CN116515000A

CN116515000A - HFE fusion proteins and uses thereof

Info

Publication number: CN116515000A
Application number: CN202310788972.0A
Authority: CN
Inventors: 邢亚非
Original assignee: Jiajin Biomedicine Shanghai Co ltd
Current assignee: Jiajin Biomedicine Shanghai Co ltd
Priority date: 2023-06-30
Filing date: 2023-06-30
Publication date: 2023-08-01

Abstract

The invention is suitable for the technical field of molecular biology, and particularly relates to an HFE fusion protein capable of being combined with TfR and application thereof. The fusion proteins include signal peptides (HFE proteins), spacers (spacers), B2M proteins, and His tags. After the HFE & B2M specific sites are connected, the defect that HFE cannot independently bind to transferrin receptor (TfR) in vitro is overcome. And the recombinant HFE & B2M fusion protein improves the affinity between the recombinant HFE & B2M fusion protein and a transferrin receptor (TfR), and can also be used for blocking experiments in antibody screening processes.

Description

HFE fusion proteins and uses thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to an HFE fusion protein capable of being combined with a transferrin receptor (TfR) and application thereof.

Background

Transferrin, also known as Transferrin (Tf), is the predominant iron-containing protein in plasma, responsible for carrying iron absorbed by the digestive tract and released by erythrocyte degradation, in a ferric complex (Tf-Fe ³⁺ ) Into the bone marrow for the production of mature red blood cells. Transferrin (Tf) carries iron by interacting with its receptor (Transferrin Receptor, tfR). TfR is a glycoprotein expressed on the cell surface and is formed by the linkage of two homodimeric subunits through disulfide bonds.

TfR is not only expressed in a variety of cells, but also highly expressed in a variety of tumors, and its role is mainly to participate in the absorption of iron ions, which are critical to the production and metabolism of cells, so that the absorption of iron ions by cells can be prevented by antibody binding TfR, which in turn leads to cell death. Currently, antibodies have been used to target TfR for the treatment of tumors with high expression of TfR. In addition, in terms of drug delivery, tfR may transport the antibody or drug across the blood brain barrier after binding to the antibody, thereby treating tumors or other diseases associated with the nervous system.

Hemochromatosis protein (hemochromatosis protein, HFE protein) is a protein that competes with Tf for binding to TfR. The HFE protein is combined with the TfR, so that the affinity of the TfR and transferrin Tf can be reduced by 5-10 times, the absorption of iron element in a human body is reduced, and the occurrence of iron overload is prevented. Iron plays a vital role in a variety of bodily functions, including helping produce blood, etc., but too much iron is harmful to the human body. In hemochromatosis, the expression of HFE proteins is affected, resulting in the body absorbing excessive iron, which is stored in major organs, especially the liver. Over the long term, the stored superfluous iron can cause serious damage to human body, possibly cause organ failure, and also possibly cause chronic diseases such as liver cirrhosis, diabetes mellitus, heart failure and the like.

Studies have shown that at neutral or slightly alkaline pH, the micromolar concentration ratio of TfR to HFE interactions in solution is 2:1 (1 TfR dimer binds to 1 HFE protein) (Lebro Â n, j.a. et al (1998), crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor, cell 95, 111±123.). The crystal structure of the complex between HFE and the extracellular portion of TfR shows that two HFE molecules grasp both sides of the bisymmetric TfR dimer (Lebro Â n j.a., west a.p. J and Pamela j.bjorkman (1999), the hemochromatosis protein HFE competes with transferrin for binding to the transferrin receptor, journal of Molecular Biology, 294 (1), 239-245). In this complex, one HFE molecule is bound to each chain of the TfR homodimer, thus forming two symmetrical complexes (Bennett M.J., lebro Â n J.A., and Pamela J.Bjorkman (2000), crystal structure of the hereditary haemochromatosis protein HFE complexed with transferrin receptor. Nature, 403 (6765), 46-53.). That is, HFE molecules alone have relatively weak binding capacity to TfR in vitro.

HFE and TfR binding were mainly referred to the three documents mentioned above, published in 1998-2000 in Cell, JMB and Nature, which investigated the binding patterns of HFE and TfR from the surface plasmon resonance (Surface Plasmon Resonance, SPR), competition of HFE and Tf, and structural aspects thereof, respectively. The data indicate that binding of TfR and HFE may require the formation of dimers of HFE and beta 2-microglobulin (beta-2 microglobulin,B2M). However, there is a lot of data that none of the in vitro experiments to incubate HFE and TfR binding, which incubated HFE and B2M first to dimerize and then bind TfR, was successful. And improves the affinity of the HFE in vitro, and is helpful for blocking the smooth progress of the experiment in the process of screening the antibody. It is therefore necessary to construct and prepare HFE fusion proteins that bind better to TfR in vitro using fusion protein technology. At the same time, in vitro synthesized HFE and B2M fusion proteins will help to alleviate symptoms in hemochromatosis patients.

Disclosure of Invention

An object of embodiments of the present invention is to provide a recombinant HFE & B2M fusion protein that better binds to TfR in vitro, the fusion protein comprising a signal peptide (signal peptide), an HFE protein, a Spacer (Spacer), a B2M protein, and a His tag, wherein the TfR is TfR1, and the fusion protein binds to TfR1 in an environment of ph 7.5.

Preferably, the amino acid residue sequence of the signal peptide is SEQ ID NO.1, or has more than 90% homology thereto, preferably more than 95% homology thereto, more preferably more than 98% homology thereto.

Preferably, the amino acid residue sequence of the HFE protein is SEQ ID NO. 2, or has more than 90% homology thereto, preferably more than 95% homology thereto, more preferably more than 98% homology thereto.

Preferably, the amino acid residue sequence of the Spacer is SEQ ID NO. 3 or SEQ ID NO. 4, or a deletion, mutation or substitution of less than 3 amino acids.

Preferably, the amino acid residue sequence of the B2M protein is SEQ ID No. 5, or has more than 90% homology thereto, preferably more than 95% homology thereto, more preferably more than 98% homology thereto.

Preferably, the amino acid residue sequence of the fusion protein is SEQ ID NO.6 or SEQ ID NO.7 or has more than 90% homology with the same.

It is another object of embodiments of the present invention to provide a nucleotide sequence encoding the recombinant HFE & B2M fusion protein described above. The nucleotide sequence comprises a signal peptide coding sequence, an HFE coding sequence, a Spacer coding sequence, a B2M coding sequence and a His tag coding sequence.

Preferably, the signal peptide has a coding sequence of SEQ ID NO. 8 or a homology of more than 90% thereto.

Preferably, the HFE sequence is SEQ ID NO.9 or has more than 90% homology thereto.

Preferably, the coding sequence of the Spacer is SEQ ID NO. 10 or SEQ ID NO. 11 or has more than 90% homology with the same.

Preferably, the B2M sequence is SEQ ID NO. 12 or has more than 90% homology thereto.

Preferably, the nucleotide sequence is SEQ ID NO.13 or SEQ ID NO.14 or has more than 90% homology therewith.

It is another object of embodiments of the present invention to provide an expression vector comprising the above nucleotide sequence.

Preferably, the expression vector is a pcdna 3.1 (+) vector.

It is another object of embodiments of the present invention to provide a host cell containing the above expression vector.

Preferably, the host cell is a human embryonic kidney cell 293 (HEK 293 cell) or chinese hamster ovary cell (CHO cell).

It is another object of embodiments of the invention to provide a kit for blocking TfR binding to an antibody thereof, the kit comprising a fusion protein as described above. The kit may also include an auxiliary agent for delivering the fusion protein into the body.

It is another object of an embodiment of the present invention to provide the use of the above fusion protein or the above cell containing the fusion protein expression vector for preparing a medicament for treating a disease, such as hemochromatosis, metabolic diseases or rare diseases, which are all related to the binding force of TfR.

Compared with the prior art, the invention has the following outstanding advantages:

1) After the HFE & B2M specific sites are connected, the defect that HFE cannot independently bind to a transferrin receptor (TfR) in vitro is overcome. And it was found that recombinant HFE & B2M fusion proteins increased their affinity to transferrin receptor (TfR) compared to co-incubation of HFE and B2M, while recombinant HFE & B2M fusion proteins with high transferrin receptor (TfR) affinity can also be used in blocking experiments in antibody screening processes.

2) After the HFE & B2M specific sites are connected, the defect that HFE cannot be independently combined with transferrin receptor (TfR) in vitro is overcome, and a theoretical and practical basis of in-vitro experiment is provided for the diagnosis and treatment means of hemochromatosis.

Drawings

FIG. 1 is a SDS-PAGE diagram of recombinant HFE & B2M fusion proteins according to an embodiment of the present invention.

FIG. 2 is a chart showing the SEC-HPLC purity of recombinant HFE & B2M fusion proteins provided in the examples of the present invention.

FIG. 3 is a graph showing the binding capacity of HFE & B2M fusion proteins to TfR1 provided in the examples of the present invention.

FIG. 4 is a schematic representation of the detection results of in vitro binding assays for HFE and B2M.

FIG. 5 is a graph showing the ability of an antibody A to compete with HFE & B2M fusion protein for binding to TfR1 according to an embodiment of the present invention.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. in the following examples are commercially available as they are.

EXAMPLE 1 Synthesis of fusion protein expression cassette and construction of expression vector

Splicing the amino acid sequence and the coding DNA expression frame of the whole recombinant HFE & B2M fusion protein according to the amino acid sequence and the coding sequence of each component of the fusion protein: the amino acid residue sequences (SEQ ID NO. 3 and SEQ ID NO. 4) of the Spacer are respectively inserted into the tail ends of the HFE amino acid residue sequences (SEQ ID NO.1 and SEQ ID NO. 2) containing the signal peptide, so that the amino acid residue sequences (SEQ ID NO. 5) of the B2M are connected in series and have His tag sequences at the tail ends of the amino acid residue sequences, and finally the amino acid residue sequences (SEQ ID NO.6 and SEQ ID NO. 7) of the corresponding recombinant HFE & B2M fusion proteins are obtained.

The whole expression cassette was synthesized by the DNA coding sequence of HFE & B2M 1 (SEQ ID NO. 13) and HFE & B2M 2 (SEQ ID NO. 14) respectively, entrusted to the Nanjing's Bonnew Biotechnology Co., ltd, inserted into the EcoRI site of the pCDNA3.1 (+) vector (Invitrogen), then transformed into E.coli, and after sequencing correctly, plasmids were extracted and purified using the plasmid purification kit of Qiagen to obtain high-quality plasmids of the corresponding two recombinant expression vectors.

Example 2 expression and purification of fusion proteins

The high quality plasmids of the two recombinant expression vectors constructed and purified in example 1 were each transfected into CHO cells (purchased from american type collection, ATCC) using Lipofectamine 2000 (Invitrogen) for expression.

One day before transfection, 8X 10 cells were seeded in 60mm cell culture dishes ⁵ Individual cells, 5ml of medium (serum-containing, antibiotic-free) was added at 37℃and 5% CO ₂ Environmental culture of (3)And (5) nourishing. On the day of transfection, adding the plasmid to be transfected, mixing uniformly and standing for 5 minutes; to the mixed mixture, 15 ul transfection reagent (Qiagen PolyFect Transfection Reagent) was added and the mixture was blown 5 times with a sample applicator. The mixture was incubated at room temperature (20-25 ℃) for 5-10 minutes. Simultaneously with the incubation, the medium in the cell culture dish was aspirated, washed once with PBS solution, and 3ml of medium (serum-containing, antibiotic-free) was added.

After 2 days, the transfected CHO cells were inoculated into new plates according to the growth of the cells and screened by limiting dilution for cell cloning. Cell lines were established that were stably transfected with the corresponding expression vectors. Then, the stably transfected cells were amplified in large numbers by shake flask culture, the culture supernatants were collected, and each fusion protein was purified by gel filtration affinity chromatography. Purified fusion proteins were confirmed by SDS-PAGE to determine molecular weight (FIG. 1) in FIG. 1: lanes M1 are protein markers, bio-rad, cat No. 1610374S, lanes R represent Reducing condition; NR lane represents Non-reducing condition; and purity was confirmed via SEC-HPLC (fig. 2). Wherein the parameters corresponding to fig. 2 are shown in table 1:

table 1 specific parameters of fig. 2

Signal: VWDIA,Wavelength=280 nm

Example 3 detection of binding Capacity of fusion proteins to TfR1

a. Comparative examples: binding Capacity detection of non-fused HFE and B2M (used in combination)

After coating 1ug/ml of C-Myc/DDK Tag-labeled HFE (cat. No. NM-139006, 100ul/well in PBS buffer) overnight at 4℃2 washes with 0.05% PBST. After 1 hour incubation at 37℃with 3% MPBS, 1 wash with 0.05% PBST was performed. Dimer formation was achieved by adding 400 nM B2M for 2 hours at room temperature, followed by 1ug/ml Biotin-human TfR1 (100 ul/well in PBST) incubation at 37℃for 1 hour and 3 washes with 0.05% PBST. The Biotin-labeled human TfR1 was captured with SA [ HRP ] (0.1 ug/ml in 0.05% PBST,100 ul/well), and the absorbance was shown to be around 0.7 in three replicates, indicating weaker binding (results see Table 2).

The inventors found that pH affects the binding effect, and experiments demonstrated that: a better binding was obtained using PBS at pH7.5 as the binding buffer, whereas the use of PBS at pH6.0 resulted in unbound. The following experiments were carried out at pH7.5 unless otherwise specified.

HFE & B2M assay results

The inventors examined the binding effect of the amino acid sequences SEQ ID NO.6 and SEQ ID NO.7 prepared in example 2, respectively, and the two detection steps were the same as follows:

1ug/ml 1V were coated overnight at 4 ℃): after HFE & B2M (100 ul/well in CBS) prepared in example 2, it was washed 2 times with 0.05% PBST. After 1 hour incubation at 37℃with 3% MPBS, 1 wash with 0.05% PBST was performed. 34 ug/ml 1D Biotin-human TfR1, his Tag (diluted 4-fold with 0.05% PBST,100 ul/well) was added and incubated for 1 hour at 37℃and washed 3 times with 0.05% PBST. Binding assays were performed at pH 7.5. With SA [ HRP ] (0.1 ug/ml in 0.05% PBST,100 ul/well), the results of Excel calculation showed an EC50 of 12.61 ug/ml. Recombinant HFE & B2M fusion proteins increased affinity to transferrin receptor (TfR) compared to co-incubation of HFE and B2M (fig. 3).

The results indicate that the attachment of specific sites on HFE & B2M alters the defect that HFE itself cannot bind to transferrin receptor (TfR) alone. And it was found that recombinant HFE & B2M fusion proteins increased affinity with transferrin receptor (TfR) compared to co-incubation of HFE and B2M.

The inventors also performed additional experiments: in vitro binding experiments were performed using HFE and B2M to verify whether heterodimers could be formed after in vitro incubation of HFE and B2M.

Scheme 1 (Format 1): coating a C-Myc/DDK Tag-labeled HFE (1U), adding a His Tag-labeled B2M (1S), incubating for a period of time, and detecting a His Tag signal;

scheme 2 (Format 2): his Tag-coated B2M (1S), HFE (1U) added with C-Myc/DDK Tag is incubated for a period of time, and the signal of C-Myc is detected. The detection results are shown in FIG. 4.

It can be seen that only when HFE is coated and B2M is added, the two are found to be bound in vitro, but not strongly.

Following in vitro binding experiments with HFE and B2M with TfR1, the inventors formed dimers by coating 1ug/ml HFE overnight, adding 400 nM B2M the next day for 2 hours at room temperature, and then adding 1ug/ml Biotin-TfR1, the secondary antibody using SA [ HRP ], and the results of three replicates showed binding values of about 0.7 and weaker binding. Specific values are shown in Table 2 below.

Table 2 binding data sheet for HFE and B2M combined incubation and human TfR1

It can be seen that HFE and B2M alone bind to human TfR1 only poorly.

Example 4 expression of fusion proteins for blocking experiments

It is another object of embodiments of the present invention to provide the use of the fusion proteins described above for blocking experiments in antibody screening processes.

A96-well plate was prepared and 1ug/ml HFE prepared in example 2 was coated overnight at 4 ℃&After B2M fusion protein (CBS, 100 ul/well), wash 2 times with 0.05% PBST. After 1 hour incubation at 37℃with 3% MPBS, 1 wash with 0.05% PBST was performed. A4-fold gradient of PBST diluted A antibody (IgG antibody, capable of specifically recognizing human TfR1 receptor, assigned to Nanjing's Boston Biotechnology Co., ltd., number CGBA-0002) and Biotin-labeled human TfR1 (25 ug/ml,50 ul/well) were then added to the plate. And washed 3 times with 0.05% PBST. By SA [ HRP ]](0.1 ug/ml in 0.05% PBST,100 ul/well) was incubated at 37℃for 30 min to capture biotin-labeled TfR1. And washed 6 times with 0.05% PBST. Detecting SA [ HRP ]]Capturing the signal intensity of the biotin label. The experimental results were plotted against the concentration according to the OD450 values, as shown in fig. 5, and the experimental results showed that: antibodies A do not block HFE&The combination of B2M and human TfR1, thereby obtaining the Fe-free alloy ³⁺ Transported a antibodies, which are relatively safer. Thus, it is shown that HFE provided by the present invention&The B2M antibody fusion protein can effectively screen better safety in the process of antibody screening, and does not influence Fe ³⁺ A transported antibody.

The amino acid residue sequences and DNA coding sequences mentioned above are listed here, respectively:

amino acid residue sequence of the signal peptide of SEQ ID NO. 1:

MGWSCIILFLVATATGVHS

amino acid residue sequence of SEQ ID No. 2 HFE:

RLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPG

amino acid residue sequence of SEQ ID NO. 3 Spacer 1:

GGGSGGGSGGSGGSGGGS

amino acid residue sequence of SEQ ID NO. 4 Spacer 2:

GGGSGGGSGGGSGGGS

amino acid residue sequence of SEQ ID No. 5 b2 m:

MGWSCIILFLVATATGVHSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

the fusion protein HFE & B2M 1 of SEQ ID NO.6 is formed by fusing signal peptide-B2M-Spacer 1-HFE-His in sequence, and the amino acid sequence is as follows:

MGWSCIILFLVATATGVHSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGSGGGSGGSGGSGGGSRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGHHHHHH*

the fusion protein HFE & B2M 2 of SEQ ID NO.7 is formed by fusing signal peptide-B2M-Spacer 2-HFE-His in sequence, and the amino acid sequence is as follows:

MGWSCIILFLVATATGVHSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGSGGGSGGGSGGGSRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGHHHHHH*

the coding sequence of the signal peptide of SEQ ID NO. 8:

ATGGGCTGGAGCTGCATCATCCTGTTTCTGGTGGCGACTGCGACTGGTGTTCATTCT

the coding sequence of SEQ ID NO.9 HFE:

CGTCTGCTGCGTTCTCACTCCCTGCACTACCTGTTTATGGGTGCCTCCGAACAAGATCTGGGCCTGTCCCTGTTCGAAGCACTGGGTTACGTAGATGACCAGCTGTTTGTTTTCTACGATCACGAATCTCGTCGTGTCGAACCGCGCACCCCATGGGTATCCAGCCGTATCTCTAGCCAGATGTGGCTGCAGCTGTCCCAGTCCCTGAAAGGTTGGGACCACATGTTCACGGTGGACTTCTGGACCATTATGGAAAACCACAACCACAGCAAGGAATCTCACACCCTGCAGGTCATCCTGGGTTGTGAAATGCAGGAAGATAACTCTACTGAAGGCTACTGGAAATACGGCTATGATGGCCAGGATCACCTGGAGTTTTGTCCGGACACCCTGGACTGGCGCGCAGCTGAACCACGTGCATGGCCGACGAAACTGGAATGGGAACGTCACAAGATCCGTGCGCGTCAAAACCGTGCTTATCTGGAACGTGACTGTCCAGCGCAGCTGCAGCAACTGCTGGAACTGGGTCGCGGCGTACTGGACCAGCAAGTTCCTCCGCTGGTTAAAGTGACCCACCACGTAACTTCTAGCGTCACCACGCTGCGTTGTCGTGCACTGAACTATTACCCGCAGAACATCACTATGAAGTGGCTGAAGGACAAGCAACCGATGGACGCTAAAGAATTCGAACCGAAAGATGTCCTGCCAAATGGCGACGGTACTTATCAGGGTTGGATTACTCTGGCGGTACCGCCGGGC

the coding sequence of SEQ ID NO. 10B 2M:

ATGGGCTGGTCCTGCATCATTCTGTTCCTGGTAGCAACCGCAACTGGTGTGCATTCTATCCAGCGCACTCCGAAAATCCAAGTTTACTCCCGTCACCCAGCCGAAAACGGCAAATCCAACTTCCTGAACTGCTACGTATCCGGCTTCCATCCGAGCGATATCGAAGTTGATCTGCTGAAAAACGGCGAACGCATCGAGAAAGTAGAACACAGCGACCTGTCTTTCTCTAAGGATTGGAGCTTCTACCTGCTGTACTACACCGAATTCACTCCGACTGAAAAGGATGAATACGCTTGTCGCGTTAACCACGTGACTCTGTCCCAGCCGAAGATTGTTAAGTGGGATCGTGATATG

the coding sequence of SEQ ID NO. 11 Spacer 1:

GGTGGTGGTTCCGGTGGCGGTTCTGGCGGTAGCGGTGGTTCTGGTGGCGGTTCT

the coding sequence of SEQ ID NO. 12 Spacer 2:

GGTGGTGGCT CTGGCGGTGG CTCTGGTGGT GGTTCCGGTG GTGGCTCC

SEQ ID NO.13 HFE & B2M 1 DNA coding sequence:

ATGGGCTGGTCTTGTATCATCCTGTTCCTGGTTGCAACCGCCACTGGTGTCCATAGCATCCAGCGTACTCCGAAAATCCAGGTTTACAGCCGTCACCCGGCCGAAAACGGCAAATCTAACTTCCTGAACTGCTATGTCTCCGGCTTCCACCCGTCTGACATCGAAGTGGATCTGCTGAAGAACGGCGAGCGTATCGAGAAAGTTGAGCACTCTGACCTGAGCTTCTCCAAAGATTGGTCCTTCTACCTGCTGTATTACACCGAGTTCACCCCGACCGAAAAAGACGAATATGCTTGCCGCGTGAACCACGTGACCCTGTCCCAACCGAAAATTGTAAAATGGGACCGTGACATGGGTGGTGGTTCCGGTGGTGGCAGCGGTGGCTCTGGCGGTTCTGGCGGTGGCTCTCGTCTGCTGCGCTCTCACAGCCTGCACTATCTGTTCATGGGTGCATCCGAACAGGATCTGGGTCTGTCCCTGTTCGAAGCGCTGGGTTATGTGGATGATCAGCTGTTCGTGTTCTACGACCATGAGTCCCGCCGTGTGGAGCCACGTACTCCGTGGGTTTCTAGCCGTATCTCTTCCCAAATGTGGCTGCAGCTGAGCCAGAGCCTGAAAGGCTGGGATCACATGTTCACCGTGGATTTCTGGACCATCATGGAGAACCATAACCACTCCAAAGAAAGCCACACCCTGCAGGTCATCCTGGGTTGTGAGATGCAGGAAGATAACTCCACCGAAGGCTACTGGAAGTATGGCTATGATGGCCAGGATCACCTGGAATTCTGTCCGGACACTCTGGACTGGCGCGCGGCTGAACCACGTGCGTGGCCGACTAAACTGGAGTGGGAACGTCACAAAATCCGCGCACGTCAAAACCGTGCTTATCTGGAGCGTGATTGCCCGGCGCAACTGCAGCAGCTGCTGGAACTGGGTCGCGGTGTACTGGATCAGCAGGTGCCGCCTCTGGTTAAGGTTACCCACCACGTTACGTCTTCTGTTACCACCCTGCGTTGCCGCGCTCTGAACTACTATCCACAGAATATCACCATGAAATGGCTGAAAGACAAACAGCCAATGGATGCAAAAGAATTCGAACCAAAAGACGTTCTGCCGAACGGTGACGGCACCTACCAGGGTTGGATCACTCTGGCTGTTCCTCCAGGTCATCACCACCACCATCAC

SEQ ID NO.14 HFE & B2M 2 DNA coding sequence:

ATGGGCTGGTCCTGCATTATTCTGTTCCTGGTTGCTACGGCTACGGGTGTGCACAGCATCCAGCGTACTCCGAAGATCCAGGTTTACTCTCGCCACCCGGCTGAAAACGGTAAGTCTAATTTCCTGAACTGCTATGTTTCTGGCTTCCACCCATCTGACATCGAAGTTGACCTGCTGAAAAATGGCGAACGTATCGAAAAAGTCGAACACTCTGACCTGAGCTTCTCCAAAGATTGGTCCTTCTACCTGCTGTATTATACTGAATTTACTCCTACCGAAAAGGACGAATACGCATGTCGCGTGAACCACGTTACCCTGAGCCAGCCGAAAATTGTCAAATGGGACCGCGACATGGGTGGTGGCTCTGGTGGTGGTTCCGGTGGCGGTTCTGGTGGTGGCTCCCGTCTGCTGCGTTCCCATTCCCTGCATTACCTGTTCATGGGTGCTAGCGAACAGGATCTGGGTCTGTCTCTGTTTGAAGCGCTGGGTTATGTTGACGACCAGCTGTTCGTGTTTTACGACCACGAATCTCGTCGCGTTGAACCACGTACCCCGTGGGTTTCCTCTCGTATCTCCTCTCAGATGTGGCTGCAACTGTCCCAGTCTCTGAAGGGTTGGGATCACATGTTCACCGTAGACTTCTGGACCATCATGGAAAACCACAATCACAGCAAGGAATCTCACACCCTGCAGGTTATCCTGGGTTGTGAAATGCAGGAAGATAACAGCACCGAAGGCTACTGGAAATACGGTTACGACGGTCAAGATCATCTGGAATTCTGCCCGGATACCCTGGACTGGCGTGCTGCGGAACCTCGTGCTTGGCCAACCAAACTGGAATGGGAACGCCACAAAATCCGCGCCCGCCAGAATCGTGCGTACCTGGAACGTGATTGTCCTGCTCAGCTGCAGCAGCTGCTGGAACTGGGCCGTGGCGTTCTGGACCAACAGGTCCCACCACTGGTTAAAGTTACCCACCATGTCACCTCTTCTGTGACTACTCTGCGCTGTCGTGCACTGAACTATTACCCGCAGAACATCACCATGAAGTGGCTGAAAGATAAACAGCCGATGGACGCGAAAGAATTCGAACCGAAAGATGTTCTGCCGAACGGCGACGGCACCTACCAGGGTTGGATCACCCTGGCTGTTCCGCCAGGCCACCACCATCATCATCAC

the above description is only of the preferred embodiments of the present invention and is not intended to limit the invention, but any modifications, equivalents, improvements, etc. within the principles of the present invention should be included in the scope of the present invention.

Claims

1. A recombinant HFE & B2M fusion protein, wherein the fusion protein is capable of binding to TfR in vitro, the fusion protein comprising a signal peptide, an HFE protein, a spacer, a B2M protein, and a His tag, the fusion protein having the amino acid residue sequence of SEQ ID No.6 or SEQ ID No.7, wherein the TfR is TfR1, and the fusion protein binds to TfR1 in an environment of ph 7.5.

2. A nucleotide sequence encoding the fusion protein of claim 1, wherein the nucleotide sequence is set forth in SEQ ID No.13 or SEQ ID No. 14.

3. An expression vector comprising the nucleotide sequence of claim 2, wherein the expression vector is a pcdna 3.1 (+) vector.

4. A host cell comprising the expression vector of claim 3, wherein the host cell is a HEK293 cell or a CHO cell.

5. A kit for blocking binding of TfR to an antibody thereof, comprising the fusion protein of claim 1.

6. Use of the fusion protein of claim 1 for the manufacture of a medicament for the treatment of a disease, wherein the disease is hemochromatosis.