Recombinant antibody as well as preparation method, nucleic acid fragment, expression vector, recombinant cell and application thereof

Abstract

The invention discloses a recombinant antibody as well as a preparation method, a nucleic acid fragment, an expression vector, a recombinant cell and application thereof, and relates to the field of antibodies, the recombinant antibody is a hexamer, the recombinant antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region; the heavy chain variable region amino acid sequence is a sheep source IgG heavy chain variable region amino acid sequence, and the heavy chain constant region amino acid sequence is a human source IgM heavy chain constant region amino acid sequence; the light chain variable region amino acid sequence is a sheep source IgG light chain variable region amino acid sequence, and the light chain constant region amino acid sequence is a human source IgM light chain constant region amino acid sequence. The recombinant antibody is stable in performance, can efficiently recognize and bind to a T3 antigen, and has higher affinity activity to the T3 antigen compared with sheep source IgG.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to recombinant antibodies and preparation methods thereof, nucleic acid fragments, expression vectors, recombinant cells and applications thereof. Background Art

[0002] Triiodothyronine (T3) is a hormone secreted by the thyroid gland. About 65% is produced directly by the thyroid gland, and 35% is formed by deiodination of tetraiodothyronine (T4) in peripheral tissues. It has a molecular weight of about 650 and is a hapten small molecule. Triiodothyronine is involved in almost every physiological process of the body, including growth and development, metabolism, regulation of body temperature and heart rate, etc. Studies have shown that total T3 levels change with the concentration of thyroid hormone binding protein (TBG). T3 is an important clinical indicator for evaluating thyroid function, and has important clinical significance for the diagnosis and efficacy testing of thyroid diseases such as hyperthyroidism and hypothyroidism.

[0003] Therefore, it is of great significance to develop antibodies with good stability and high activity that can specifically bind to T3 and then use them to prepare diagnostic reagents for thyroid diseases. Summary of the invention

[0004] Based on the above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a recombinant antibody and a preparation method thereof, a nucleic acid fragment, an expression vector, a recombinant cell and an application thereof, aiming to provide a recombinant antibody with good stability, high activity and the ability to specifically bind to the T3 antigen.

[0005] The technical solution of the present invention is as follows:

[0006] The first aspect of the present invention provides a recombinant antibody, wherein the recombinant antibody is a hexamer, the recombinant antibody includes a heavy chain and a light chain, the heavy chain includes a heavy chain variable region and a heavy chain constant region, the light chain includes a light chain variable region and a light chain constant region; the heavy chain variable region amino acid sequence is the amino acid sequence of the heavy chain variable region of sheep IgG, and the heavy chain constant region amino acid sequence is the amino acid sequence of the heavy chain constant region of human IgM; the light chain variable region amino acid sequence is the amino acid sequence of the light chain variable region of sheep IgG, and the light chain constant region amino acid sequence is the amino acid sequence of the light chain constant region of human IgM.

[0007] Optionally, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, the amino acid sequence of the CH1 domain of the heavy chain constant region is as shown in SEQ ID NO: 2; the amino acid sequences of the CH2 domain, CH3 domain and CH4 domain of the heavy chain constant region are as shown in SEQ ID NO: 3;

[0008] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:5.

[0009] The second aspect of the present invention provides a nucleic acid fragment, wherein the nucleic acid fragment contains a nucleotide sequence encoding the recombinant antibody of the present invention as described above.

[0010] The third aspect of the present invention provides an expression vector, wherein the expression vector contains the nucleic acid fragment of the present invention as described above.

[0011] A fourth aspect of the present invention provides a recombinant cell, wherein the recombinant cell contains the expression vector of the present invention as described above.

[0012] A fifth aspect of the present invention provides a method for preparing the recombinant antibody of the present invention as described above, comprising the following steps:

[0013] inserting a nucleotide sequence encoding a heavy chain into the first plasmid to obtain a recombinant heavy chain plasmid;

[0014] inserting the nucleotide sequence encoding the light chain into the second plasmid to obtain a recombinant light chain plasmid;

[0015] The recombinant heavy chain plasmid and the recombinant light chain plasmid are mixed, then transfected into cells for eukaryotic expression, and then sequentially subjected to salting-out purification and molecular sieve purification to obtain the recombinant antibody.

[0016] Optionally, the step of inserting the nucleotide sequence encoding the heavy chain into the first plasmid to obtain the recombinant heavy chain plasmid specifically comprises:

[0017] Connecting the nucleotide sequence encoding the variable region of the sheep IgG heavy chain with the nucleotide sequence encoding the constant region of the human IgM heavy chain to obtain a nucleotide sequence encoding the heavy chain;

[0018] Inserting the nucleotide sequence encoding the heavy chain into the first plasmid to obtain a recombinant heavy chain plasmid;

[0019] Among them, the nucleotide sequence encoding the variable region of the sheep IgG heavy chain is shown as SEQ ID NO: 6, the nucleotide sequence encoding the CH1 domain of the human IgM heavy chain constant region is shown as SEQ ID NO: 7, and the nucleotide sequence encoding the CH2 domain, CH3 domain and CH4 domain of the human IgM heavy chain constant region is shown as SEQ ID NO: 8.

[0020] Optionally, the step of inserting the nucleotide sequence encoding the light chain into the second plasmid to obtain the recombinant light chain plasmid specifically comprises:

[0021] Connecting a nucleotide sequence encoding a sheep IgG light chain variable region with a nucleotide sequence encoding a human IgM light chain constant region to obtain a nucleotide sequence encoding a light chain;

[0022] inserting the nucleotide sequence encoding the light chain into a second plasmid to obtain a recombinant light chain plasmid;

[0023] The nucleotide sequence encoding the sheep IgG light chain variable region is shown in SEQ ID NO: 9; the nucleotide sequence encoding the human IgM light chain constant region is shown in SEQ ID NO: 10.

[0024] Optionally, the steps of performing salting-out purification and molecular sieve purification specifically include:

[0025] Add ammonium sulfate to the expressed product, centrifuge, and take the supernatant;

[0026] The supernatant is purified by molecular sieve using a gel filtration chromatography column.

[0027] In a sixth aspect, the present invention provides a recombinant antibody as described above, a nucleic acid fragment as described above, an expression vector as described above, a recombinant cell as described above, or a recombinant antibody prepared by the preparation method as described above in the preparation of a detection reagent for diagnosing thyroid diseases.

[0028] Beneficial effects: The recombinant antibody provided by the present invention is a hexamer recombined from immunoglobulins of different species, and the sheep-derived antibody is humanized, specifically, the variable region of the Fab segment of the antigen-binding part of the sheep-derived IgG is recombinantly fused with the constant region of the human IgM to obtain the recombinant antibody, that is, the human-sheep chimeric antibody. The degree of humanization of the recombinant antibody reaches 70%, and the variable region of the sheep-derived antibody is completely retained. The performance of the recombinant antibody is stable, and it has good stability at both high and low temperatures, and can efficiently recognize and bind to the T3 antigen. Compared with the sheep-derived IgG, the recombinant antibody has a higher affinity activity to the T3 antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the recombinant heavy chain plasmid map.

[0030] Figure 2 This is the recombinant light chain plasmid map.

[0031] Figure 3 This is the electrophoresis result of the IgG / IgM hybrid recombinant antibody prepared in Example 1.

[0032] Figure 4The DOT-ELISA test results of the activity of the IgG / IgM hybrid recombinant antibody in Example 2, wherein (a) is the result of the IgG / IgM hybrid recombinant antibody diluted 1:10000, (b) is the result of the IgG / IgM hybrid recombinant antibody diluted 1:20000, (c) is the result of the IgG positive control, and (d) is the result of the PBS negative control.

[0033] Figure 5 The stability test results of the IgG / IgM hybrid recombinant antibody in Example 3. DETAILED DESCRIPTION

[0034] The present invention provides a recombinant antibody and a preparation method thereof, a nucleic acid fragment, an expression vector, a recombinant cell and an application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0036] The inventors found in the study that although the IgG monomer can specifically bind to the T3 antigen, its affinity activity needs to be improved. Although the antibody activity of the multimeric antibody in the application process of the detection reagent is much higher than that of the monomeric antibody, the IgG polymer occurring in nature is unstable and cannot be used in practice. Based on this, an embodiment of the present invention provides a recombinant antibody, wherein the recombinant antibody is a hexamer, the recombinant antibody includes a heavy chain and a light chain, the heavy chain includes a heavy chain variable region and a heavy chain constant region, and the light chain includes a light chain variable region and a light chain constant region; the amino acid sequence of the heavy chain variable region is the amino acid sequence of the heavy chain variable region of sheep IgG, and the amino acid sequence of the heavy chain constant region is the amino acid sequence of the heavy chain constant region of human IgM; the amino acid sequence of the light chain variable region is the amino acid sequence of the light chain variable region of sheep IgG, and the amino acid sequence of the light chain constant region is the amino acid sequence of the light chain constant region of human IgM. The recombinant antibody provided in the embodiment of the present invention is a hexamer recombined from immunoglobulins of different species, and the sheep-derived antibody is humanized, specifically, the variable region of the Fab segment of the antigen-binding part of the sheep-derived IgG is recombinantly fused with the human IgM constant region, more specifically, the lambda variable region of the sheep-derived IgG light chain is fused with the lambda constant region of the human IgM light chain, and the variable region of the sheep-derived IgG heavy chain is fused with the human IgM heavy chain constant region (CH1, CH2, CH3, CH4) to obtain the recombinant antibody, that is, the human-sheep chimeric antibody, the degree of humanization of the recombinant antibody reaches 70%, and the variable region of the sheep-derived monoclonal antibody is completely retained. The recombinant antibody has stable performance and good stability at both high and low temperatures, can efficiently recognize and bind to the T3 antigen, and compared with the sheep-derived IgG, the recombinant antibody has a higher affinity activity for the T3 antigen (that is, the recombinant antibody retains the stability of the human IgM, and at the same time has better binding activity with the T3 antigen than the sheep-derived Ig).

[0037] In some embodiments, the amino acid sequence of the heavy chain variable region (i.e., the amino acid sequence of the sheep IgG heavy chain variable region, i.e., the amino acid sequence of the sheep IgG-VH region) is as shown in SEQ ID NO: 1, and the amino acid sequence of the CH1 domain of the heavy chain constant region (i.e., the human IgM-CH1 domain) is as shown in SEQ ID NO: 2 (PDB ID: 2RCJ); the amino acid sequences of the CH2 domain, CH3 domain and CH4 domain of the heavy chain constant region (i.e., the human IgM-CH2-CH4 region) are as shown in SEQ ID NO: 3 (PDBID: 8AE3); the amino acid sequence of the light chain variable region (sheep IgG light chain variable region, i.e., sheep IgG-VL region) is as shown in SEQ ID NO: 4, and the amino acid sequence of the light chain constant region (i.e., the human IgM light chain constant region, i.e., the light chain Lambda CL region) is as shown in SEQ ID NO: 5 (P0CG04·IGLC1_HUMAN).

[0038] An embodiment of the present invention further provides a nucleic acid fragment, wherein the nucleic acid fragment contains a nucleotide sequence encoding the recombinant antibody of the present invention as described above.

[0039] The present invention also provides an expression vector, wherein the expression vector contains the nucleic acid fragment described above in the present invention. Specifically, the expression vector can be constructed by molecular biological methods.

[0040] The present invention also provides a recombinant cell, wherein the recombinant cell contains the expression vector described above in the present invention. Specifically, the expression vector can be used to construct a host cell (i.e., a recombinant cell) capable of effectively expressing the recombinant antibody by cell biology methods.

[0041] Biotherapeutic proteins including immunoglobulins can be expressed in a variety of expression host cells. Mammalian cells are often used as host cells for IgM expression to maintain the best glycosylation pattern for biological activity or pharmacokinetic properties. IgM purification for good manufacturing practice (GMP) production cannot utilize affinity fillers, such as protein A (IgM does not bind to protein A), and protein A has always been the standard recovery method for IgG. In addition, the solubility range of IgM antibodies is relatively narrow, which brings great difficulties to the purification of antibodies. Early IgM antibody (antibody produced by hybridoma cells) purification was basically purified by isoelectric precipitation combined with gel chromatography columns. Some scholars have developed a purification method combining hydroxyapatite chromatography columns with anion and cation chromatography columns. This method has low specificity, is easily interfered by other proteins, and has cumbersome steps and is time-consuming. The present invention provides a two-step rapid purification method, which is simple and efficient to obtain recombinant humanized hexamer. Specifically, an embodiment of the present invention also provides a method for preparing a recombinant antibody as described above in an embodiment of the present invention, which includes the following steps:

[0042] inserting a nucleotide sequence encoding a heavy chain into the first plasmid to obtain a recombinant heavy chain plasmid;

[0043] inserting the nucleotide sequence encoding the light chain into the second plasmid to obtain a recombinant light chain plasmid;

[0044] The recombinant heavy chain plasmid and the recombinant light chain plasmid are mixed, then transfected into cells for eukaryotic expression, and then sequentially subjected to salting-out purification and molecular sieve purification to obtain the recombinant antibody.

[0045] The present invention combines the characteristics of IgG and IgM antibodies and the hydrophilic and hydrophobic properties of their constant regions, recombines and fuses the variable region of the antigen-binding part Fab segment of sheep-derived IgG with the constant region of human-derived IgM, transforms cells (such as mammalian cells, specifically 293 cells) for expression, and then uses salting-out purification and molecular sieve purification in two steps to simply and efficiently obtain recombinant antibodies that can efficiently recognize and bind to T3 antigens, have stable performance, high purity, and high expression. The degree of humanization of the recombinant antibody reaches about 70%, completely retains the variable region of the sheep-derived antibody, improves the affinity activity, and the introduction of the constant region of the humanized antibody greatly reduces the immunogenicity.

[0046] The recombinant antibody provided by the present invention can be efficiently expressed by mammalian cells.

[0047] In some embodiments, the step of inserting the nucleotide sequence encoding the heavy chain into the first plasmid to obtain the recombinant heavy chain plasmid specifically comprises:

[0048] Connecting the nucleotide sequence encoding the variable region of the sheep IgG heavy chain with the nucleotide sequence encoding the constant region of the human IgM heavy chain to obtain a nucleotide sequence encoding the heavy chain;

[0049] Inserting the nucleotide sequence encoding the heavy chain into the first plasmid to obtain a recombinant heavy chain plasmid;

[0050] Among them, the nucleotide sequence encoding the variable region of the sheep IgG heavy chain is shown as SEQ ID NO: 6, the nucleotide sequence encoding the CH1 domain of the human IgM heavy chain constant region is shown as SEQ ID NO: 7, and the nucleotide sequence encoding the CH2 domain, CH3 domain and CH4 domain of the human IgM heavy chain constant region is shown as SEQ ID NO: 8.

[0051] In some embodiments, the nucleotide sequence of the first plasmid is shown in SEQ ID NO:11.

[0052] In some embodiments, the step of inserting the nucleotide sequence encoding the light chain into the second plasmid to obtain the recombinant light chain plasmid specifically comprises:

[0053] Connecting a nucleotide sequence encoding a sheep IgG light chain variable region with a nucleotide sequence encoding a human IgM light chain constant region to obtain a nucleotide sequence encoding a light chain;

[0054] inserting the nucleotide sequence encoding the light chain into a second plasmid to obtain a recombinant light chain plasmid;

[0055] The nucleotide sequence encoding the sheep IgG light chain variable region is shown in SEQ ID NO: 9; the nucleotide sequence encoding the human IgM light chain constant region is shown in SEQ ID NO: 10.

[0056] In some embodiments, the nucleotide sequence of the second plasmid is shown in SEQ ID NO:12.

[0057] In some embodiments, the steps of performing salting-out purification and molecular sieve purification specifically include:

[0058] Add ammonium sulfate to the expressed product, centrifuge, and take the supernatant;

[0059] The supernatant is purified by molecular sieve using a gel filtration chromatography column.

[0060] The present invention also provides a use of the recombinant antibody, nucleic acid fragment, expression vector, or recombinant cell as described above, or the recombinant antibody prepared by the preparation method described above, in the preparation of a thyroid disease diagnostic test reagent (or a thyroid disease efficacy test reagent). Specifically, the thyroid disease includes but is not limited to hyperthyroidism, hypothyroidism, etc.

[0061] The recombinant antibody provided in the embodiment of the present invention is stable in nature, can specifically bind to T3 antigen, and can be used to prepare a detection reagent for diagnosing thyroid diseases.

[0062] The present invention will be further described below by means of specific examples.

[0063] Example 1

[0064] (1) Search the following sequences through NCBI:

[0065] Sheep IgG uses sheep monoclonal antibodies, and through gene comparison, finds the nucleotide fragment with the highest homology to human IgM antibodies. The amino acid sequence of the sheep IgG heavy chain variable region (i.e., sheep IgG-VH region) from N-terminus to C-terminus is shown in SEQ ID NO: 1; the nucleotide sequence encoding the sheep IgG heavy chain variable region is shown in SEQ ID NO: 6.

[0066] The human IgM heavy chain constant region (i.e., the human IgM heavy chain CH region) includes the CH1 domain, as well as the CH2, CH3 and CH4 domains. The amino acid sequence of the CH1 domain (referred to as the human IgM-CH1 region) is shown in SEQ ID NO: 2 from N-terminus to C-terminus (PDBID: 2RCJ); the amino acid sequence of the CH2 domain, CH3 domain and CH4 domain (referred to as the IgM-CH2-CH4 region) is shown in SEQ ID NO: 3 from N-terminus to C-terminus (PDB ID: 8AE3); the nucleotide sequence encoding the IgM-CH1 region is shown in SEQ ID NO: 7, and the nucleotide sequence encoding the IgM-CH2-CH4 region is shown in SEQ ID NO: 8.

[0067] The amino acid sequence of the sheep IgG light chain variable region (i.e., sheep IgG-VL region) from N-terminus to C-terminus is shown in SEQ ID NO: 4, and the nucleotide sequence encoding the sheep IgG light chain variable region is shown in SEQ ID NO: 9.

[0068] The amino acid sequence of the human IgM light chain constant region (i.e., the human IgM light chain Lambda CL region) from N-terminus to C-terminus is shown in SEQ ID NO: 5 (P0CG04·IGLC1_HUMAN), and the nucleotide sequence encoding the human IgM light chain constant region is shown in SEQ ID NO: 10.

[0069] (2) Construction of expression plasmid

[0070] The IgM sequence itself has the characteristics of a polymer. The nucleotide sequence encoding the sheep IgG-VH region (as shown in SEQ ID NO: 6) and the nucleotide sequence encoding the human IgM heavy chain CH region (as shown in SEQ ID NO: 7 and SEQ ID NO: 8) were connected and then inserted into the pM06 plasmid (nucleotide sequence as shown in SEQ ID NO: 11). This process was entrusted to General Bio (Anhui) Co., Ltd., and the recombinant heavy chain plasmid (as shown in Figure 1 shown);

[0071] The nucleotide sequence of the sheep IgG-VL region (as shown in SEQ ID NO: 9) and the nucleotide sequence encoding the human IgM light chain Lambda CL region (as shown in SEQ ID NO: 10) were connected and then inserted into the pM06.1 plasmid (the nucleotide sequence is shown in SEQ ID NO: 12). This process was commissioned to GenScript Biotech Co., Ltd. to obtain the recombinant light chain plasmid (as shown in Figure 2 shown).

[0072] The synthesized recombinant heavy chain plasmid and recombinant light chain plasmid were extracted using the Omega plasmid extraction kit for later use.

[0073] (3) Stable transfection and purification of recombinant antibody (i.e. IgG / IgM hybrid recombinant antibody) expression plasmid

[0074] First, determine the cell (specifically 293 cells, the same below) density and viability to ensure that the viability is >98%, and adjust the cell density to 2.0×10 6 / mL, placed in CO2 constant temperature shaker and cultured for at least 10 minutes;

[0075] Prepare two sterile centrifuge tubes, add 20mL serum-free cell transfection buffer solution (KPM) and 50μg recombinant heavy chain plasmid and 50μg recombinant light chain plasmid to one tube, and gently blow and mix; add 20mL KPM and 2mL TA 293 transfection reagent to the other tube, and gently blow and mix; transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently blow and mix; let it stand at room temperature for 10 minutes to obtain a plasmid-vector complex; take out the cells from the CO2 constant temperature shaker to the A2 biosafety cabinet, add the prepared plasmid-vector complex while shaking, and then put it back into the CO2 constant temperature shaker for shaking culture; after 24 hours, add 1% p / s (double antibody, i.e. penicillin plus streptomycin), 2.4mL of KE-293 protein expression enhancer and 8mL transient transfection nutrient additive (KT-Feed 50×) protein expression enhancer; collect the cells 6 days after transfection.

[0076] The purification was carried out by using salting-out method (ammonium sulfate precipitation method) and molecular sieve method. Specifically, in the first step, a 40% ammonium sulfate solution was added for precipitation, and the supernatant was collected after centrifugation at 20,000×g and 4°C. In the second step, the supernatant was purified by molecular sieve using a SUPERDEX 200INCREASE gel filtration chromatography column to collect the target peak sample.

[0077] The multimeric antibodies of IgG / IgM hybrid recombinant antibodies were obtained by two-step purification.

[0078] Take 10 μL of purified IgG / IgM hybrid recombinant antibody (concentration of 1 mg / mL) and perform reducing SDS-PAGE electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The results are as follows Figure 3 shown. Figure 3 Lane M on the left side represents the protein molecular weight standard; Lanes 1 to 3 on the right side are bands of recombinant antibodies (3 sets of parallel samples), and two bands are displayed on each lane from Lane 1 to Lane 3, one with a relative molecular mass (Mr) of 65-75kD (heavy chain) and the other with a relative molecular mass of 20-35kD (light chain).

[0079] Example 2

[0080] The activity detection of the IgG / IgM hybrid recombinant antibody (prepared in Example 1) by dot immunoenzyme staining (DOT-ELISA) comprises the following steps:

[0081] (1) After the transfer membrane is activated by soaking in methanol, it is taken out and placed in PBS (phosphate buffered saline) for washing, and then dried by filter paper.

[0082] (2) Place 8 transfer membranes on filter paper, add 2 μL (1 mg / mL) of T3 antigen to each membrane, and dry in a 37°C oven for 30 min.

[0083] (3) Add blocking solution and shake at 37°C and 100 rpm for 1.5 h.

[0084] (4) Wash three times with washing buffer, shaking for 5 min each time.

[0085] (5) diluting the IgG / IgM hybrid recombinant antibody (1 mg / mL, prepared in Example 1) at 1:10,000 and 1:20,000, respectively, to obtain recombinant antibody dilutions;

[0086] Add 2 μL of the recombinant antibody dilution solution (1:10000) to the first and second transfer membranes respectively;

[0087] Add 2 μL of the recombinant antibody dilution solution (1:20000) to the third and fourth transfer membranes respectively;

[0088] PBS (2 μL) was added to the 5th and 6th transfer membranes as a negative control;

[0089] IgG (1 mg / mL, 2 μL) was added to the 7th and 8th transfer membranes as a positive control;

[0090] The blocking solution was shaken at 37°C and 100 r / min for 1 h.

[0091] (6) Wash three times with washing buffer, shaking for 5 min each time.

[0092] (7) diluting the secondary antibody rabbit anti-human-HRP (1 mg / mL, i.e., horseradish peroxidase-labeled secondary antibody) 1:20,000 to obtain an antibody diluent;

[0093] Then, 2 μL of antibody diluent was added to the 6 transfer membranes except the positive control (the positive control was added with rabbit anti-goat-HRP diluent, which was obtained by diluting 1 mg / mL rabbit anti-goat-HRP at 1:20000), and the blocking solution was shaken at 37°C and 100 r / min for 1 h.

[0094] (8) Wash three times with washing buffer, shaking for 5 min each time.

[0095] (9) Add TMB (3,3',5,5'-tetramethylbenzidine dihydrochloride, a peroxidase substrate) working solution and color develop for 15 min at room temperature in the dark.

[0096] The results are as follows Figure 4 As shown, it can be seen that the IgG / IgM hybrid recombinant antibody can efficiently recognize and bind to the T3 antigen and has higher activity than IgG.

[0097] Example 3

[0098] The stability test of the IgG / IgM hybrid recombinant antibody (prepared in Example 1) comprises the following steps:

[0099] (1) 0.5 mg of IgG / IgM hybrid recombinant antibody was placed at -80°C, -20°C, 4°C and 37°C for 14 days and then collected. The samples were repeatedly frozen and thawed at -20°C for 14 times.

[0100] (2) Perform reduction electrophoresis on the recovered samples (5 μg of each sample) to observe whether the bands are correct and verify the stability of the antibody.

[0101] The results are as follows Figure 5 As shown, lane M represents a protein molecular weight standard (Marker), lane 1 represents an electrophoresis band of an IgG control, lane 2 represents an electrophoresis band of an IgG / IgM hybrid recombinant antibody at -80°C, lane 3 represents an electrophoresis band of an IgG / IgM hybrid recombinant antibody at -20°C, lane 4 represents an electrophoresis band of an IgG / IgM hybrid recombinant antibody at 4°C, and lane 5 represents an electrophoresis band of an IgG / IgM hybrid recombinant antibody at 37°C. It can be seen that the IgG / IgM hybrid recombinant antibody has good stability.

[0102] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A recombinant antibody, characterized in that: The recombinant antibody is a hexamer, and the recombinant antibody includes a heavy chain and a light chain, the heavy chain includes a heavy chain variable region and a heavy chain constant region, and the light chain includes a light chain variable region and a light chain constant region; the amino acid sequence of the heavy chain variable region is the amino acid sequence of the heavy chain variable region of sheep IgG, and the amino acid sequence of the heavy chain constant region is the amino acid sequence of the heavy chain constant region of human IgM; the amino acid sequence of the light chain variable region is the amino acid sequence of the light chain variable region of sheep IgG, and the amino acid sequence of the light chain constant region is the amino acid sequence of the light chain constant region of human IgM.

2. The recombinant antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, the amino acid sequence of the CH1 domain of the heavy chain constant region is shown in SEQ ID NO: 2; the amino acid sequences of the CH2 domain, CH3 domain and CH4 domain of the heavy chain constant region are shown in SEQ ID NO: 3; The amino acid sequence of the light chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

5.

3. A nucleic acid fragment, characterized in that The nucleic acid fragment contains a nucleotide sequence encoding the recombinant antibody according to any one of claims 1-2.

4. An expression vector, characterized in that: The expression vector contains the nucleic acid fragment according to claim 3.

5. A recombinant cell, characterized in that The recombinant cell contains the expression vector according to claim 4.

6. A method for preparing the recombinant antibody according to claim 1, characterized in that: The steps include: inserting a nucleotide sequence encoding a heavy chain into the first plasmid to obtain a recombinant heavy chain plasmid; inserting the nucleotide sequence encoding the light chain into the second plasmid to obtain a recombinant light chain plasmid; The recombinant heavy chain plasmid and the recombinant light chain plasmid are mixed, then transfected into cells for eukaryotic expression, and then sequentially subjected to salting-out purification and molecular sieve purification to obtain the recombinant antibody.

7. The preparation method according to claim 6, characterized in that: The step of inserting the nucleotide sequence encoding the heavy chain into the first plasmid to obtain the recombinant heavy chain plasmid specifically comprises: Connecting the nucleotide sequence encoding the variable region of the sheep IgG heavy chain with the nucleotide sequence encoding the constant region of the human IgM heavy chain to obtain a nucleotide sequence encoding the heavy chain; Inserting the nucleotide sequence encoding the heavy chain into the first plasmid to obtain a recombinant heavy chain plasmid; Among them, the nucleotide sequence encoding the variable region of the sheep IgG heavy chain is shown as SEQ ID NO: 6, the nucleotide sequence encoding the CH1 domain of the human IgM heavy chain constant region is shown as SEQ ID NO: 7, and the nucleotide sequence encoding the CH2 domain, CH3 domain and CH4 domain of the human IgM heavy chain constant region is shown as SEQ ID NO:

8.

8. The preparation method according to claim 6, characterized in that: The step of inserting the nucleotide sequence encoding the light chain into the second plasmid to obtain the recombinant light chain plasmid specifically comprises: Connecting a nucleotide sequence encoding a sheep IgG light chain variable region with a nucleotide sequence encoding a human IgM light chain constant region to obtain a nucleotide sequence encoding a light chain; inserting the nucleotide sequence encoding the light chain into a second plasmid to obtain a recombinant light chain plasmid; The nucleotide sequence encoding the sheep IgG light chain variable region is shown in SEQ ID NO: 9; the nucleotide sequence encoding the human IgM light chain constant region is shown in SEQ ID NO:

10.

9. The preparation method according to claim 6, characterized in that: The steps of performing salting-out purification and molecular sieve purification specifically include: Add ammonium sulfate to the expressed product, centrifuge, and take the supernatant; The supernatant is purified by molecular sieve using a gel filtration chromatography column.

10. Use of the recombinant antibody according to any one of claims 1 to 2, the nucleic acid fragment according to claim 3, the expression vector according to claim 4, the recombinant cell according to claim 5, or the recombinant antibody prepared by the preparation method according to any one of claims 6 to 9 in the preparation of a diagnostic reagent for thyroid diseases.

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