Magnetic resonance contrast agent as well as preparation method and application thereof
The preparation of lanthanide-binding protein mutants combined with gadolinium ions through genetic engineering technology to form a high-relaxation magnetic resonance contrast agent, solving the problems of low relaxation and toxic side effects of existing contrast agents, and achieving high-efficiency imaging and safety improvement.
Patent Information
- Application Number
- CN202311762570.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-20
- Publication Date
- 2025-06-20
AI Technical Summary
In clinical applications, existing MRI contrast agents have toxic side effects such as low relaxation, short circulation time, gadolinium deposition, skin fibrosis, deterioration of renal function and patient allergies, which are difficult to meet the requirements of high-efficiency imaging and safety.
The lanthanide-binding protein mutants are prepared through genetic engineering technology. The specific steps include preparing lanthanide-binding protein mutant gene fragments, introducing host expression and isolation and collection of proteins, and then mixing and incubating with lanthanide ions (such as gadolinium ions) to form a high relaxation magnetic resonance contrast agent.
It improves the relaxation of magnetic resonance contrast agents, extends its in vivo metabolism time, overcomes the problems of low relaxation of existing gadolinium contrast agents and short imaging time windows. At the same time, the lanthanide-binding protein gadolinium can be metabolized by the kidney and is used for renal function evaluation.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of pharmaceutical technology, and particularly to magnetic resonance contrast agents and their preparation methods and applications. Background Art
[0002] The kidney has key metabolic functions, which can regulate the balance of water, electrolytes and acid-base, produce erythropoietin, and affect bone and mineral metabolism. Due to its diverse functions, the kidney is vulnerable to threats such as toxins, infections, genetic factors and hemodynamic changes, which may lead to dysfunction. Kidney diseases may evolve into chronic kidney diseases and renal failure. Therefore, early diagnosis of kidney diseases is of great significance. Current diagnostic methods, such as blood tests measuring blood urea nitrogen and serum creatinine, are difficult to provide the real-time status of the kidney. Although biopsy provides a definite diagnosis, as an invasive method, it has the risk of damaging adjacent organs. Some non-invasive imaging techniques, including single photon emission computed tomography, positron emission tomography or computed tomography, all have radiation. And due to the limited tissue penetration ability of fluorescence technology, it is difficult to be applied in the human body. Therefore, there is an urgent need for a non-invasive and radiation-free imaging method for clinical evaluation of renal dysfunction.
[0003] Magnetic Resonance Imaging (MRI), as an important tool in the field of medical imaging, plays a great role in the diagnosis of kidney diseases. By injecting a contrast agent to enhance the imaging contrast, the imaging sensitivity can be further improved. Currently, the commonly used MRI contrast agents in clinics mainly include Magnevist, Dotarem, Gadavist, etc. However, these contrast agents based on lanthanide element gadolinium (Gd) still face some challenges in clinical applications: i) relatively low relaxivity (<10 mM-1s-1); ii) short circulation time (<30 minutes); iii) side effects such as gadolinium deposition, skin fibrosis, deterioration of renal function and patient allergy. Therefore, it is of great significance to develop new contrast agents with kidney imaging function.
[0004] Lanmodulin is found in methylotrophic bacteria and has three binding sites for lanthanide elements (Gd, Nd, Eu, etc.), with an affinity reaching the picomolar level. It is a newly discovered molecule with lanthanide element selectivity characteristics. Currently, it is mainly applied to rare earth element separation, and there is still little research in the field of magnetic resonance imaging applications. Some studies disclose metal-binding proteins that have high selective affinity for certain trivalent cations and / or tetravalent cations, where the trivalent cations and / or tetravalent cations are derived from rare earth elements or their ions (such as lanthanide elements) and actinide elements or their ions, as well as from hafnium and zirconium elements or their compounds, and sensors comprising the metal-binding proteins, and methods for using the metal-binding proteins to capture and separate such trivalent cations and / or tetravalent cations. Some studies describe rare earth element (REE)-binding proteins (e.g., lanthanide-binding proteins), host cells expressing REE-binding proteins, and methods for recovering REE. Some studies prepared a gadolinium-based magnetic resonance contrast agent loaded with bovine serum albumin (BSA-Gd), which can be metabolized by the liver, and the synthesized albumin gadolinium-based magnetic resonance contrast agent can not only well display blood vessels but also avoid renal metabolism, perfectly solving existing clinical problems. Some studies disclose a low-renal-toxicity protein-iron oxide composite nanomagnetic resonance contrast agent and its preparation method and application. The contrast agent has iron oxide as the core and functionalized protein coated thereon, has high uniformity, hydrophilicity and monodispersity, and has low renal toxicity and good biocompatibility, and can be used as a safe magnetic resonance contrast agent. Some studies disclose a magnetic resonance contrast agent using an endogenous protein in the human body as a chelating agent and its preparation. The contrast agent includes a chelating agent and a paramagnetic metal ion coordinated with the chelating agent, and the chelating agent is an endogenous protein in the human body. Some studies disclose a protamine-stabilized manganese dioxide nanoparticle complex (PS@MnO2) and its preparation method. The invention uses the positively charged protein - protamine to prepare PS@MnO2 by a one-step redox method. The prepared PS@MnO2 is spherical with a particle size of 90 - 350 nm. The relaxation degrees of the magnetic resonance contrast agents disclosed in these studies are relatively low and difficult to meet the requirements. Summary of the Invention
[0005] Based on this, the present application provides a magnetic resonance contrast agent with a relatively high relaxation degree, and its preparation method and application.
[0006] A magnetic resonance contrast agent, comprising a lanmodulin mutant, where the lanmodulin mutant is obtained by mutating asparagine at position 108 of lanmodulin to aspartic acid.
[0007] Among the above magnetic resonance contrast agents, the lanthanide-binding protein mutant is obtained by mutating asparagine at position 108 of the lanthanide-binding protein to aspartic acid, which is beneficial to improving the relaxation rate. Through experimental verification, the effective relaxation rate (about 52 mM -1 s -1 ) of the above magnetic resonance contrast agent is 10 times that of Magnevist (about 5 mM -1 s -1 ) and can be applied to magnetic resonance imaging detection.
[0008] In one embodiment, the amino acid sequence of the lanthanide-binding protein is as shown in SEQ ID No.1.
[0009] In one embodiment, the lanthanide-binding protein mutant binds to lanthanide ions, and the lanthanide ions include gadolinium ions.
[0010] A method for preparing a magnetic resonance contrast agent includes the following steps: preparing the magnetic resonance contrast agent by using genetic engineering technology, wherein the magnetic resonance contrast agent includes a lanthanide-binding protein mutant, and the lanthanide-binding protein mutant is obtained by mutating asparagine at position 108 of the lanthanide-binding protein to aspartic acid.
[0011] In one embodiment, the step of preparing the magnetic resonance contrast agent by using genetic engineering technology includes:
[0012] Preparing the gene fragment of the lanthanide-binding protein mutant;
[0013] Introducing the gene fragment of the lanthanide-binding protein mutant into a host for expression, and separating and collecting the lanthanide-binding protein mutant;
[0014] Mixing and incubating the lanthanide-binding protein mutant with lanthanide ions to obtain the magnetic resonance contrast agent.
[0015] In one embodiment, the asparagine at position 108 of the lanthanide-binding protein is mutated to aspartic acid by using overlapping PCR amplification technology to obtain the gene fragment of the lanthanide-binding protein mutant.
[0016] In one embodiment, the amino acid sequence of the lanthanide-binding protein is as shown in SEQ ID No.1.
[0017] In one embodiment, the host is Escherichia coli. The steps of introducing the lanthanide element-binding protein mutant gene fragment into the host for expression, separating and collecting the lanthanide element-binding protein mutant include: introducing the lanthanide element-binding protein mutant gene fragment into the Escherichia coli host for expression, disrupting the bacterial cells, separating and purifying to obtain the lanthanide element-binding protein mutant.
[0018] In one embodiment, the lanthanide element ion includes gadolinium ion;
[0019] and / or, the molar ratio of the lanthanide element ion to the lanthanide element-binding protein mutant is 1 to 4.
[0020] Use of the above magnetic resonance contrast agent, or a magnetic resonance contrast agent prepared by the preparation method of the above magnetic resonance contrast agent, in magnetic resonance imaging. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 SDS-PAGE diagrams before and after mutation of the lanthanide element protein;
[0022] Figure 2 Graphs showing the evaluation results of the binding performance of the lanthanide-binding protein before and after mutation to gadolinium ions;
[0023] Figure 3 Photographs and SDS-PAGE diagrams before and after mutation of the lanthanide element protein and before and after binding to gadolinium ions;
[0024] Figure 4 Graph showing the evaluation results of the magnetic resonance imaging ability of the point-mutated lanthanide element-binding protein-gadolinium;
[0025] Figure 5 Graph showing the evaluation results of the magnetic resonance in vivo imaging ability and metabolism of the point-mutated lanthanide element-binding protein-gadolinium. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0026] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following detailed description of the specific embodiments of the present invention will be given in conjunction with specific embodiments and the accompanying drawings. Many specific details are set forth in the following description to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.
[0027] One embodiment of the present application provides a magnetic resonance contrast agent, which includes a lanthanide element-binding protein mutant, and the lanthanide element-binding protein mutant is obtained by mutating asparagine at position 108 of the lanthanide element-binding protein to aspartic acid.
[0028] In one embodiment, the amino acid sequence of the lanthanide-binding protein is as shown in SEQ ID No.1. Specifically, the sequence shown in SEQ ID No.1 is MAFRLSSAVLLAALVAAPAYAAPTTTTKVDIAAFDPDKDGTIDLKEALAA GSAAFDKLDPDKDGTLDAKELKGR VSEADLKKLDPDNDGTLDKKEYLAAVEAQFKAANPDNDGTIDARELASPA GSALVNLIR.
[0029] In some embodiments, the lanthanide-binding protein mutant binds to lanthanide ions. Lanthanide ions include gadolinium ions. It should be noted that the lanthanide ions are not limited to gadolinium ions, and can also be other lanthanide ions, such as lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, lutetium, etc.
[0030] In the above magnetic resonance contrast agent, the lanthanide-binding protein mutant is mutated from asparagine at position 108 of the lanthanide-binding protein to aspartic acid, which is beneficial to improving the relaxation degree and can be applied to magnetic resonance imaging. After experimental verification, the effective relaxation degree of the above magnetic resonance contrast agent (about 52 mM -1 s -1 ) is 10 times that of Magnevist (about 5 mM -1 s -1).
[0031] Furthermore, the above magnetic resonance contrast agent modifies the lanthanide-binding protein through single-point gene mutation to increase the binding performance of the protein to gadolinium ions. Utilizing the characteristics of the binding protein, it inhibits the molecular rotation correlation time, improves the relaxation degree of the contrast agent and prolongs its in vivo metabolism time; thus overcoming the problems of low relaxation degree and short imaging time window of existing gadolinium contrast agents. At the same time, lanthanide-binding protein-gadolinium can be metabolized by the kidneys, so it can be used for kidney function evaluation.
[0032] One embodiment of the present application also provides a method for preparing the above magnetic resonance contrast agent, including the following steps: preparing the magnetic resonance contrast agent by using genetic engineering technology. Among them, the magnetic resonance contrast agent includes a lanthanide-binding protein mutant, and the lanthanide-binding protein mutant is mutated from asparagine at position 108 of the lanthanide-binding protein to aspartic acid.
[0033] The specific description of the magnetic resonance contrast agent can be found in the above text and will not be repeated here.
[0034] In some embodiments, the steps of preparing the magnetic resonance contrast agent by using genetic engineering technology include S110 - S130:
[0035] S110. Prepare the gene fragment of the lanthanide-binding protein mutant.
[0036] Among them, the 108th asparagine of the lanthanide-binding protein was mutated to aspartic acid by overlapping PCR amplification technology to obtain a lanthanide-binding protein mutant gene fragment.
[0037] In one specific example, the amino acid sequence of the lanthanide-binding protein is shown in SEQ ID No.1. Specifically, the sequence shown in SEQ ID No.1 is MAFRLSSAVLLAALVAAPAYAAPTTTTKVDIAAFDPDKDGTIDLKEALAA GSAAFDKLDPDKDGTLDAKELKGR VSEADLKKLDPDNDGTLDKKEYLAAVEAQFKAADPDNDGTIDARELASPAGSALVNLIR.
[0038] S120. Introduce the lanthanide-binding protein mutant gene fragment into a host for expression, and isolate and collect the lanthanide-binding protein mutant.
[0039] In some of these embodiments, the host is Escherichia coli. S120 includes the following steps: introduce the lanthanide-binding protein mutant gene fragment into an Escherichia coli host for expression, break the bacterial cells, and isolate and purify to obtain the lanthanide-binding protein mutant.
[0040] Among them, the method of breaking the bacterial cells is not limited. For example, it can be ultrasonic disruption. The method of isolation and purification is not limited. For example, it can be Ni column affinity chromatography.
[0041] S130. Mix the lanthanide-binding protein mutant with lanthanide ions and incubate to obtain a magnetic resonance contrast agent.
[0042] In one embodiment, the lanthanide ions include gadolinium ions.
[0043] In some of these embodiments, the molar ratio of lanthanide ions to the lanthanide-binding protein mutant is 1-4.
[0044] In the above method for preparing the magnetic resonance contrast agent of the present application, the engineered lanthanide-binding protein is expressed by introducing the constructed point mutation gene into Escherichia coli through genetic engineering technology, making full use of synthetic biology technology. The preparation method is simple and has the prospect of large-scale production; in terms of purification and separation, compared with the separation of small molecule chelates and unchelated gadolinium ions, the separation of protein macromolecules and unchelated gadolinium ions is easier due to the large difference in molecular weight and size.
[0045] Existing protein-based magnetic resonance contrast agents (such as albumin-gadolinium, protein-iron oxide, protein-manganese dioxide, etc.) are difficult to be metabolized by the kidneys. The preparation method of the magnetic resonance contrast agent of the present application obtains a magnetic resonance contrast agent with high relaxivity, long circulation time, and a genetically mutated lanthanide element protein with high affinity for gadolinium ions; the genetically mutated lanthanide element protein-gadolinium magnetic resonance contrast agent has kidney metabolism function and can be applied to magnetic resonance imaging.
[0046] Traditional small molecule contrast agents need to be constructed through complex coupling and purification to build contrast agents with active targeting imaging functions. However, the engineered lanthanide-binding proteins constructed by the biological method of the present application can be customized through underlying gene design to synthesize molecules with different targeting functions, laying a foundation for expanding the application scope of lanthanide-binding proteins.
[0047] The following are specific examples.
[0048] Unless otherwise specified, the drugs and instruments used in the examples are all conventional selections in the art. For the experimental methods without specific conditions noted in the examples, they are usually carried out under conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the kit manufacturers.
[0049] Example 1
[0050] 1. Design site-directed mutagenesis primers, amplify and clone the lanthanide element protein with asparagine at position 108 mutated to aspartic acid by overlapping PCR, and sequence and compare the PCR fragments.
[0051] 2. Introduce the mutated lanthanide element-binding protein gene into the Escherichia coli BL21(DE3) strain with expression ability. Then, the bacteria are evenly spread on an LB agar plate containing 50 μg / mL kanamycin and cultured at 37 °C. Select a single colony for inoculation, culture it at 37 °C with a shaker speed of 200 rpm for about 16 hours, and then inoculate the mixture for large-scale culture. When the optical density (OD 600 ) at 600 nm reaches 0.6, set the culture temperature to 16 °C and add IPTG at a final concentration of 0.6 mM to induce the expression of the recombinant protein. After further overnight shaker incubation, harvest the bacterial cells by centrifugation at 6000 rpm for 20 minutes at 4 °C.
[0052] 3. The bacteria were lysed by sonication in a buffer containing 150 mM NaCl, 10 mM Tris·HCl (pH 7.5), and 100 μM PMSF. Subsequently, the cell lysate was centrifuged at 15,000 rpm for 30 minutes at 4 °C, and the supernatant was collected and co-incubated with Ni-NTA agarose 6FF beads pre-equilibrated in a buffer of 150 mM NaCl, 10 mM Tris·HCl (pH 7.5) (buffer A) for 1 hour at 4 °C. It should be noted that the Ni-NTA beads were pre-equilibrated in buffer A containing 150 mM NaCl and 10 mM Tris·HCl (pH 7.5). Then, the mixed solution was transferred to a column and eluted sequentially with buffer A containing 20 mM imidazole. Subsequently, the protein bound to the beads was eluted with buffer A containing 200 mM imidazole, and the eluted protein was further concentrated by a 3-kD centrifugal filter device.
[0053] 4. To load Gd 3+ into the protein, GdCl3 was added to the purified protein at a molar ratio of 5:1, and then they were co-incubated at 4 °C for 30 minutes to ensure that the metal ions were fully bound to the protein. The unbound Gd in the mixture was removed by ultrafiltration centrifugation 3+ , and the Gd 3+ content in the protein was quantitatively determined by inductively coupled plasma - optical emission spectrometry (ICP-OES).
[0054] 5. Before the competition experiment, the buffer A of the point-mutated lanthanide-binding protein (LanND) and the non-mutated lanthanide-binding protein (LanM) was changed to a buffer containing 20 mM MOPS, 100 mM NaCl, pH 6.0 (buffer B). LanM and LanND were diluted to the same concentration (100 μM) with buffer B. GdCl3 and xylenol orange (XO) were also diluted to 100 μM with buffer B. When conducting the competition test, three groups were set up for the control group, the LanM group, and the LanND group respectively. Then, first, 10 μL of XO was added to a 96-well plate, and then 10 μL of buffer B, LanM, or LanND was added to the corresponding wells and mixed with XO. Subsequently, GdCl3 was added dropwise to the wells in 10 μM alternating steps to a final concentration of 0 to 70 μM. Different volumes of buffer B were added to ensure that the final volume of each well was 100 μL. After all components were completely mixed, the plate was transferred to a microplate absorbance reader, and the absorbance was read at 570 nm. The absorbance was plotted against the Gd 3+ concentration, and fitting was performed using Boltzmann fitting. The Gd 3+ concentration at the point where the XO saturation was 10% was regarded as an estimated point for saturation of the tight-binding sites within the protein.
[0055] 6. Lanthanide element protein-gadolinium solutions with different concentrations were prepared to measure the T1-weighted relaxation time. The T1WI and T2WI scanning parameters under 3T conditions (uMR790, United Imaging Healthcare) were as follows: The radiofrequency coil was a 48-channel body receive head coil; TR was 500 milliseconds; TE was 20 milliseconds; FOV was 150 mm × 250 mm; FA was 145°; the matrix size was 512 × 308; the bandwidth / pixel was 200 Hz / pixel; the slice thickness was 3 mm. For T1-weighted imaging of animals, a 3.0T preclinical magnetic resonance imaging system was used, and the specific specifications were as follows: The radiofrequency coil was a dedicated small animal coil (mouse coil); TR was 600 milliseconds; TE was 12.16 milliseconds; FOV was 60 mm × 30 mm; FA was 145°; the voxel size was 0.47 mm × 0.23 mm × 0.6 mm; the matrix size was 256 × 256; the bandwidth / pixel was 260 Hz / pixel; the slice thickness was 0.6 mm; the number of averages was 8; the number of slices was 10.
[0056] The detection results are as Figures 1 to 5 shown. Figure 1 are the SDS-PAGE diagrams before and after the mutation of the lanthanide element protein; Figure 2 are the diagrams of the evaluation results of the binding performance of the lanthanide-binding protein before and after the mutation to gadolinium ions; Figure 3 are the photos and SDS-PAGE diagrams before and after the mutation of the lanthanide element protein and before and after binding to gadolinium ions; Figure 4 are the diagrams of the evaluation results of the magnetic resonance imaging ability of the point-mutated lanthanide-binding protein-gadolinium; Figure 5 are the diagrams of the evaluation results of the magnetic resonance in vivo imaging ability and metabolism of the point-mutated lanthanide-binding protein-gadolinium.
[0057] From Figures 1 to 3 it can be seen that the weakly bound ions in one of the binding sites of the lanthanide-binding protein may easily dissociate from the protein, resulting in potential toxicity. Therefore, in this example, a single point mutation N108D was introduced into the fourth EF hand structure to form a mutant engineered lanthanide-binding protein (LanND). The SDS-PAGE gel bands showed that the molecular weights of the purified wild-type LanM and the mutant LanND were similar, approximately 12 KDa. We used xylenol orange as an indicator for free Gd3+ ions to evaluate their Gd 3+ binding ability. The results showed that the single point mutation in LanND increased its number of Gd 3+ binding sites compared with LanM. The binding Gd3+ stoichiometry increased from approximately 2.1 to 3.8. Moreover, in the presence or absence of Gd 3+ this mutant did not cause any abnormal aggregation or degradation.
[0058] From Figure 4 andFigure 5 It can be seen that at a magnetic field strength of 3.0 T, LanND-Gd exhibits a T1 relaxation rate (r1) of 13.25 mM -1 s -1 which is 2.7 times that of Magnevist (4.97 mM -1 s -1 ) clinically used. Moreover, a single LanND-Gd molecule contains four Gd 3+ binding sites, while Magnevist has only one. Therefore, the effective relaxation rate of LanND-Gd (about 52 mM -1 s -1 ) is 10 times that of Magnevist (about 5 mM-1s-1). To develop a contrast agent with clinical translation potential, it is first necessary to determine its metabolic pathway in the body. Traditional nanoparticle-based contrast agents usually have too long a circulation time in the blood and may accumulate in organs, causing potential side effects or toxicity. Therefore, this application studied the metabolism of engineered protein contrast agents. In this example, LanND-Gd was intravenously injected into mice at a drug dose of 2.5 μmol / kg. MRI images showed obvious MRI signals in the kidneys and bladder, indicating that the protein was filtered by the kidneys and excreted through the urethra. Within the time period of 0-75 minutes, the MRI signal of the bladder from transverse and longitudinal abdominal imaging profiles increased significantly, confirming that the engineered protein was cleared by the kidneys through urine. Compared with clinical Magnevist, the in vivo metabolism time of LanND-Gd was significantly prolonged.
[0059] In summary, this application discloses a magnetic resonance contrast agent and its preparation method. The contrast agent modifies a lanthanide-binding protein through single-point gene mutation to increase the binding performance of the protein to gadolinium ions. Utilizing the characteristics of the binding protein, it inhibits the molecular rotation correlation time, improves the relaxation rate of the contrast agent, and prolongs its in vivo metabolism time; thereby overcoming the problems of low relaxation rate and short imaging time window of existing gadolinium contrast agents. At the same time, the lanthanide-binding protein-gadolinium can be metabolized by the kidneys, and thus can be used for kidney function evaluation.
[0060] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.
[0061] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.
Claims
1. A magnetic resonance contrast agent, characterized in that, Comprising a lanthanide-binding protein mutant, wherein the lanthanide-binding protein mutant is obtained by mutating asparagine at position 108 of the lanthanide-binding protein to aspartic acid.
2. The magnetic resonance contrast agent according to claim 1, characterized in that, The amino acid sequence of the lanthanide-binding protein is shown in SEQ ID No.
1.
3. The magnetic resonance contrast agent according to any one of claims 1-2, characterized in that, The lanthanide-binding protein mutant binds to lanthanide ions, and the lanthanide ions include gadolinium ions.
4. A method for preparing a magnetic resonance contrast agent, characterized in that, Comprising the following steps: preparing the magnetic resonance contrast agent by genetic engineering techniques, wherein the magnetic resonance contrast agent comprises a lanthanide-binding protein mutant, and the lanthanide-binding protein mutant is obtained by mutating asparagine at position 108 of the lanthanide-binding protein to aspartic acid.
5. The preparation method according to claim 4, characterized in that, The steps of preparing the magnetic resonance contrast agent by genetic engineering techniques include: Preparing the gene fragment of the lanthanide-binding protein mutant; Introducing the gene fragment of the lanthanide-binding protein mutant into a host for expression, and separating and collecting the lanthanide-binding protein mutant; Mixing and incubating the lanthanide-binding protein mutant with lanthanide ions to obtain the magnetic resonance contrast agent.
6. The preparation method according to claim 5, characterized in that, Using the overlapping PCR amplification technique to mutate asparagine at position 108 of the lanthanide-binding protein to aspartic acid to obtain the gene fragment of the lanthanide-binding protein mutant.
7. The preparation method according to claim 6, characterized in that, The amino acid sequence of the lanthanide-binding protein is shown in SEQ ID No.
1.
8. The preparation method according to claim 5, characterized in that, The host is Escherichia coli, and the steps of introducing the gene fragment of the lanthanide-binding protein mutant into the host for expression, and separating and collecting the lanthanide-binding protein mutant include: introducing the gene fragment of the lanthanide-binding protein mutant into the Escherichia coli host for expression, disrupting the cells, and separating and purifying to obtain the lanthanide-binding protein mutant.
9. The preparation method according to any one of claims 5-7, characterized in that, The lanthanide ions include gadolinium ions; And / or, the molar ratio of the lanthanide ions to the lanthanide-binding protein mutant is 1 to 4.
10. Use of the magnetic resonance contrast agent according to any one of claims 1-3, or the magnetic resonance contrast agent prepared by the preparation method according to any one of claims 4-9, in magnetic resonance imaging.