Anti-human h-fabp recombinant antibodies, nucleic acids, recombinant expression vectors, host cells and uses thereof

Abstract

The application relates to anti-human H-FABP recombinant antibodies, nucleic acids, recombinant expression vectors, host cells and applications thereof, a heavy chain variable region of the anti-human H-FABP recombinant antibody comprises H-CDR1, H-CDR2 and H-CDR3, a light chain variable region of the anti-human H-FABP recombinant antibody comprises L-CDR1, L-CDR2 and L-CDR3; the amino acid sequence of H-CDR1 is shown in SEQ ID No. 1, the amino acid sequence of H-CDR2 is shown in SEQ ID No. 2, the amino acid sequence of H-CDR3 is shown in SEQ ID No. 3, the amino acid sequence of L-CDR1 is shown in SEQ ID No. 4, the amino acid sequence of L-CDR2 is shown in SEQ ID No. 5, and the amino acid sequence of L-CDR3 is shown in SEQ ID No. 6.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a recombinant antibody against human H-FABP, nucleic acid, recombinant expression vector, host cell, and their applications. Background Technology

[0002] Fatty acid-binding proteins (FABPs) are low-molecular-weight (12-15 kDa) non-enzymatic intracellular proteins widely distributed in the cells of mammalian tissues such as cardiomyocytes, small intestines, livers, adipose tissue, and brain epithelial cells. Currently, the FABP family comprises at least nine different types, each exhibiting a characteristic tissue distribution pattern and substantial sequence homology. Common FABPs include cardiac fatty acid-binding protein (H-FABP), liver fatty acid-binding protein (L-FABP), kidney fatty acid-binding protein (K-FABP), and skeletal fatty acid-binding protein (S-FABP). Studies have found that FABPs constitute 4%–8% of the proteins in the cytoplasm of cardiac muscle cells and are believed to play a role in lipid metabolism by participating in the uptake and intracellular transport of long-chain fatty acids and their lipolysis.

[0003] Acute myocardial infarction (AMI) refers to the sudden and complete occlusion of a coronary artery, leading to myocardial ischemia, damage, and necrosis, and is characterized by severe chest pain, dynamic changes in electrocardiogram and myocardial enzymes. Therefore, serum H-FABP levels are an important parameter for the early diagnosis of suspected AMI patients.

[0004] Developing anti-human H-FABP antibodies can lay the material foundation for further research on the role of this molecule in acute myocardial infarction. Traditional monoclonal antibodies typically involve using B cells from immunized animals to create immortalized hybridoma cells that secrete the desired antibodies. While monoclonal antibodies can improve antibody specificity to some extent, over time, hybridoma cell lines undergo genetic drift, leading to slight changes in the produced antibodies and making them unstable. Furthermore, due to individual differences in mice, monoclonal antibodies are prone to batch-to-batch variations. Summary of the Invention

[0005] Based on this, this application provides a recombinant antibody against human H-FABP, which has good affinity and specificity and high batch-to-batch consistency.

[0006] The technical solution to the above-mentioned technical problems in this application is as follows:

[0007] This application provides a recombinant antibody against human H-FABP, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises H-CDR1, H-CDR2, and H-CDR3, and the light chain variable region comprises L-CDR1, L-CDR2, and L-CDR3. The amino acid sequence of H-CDR1 is shown in SEQ ID No. 1, the amino acid sequence of H-CDR2 is shown in SEQ ID No. 2, the amino acid sequence of H-CDR3 is shown in SEQ ID No. 3, the amino acid sequence of L-CDR1 is shown in SEQ ID No. 4, the amino acid sequence of L-CDR2 is shown in SEQ ID No. 5, and the amino acid sequence of L-CDR3 is shown in SEQ ID No. 6.

[0008] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 8.

[0009] In one embodiment, the anti-human H-FABP recombinant antibody further includes a heavy chain constant region and a light chain constant region, the amino acid sequence of which is shown in SEQ ID No. 11; the amino acid sequence of which is shown in SEQ ID No. 12.

[0010] A recombinant antibody against human H-FABP, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises H-CDR1, H-CDR2, and H-CDR3, and the light chain variable region comprises L-CDR1, L-CDR2, and L-CDR3; the amino acid sequence of H-CDR1 is shown in SEQ ID No. 13, the amino acid sequence of H-CDR2 is shown in SEQ ID No. 14, the amino acid sequence of H-CDR3 is shown in SEQ ID No. 15, the amino acid sequence of L-CDR1 is shown in SEQ ID No. 16, the amino acid sequence of L-CDR2 is shown in SEQ ID No. 17, and the amino acid sequence of L-CDR3 is shown in SEQ ID No. 18.

[0011] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 20.

[0012] In one embodiment, the anti-human H-FABP recombinant antibody further includes a heavy chain constant region and a light chain constant region, the amino acid sequence of which is shown in SEQ ID No. 23; the amino acid sequence of which is shown in SEQ ID No. 24.

[0013] A nucleic acid used to encode the recombinant anti-human H-FABP antibody as described above.

[0014] A vector containing the aforementioned nucleic acid.

[0015] A host cell, transformed with the aforementioned vector.

[0016] A diagnostic reagent comprising the aforementioned recombinant anti-human H-FABP antibody.

[0017] A diagnostic kit comprising the aforementioned recombinant anti-human H-FABP antibody.

[0018] This application provides a recombinant anti-human H-FABP antibody. Since the antibody gene is isolated separately and the sequence is known, the batch-to-batch consistency is high, and large-scale antibody expression can be performed. This antibody can be used for immunohistochemical detection of acute myocardial infarction, thereby improving the accuracy of immunohistochemical detection of myocardial infarction serum samples. Attached Figure Description

[0019] Figure 1 This is a graph showing the results of monoclonal antibody screening in Example 1;

[0020] Figure 2 Analysis of the pairing performance of the mouse monoclonal antibody in Example 1;

[0021] Figure 3 This is a schematic diagram of the hard matrix chip-coated antibody principle in Example 1;

[0022] Figure 4 Analysis of the pairing performance of the recombinant antibody in Example 1;

[0023] Figure 5 This is an SDS-PAGE image of the purified anti-human H-FABP recombinant antibody 1 from Example 1;

[0024] Figure 6 This is an SDS-PAGE image of the purified anti-human H-FABP recombinant antibody 2 from Example 1. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more comprehensive description will be provided below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the present invention will be thorough and complete.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0027] Terminology Explanation:

[0028] Antibodies are a class of immunoglobulins that specifically bind to antigens. Generally, antibodies exist as one or more Y-shaped monomers. Each Y-shaped monomer consists of four polypeptide chains, including two identical heavy chains and two identical light chains, named according to their molecular weight. The apex of the Y-shaped structure is the variable region, the antigen-binding site. Each heavy chain has two regions: a constant region and a variable region. All antibodies of the same type share the same constant region, while different types of antibodies differ. Each light chain also has two connected domains: a constant region and a variable region.

[0029] A "vector" is a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0030] "Host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0031] One embodiment of the present invention provides a recombinant antibody against human H-FABP. The recombinant antibody against human H-FABP includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes H-CDR1, H-CDR2, and H-CDR3, and the light chain variable region includes L-CDR1, L-CDR2, and L-CDR3. The amino acid sequence of H-CDR1 is shown in SEQ ID No. 1, the amino acid sequence of H-CDR2 is shown in SEQ ID No. 2, the amino acid sequence of H-CDR3 is shown in SEQ ID No. 3, the amino acid sequence of L-CDR1 is shown in SEQ ID No. 4, the amino acid sequence of L-CDR2 is shown in SEQ ID No. 5, and the amino acid sequence of L-CDR3 is shown in SEQ ID No. 6.

[0032] Specifically, the amino acid sequence shown in SEQ ID No. 1 is HIYMK; the amino acid sequence shown in SEQ ID No. 2 is PSRNKANYYTTDYSTSVQS; the amino acid sequence shown in SEQ ID No. 3 is HYEYGMEN; the amino acid sequence shown in SEQ ID No. 4 is TSCSSVNYMYWY; the amino acid sequence shown in SEQ ID No. 5 is HTSNLAA; and the amino acid sequence shown in SEQ ID No. 6 is KQWSGYPLS.

[0033] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 8. The light chain variable region and the heavy chain variable region are linked by disulfide bonds. Specifically, the amino acid sequence shown in SEQ ID No. 7 is: EVQLQESGGDLAQPGGSLRLSCATSGFTFSHIYMKWVRQPPGQRLEWIAP SRNKANYYTTDYSTSVQSRFIVSRDASQSILYLQMNALRTEDTAIYYCAR HYEYGMENWGPGTSVTVSS. The amino acid sequence shown in SEQ ID No. 8 is: DIQLTQSPAIMSASPGEKVTMTCTSCSSVNYMYWYQQKPGSSPRLLIHHT SNLAAGVPVRFSGSGSGTSYSLTISRMEAEDAATYSCKQWSGYPLSFGAG TKLEIK.

[0034] It is understood that in other embodiments, the heavy chain variable region is not limited to the polypeptide with the amino acid sequence shown in SEQ ID No. 7, but may also be other polypeptides containing the above-mentioned heavy chain complementarity-determining region (amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 3); the light chain variable region is not limited to the polypeptide with the amino acid sequence shown in SEQ ID No. 8, but may also be other polypeptides containing the above-mentioned light chain complementarity-determining region (amino acid sequences shown in SEQ ID No. 4 to SEQ ID No. 6).

[0035] Optionally, the nucleotide sequence encoding the variable region of the heavy chain is as shown in SEQ ID No. 9, and the nucleotide sequence shown in SEQ ID No. 9 is: 5'-GAGGTGCAGCTGCAGGAGTCCGGAGGCGACCTGGCACAGCCAGGAGGCAGCCTGAGGCTGTCCTGCGCCACCTCTGGCTTCACCTTCAGCCATATCTACATGAAGTGGGTGAGGCAGCCACCTGGACAGCGCCTGGAGTGGATCGCCCCCTCCAGAAACAAGGCCAATTACTATACCACAGACTACTCTACCAGCGTGCAGAGCCGGTTTATCGTGTCTAGAGATGCCTCCCAGTCTATCCTGTATCTGCAGATGAATGCCCTGCGGACCGAGGACACAGCCATCTACTATTGTGCCAGACATTACGAGTATGGCATGGA GAATTGGGGC CCCGGCACCA GCGTGACAGTGAGCTCC-3'.

[0036] Optionally, the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 10, and the nucleotide sequence shown in SEQ ID No. 10 is: 5'-GATATTCAGCTGACACAGAGCCCCGCCATCATGAGCGCCTCCCCTGGCGAGAAGGTGACAATGACCTGTACCTCCTGCTCCTCCGTGAACTACATGTACTGGTACCAGCAGAAGCCTGGCAGCTCCCCTAGGCTGCTGATCCACCATACCTCCAATCTGGCCGCCGGCGTGCCTGTGAGATTTTCCGGCAGCGGCTCCGGCACCTCCTACAGCCTGACCATCTCCAGAATGGAGGCCGAGGATGCCGCCACCTACTCCTGTAAGCAGTGGAGCGGCTACCCCCTGTCATTCGGCGCCGGCACCAAGCTGGAGATCAAG-3'.

[0037] It is understood that, based on the degeneracy of codons, in other embodiments, the nucleic acid encoding the heavy chain variable region is not limited to the above, but may also be other nucleic acids that can encode the heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 7; the nucleic acid encoding the light chain variable region is not limited to the above, but may also be other nucleic acids that can encode the light chain variable region with an amino acid sequence as shown in SEQ ID No. 8.

[0038] In one optional specific example, the aforementioned anti-human H-FABP recombinant antibody consists of a heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 7 and a light chain variable region with an amino acid sequence as shown in SEQ ID No. 8, wherein the light chain variable region and the heavy chain variable region are linked by disulfide bonds. In this case, the heavy chain variable region and the light chain variable region form an FV region with all antigen-binding sites through non-covalent bonding, thus possessing antigen-binding capability.

[0039] In some embodiments, the anti-human H-FABP recombinant antibody further includes a heavy chain constant region and a light chain constant region. The heavy chain constant region is connected to a heavy chain variable region, the light chain constant region is connected to a light chain variable region, and the heavy chain constant region is also connected to the light chain constant region to form a Y-shaped monomer structure.

[0040] In one optional specific example, the amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 11; the amino acid sequence of the light chain constant region is shown in SEQ ID No. 12; it is understood that in other embodiments, the amino acid sequences of the heavy chain constant region and the light chain constant region are not limited to the above, and may also be other polypeptide fragments that can serve as constant regions.

[0041] Furthermore, one embodiment of the present invention also provides another recombinant antibody against human H-FABP, which includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes H-CDR1, H-CDR2, and H-CDR3, and the light chain variable region includes L-CDR1, L-CDR2, and L-CDR3. The amino acid sequence of H-CDR1 is shown in SEQ ID No. 13, the amino acid sequence of H-CDR2 is shown in SEQ ID No. 14, the amino acid sequence of H-CDR3 is shown in SEQ ID No. 15, the amino acid sequence of L-CDR1 is shown in SEQ ID No. 16, the amino acid sequence of L-CDR2 is shown in SEQ ID No. 17, and the amino acid sequence of L-CDR3 is shown in SEQ ID No. 18.

[0042] Specifically, the amino acid sequence shown in SEQ ID No. 13 is CRNYWN; the amino acid sequence shown in SEQ ID No. 14 is DIRYDGNNNYNPSLK; the amino acid sequence shown in SEQ ID No. 15 is AGILFDN; the amino acid sequence shown in SEQ ID No. 16 is ATDSSVNFMYWY; the amino acid sequence shown in SEQ ID No. 17 is VASNLAS; and the amino acid sequence shown in SEQ ID No. 18 is EQSSSDPPS.

[0043] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 20. The light chain variable region and the heavy chain variable region are linked by disulfide bonds. Specifically, the amino acid sequence shown in SEQ ID No. 19 is: QVKLEESGPGLVKPSQSLSLTCSVTGYSVTCRNYWNWIRQFPGNRLEWMGDIRYDGNNNYNPSLKDRISVTRDTSQNQFFLKLNSVTSEDTATYYCARAGILFDNWGQGTTLTVSS. The amino acid sequence shown in SEQ ID No. 20 is: DIQLTQSPALMSASPGEKVTMTCATDSSVNFMYWYQQKPRSSPKPWIFVASNLASGVPARFSGSRSGTSYSLTISSMEAEDAATYYCEQSSSDPPSFGAGTKLEIK.

[0044] It is understood that in other embodiments, the heavy chain variable region is not limited to the polypeptide with the amino acid sequence shown in SEQ ID No. 19, but may also be other polypeptides containing the above-mentioned heavy chain complementarity-determining region (amino acid sequences shown in SEQ ID No. 13 to SEQ ID No. 15); the light chain variable region is not limited to the polypeptide with the amino acid sequence shown in SEQ ID No. 20, but may also be other polypeptides containing the above-mentioned light chain complementarity-determining region (amino acid sequences shown in SEQ ID No. 16 to SEQ ID No. 18).

[0045] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 21. The nucleotide sequence shown in SEQ ID No. 21 is: 5'-CAGGTGAAGCTGGAGGAGAGCGGAC CAGGACTGGTGAAGCCTTCTCAGAGCCTGTCCCTGACCTG CTCCG TGACA GGCTACTCTGTGACCTGCCGCAACTATTGGAATTGGATCAGGCAGTTCCCAGGCAACCGCCTGGAGTGGATGGGCGACATCCGGTATGACGGCAACAATAACTATAATCCCAGCCTGAAGGACCGGATCTCCGTGACCAGAGATACATCTCAGAATCAGTTCTTTCTGAAGCTGAACTCCGTGACCTCTGAGGACACCGCCACATACTATTGTGCCAGGGCTGGCATCCTGTTTGATAACTGGGGCCAGGGCACC ACACT GACAG TGAGCTCC-3'.

[0046] Optionally, the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 22, and the nucleotide sequence shown in SEQ ID No. 22 is: 5'-GATATTCAGCTGACACAGTCCCCTGCCCTGATGTCCGCCAGCCCTGGCGAGAAGGTGACAATGACCTGCGCCACCGATTCCTCCGTGAACTTCATGTACTGGTACCAGCAGAAGCCTAGAAGCAGCCCCAAGCCCTGGATCTTTGTGGCCAGCAACCTGGCCTCCGGCGTGCCCGCTAGATTTTCCGGCTCCAGGAGCGGCACCAGCTACTCCCTGACCATCTCCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGTGAGCAGAGCAGCTCCGATCCCCCCTCCTTCGGCGCCGGAACCAAGCTGGAGATCAAG-3'.

[0047] It is understood that, based on the degeneracy of codons, in other embodiments, the nucleic acid encoding the heavy chain variable region is not limited to the above, but may also be other nucleic acids that can encode the heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 19; the nucleic acid encoding the light chain variable region is not limited to the above, but may also be other nucleic acids that can encode the light chain variable region with an amino acid sequence as shown in SEQ ID No. 20.

[0048] In one optional specific example, the aforementioned anti-human H-FABP recombinant antibody consists of a heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 19 and a light chain variable region with an amino acid sequence as shown in SEQ ID No. 20, wherein the light chain variable region and the heavy chain variable region are linked by disulfide bonds. In this case, the heavy chain variable region and the light chain variable region form an FV region with all antigen-binding sites through non-covalent bonding, thus possessing antigen-binding capability.

[0049] In some embodiments, the anti-human H-FABP recombinant antibody further includes a heavy chain constant region and a light chain constant region. The heavy chain constant region is connected to a heavy chain variable region, the light chain constant region is connected to a light chain variable region, and the heavy chain constant region is also connected to the light chain constant region to form a Y-shaped monomer structure.

[0050] In one optional specific example, the amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 23; the amino acid sequence of the light chain constant region is shown in SEQ ID No. 24; it is understood that in other embodiments, the amino acid sequences of the heavy chain constant region and the light chain constant region are not limited to the above, and may also be other polypeptide fragments that can serve as constant regions.

[0051] Furthermore, one embodiment of the present invention also provides a nucleic acid of the above-mentioned anti-human H-FABP recombinant antibody.

[0052] In some embodiments, the nucleic acid comprises a nucleic acid fragment encoding the heavy chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 7 and a nucleic acid fragment encoding the light chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 8. Further, the nucleic acid also comprises a nucleic acid fragment encoding the heavy chain constant region with the amino acid sequence as shown in SEQ ID No. 11 and a nucleic acid fragment encoding the light chain constant region with the amino acid sequence as shown in SEQ ID No. 12.

[0053] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence shown in SEQ ID No. 7 is shown in SEQ ID No. 9. The nucleotide sequence of the nucleic acid encoding the light chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence shown in SEQ ID No. 8 is shown in SEQ ID No. 10. It is understood that, based on codon degeneracy, in other embodiments, the nucleotide sequences of the nucleic acid fragments encoding the heavy chain variable region and light chain variable region with the amino acid sequences shown in SEQ ID No. 7 and SEQ ID No. 8 are not limited to the above, and may be other sequences.

[0054] It should be noted that, among the above nucleic acids, the nucleic acid fragment encoding the heavy chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 7 and the nucleic acid fragment encoding the light chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 8 exist in different reagent systems (for example, the above two nucleic acid fragments are located on different expression vectors), or they can exist simultaneously in the same reagent system (for example, the above two nucleic acid fragments are located on the same expression vector).

[0055] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain constant region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 11 is shown in SEQ ID No. 25. The nucleotide sequence of the nucleic acid encoding the light chain constant region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 12 is shown in SEQ ID No. 26. It is understood that, based on codon degeneracy, in other embodiments, the nucleotide sequences of the nucleic acid fragments encoding the heavy chain constant region and light chain constant region with the amino acid sequences as shown in SEQ ID No. 11 and SEQ ID No. 12 are not limited to the above, and may be other sequences.

[0056] In other embodiments, the nucleic acid comprises a nucleic acid fragment encoding the heavy chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 19 and a nucleic acid fragment encoding the light chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 20. Further, the nucleic acid also includes a nucleic acid fragment encoding the heavy chain constant region with the amino acid sequence as shown in SEQ ID No. 23 and a nucleic acid fragment encoding the light chain constant region with the amino acid sequence as shown in SEQ ID No. 24.

[0057] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 19 is shown in SEQ ID No. 21. The nucleotide sequence of the nucleic acid encoding the light chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 20 is shown in SEQ ID No. 22. It is understood that, based on codon degeneracy, in other embodiments, the nucleotide sequences of the nucleic acid fragments encoding the heavy chain variable region and light chain variable region with the amino acid sequences as shown in SEQ ID No. 19 and SEQ ID No. 20 are not limited to the above, and may be other sequences.

[0058] It should be noted that, among the above nucleic acids, the nucleic acid fragment encoding the heavy chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 19 and the nucleic acid fragment encoding the light chain variable region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 20 exist in different reagent systems (e.g., the above two nucleic acid fragments are located on different expression vectors), or they can exist simultaneously in the same reagent system (e.g., the above two nucleic acid fragments are located on the same expression vector).

[0059] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain constant region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 23 is shown in SEQ ID No. 27. The nucleotide sequence of the nucleic acid encoding the light chain constant region of the anti-human H-FABP recombinant antibody with the amino acid sequence as shown in SEQ ID No. 24 is shown in SEQ ID No. 28. It is understood that, based on codon degeneracy, in other embodiments, the nucleotide sequences of the nucleic acid fragments encoding the heavy chain constant region and light chain constant region with the amino acid sequences as shown in SEQ ID No. 23 and SEQ ID No. 24 are not limited to the above, and may be other sequences.

[0060] In some embodiments, the nucleic acid of any of the above embodiments further includes transcription elements, such as promoters and terminators. It is understood that in some embodiments, the nucleic acid of any of the above embodiments may not include transcription elements. In this case, during use, the nucleic acid can be inserted into a vector having the corresponding transcription elements for expression.

[0061] Furthermore, one embodiment of the present invention also provides a carrier containing the nucleic acid of any of the above embodiments. The nucleic acid fragment encoding the heavy chain variable region as shown in SEQ ID No. 7, the nucleic acid fragment encoding the light chain variable region as shown in SEQ ID No. 8, the nucleic acid fragment encoding the heavy chain constant region as shown in SEQ ID No. 11, and the nucleic acid fragment encoding the light chain constant region as shown in SEQ ID No. 12 are all described above.

[0062] In other embodiments, the vector contains the nucleic acid of any of the above embodiments. The nucleic acid fragment encoding the heavy chain variable region as shown in SEQ ID No. 19, the nucleic acid fragment encoding the light chain variable region as shown in SEQ ID No. 20, the nucleic acid fragment encoding the heavy chain constant region as shown in SEQ ID No. 23, and the nucleic acid fragment encoding the light chain constant region as shown in SEQ ID No. 24.

[0063] In one embodiment, the vector is a pCDNA3.1 vector. Of course, in other embodiments, the vector is not limited to the pCDNA3.1 vector, but can be other vectors.

[0064] Furthermore, one embodiment of the present invention also provides a host cell transformed with a vector comprising any of the above embodiments. The recombinant expression vectors are the nucleic acid fragments encoding the heavy chain variable region as shown in SEQ ID No. 7 and SEQ ID No. 19 and the nucleic acid fragments encoding the light chain variable region as shown in SEQ ID No. 8 and SEQ ID No. 20.

[0065] In another embodiment, the host cells are transformed with a recombinant expression vector containing a nucleic acid fragment encoding the heavy chain variable region of the anti-human H-FABP recombinant antibody and a recombinant expression vector containing a nucleic acid fragment encoding the light chain variable region of the anti-human H-FABP recombinant antibody. Placing the nucleic acid fragments encoding the light chain variable region and the heavy chain variable region of the anti-human H-FABP recombinant antibody on different expression vectors and co-transfecting them into the host cells facilitates control over the amount of light and heavy chain variable regions of the generated anti-human H-FABP recombinant antibody.

[0066] In one embodiment, the host cell is HEK293. Of course, in other embodiments, the host cell is not limited to HEK293 and can be other cells.

[0067] In addition, one embodiment of the present invention provides an H-FABP detection reagent, which includes the anti-human H-FABP recombinant antibody of any of the above embodiments.

[0068] One embodiment of the present invention also provides an H-FABP detection kit, which includes the recombinant anti-human H-FABP antibody from any of the above embodiments, for detecting H-FABP and improving the diagnostic accuracy of acute myocardial infarction. Optionally, the above-mentioned recombinant anti-human H-FABP antibody can be used as a capture antibody or as a labeling antibody.

[0069] In some embodiments, the H-FABP detection kit includes antibody 1 and antibody 2. Antibody 1 includes a light chain variable region and a heavy chain variable region. The heavy chain variable region of antibody 1 includes a complement-determining region (CDR) with an amino acid sequence as shown in SEQ ID Nos. 1-3, and the light chain variable region of antibody 1 includes a CDR with an amino acid sequence as shown in SEQ ID Nos. 4-6. Antibody 2 includes a light chain variable region and a heavy chain variable region. The heavy chain variable region of antibody 2 includes a CDR with an amino acid sequence as shown in SEQ ID Nos. 13-15, and the light chain variable region of antibody 2 includes a CDR with an amino acid sequence as shown in SEQ ID Nos. 16-18.

[0070] In one embodiment, the H-FABP detection kit further includes at least one of a solid support and a buffer solution. Optionally, the solid support is a magnetic bead or a resin. The buffer solution includes phosphate buffer and citric acid solution, used as a sample dilution buffer and an elution buffer, respectively.

[0071] The above-mentioned H-FABP detection kit includes the aforementioned recombinant anti-human H-FABP antibody, which has good affinity and specificity for H-FABP and high batch-to-batch consistency, and can be used for the development of H-FABP indicator diagnostic products. Specific Implementation

[0072] The following detailed description is provided with reference to specific embodiments. Unless otherwise specified, the embodiments do not include components other than unavoidable impurities. Unless otherwise specified, the reagents and instruments used in the embodiments are conventionally selected in the art. Experimental methods not specifying specific conditions in the embodiments were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer. The recombinant H-FABP protein used below is from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0073] Example 1

[0074] 1. Preparation of H-FABP monoclonal antibody:

[0075] 1.1 Animal Immunization: The recombinant human myocardial fatty acid-binding protein (H-FABP) antigen produced by expression was diluted to 0.5 mg / mL and mixed with an equal volume of Freund's complete adjuvant. The mixture was then fully emulsified using an emulsifier. BALB / c mice were subcutaneously injected with 0.2 mL per mouse. Female mice were 6-8 weeks old, with 5 mice per group. Two weeks later, booster immunizations were performed by mixing 0.05 mg of antigen per mouse with an equal volume of Freund's incomplete adjuvant. After three booster immunizations, tail blood was collected for titer testing until fusion titer was reached. Three days before fusion, the same dose of antigen and an equal volume of PBS buffer were mixed and used for intraperitoneal immunization.

[0076] 2. Construction of hybridoma cell lines

[0077] 2.1 Preparation of feeder cells: Peritoneal macrophages from BALB / c mice were used as feeder cells. One day before fusion, BALB / c mice were euthanized by cervical dislocation and disinfected in 75% alcohol for 5 min. The peritoneum was then opened with ophthalmic scissors in a clean bench to expose the peritoneum. 5 mL of RPMI 1640 medium was injected into the peritoneal cavity using a syringe. The cells were repeatedly flushed with the syringe, and the flushing fluid was collected. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 medium (containing HAT). The cell density was adjusted to 1 × 10⁶ cells / mL. 5 150 μL / well was added to a 96-well cell culture plate and incubated at 37°C in a 5% CO2 incubator.

[0078] 2.2 Preparation of myeloma cell suspension: 36 h before fusion, bone marrow cells were cultured to expand their volume. On the day of fusion, cells were gently pipetted from the culture dish and collected in sterile 50 mL centrifuge tubes. Centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 30 mL of RPMI 1640 medium was added, and the cells were washed once using the same method. The cells were then resuspended in 10 mL of RPMI 1640 medium and mixed thoroughly. The myeloma cell suspension was then treated with 0.4% trypan blue for viable cell counting before use.

[0079] 2.3 Preparation of Splenic Lymphocytes: Immunized BALB / c mice were used. Blood was collected by enucleation, and the serum was separated to serve as a positive control serum for antibody detection. Simultaneously, mice were euthanized by cervical dislocation, disinfected in 75% alcohol for 5 minutes, fixed on a dissection board, and the abdominal cavity was opened with ophthalmic scissors. The spleen was removed and placed in a culture dish containing 10 mL of RPMI 1640 medium. It was gently washed, and the surrounding connective tissue was carefully removed. The spleen was transferred to another culture dish containing 10 mL of RPMI 1640 medium. 1 mL of medium was drawn up and injected into the spleen using a syringe. The cells were repeatedly pipetted and collected. The cell suspension was centrifuged at 1000 rpm for 5 minutes, washed twice with RPMI 1640 medium, and then resuspended in 10 mL of RPMI 1640 medium. The resulting suspension was then stained with phenol blue for viable cell counting and used for later use.

[0080] 2.4. Cell fusion and selective culture of hybridoma cells: 1×10 8 One spleen cell and 2×10 7 One SP2 / 0 myeloma cell line was mixed in a 50 mL fusion tube, and RPMI 1640 medium was added to a final volume of 30 mL. The mixture was thoroughly mixed. Centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated as completely as possible. The bottom of the fusion tube was gently tapped on the palm of the hand to loosen and evenly distribute the cell pellet. 1 mL of 50% PEG (pH 8.0) solution preheated to 37°C was added, followed by 25 mL of RPMI 1640 medium preheated to 37°C within 90 s. The mixture was incubated at 37°C for 10 min, then centrifuged at 1000 rpm for 5 min; the supernatant was discarded. 5 mL of RPMI 1640 medium (containing HAT) was added, and the cell pellet was gently aspirated to mix. The cell suspension was aliquoted into 96-well cell culture plates, 0.10 mL per well. The plates were then incubated at 37°C in a 5% CO2 incubator. After 7 days of culture, the medium was replaced with RPMI 1640 medium containing HT. Regularly observe the growth of hybridoma cells, and when they grow to more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for antibody detection.

[0081] 2.5. Screening of hybridoma cells: Recombinant myocardial fatty acid-binding protein (H-FABP) antigen was diluted to 2 μg / mL with 50 mM (pH 9.5) carbonate buffer. 0.1 mL was added to each well of a 96-well ELISA plate and incubated overnight at 4 ℃. The next day, 200 μL of 2% BSA blocking solution was added to each well and the plate was blocked at 37 ℃ for 2 h. On day 7 of cell fusion culture, 0.1 mL of cell culture supernatant was added to the coated 96-well plate and incubated at 37 ℃ for 90 min. After washing the plate 6 times with PBST buffer, 100 μL of 1:10000 diluted HRP-Goat anti-Mouse IgG secondary antibody was added and incubated at 37 ℃ for 30 min. After washing the plate 6 times with PBST buffer, 100 μL of TMB chromogenic solution was added and the plate was developed in the dark for 10 min. 50 μL of 2 M sulfuric acid solution was added, and the absorbance at 450 nm was measured using a UV spectrophotometer. RPMI 1640 medium was used as a negative control, and wells with an OD ratio ≥2 between the measured value and the control group were considered positive.

[0082] Cells from positive clone wells were selected for screening monoclonal cell lines using a limiting dilution method. The selected positive monoclonal cells were then expanded into larger cultures and cryopreserved in 40% RPMI 1640 medium + 50% FBS + 10% DMSO at a density of 1×10⁻⁶ cells / well. 6 pcs / ml. For example... Figure 1 As shown, a total of 18 plants were screened. Figure 1 Monoclonal cells with good affinity (OD450>0.7).

[0083] 3. Small-scale antibody production and purification

[0084] Balb / c mice were first treated with 0.3 mL of liquid paraffin via intraperitoneal injection for approximately 10 days. The selected 18 hybridoma cell lines with strong affinity were then expanded and cultured, followed by inoculation with 1×10⁻⁶ cells. 6Hybridoma cells were injected into the peritoneal cavity of Balb / c mice. Approximately 7 days after injection, the mice's abdomens began to swell. Daily observation was necessary to prevent sudden death. When mice exhibited difficulty eating, gait impairment, and dull fur, they were euthanized, and ascites was extracted once. Mice were euthanized by cervical dislocation, disinfected by immersion in 75% alcohol for 5 minutes, and a small incision was made in the abdomen with surgical scissors. The surrounding skin was peeled off to expose the peritoneal cavity. A small incision was then made in the peritoneal cavity, and a dropper was inserted to aspirate the fluid into a clean 15 mL centrifuge tube. An equal volume of saturated ammonium sulfate was added to the centrifuge tube for precipitation. The precipitate was centrifuged at 12000 rpm for 10 minutes, then resuspended in pH 7.4 PBS buffer and centrifuged again at 12000 rpm for 10 minutes. The rProtein A affinity chromatography column was first connected to an AKTA pure 150L instrument, and equilibration buffer (PBS, pH 7.4) was used for 5 column volumes (CV) of the column. 7.4) Perform column equilibration, then load the antibody supernatant after centrifugation. After loading, continue equilibration with equilibration buffer until the UV baseline is stable. Elute the antibody on the chromatography column with citric acid at pH 3.0, collect the elution peak, adjust the pH of the eluent to 7.2 with Tris-HCl at pH 9.0, and then replace the antibody buffer with PBS at pH 7.4 using a dialysis bag.

[0085] 4. Mouse monoclonal antibody pairing screening test

[0086] The 18 mouse monoclonal antibodies screened in the previous step were diluted with spotting buffer (20 mM Tris-HCl, 30% glycerol, pH 8.0) to 0.5 mg / mL, and then spotted onto a hard matrix chip using a spotting instrument. The specific implementation method is as follows: Figure 3 As shown, after spotting, the chip was blocked with 2% BSA and then packaged into a finished chip for experimentation. The SLXP-001B (Sunlant) fully automated biochip reader was used for chip detection. The chip prepared in the previous step was loaded into the chip tray. The recombinant H-FABP protein was diluted and added to the sample tray. In this case, antibody 2 (clone number F2-243) labeled with HRP enzyme and the luminescent solution were simultaneously loaded into the corresponding positions. After the instrument self-tested, the fully automated analysis experiment could begin. This experiment was repeated three times, and the average value was calculated. After 18 pairs of screening experiments, as shown... Figure 2 As shown, four antibody pairs were finally screened, which had a high signal-to-noise ratio. A high signal-to-noise ratio has advantages in terms of detection sensitivity, detection range, and specificity. Therefore, in this case, the antibody with the highest signal-to-noise ratio (clone number F2-36) was selected for humanization.

[0087] 5. Preparation of recombinant anti-human H-FABP antibody

[0088] 5.1 Obtaining the antibody variable region gene sequence: The monoclonal cells corresponding to the pair of antibodies with the highest signal-to-noise ratio selected above were cultured in 24-well plates. When the cells in the 24-well plates reached a confluence of more than 70%, the cells were collected, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Total RNA was extracted from the cell pellet using an RNA extraction kit, and then reverse transcribed into total cDNA. The antibody heavy chain variable region sequence was amplified using VH-F and VH-R, and the antibody light chain variable region sequence was amplified using ML-F and ML-R. After amplification, the heavy and light chain variable region gene sequences were ligated to the pCE2 TA / Blunt-Zero vector through blunt ends. Positive clones were selected and sequenced using the primer sequences of M13F. The specific primer sequences used are shown in Table 1.

[0089] Table 1

[0090]

[0091] The sequencing results were analyzed to obtain the variable region sequence of the antibody. The recombinant vector was sequenced by Sangon Biotech (Shanghai) Co., Ltd. The nucleic acid fragments of the variable region of the anti-human H-FABP recombinant antibody and the sequencing results of the variable region of the anti-human H-FABP recombinant antibody are shown in Table 2.

[0092] Table 2

[0093]

[0094] 5.2 Expression and purification of recombinant antibodies: The successfully sequenced variable region sequences were constructed into the eukaryotic expression vector pCDNA3.1. After construction, plasmids were extracted in large quantities using an endotoxin-free plasmid extraction kit. Suspension HEK293 cells were cultured in Freestyle293 medium (Gibco), and the cell density was seeded at 1×10⁶ cells / year 24 h before transfection. 6 On the day of transfection, preheat 1 / 15 volume of Freestyle293 medium at 37°C. Add transfection plasmid at a rate of 1 μg DNA / mL of culture, followed by PEI transfection reagent (1 mg / mL) at twice the amount of plasmid. After mixing by inversion, let stand at room temperature for 20 min. Then slowly add the DNA & PEI mixture to the cultured cells. On days 2 and 4 of culture, supplement with 1% glucose (2 M) and glutamine solution (200 mM). Collect the cell expression supernatant after 7 days of culture and purify it.

[0095] First, the rProtein A affinity chromatography column was connected to an AKTA pure 150L instrument. The column was equilibrated with 5 CV equilibration buffer (PBS, pH 7.4). The antibody supernatant was then loaded after centrifugation. After loading, equilibration was continued until the UV baseline stabilized. The antibody on the column was eluted with 20 mM citric acid (pH 3.0), and the elution peak was collected. The pH of the eluent was adjusted to 7.2 with Tris-HCl (pH 9.0). The antibody buffer was then replaced with PBS at pH 7.4 using a dialysis bag. The purity of the target protein was controlled by SDS-PAGE. The SDS-PAGE analysis of the purified recombinant antibody 1 (F2-36-hIgG1) is shown below. Figure 5 As shown in the figure, the SDS-PAGE analysis of the purified recombinant antibody 2 (F2-243-hIgG1) is as follows. Figure 6 As shown, from left to right, the images are: standard molecular weight marker, marker, and target protein electrophoresis bands.

[0096] 6. Analysis of the pairing performance of recombinant antibodies

[0097] The recombinant antibody 1 (clone number F2-36) in this case was diluted with spotting buffer (20 mM Tris-HCl, 30% glycerol, pH 8.0) to 0.5 mg / mL. The antibody was then spotted onto a hard matrix chip using a spotting instrument. After spotting, the chip was blocked with 2% BSA and packaged into a finished chip for experimentation. An automated biochip reader, SLXP-001B (Sunlant), was used for chip detection. The chip prepared in the previous step was loaded into the chip tray. A serially diluted H-FABP serum sample was added to the sample tray. The HRP enzyme-labeled antibody 2 (clone number F2-243) and the luminescent solution were simultaneously loaded into the corresponding positions. After the instrument self-tested, the fully automated analysis experiment could begin. This experiment was repeated three times, and the average value was calculated. After three sets of experiments, as... Figure 4 As shown, the final results indicate that this pair of recombinant anti-human H-FABP antibodies exhibits good linearity for the detection of gradient samples, with R... 2 =0.9881.

[0098] 7. Sample evaluation of antibody activity

[0099] Unlabeled H-FABP paired antibodies were diluted with spotting buffer (20 mM Tris-HCl, 30% glycerol, pH 8.0) to 0.1 mg / ml, and then the paired antibodies were spotted onto the hard matrix chip using a spotting instrument. The specific implementation method is as follows: Figure 3As shown, after spotting, the chips were blocked with 2% BSA and then packaged into finished chips for experimentation. The SLXP-001B (Sunlant) fully automated biochip reader was used for chip detection. The prepared chips were loaded into the chip tray, the diluted serum samples were added to the sample tray, and the enzyme-labeled secondary antibody and chemiluminescent solution were loaded into the corresponding positions. After self-testing, the instrument could begin fully automated analysis. Thirty-five H-FABP positive serum samples and ten negative serum samples were tested. The results are shown in Table 3. All 35 positive samples showed positive signal values, and no false positives were observed in the negative samples.

[0100] Table 3

[0101]

[0102] Table 3 shows that 35 positive samples of myocardial infarction were detected by the screened H-FABP paired antibody, with a detection rate of 100%. No false positives were found in the negative samples, which proves that the screened H-FABP pair has good specificity for the detection of myocardial infarction samples and can be used for the development of H-FABP indicator diagnostic products.

[0103] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. An anti-human H-FABP recombinant antibody, characterized in that, The anti-human H-FABP recombinant antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3; the amino acid sequence of the H-CDR1 is shown as SEQ ID No. 1, the amino acid sequence of the H-CDR2 is shown as SEQ ID No. 2, the amino acid sequence of the H-CDR3 is shown as SEQ ID No. 3, the amino acid sequence of the L-CDR1 is shown as SEQ ID No. 4, the amino acid sequence of the L-CDR2 is shown as SEQ ID No. 5, and the amino acid sequence of the L-CDR3 is shown as SEQ ID No.

6.

2. The anti-human H-FABP recombinant antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.

8.

3. The recombinant antibody against human H-FABP according to claim 1 or 2, characterized in that, The anti-human H-FABP recombinant antibody further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is shown as SEQ ID No. 11, and the amino acid sequence of the light chain constant region is shown as SEQ ID No.

12.

4. An anti-human H-FABP recombinant antibody, characterized in that, The anti-human H-FABP recombinant antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, and the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3; the amino acid sequence of the H-CDR1 is shown as SEQ ID No. 13, the amino acid sequence of the H-CDR2 is shown as SEQ ID No. 14, the amino acid sequence of the H-CDR3 is shown as SEQ ID No. 15, the amino acid sequence of the L-CDR1 is shown as SEQ ID No. 16, the amino acid sequence of the L-CDR2 is shown as SEQ ID No. 17, and the amino acid sequence of the L-CDR3 is shown as SEQ ID No.

18.

5. The anti-human H-FABP recombinant antibody of claim 4, wherein the antibody is selected from the group consisting of: and. The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 19, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.

20.

6. The recombinant antibody against human H-FABP according to claim 4 or 5, characterized in that, The anti-human H-FABP recombinant antibody further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is shown as SEQ ID No. 23, and the amino acid sequence of the light chain constant region is shown as SEQ ID No.

24.

7. A nucleic acid, characterized in that, A nucleic acid for encoding the anti-human H-FABP recombinant antibody according to any one of claims 1-6.

8. A vector, characterized in that, The nucleic acid according to claim 7.

9. A host cell, characterized in that, The vector according to claim 8.

10. A detection reagent or a detection kit, characterized by, The anti-human H-FABP recombinant antibody according to any one of claims 1-6.

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