 2; a bifunctional antibody; a linear antibody; monovalent antibodies, single chain antibody molecules (e.g., scFv). These functional fragments may be made using methods well known in the art that retain the ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody from which they are derived.
As used herein, the term "pharmaceutical instructions" is used to refer to instructions that are typically contained in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, combination therapies, contraindications and/or warnings associated with the use of such therapeutic products.
As used herein, the term "pharmaceutical composition" refers to a formulation in a form that allows the biological activity of the antibody hsa mab contained therein to be effectively exerted and that is free of other components that have unacceptable toxicity to the individual to whom the composition is to be administered.
As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition that is nontoxic to the individual to whom it is exposed, at the dosages and concentrations employed, in addition to the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
The term "variable region" of an antibody as used herein refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". These variable regions are typically the majority of the variable portions of the antibody and contain antigen binding sites.
As used herein, the term "vector" refers to a nucleotide molecule capable of propagating to another nucleotide to which it is attached. The term encompasses vectors that are self-replicating nucleotide structures and that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleotides to which they are operably linked.
The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present invention. However, one skilled in the art will readily recognize that some of the features may be omitted, or replaced by other elements, materials, or methods in different situations.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
The invention provides a human serum albumin monoclonal antibody, which comprises a heavy chain variable region with an amino acid sequence SEQ ID NO.1 and an antibody with a light chain variable region with an amino acid sequence SEQ ID NO. 2. Since the specific binding activity of an antibody is determined by a variable region, the present invention defines an antibody sequence by defining the amino acid sequence of the variable region, and may further include other sequences such as a linker sequence, for example, a sequence in which the amino acid sequence of the light chain variable region is linked to the constant region sequence, without affecting the specific binding activity of the antibody.
Taking a female Balb/c mouse with the age of 6-8 weeks, using human serum albumin with Freund's adjuvant, performing intraperitoneal injection once every two weeks, taking blood after three times of immunization, measuring antibody titer in serum, taking spleen of the mouse with the highest titer, separating B lymphocytes, performing electrofusion with myeloma cells, and then screening out hybridoma cells successfully fused by using a HAT hybridoma cell screening system. Subcloning and culturing the hybridoma cells which are successfully fused through a limiting dilution method, screening out positive holes with highest antibody titer through indirect ELISA, carrying out subsequent subcloning and screening, and obtaining a hybridoma cell line secreting the anti-human serum albumin antibody after three rounds of subcloning. Extracting total RNA of a human serum albumin hybridoma cell line by using a Trizol method, performing reverse transcription to obtain a cDNA library, constructing a second-generation sequencing library, and sequencing, wherein the sequencing result is analyzed by using a BASIC+rnaSPades+Igfinder bioinformatics pipeline flow to obtain an antibody light and heavy chain sequence. Designing a primer according to an analysis result, carrying out PCR by taking the cDNA library as a template, and carrying out sanger sequencing on a PCR product to verify the correctness of the sequence of the light chain and the heavy chain of the antibody obtained by analysis. Constructing and displaying a phage library containing human serum albumin, breaking the protein into 56 polypeptides by using the library display fragments, overlapping the 56 polypeptides, incubating for 18 hours through an antibody-phage library, precipitating protein A protein G magnetic beads, obtaining an enrichment, constructing a second-generation sequencing library, sequencing, and finally obtaining enriched peptide members through differential analysis with a PBS control group without antibody input. To verify that the enriched peptide members were epitopes of the hsa mab, we also performed 5 rounds of biopanning and second generation sequencing of the phage library, showing that the counts of the enriched peptide members after 5 rounds of enrichment were continuously rising, further proving the correctness of the previous single round immunoprecipitation.
In one embodiment of the invention, a functional fragment of the human serum albumin monoclonal antibody is provided, said functional fragment being selected from the group consisting of Fab, F (ab) 2, fab' and Fv.
In one embodiment of the invention, a nucleic acid sequence for encoding the amino acid sequences of the heavy chain variable region and the light chain variable region of the human serum albumin monoclonal antibody is provided, and is specifically shown as SEQ ID NO.2 and SEQ ID NO. 4. It should be noted that, based on the principle of base degeneracy, the nucleic acid sequence encoding the amino acid sequence of the heavy chain variable region is not the only constant sequence, and any nucleic acid sequence capable of encoding the same heavy chain variable region amino acid sequence is within the scope of the present invention.
In one embodiment of the invention, an expression vector is provided comprising a nucleic acid sequence encoding an antibody fragment or antibody of the invention. The vector backbone in the present invention is not particularly limited, and one skilled in the art can incorporate a nucleic acid sequence encoding the antibody fragment or antibody of the present invention into a suitable vector backbone as required to obtain the expression vector of the present invention. The antibody molecule consists of a light chain and a heavy chain, wherein the sequences 1-4 in the sequence table are respectively the amino acid and the nucleic acid sequence of the light chain and the heavy chain of the antibody, the vector is a vector containing an Ef1 alpha promoter, and a target antibody fragment is added into a biological common eukaryotic expression vector.
In one embodiment of the invention a recombinant host cell is provided comprising said expression vector. The host cell of the present invention is not particularly limited, and one skilled in the art can introduce an expression vector into an appropriate host cell for expressing the antibody fragment or antibody of the present invention as needed.
In one embodiment of the invention, a modified anti-human serum albumin antibody or functional fragment of an antibody is provided, comprising said antibody or functional fragment of an antibody and at least one moiety selected from the group consisting of oligosaccharides, non-protein moieties, therapeutic agents, prophylactic agents and diagnostic agents.
In one embodiment of the present invention, there is provided a method for producing an antibody fragment or an antibody, comprising the steps of culturing the host cell, and collecting the antibody of interest or a functional fragment of the antibody from the culture obtained by the above steps.
In one embodiment of the invention, a pharmaceutical composition is provided comprising an antibody or functional fragment of an antibody, or a modified anti-human serum albumin antibody or functional fragment of an antibody, as described herein, and a pharmaceutically acceptable carrier.
In one embodiment of the invention a kit for diagnosis or treatment is provided, comprising the antibody or antibody fragment, or a functional fragment of the modified anti-human serum albumin antibody or antibody, or the pharmaceutical composition, and instructions for using the antibody or antibody fragment, the modified antibody or antibody fragment, or the pharmaceutical composition for diagnosis or treatment.
Example 1 bioinformatic analysis of human serum albumin antibody sequences.
The specific implementation process is as described in the summary of the invention, and the general flow is as follows: taking a female Balb/c mouse with the age of 6-8 weeks, using human serum albumin with Freund's adjuvant, performing intraperitoneal injection once every two weeks, taking blood after three times of immunization, measuring antibody titer in serum, taking spleen of the mouse with the highest titer, separating B lymphocytes, performing electrofusion with myeloma cells, and then screening out hybridoma cells successfully fused by using a HAT hybridoma cell screening system. Subcloning and culturing the hybridoma cells which are successfully fused through a limiting dilution method, screening out positive holes with highest antibody titer through indirect ELISA, carrying out subsequent subcloning and screening, and obtaining a hybridoma cell line secreting the anti-human serum albumin antibody after three rounds of subcloning.
Extracting total RNA of a human serum albumin hybridoma cell line by using a Trizol method, performing reverse transcription to obtain a cDNA library, constructing a second-generation sequencing library, and sequencing, wherein the sequencing result is analyzed by using a written BASIC+rnaSPades+Igfinder bioinformatics pipeline flow to obtain an antibody light and heavy chain sequence as follows:
the nucleic acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1;
the nucleic acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 4; the amino acid sequence of the antibody light chain variable region is shown as SEQ ID NO. 3.
Example 2, validation of human serum albumin antibody sequences.
The full-length heavy and light chain sequences of the HSA mAb were cloned into expression vectors containing the promoter EF 1. Alpha. (cleavage sites: ndeI and XhoI), respectively, and 293FT cells were co-transfected. After 4 days of cell culture, the culture solution is centrifuged at 1000rpm/min for 5min to collect the supernatant, and after filtration through a 0.45 mu m filter membrane, the recombinant antibody is obtained by purifying with ProteinA magnetic beads, and SDS-PAGE electrophoresis detection is carried out. The results are shown in FIG. 1, where the target protein is expected to be at approximately 55kD and 24 kD.
EXAMPLE 3 Western-blot detection of binding Activity of HSA mAb to human serum albumin
1. Mu.g of human serum albumin and bovine serum albumin were taken, and SDS-PAGE protein loading buffer (5X) was added to the working concentration, and the proteins were denatured thoroughly in a water bath at 100℃for 5-10 minutes. 12% SDS-PAGE separation gel and 5% concentration gel were prepared according to the kit. Loading the gel into SDS-PAGE gel loading holes according to a certain sequence, concentrating the gel at 90V, performing constant-pressure electrophoresis on the separation gel at 120V for about 1 hour, and stopping electrophoresis when the blue dye reaches the position near the bottom end of the gel;
3. transferring the sample to NC membrane according to instruction operation after electrophoresis, and sealing with 5% skimmed milk powder for 2h;
4. hsa mab antibody (1:100 diluted in blocking solution) was added and incubated overnight at 4 ℃;
5. the next day, washing with TBST shaking for 3 times each for 10 minutes, then adding horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (PBST 1:2000 dilution), standing in a shaking table at room temperature for 1 hour, and washing again thoroughly;
6. developing color;
7. as a result, as shown in FIG. 2, the band of the target protein was detected in human serum albumin at about 72kD, and the band was not detected in bovine serum albumin.
Example 4 selection of epitopes of hybridoma antibody sequences.
Constructing and displaying a phage library containing human serum albumin, breaking the protein into 56 polypeptides by using the library display fragments, overlapping the 56 polypeptides, incubating for 18 hours through an antibody-phage library, precipitating protein A protein G magnetic beads, obtaining an enrichment, constructing a second-generation sequencing library, sequencing, and finally obtaining enriched peptide members through differential analysis with a PBS control group without antibody input. To verify that the enriched peptide members were epitopes of the hsa mab, phage libraries were biopanning for 5 rounds and sequencing for the second generation, the results showed that the counts of the enriched peptide members after 5 rounds of enrichment were continuously rising, verifying the correctness of single round immunoprecipitation.
1. Thawing the phage library and combining into a large enough container, and diluting with PBS to an input of 105pfu per phage library member;
2. phage were thoroughly mixed by pipetting up and down with a pipette and 1ml of the dilution was dispensed into a 1.5ml centrifuge tube;
3. mu.g of HSA mAb was added to a centrifuge tube containing phage dilutions (i.e., IP mix). The mixture was rotated upside down at 4 ℃ for about 18 hours;
4. adding 20 μl of each of the protein A and protein G magnetic beads to the IP mixture;
5. the IP mixture was spun upside down in 4 ℃ for about 4 hours and washed 3 times on a magnetic rack with IP wash to obtain immunoprecipitates;
6. designing a linker conforming to the two-to-sequencing library, adding the linker to the two ends of the library by using a PCR technique with the precipitate as a template;
7. sequencing by a Inonosethoxydim source company;
8. sequencing data were subjected to differential analysis using EdgeR to yield enriched library members.
Example 5, validation of hybridoma antibody sequence epitopes.
1. Thawing the phage library and combining into a large enough container, and diluting with PBS to an input of 105pfu per phage library member;
2. phage were thoroughly mixed by pipetting up and down with a pipette and 1ml of the dilution was dispensed into a 1.5ml centrifuge tube;
3. mu.g of HSA mAb was added to a centrifuge tube containing phage dilutions (i.e., IP mix). The mixture was rotated upside down at 4 ℃ for about 18 hours;
4. adding 20 μl of each of the protein A and protein G magnetic beads to the IP mixture;
5. the IP mixture was spun upside down in 4 ℃ for about 4 hours and washed 3 times on a magnetic rack with IP wash to obtain immunoprecipitates;
6. culturing BLT5403 host bacteria in M9TB medium in advance to logarithmic phase;
7. adding the immunoprecipitate into BLT5403 host bacteria in logarithmic phase, culturing at 37 deg.C until culture solution is clear and filiform lysate appears;
8. centrifuging the lysate at 8000r/min for 15min to remove cell debris, and obtaining phage lysate.
9. Repeating the steps 1-85 times;
10. constructing a second generation sequencing library by using phage lysate as a template and sequencing;
11. sequencing data were aligned to a reference library using bowtie2 and normalized counts were calculated.
As shown in fig. 3 and fig. 4, the epitope of the present antibody, that is, the region where the antibody binds to the antigen, was detected and further demonstrated, the sequence difference between the epitope and the common bovine serum albumin was large, and the present antibody did not bind to bovine serum albumin by Western Blot (as shown in fig. 2).
The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.
Sequence listing
<120> monoclonal antibody specifically binding to human serum albumin and use thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 138
<212> DNA
<213> mice (Mus musculus)
<220>
<223> heavy chain variable region amino acid sequence
<400> 1
Gln Ile Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly
1 5 10 15
Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Gly
20 25 30
Ser His Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
35 40 45
Glu Trp Ile Gly Gln Ile Tyr Pro Gly Asp Gly Asp Ser Gln Tyr
50 55 60
Asn Val Lys Phe Arg Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser
65 70 75
Ser Asn Thr Ala Phe Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp
80 85 90
Ser Ala Val Tyr Phe Cys Ala Arg Thr Asn Phe Phe Gly Ser Thr
95 100 105
Tyr Leu Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Pro Leu Thr
110 115 120
Val Ser Ser
<210> 2
<211> 369
<212> DNA
<213> mice (Mus musculus)
<220>
<223> heavy chain variable region DNA sequence
<400> 2
caaattcagc tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc agtgaagatt 60
tcctgcaagg cttctggcta tgcattcggt agccactgga tgaactgggt gaaacagagg 120
cctggacagg gtcttgagtg gattggacag atttatcctg gagatggtga ttctcagtac 180
aatgtaaagt tcagggataa agccacactg actgcggaca agtcctccaa cacagccttc 240
ttgcagctca gcagcctaac atctgaggac tctgcggtct atttctgtgc acgaacgaat 300
ttcttcggta gtacctactt gtacttcttt gactactggg gccaaggcac ccctctcaca 360
gtctcctca 381
<210> 3
<211> 106
<212> PRT
<213> mice (Mus musculus)
<220>
<223> light chain variable region amino acid sequence
<400> 3
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Gly Val Ser Tyr Ile
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Cys
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Arg Trp Gly Gly Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg
100 105
<210> 4
<211> 318
<212> DNA
<213> mice (Mus musculus)
<220>
<223> light chain variable region DNA sequence
<400> 4
caaattgttc tctcccagtc tccagcaatc ctgtctgcat ctccagggga gaaggtcaca 60
atgacttgca gggccagctc aggtgtaagt tacatacact ggtatcagca gaagccagga 120
tcctccccca aaccctggat ttgtgccaca tccaacctgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgag 240
gatgctgcca cttattactg ccagcggtgg ggtggtaacc cactcacgtt cggtgctggg 300
accaagctgg agctgaga 328
<210> 5
<211> 56
<212> PRT
<213> human (homosapiens)
<220>
<223> epitope sequence
<400> 5
Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr
1 5 10 15
Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys
20 25 30
Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile
35 40 45
Lys Gln Asn Cys Glu Leu Phe Glu
Claims (10)
1. A monoclonal antibody that specifically binds human serum albumin, characterized in that: an antibody comprising a heavy chain variable region having the amino acid sequence SEQ ID NO.1 and a light chain variable region having the amino acid sequence SEQ ID NO. 3.
2. A functional fragment of the antibody of claim 1, wherein: the functional fragment is selected from the group consisting of Fab, F (ab) 2, fab' and Fv.
3. A nucleic acid sequence characterized by: a nucleic acid sequence as set forth in claim 1 or 2 encoding an antibody or antibody functional fragment comprising a nucleic acid sequence as set forth in SEQ ID No.2 encoding a heavy chain variable region and a nucleic acid sequence as set forth in SEQ ID No.4 encoding a light chain variable region.
4. The epitope peptide to which the antibody of claim 1 binds, wherein: the amino acid sequence is shown as SEQ ID NO. 5.
5. An expression vector, characterized in that: a nucleic acid sequence according to claim 3.
6. A host cell, characterized in that: transformation using the expression vector of claim 5.
7. A method for producing an antibody or functional fragment thereof of interest, characterized by: comprising the steps of culturing the host cell according to claim 6, and collecting the antibody of interest or a functional fragment of said antibody from the culture obtained by the above steps.
8. A modified anti-human serum albumin antibody or functional fragment of an antibody, characterized in that: an antibody or functional fragment of an antibody as claimed in claim 1 or 2 and at least one moiety selected from the group consisting of oligosaccharides, non-protein moieties, therapeutic agents, prophylactic agents and diagnostic agents.
9. A pharmaceutical composition characterized by: comprising an antibody or functional fragment of an antibody as defined in claim 1 or 2, or a modified anti-human serum albumin antibody or functional fragment of an antibody as defined in claim 8, and a pharmaceutically acceptable carrier.
10. A kit for diagnosis or treatment comprising an antibody or antibody fragment according to any one of claims 1 to 2, or a modified anti-human serum albumin antibody or functional fragment of an antibody according to claim 8, or a pharmaceutical composition according to claim 9, and instructions for using the antibody or antibody fragment, the modified antibody or antibody fragment, or the pharmaceutical composition for diagnosis or treatment.
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| CN102453091A (en) * | 2010-10-20 | 2012-05-16 | 上海生物制品研究所有限责任公司 | Tetanus toxoid monoclonal antibody and preparation method and application thereof |
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| CN108409842A (en) * | 2018-03-15 | 2018-08-17 | 山东省农业科学院畜牧兽医研究所 | A kind of monoclonal antibody and preparation method thereof |
| CN108586611A (en) * | 2018-04-28 | 2018-09-28 | 暨南大学 | Recombinate full humanized anti-spasmotoxin monoclone antibody |
| CN110305216A (en) * | 2018-03-20 | 2019-10-08 | 无锡智康弘义生物科技有限公司 | Novel anti-TIM-3 antibody |
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2021
- 2021-11-07 CN CN202111309824.3A patent/CN114702580B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102453091A (en) * | 2010-10-20 | 2012-05-16 | 上海生物制品研究所有限责任公司 | Tetanus toxoid monoclonal antibody and preparation method and application thereof |
| CN104936980A (en) * | 2012-12-31 | 2015-09-23 | 生物控股有限公司 | Recombinant human antibodies for therapy and prevention of polyomaviruses-related diseases |
| CN107674123A (en) * | 2016-08-02 | 2018-02-09 | 厦门大学 | A kind of anti-idiotype and its application |
| CN108409842A (en) * | 2018-03-15 | 2018-08-17 | 山东省农业科学院畜牧兽医研究所 | A kind of monoclonal antibody and preparation method thereof |
| CN110305216A (en) * | 2018-03-20 | 2019-10-08 | 无锡智康弘义生物科技有限公司 | Novel anti-TIM-3 antibody |
| CN108586611A (en) * | 2018-04-28 | 2018-09-28 | 暨南大学 | Recombinate full humanized anti-spasmotoxin monoclone antibody |
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