Vitamin B12 immunodetection recombinant protein and application thereof

Abstract

The invention provides a vitamin B12 immunodetection recombinant protein and application thereof, and the vitamin B12 immunodetection recombinant protein comprises a first recombinant protein and a second recombinant protein, the first recombinant protein is formed by fusing a functional structural domain of a human endofactor and an IgG Fc structural domain, and the functional structural domain can be specifically combined with vitamin B12; the second recombinant protein is formed by fusing a binding polypeptide and an IgM Fc structural domain, the sequence of the binding polypeptide is as shown in SEQ ID NO.2, and the binding polypeptide can be specifically bound with a compound formed by the first recombinant protein and vitamin B12.

Description

Technical Field

[0001] The invention belongs to the technical field of biological immunology, and in particular relates to a vitamin B12 immunoassay recombinant protein and an application thereof. Background Art

[0002] Vitamin B12 (VB12), also known as cobalamin, is an essential micronutrient for maintaining normal metabolism and function of the human body. As a cofactor of methyltransferase, it participates in the synthesis of methionine, thymine, etc., as well as the transfer and storage of folic acid in cells. It is an important coenzyme in the process of DNA synthesis. If VB12 is deficient, it will affect the body's utilization of folic acid, and may also lead to peripheral neuritis and anemia in children. It is also related to Alzheimer's disease, depression and coronary heart disease.

[0003] At present, the clinical detection methods for VB12 in biological samples include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescent immunoassay, liquid chromatography tandem mass spectrometry, etc. At present, chemiluminescent immunoassay and immunofluorescence assay are mainly used to detect VB12, and the competition method is used. During the detection, the VB12 antigen is coated, and when the sample is added, the antigen to be tested in the sample competes with the antigen coupled to the magnetic beads or microspheres to bind to the labeled detection VB12 antibody or VB12 specific binding protein. Due to the diversity of VB12 active forms in organisms, its affinity with antibodies or specific binding proteins is different, resulting in the competition method still having the problems of low sensitivity, poor specificity, poor reproducibility, and narrow detection range.

[0004] The sandwich method theoretically has a higher detection sensitivity than the competition method. The sensitivity of the competition method mainly depends on the binding ability (affinity) of the antibody / specific binding protein. The sensitivity of the sandwich method is less affected by affinity and mainly depends on the signal gain amplitude when the analyte is present and the measurement error when the analyte is not present. From the signal response curve, the locations of the hook effect of the sandwich method and the competition method are different. The hook effect of the sandwich method occurs at a high dose of the antibody / specific binding protein, and the competition method occurs at a low dose of the antibody / specific binding protein. Generally speaking, it is much easier to measure a strong signal under a low background than to measure the difference between two weak signals. In addition, in terms of kinetics, the excessive use of capture and tracer antibodies / specific binding proteins is conducive to the formation of the "antibody / specific binding protein-antigen-antibody / specific binding protein" sandwich complex, thereby improving sensitivity.

[0005] Therefore, developing a sandwich-based immunoassay for VB12 is helpful to improve the detection sensitivity of VB12 in biological samples. Summary of the invention

[0006] The present invention provides a vitamin B12 immunoassay recombinant protein and application thereof, which can effectively solve the above problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a vitamin B12 immunoassay recombinant protein, comprising a first recombinant protein and a second recombinant protein;

[0009] The first recombinant protein is formed by fusing the functional domain of human intrinsic factor with the Fc domain of IgG, and the functional domain can specifically bind to vitamin B12;

[0010] The second recombinant protein is formed by the fusion of a binding polypeptide and an IgM Fc domain. The sequence of the binding polypeptide is shown in SEQ ID NO.2. The binding polypeptide can specifically bind to a complex formed by the first recombinant protein and vitamin B12.

[0011] In a second aspect, the present invention provides a recombinant protein, wherein the recombinant protein is formed by the fusion of the functional domain of human intrinsic factor and the Fc domain of IgG, and the functional domain can specifically bind to vitamin B12.

[0012] In a third aspect, the present invention provides a recombinant protein, which is formed by a fusion of a binding polypeptide and an IgM Fc domain, wherein the sequence of the binding polypeptide is shown in SEQ ID NO.2, and the binding polypeptide can specifically bind to a complex formed by the recombinant protein described in the second aspect and vitamin B12.

[0013] In a fourth aspect, the present invention provides a vitamin B12 sandwich immunoassay reagent, comprising the vitamin B12 immunoassay recombinant protein described in the first aspect.

[0014] In a fifth aspect, the present invention provides a binding polypeptide, the sequence of which is shown in SEQ ID NO.2, and the binding polypeptide can specifically bind to the complex formed by the recombinant protein and vitamin B12 described in the second aspect.

[0015] In a sixth aspect, the present invention provides a method for preparing the binding polypeptide according to the fifth aspect, comprising:

[0016] A polypeptide having affinity to the complex formed by the recombinant protein and vitamin B12 is obtained by screening a random twelve-peptide phage library, and then sequence determination is performed to obtain the binding polypeptide, wherein the preparation method of the complex comprises:

[0017] The recombinant protein and vitamin B12 are mixed evenly in a pH 5.5 acetate buffer, and then dialyzed and replaced and stored in a pH 7.4 phosphate buffer to obtain the complex. The molar ratio of the recombinant protein and vitamin B12 is 1:1.1-1.5.

[0018] The beneficial effects of the present invention are:

[0019] The present invention provides a vitamin B12 immunoassay recombinant protein, comprising a first recombinant protein rhIFb-Fc and a second recombinant protein PepVB12-Fc, wherein the first recombinant protein rhIFb-Fc can form a stable complex with VB12, and the second recombinant protein PepVB12-Fc can specifically recognize the rhIFb / VB12 complex to form a "sandwich" sandwich complex combination. The vitamin B12 immunoassay recombinant protein can be used for a complex-specific binding protein sandwich method, greatly improving the sensitivity of vitamin B12 detection.

[0020] Furthermore, the first recombinant protein rhIFb-Fc uses the C-terminal functional domain of human intrinsic factor, which has a stronger affinity with VB12 and binds more firmly. The formed complex can expose more VB12 molecular structures, which is more conducive to the screening of sandwich pairing ligands.

[0021] Furthermore, the present invention screened a twelve-peptide strain that can specifically recognize the rhIFb / VB12 complex and does not recognize the first recombinant protein rhIFb-Fc through a random twelve-peptide phage library, and fused and recombinantly expressed it as the second recombinant protein PepVB12-Fc.

[0022] Furthermore, the first recombinant protein and the second recombinant protein provided by the present invention are prepared by recombinant expression and purification of mammalian cells, and have the advantages of simple production and preparation, high purity, high uniformity, and small batch-to-batch differences. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a schematic diagram of the structure of the complex formed by the combination of intrinsic factor and VB12.

[0025] Figure 2 This is the test result of the sandwich ELISA method in the embodiment of the present invention for detecting the activity of the recombinant protein rhIFb-Fc and the recombinant protein PepVB12-Fc. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] An embodiment of the present invention provides a vitamin B12 immunoassay recombinant protein, including a first recombinant protein and a second recombinant protein;

[0028] The first recombinant protein is formed by the fusion of the functional domain of human intrinsic factor and the Fc domain of human IgG1, the sequence of the functional domain is shown in SEQ ID NO.1, and the functional domain can specifically bind to vitamin B12;

[0029] The second recombinant protein is formed by the fusion of a binding polypeptide and a human IgM Fc domain. The sequence of the binding polypeptide is shown in SEQ ID NO.2. The binding polypeptide can specifically bind to a complex formed by the first recombinant protein and vitamin B12.

[0030] Human intrinsic factor (IF) is a VB12 transporter secreted by the stomach and is a factor necessary for VB12 intestinal absorption. The structural diagram of the complex formed by the combination of intrinsic factor and VB12 is shown in Figure 1 As shown (PDB ID: 2PMV).

[0031] After the intrinsic factor binds to VB12 to form a complex, VB12 is buried inside the binding region, with only 19% of the surface area exposed to solvent, and the conformational change of the intrinsic factor is small. It is difficult to screen sandwich pairing ligands using this complex as a screening source.

[0032] The functional domain of human intrinsic factor is the C-terminal domain of human intrinsic factor.

[0033] The intrinsic factor is composed of the N-terminal domain and the C-terminal domain, both of which have VB12 binding sites. The C-terminal domain has a stronger affinity and a more firm binding. The formed complex can expose more VB12 molecular structures, which is more conducive to the screening of sandwich pairing ligands.

[0034] The present invention intercepts the protein sequence of the C-terminal domain of human intrinsic factor to form the VB12 binding region, fuses it with the human IgG1 Fc domain through recombinant expression technology, and provides a recombinant protein rhIFb-Fc that specifically binds to VB12.

[0035] The sequence of the C-terminal domain of intrinsic factor is shown in SEQ ID NO.1.

[0036] SEQ ID NO.1:

[0037] HEVQPTLPSNPGPGPTSASNITVIYTINNQLRGVELLFNETINVSVKSGSVLLVVLEEAQRKNPMFKFETTMTSWGLVVSSINNIAENVNHKTYWQFLSGVTPLNEGVADYIPFNHEHITANFTQY

[0038] In some embodiments, the first recombinant protein comprises:

[0039] A first connecting peptide, the sequence of which is shown in SEQ ID NO. 3, and which is used to connect the functional domain of human intrinsic factor with the Fc domain of human IgG1.

[0040] Linkers play an important role in fusion protein design. They are usually used to connect two or more proteins or peptides to form fusion proteins. The choice of peptide linkers is crucial to the expression, stability and functional activity of fusion proteins.

[0041] The present invention uses a flexible linker, in which Gly and Ser provide flexibility, allowing a certain amount of mobility between the linked protein domains.

[0042] SEQ ID NO.3:

[0043] GGGGSGGGGSGGGGGSGGGGS

[0044] The IgG1 Fc domain contains the heavy chain CH2 and CH3 sequences.

[0045] The present invention fuses the Fc segment with the functional protein, which can improve the expression and secretion of the fusion protein in mammalian cells, enhance the stability of the fusion protein, and facilitate its labeling coupling with various immune detection signal molecules, microspheres, and magnetic beads. In addition, the Fc segment can specifically bind to protein A, which is beneficial to the purification of the fusion protein.

[0046] The sequence of the human IgG1 Fc domain is shown in SEQ ID NO.4.

[0047] SEQ ID NO.4:

[0048] VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTK GRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0049] The second recombinant protein is the recombinant protein PepVB12-Fc formed by fusing the binding polypeptide that specifically recognizes the rhIFb-Fc / VB12 complex with the human IgM Fc domain.

[0050] In the present invention, the binding polypeptide is fused with IgM Fc, so that the polypeptide sequence can be recombinantly expressed in mammalian cells and can form pentamers, thereby improving its sensitivity as an immunodetection reagent.

[0051] In some embodiments, a polypeptide having affinity to the complex formed by the recombinant protein rhIFb-Fc and vitamin B12 is obtained by screening a random twelve-peptide phage library, and then sequence determination is performed to obtain the binding polypeptide.

[0052] The complex formed by the combination of recombinant protein rhIFb-Fc and VB12 was screened for polypeptides of specific affinity complexes using a phage display library of random twelve peptides.

[0053] The binding polypeptide recognition site is a new epitope formed after the recombinant protein rhIFb-Fc and VB12 bind, and does not recognize free recombinant protein rhIFb-Fc and free VB12.

[0054] After sequencing, the twelve peptide sequence is shown as SEQ ID NO.2.

[0055] SEQ ID NO.2:

[0056] ASEYQTFNPRTV

[0057] In some embodiments, the second recombinant protein comprises:

[0058] A second connecting peptide, the sequence of which is shown in SEQ ID NO. 3, is used to connect the binding polypeptide to the human IgM Fc domain.

[0059] The sequence of the human IgM Fc domain is shown in SEQ ID NO.5.

[0060] SEQ ID NO.5:

[0061] AVAEMNPNVNVNFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHP NGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY

[0062] In some embodiments, the method for preparing the first recombinant protein or the second recombinant protein comprises:

[0063] 1) constructing an expression vector containing the gene encoding the recombinant protein;

[0064] 2) transfecting cells with the expression vector;

[0065] 3) After transfection, the cells are cultured and the recombinant protein is obtained after expression and purification.

[0066] An embodiment of the present invention provides a recombinant protein, wherein the recombinant protein is formed by fusion of a functional domain of human intrinsic factor and a human IgG1 Fc domain, wherein the sequence of the functional domain is shown in SEQ ID NO.1, and the functional domain can specifically bind to vitamin B12.

[0067] The sequence of the recombinant protein rhIFb-Fc is shown in SEQ ID NO.6.

[0068] SEQ ID NO.6:

[0069] HEVQPTLPSNPGPGPTSASNITVIYTINNQLRGVELLFNETINVSVKSGSVLLVVLEEAQRKNPMFKFETTMTSWGLVVSSINNIAENVNHKTYWQFLSGVTPLNEGVADYIPFNHEHITANFTQYGGGGSGGGGSGGGGGSGGGGSVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDI SKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0070] An embodiment of the present invention provides a recombinant protein, which is formed by fusion of a binding polypeptide and a human IgM Fc domain. The sequence of the binding polypeptide is shown in SEQ ID NO.2, and the binding polypeptide can specifically bind to a complex formed by the first recombinant protein and vitamin B12.

[0071] The sequence of the recombinant protein PepVB12-Fc is shown in SEQ ID NO.7.

[0072] SEQ ID NO.7:

[0073] ASEYQTFNPRTVGGGGSGGGGSGGGGGSGGGGSAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWAS QSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY

[0074] An embodiment of the present invention provides a vitamin B12 sandwich immunoassay reagent, comprising the above-mentioned vitamin B12 immunoassay recombinant protein.

[0075] The vitamin B12 sandwich immunoassay reagent comprises the recombinant protein rhIFb-Fc and the recombinant protein PepVB12-Fc.

[0076] In some embodiments, the first recombinant protein is coupled to a labeling enzyme.

[0077] In some embodiments, the labeling enzyme is horseradish peroxidase.

[0078] An embodiment of the present invention provides a binding polypeptide, the sequence of which is shown in SEQ ID NO.2, and the binding polypeptide can specifically bind to a complex formed by the recombinant protein rhIFb-Fc and vitamin B12.

[0079] In some embodiments, the method for preparing the binding polypeptide comprises:

[0080] A polypeptide having affinity with the complex formed by the recombinant protein rhIFb-Fc and vitamin B12 is obtained by screening a random twelve-peptide phage library, and then sequence determination is performed to obtain the binding polypeptide.

[0081] In some embodiments, the method for preparing the complex comprises:

[0082] The recombinant protein rhIFb-Fc and vitamin B12 are evenly mixed in 20mM pH 5.5 acetate buffer, and the dialyzed replacement buffer is 20mM pH 7.4 phosphate buffer containing 120mM sodium chloride to obtain the complex. The molar ratio of the recombinant protein rhIFb-Fc to vitamin B12 is 1:1.1-1.5.

[0083] Example 1

[0084] Preparation of recombinant protein rhIFb-Fc

[0085] 1. Sequence synthesis and expression

[0086] The recombinant protein rhIFb-Fc is in the form of human intrinsic factor C-terminal domain-first connecting peptide-human IgG1 Fc domain.

[0087] 1) Construct an expression vector containing the gene encoding the recombinant protein rhIFb-Fc. Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. was commissioned to synthesize the DNA sequence of the gene encoding the recombinant protein rhIFb-Fc and insert it into the PTT5 vector.

[0088] 2) Transfect cells with the expression vector. TM 293Expression System, transfect the vector connected to the target gene into HEK293-F cells.

[0089] 3) After transfection, the cells are cultured and the fusion protein is obtained by expression and purification.

[0090] The cell supernatant expressed by HEK293-F cells was taken and added to the Protein A affinity chromatography balanced with 20 mM pH 7.4 phosphate buffer at a flow rate of 200-400 cm / h to enrich the target protein.

[0091] After loading, elution was performed using 20 mM pH 3.5 acetate buffer, and the eluted fractions were collected.

[0092] The eluted fractions were taken and concentrated by ultrafiltration using a 10 kD ultrafiltration tube and buffer exchange was performed, and the recombinant protein was stored in a pH 7.4 phosphate buffer.

[0093] Example 2

[0094] Preparation of rhIFb-Fc / VB12 complex

[0095] The recombinant protein rhIFb-Fc and cyanocobalamin prepared in Example 1 were mixed in a 20 mM pH 5.5 acetate buffer at a molar ratio of 1:1.2, and then dialyzed and replaced in a 20 mM pH 7.4 phosphate buffer containing 120 mM sodium chloride to obtain an rhIFb-Fc / VB12 complex.

[0096] Preparation of recombinant protein PepVB12-Fc

[0097] 1. Screening

[0098] Using a Ph.D. TM -12 Phage Display Peptide LibraryKit v2 (NEB#E8110S), using the above complex as the screening source, screened for twelve peptides that specifically recognized the complex but did not recognize the recombinant protein rhIFb-Fc.

[0099] After sequencing, the amino acid sequence of the twelve peptide was screened as shown in SEQ ID NO.2.

[0100] The twelve peptide was fused with the human IgM Fc domain to obtain the recombinant protein PepVB12-Fc. The fusion form was twelve peptide-second connecting peptide-human IgM Fc domain.

[0101] 2. Sequence Synthesis and Expression

[0102] 1) Construct an expression vector containing the gene encoding the recombinant protein PepVB12-Fc. Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. was commissioned to synthesize the DNA sequence of the gene encoding the recombinant protein PepVB12-Fc and insert it into the PTT5 vector.

[0103] 2) Transfect cells with the expression vector. TM 293Expression System, transfect the vector connected to the target gene into HEK293-F cells.

[0104] 3) After transfection, the cells are cultured and the fusion protein is obtained by expression and purification.

[0105] The cell supernatant expressed by HEK293-F cells was taken, the pH was adjusted to 7.0, the conductivity was 5.0 mS / cm, and the supernatant was added to an anion exchange chromatography column balanced with 20 mM, pH 7.0 phosphate buffer at a linear flow rate of 200-400 cm / h to enrich the target protein.

[0106] After loading, washing was performed with 20 mM pH 7.0 phosphate buffer containing 0.1 M sodium chloride, followed by elution with 20 mM pH 7.0 phosphate buffer containing 0.3 M sodium chloride, and the eluted fractions were collected.

[0107] The eluted fractions were taken and concentrated by ultrafiltration using a 50 kD ultrafiltration tube and buffer exchange was performed. The recombinant protein was stored in a 20 mM pH 7.4 phosphate buffer containing 120 mM sodium chloride.

[0108] Example 3

[0109] Detection of the activity of recombinant protein rhIFb-Fc and recombinant protein PepVB12-Fc using sandwich ELISA

[0110] The specific steps include:

[0111] (1) The recombinant protein PepVB12-Fc prepared in Example 2 was diluted to 100 μg / mL with coating solution PBS pH 7.4 (20 mM), added to a microplate, 100 μL per well, and incubated at 4° C. overnight.

[0112] (2) Each well of the microplate was washed twice with washing solution PBST (PBS containing 0.05% Tween-20), patted dry, and then added with blocking solution (PBS containing 5% BSA), 200 μL per well, and incubated at 37° C. for 2 h.

[0113] (3) Each well of the microplate was washed three times with washing solution. The VB12 sample was diluted 3-fold with the above coating solution, mixed and added to the microplate, 100 μL per well, and incubated at 37°C for 30 min.

[0114] (4) The recombinant protein rhIFb-Fc prepared in Example 1 was labeled with horseradish peroxidase HRP at a mass ratio of 1:1 to obtain labeled protein rhIFb-Fc-HRP, which was then diluted to 1 μg / mL with the above coating solution.

[0115] (5) Each well of the microplate was washed five times with washing solution, and 100 μL of the diluted marker protein rhIFb-Fc-HRP was added to each well and incubated at 37°C for 30 min.

[0116] (6) Each well of the microplate was washed five times with washing solution, 100 μL of color development solution was added to each well, incubated at 37°C for 10 min, and then 50 μL of stop solution was added to each well.

[0117] (7) Read the absorbance of the liquid in each well at 450 / 630 nm using an ELISA reader.

[0118] The curve of the test results is as follows Figure 2 shown.

[0119] The test results showed that the recombinant protein rhIFb could form a complex with VB12 and be recognized by the affinity polypeptide fusion protein PepVB12-Fc that specifically recognizes the complex, forming a "sandwich" complex.

[0120] The recombinant protein rhIFb and the recombinant protein PepVB12-Fc are paired and can be applied to VB12 sandwich immunoassay reagents.

[0121] The embodiment of the present invention provides a VB12 sandwich immunoassay reagent, comprising the recombinant protein rhIFb prepared in Example 1 and the recombinant protein PepVB12-Fc prepared in Example 2, which is used for VB12 detection using a complex-specific binding protein sandwich method.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A vitamin B12 immunoassay recombinant protein, characterized in that: comprising a first recombinant protein and a second recombinant protein; The first recombinant protein is formed by fusing the functional domain of human intrinsic factor with the Fc domain of IgG, and the functional domain can specifically bind to vitamin B12; The second recombinant protein is formed by the fusion of a binding polypeptide and an IgM Fc domain. The sequence of the binding polypeptide is shown in SEQ ID NO.

2. The binding polypeptide can specifically bind to a complex formed by the first recombinant protein and vitamin B12.

2. The vitamin B12 immunoassay recombinant protein according to claim 1, characterized in that The IgG Fc domain is a human IgG1 Fc domain; The IgM Fc domain is a human IgM Fc domain.

3. The vitamin B12 immunoassay recombinant protein according to claim 1, characterized in that The first recombinant protein comprises: The first connecting peptide is used to connect the functional domain of human intrinsic factor and the IgG Fc domain.

4. The vitamin B12 immunoassay recombinant protein according to claim 1, characterized in that The second recombinant protein comprises: A second connecting peptide, wherein the second connecting peptide is used to connect the binding polypeptide to the IgM Fc domain.

5. The vitamin B12 immunoassay recombinant protein according to claim 1, characterized in that: The functional domain of human intrinsic factor is the C-terminal functional domain of human intrinsic factor, and the sequence of the functional domain is shown in SEQ ID NO.

1.

6. A recombinant protein, characterized in that The first recombinant protein is formed by the fusion of the functional domain of human intrinsic factor and the Fc domain of IgG, and the functional domain can specifically bind to vitamin B12.

7. A recombinant protein, characterized in that The second recombinant protein is formed by the fusion of a binding polypeptide and an IgM Fc domain, the sequence of the binding polypeptide is shown in SEQ ID NO.2, and the binding polypeptide can specifically bind to the complex formed by the recombinant protein according to claim 6 and vitamin B12.

8. A vitamin B12 sandwich immunoassay reagent, characterized in that: The invention comprises the vitamin B12 immunoassay recombinant protein according to any one of claims 1 to 5.

9. A binding polypeptide, characterized in that The sequence of the binding polypeptide is shown in SEQ ID NO.2, and the binding polypeptide can specifically bind to the complex formed by the recombinant protein according to claim 6 and vitamin B12.

10. A method for preparing the binding polypeptide according to claim 9, characterized in that: include: A polypeptide having affinity to the complex formed by the recombinant protein and vitamin B12 is obtained by screening a random twelve-peptide phage library, and then sequence determination is performed to obtain the binding polypeptide, wherein the preparation method of the complex comprises: The recombinant protein and vitamin B12 are mixed evenly in a pH 5.5 acetate buffer, and then dialyzed and replaced and stored in a pH 7.4 phosphate buffer to obtain the complex. The molar ratio of the recombinant protein and vitamin B12 is 1:1.1-1.5.

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