Recombinant human light chain ferritin and application thereof

Abstract

The invention discloses recombinant human light chain ferritin and application thereof, and relates to the technical field of nano biological materials. The recombinant human light chain ferritin comprises a functional tag combination inserted into an N end, a self-assembly body, and a functional structure domain inserted into a disordered amino acid sequence region between a fourth alpha helix and a fifth alpha helix of a subunit of the self-assembly body, the functional tag combination consists of a functional structure domain which is inserted into an N end and is used for promoting expression and a functional structure domain which is inserted into the N end and is used for promoting purification; the functional structural domain of the disordered amino acid sequence region is polypeptide with a targeted anti-inflammatory effect. And removing the functional tag combination inserted into the N end through enzyme digestion to obtain the functionally modified human light chain ferritin with the anti-inflammatory effect. The functional ferritin with a complete structure is obtained by performing solubilization expression transformation on the functionally modified human light-chain ferritin, reducing the formation of inclusion bodies in the protein expression process by using a tag combination at the N end of the human light-chain ferritin and improving the purification efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-biomaterials, and in particular to a functionally modified human light-chain ferritin and an application thereof. Background Art

[0002] Human light chain ferritin (LFn) is a hollow cage-like protein formed by the self-assembly of 24 protein subunits, with an outer diameter of 12 nm and an inner diameter of 8 nm. Due to its stable physical and chemical properties such as good water solubility, thermal stability and biocompatibility, it is widely used in nano-drug delivery and disease treatment, becoming a highly potential nano-protein drug platform.

[0003] The prokaryotic expression system is the most mature expression system currently studied and has been widely used in the field of exogenous protein expression. However, the functionalized ferritin nanoparticles have always been unstable after genetic modification during prokaryotic expression, and inclusion bodies are easily formed. The conventional purification method is to denature and renature the inclusion bodies with high concentrations of urea, and then further purify the renatured protein. This method has a low protein yield and is very likely to cause irreversible structural damage to ferritin, a protein with a special spatial structure.

[0004] Therefore, it is urgent to design and develop a functional modified ferritin solubilization expression modification and purification method, which can optimize the modified ferritin at the molecular level, and then form supernatant expression during protein expression, remove protein loss during purification caused by inclusion bodies, effectively improve protein recovery rate, and reduce production costs. This method effectively solves the instability and other problems in the expression process of functionalized ferritin, making it more widely used in functional modified recombinant protein drugs and nano drug delivery, and is also of great reference significance in exploring the prokaryotic expression of other recombinant proteins. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a functionally modified human light chain ferritin and its application, which is not only safe and efficient, but also the entire transfection process is convenient and controllable.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides a functionally modified human light chain ferritin, comprising a functional tag combination inserted at the N-terminus, a self-assembly, and a functional domain inserted in the disordered amino acid sequence region between the fourth and fifth α helices of the subunit of the self-assembly; wherein the functional tag combination consists of a functional domain for promoting expression inserted at the N-terminus and a functional domain for promoting purification inserted at the N-terminus; the functional domain in the disordered amino acid sequence region is a polypeptide with targeted anti-inflammatory effect; and the self-assembly is a homologous twenty-tetrameric cage protein formed by self-assembly of self-assembly monomers.

[0007] The present invention utilizes a combination of SUMO-tag and His-tag to achieve expression and purification of functionally modified human light-chain ferritin, and then removes the front-end functional tag combination through SUMO protease, so that human light-chain ferritin has an original structure close to that of wild-type human light-chain ferritin, thereby achieving its role in targeted treatment of vascular inflammation.

[0008] As one of the preferred embodiments, the functionally modified human light chain ferritin includes a His-tag functional domain inserted at the N-terminus to promote purification; a SUMO-tag functional domain inserted at the N-terminus to promote expression; the amino acid sequence of the naturally self-assembled human light chain ferritin subunit and the functional domain inserted in the disordered amino acid sequence region between the fourth and fifth α helices of the self-assembling protein subunit is a VHP peptide targeting VCAM-1.

[0009] Preferably, the functional domain of the disordered amino acid sequence region is selected from any one of a VHP peptide targeting vascular cell adhesion molecule-1, a GHK peptide, an RGD peptide targeting multiple integrin receptors, or an NGR peptide targeting aminopeptidase N (APN / CD13).

[0010] Preferably, the human light chain ferritin self-assembling monomer is a natural human light chain ferritin subunit.

[0011] Preferably, the functional domain promoting purification is a His-tag; the amino acid sequence is shown in SEQ ID No.4.

[0012] Preferably, the functional domain promoting expression is SUMO-tag; the amino acid sequence is shown in SEQ ID No.5.

[0013] Preferably, the amino acid sequence of the naturally self-assembled human light chain ferritin single subunit is as shown in SEQ ID No.3.

[0014] Preferably, the amino acid sequence of the recombinant human light chain ferritin is as shown in SEQ ID No.1.

[0015] Preferably, the amino acid sequence of the functional domain in the disordered amino acid sequence region is as shown in SEQ ID No. 2; As a preferred embodiment, the functionally modified human light chain ferritin further comprises connecting a fluorescent substance to the functionally modified human light chain ferritin by chemical modification.

[0016] As one aspect of the invention, the present invention also provides a nucleic acid molecule encoding the above-mentioned functionally modified human light chain ferritin.

[0017] As one of the aspects of the invention, the present invention also provides a recombinant expression vector into which the nucleic acid molecule as described above is inserted, and the expression vector is capable of causing the transformed or transfected host cells to express the functionally modified human light chain ferritin after transforming or transfecting the host cells.

[0018] As one aspect of the invention, the present invention also provides a recombinant cell, which comprises the above-mentioned recombinant expression vector, or the above-mentioned nucleic acid molecule is integrated into the genome of the recombinant cell.

[0019] Preferably, the recombinant cell comprises Escherichia coli.

[0020] As one aspect of the invention, the present invention provides a method for preparing the above-mentioned functionally modified human light chain ferritin, the specific steps comprising: The natural human light chain ferritin gene sequence is used as a template to construct the recombinant expression vector; the recombinant expression vector is transferred into a host cell and expressed to obtain a recombinant human light chain ferritin fusion protein; the His-tag domain and the SUMO-tag domain at the N-terminus of the recombinant human light chain ferritin fusion protein are removed by SUMO protease cleavage; and the functionally modified human light chain ferritin is obtained.

[0021] Preferably, the recombinant expression vector is formed by enzyme-linking a pET-30a(+) plasmid that has been digested with enzymes and a target sequence that has been digested with enzymes.

[0022] Preferably, the amino terminus of the recombinant human light chain ferritin fusion protein carries a His-tag and a SUMO-tag, and the fourth and fifth α-helical disordered regions at the carboxyl terminus carry a polypeptide with a targeted anti-inflammatory effect.

[0023] As one aspect of the invention, the present invention provides a pharmaceutical composition, the active ingredient of which includes the functionally modified human light chain ferritin as described above. By adopting the technical solution of the present invention, the front-end tag is removed by SUMO protease, so that the functionally modified human light chain ferritin differs from the wild-type protein only in the required functional peptide domain, thereby eliminating other possible influencing factors.

[0024] Preferably, the functionally modified light chain ferritin is administered by intravenous injection.

[0025] As one aspect of the invention, the present invention provides the use of the above-mentioned functionally modified human light chain ferritin in the preparation of drugs for treating vascular inflammation, chronic inflammation and other related diseases.

[0026] Beneficial technical effects obtained by the present invention: (1) The method for promoting the expression and purification of functionalized modified ferritin provided by the technical solution of the present invention optimizes the molecular level of the modified ferritin, thereby forming a supernatant expression during the protein expression process, eliminating the protein loss during the purification process caused by inclusion bodies, effectively improving the protein recovery rate, and reducing the production cost; it effectively solves the problems of instability in the expression process of functionalized ferritin, so that it can be more widely used in the delivery of functionally modified recombinant protein drugs and nanodrugs, especially for the prokaryotic expression process of other recombinant proteins. It also has important reference significance.

[0027] (2) The technical scheme of the present invention is adopted, and the functional tag combination of SUMO-tag and His-tag is used to realize the supernatant expression and purification of anti-inflammatory functional modified ferritin. The front functional tag combination is removed by SUMO protease, so that the protein is restored to the original structure close to the wild-type human light chain ferritin, and targeted treatment of vascular inflammation is performed. The method of the present invention is novel, precise in regulation, and mild in conditions. It is suitable for the optimization of recombinant protein expression that is easy to form inclusion bodies during the expression process of most prokaryotic systems, and thus has broad application prospects.

[0028] (3) The method for preparing the functionally modified human light chain protein provided by the present invention has simple steps and mild conditions, which solves the technical problems of difficult modification and expression of human light chain ferritin; and the obtained functional protein has good dispersibility, stability and biosafety, and has important application value in the treatment of vascular inflammation and functional modification of human ferritin.

[0029] (4) The preparation of the functionalized modified human light chain ferritin provided by the present invention has the advantages of simple and easy availability of raw materials, safety and reliability, and a convenient and controllable preparation process, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an SDS-PAGE image of the purified human light chain ferritin after functional modification with a tag constructed in Example 1 of the present invention.

[0031] Figure 2 This is an SDS-PAGE image of the functionally modified human light chain ferritin tag digested in Example 2 of the present invention.

[0032] Figure 3 This is a transmission electron micrograph of the functionally modified human light chain ferritin constructed in Example 2 of the present invention.

[0033] Figure 4 This is the spectrum of the functionalized modified human light chain ferritin protein constructed in Example 2 of the present invention.

[0034] Figure 5 This is the flow cytometric count and quantitative graph of the inflammatory cell experiment in Example 4 of the present invention.

[0035] Figure 6 This is the cell fluorescence and quantitative graph of the inflammatory cell experiment in Example 4 of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The present invention provides a functionally modified human light chain ferritin with anti-vascular inflammation effect, which is obtained by proteolytic cleavage of a functional tag domain inserted into the amino terminal of a self-assembly subunit by recombinant human light chain ferritin.

[0038] In the first aspect provided by the embodiment of the present invention, the recombinant human light chain ferritin comprises a human light chain ferritin self-assembly monomer, a functional tag domain inserted into the amino terminal of the self-assembly subunit, and a functional domain inserted into the disordered amino acid sequence region between the fourth and fifth α helices of the self-assembly protein subunit; finally, the recombinant human light chain ferritin can be obtained. The amino terminal domain of the human light chain ferritin self-assembly monomer is a His-tag, which is used to promote the purification function.

[0039] The functional domain at the amino terminus of the self-assembling monomer of human light chain ferritin is a SUMO-tag, which is used to promote protein expression.

[0040] The functional domain of the disordered amino acid sequence region between the fourth and fifth alpha helices of the self-assembling monomer of human light chain ferritin is a polypeptide with targeted anti-inflammatory effect.

[0041] Preferably, the polypeptide with anti-inflammatory effect is selected from any one of VHP peptide targeting vascular cell adhesion molecule-1, GHK peptide, RGD peptide targeting multiple integrin receptors or NGR peptide targeting aminopeptidase N (APN, CD13).

[0042] As a preferred embodiment, the human light chain ferritin self-assembly monomer is a natural human light chain ferritin subunit, and the self-assembly body is a homologous twenty-tetrameric cage protein formed by self-assembly of the self-assembly monomer.

[0043] As a preferred embodiment, the present invention utilizes a combination of SUMO-tag and His-tag to achieve the supernatant expression and purification of anti-inflammatory functional modified ferritin, and the functional tag combination at the front end is removed by SUMO protease to restore the protein to an original structure close to that of wild-type human light chain ferritin, and to perform targeted treatment of vascular inflammation. A tag structure is inserted into the front end of the recombinant human light chain ferritin, making the recombinant human light chain ferritin larger than the wild-type human light chain ferritin, and the tag structure extends outward to form a structure similar to a sea urchin. After the tag is removed, the overall structure of the protein remains consistent with the wild-type protein, and is a regular spherical cage.

[0044] Preferably, the polypeptide having an anti-inflammatory effect is selected from any one of a VHP peptide targeting vascular cell adhesion molecule-1, a GHK peptide, an RGD peptide targeting multiple integrin receptors, or an NGR peptide targeting aminopeptidase N (APN, CD13).

[0045] In some specific embodiments, the recombinant human light chain ferritin includes a His-tag functional domain inserted at the N-terminus to promote purification; a SUMO-tag functional domain inserted at the N-terminus to promote expression; a naturally self-assembling human light chain ferritin subunit amino acid sequence and a functional domain inserted in the disordered amino acid sequence region between the fourth and fifth α helices of the self-assembling protein subunit, which is a VHP peptide targeting VCAM-1.

[0046] Preferably, the amino acid sequence of the functional domain SUMO-tag inserted at the N-terminus for promoting expression is shown as SEQ ID No.5.

[0047] Preferably, the amino acid sequence of the functional domain His-tag inserted at the N-terminus for promoting purification is as shown in SEQ ID No.4.

[0048] Preferably, the amino acid sequence of the naturally self-assembled human light chain ferritin single subunit is as shown in SEQ ID No.3.

[0049] Preferably, the amino acid sequence of the functional domain inserted into the disordered amino acid sequence region between the fourth and fifth α helices of the self-assembling protein subunit is as shown in SEQ ID No.2.

[0050] Preferably, the single subunit amino acid sequence of the recombinant human light chain ferritin sequence with a His-tag and a SUMO-tag at the N-terminus is as shown in SEQ ID No.1.

[0051] In a second aspect, the present invention provides a nucleic acid molecule encoding the functionally modified human light chain ferritin described in the first aspect.

[0052] In a third aspect, the present invention provides an expression vector, into which the nucleic acid molecule described in the second aspect is inserted, and after transforming or transfecting the host cell, the expression vector can make the transformed or transfected host cell express the functionally modified human light chain ferritin described in the first aspect.

[0053] In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell comprises the expression vector described in the third aspect, or the nucleic acid molecule described in the second aspect is integrated into the genome of the recombinant cell.

[0054] Preferably, the recombinant cell comprises Escherichia coli.

[0055] In a fifth aspect, the present invention provides a method for preparing the functionally modified human light chain ferritin according to the first aspect, the preparation method comprising: Construct the expression vector described in the third aspect; transfer the expression vector into a host cell and express it; obtain the target protein with a His-tag and a SUMO-tag at the amino terminus; use SUMO protease to identify the cleavage site and perform cleavage; and obtain recombinant human light chain ferritin.

[0056] The subunit amino acid sequence of the functionally modified human light chain ferritin with His-tag and SUMO-tag described in the present invention is shown in SEQ ID No.1.

[0057] The present invention also provides a method for constructing recombinant human light chain ferritin, the specific steps of which include: (1) Using the natural human light chain ferritin gene sequence as a template, a fusion protein recombinant expression vector with a His-tag and a SUMO-tag at the amino terminus and a VHP peptide at the fourth and fifth α-helical disordered regions at the carboxyl terminus was constructed, the sequence of which is shown in SEQ ID No. 1; (2) The recombinant vector is transferred into a host cell for expression to obtain a recombinant human light chain ferritin fusion protein having a His-tag and a SUMO-tag at the amino terminus and a VHP peptide at the fourth and fifth α-helical disordered regions at the carboxyl terminus.

[0058] In a sixth aspect, the present invention provides a functionally modified human light chain ferritin, which is obtained by enzymatically removing the His-tag and SUMO-tag domains with SUMO protease in a buffer solution, and after purification, a functionally modified human light chain ferritin (LFn-VHP) with anti-vascular inflammation is obtained.

[0059] Preferably, the purification treatment includes ammonium sulfate precipitation, dialysis purification, affinity chromatography, ion chromatography, etc., but is not limited thereto.

[0060] Preferably, the storage condition screening of the functionalized modified human light chain ferritin includes different buffer systems, pH values, salt ion concentrations, temperatures, etc., but is not limited thereto.

[0061] Preferably, the method for preparing the functionalized modified human light-chain ferritin further comprises: connecting a fluorescent substance to the functionalized modified human light-chain ferritin by chemical modification.

[0062] As a preferred technical solution of the present invention, the preparation method specifically comprises: (1) Construction and expression of functional proteins The recombinant expression vector is transferred into the host cell by the CaCl2 method, a monoclonal bacterium is picked and inoculated into a culture medium, kanamycin sulfate is added, and the culture is carried out at 37°C for 12-20 hours, antibiotics are added, and the culture is carried out at 37°C for 2-4 hours, isopropyl thiogalactoside is added, and the induction culture is continued at 27°C for 12-20 hours, and the bacteria are collected by centrifugation, and the collected bacteria are resuspended in a lysis buffer, placed on ice for ultrasonic disruption, centrifuged at 4°C to obtain the supernatant, and dialysis, liquid exchange, filtration and impurity removal, and affinity chromatography are performed in sequence to obtain recombinant human light chain ferritin with His-tag and SUMO-tag.

[0063] (2) Preparation of functional proteins The recombinant human light chain ferritin with His-tag and SUMO-tag obtained in step (1) is placed in tris(hydroxymethyl)aminomethane, sodium chloride, β-mercaptoethanol buffer and SUMO protease for enzyme digestion at 4°C for 12-20 hours, and then subjected to high-speed centrifugation, filtration to remove impurities, affinity chromatography, and dialysis purification in sequence to obtain the target protein. Qualitative analysis is performed by SDS-PAGE, and then polyethylene glycol 20000 (PEG 20000) dialysis concentration and ultrafiltration centrifuge tube concentration are used. The protein concentration is determined using a nucleic acid protein quantifier and a BCA protein concentration detection kit, and then sterilized and filtered again to obtain a higher concentration of functionalized human light chain ferritin.

[0064] More specifically, the method for preparing the functionalized modified human light chain ferritin mainly includes the following three steps: Step E1: inserting His-tag (shown in SEQ ID No.4) and SUMO-tag (shown in SEQ ID No.5) into the amino terminus of natural human light chain ferritin, inserting VHP functional peptide (shown in SEQ ID No.2) into the disordered structure region of the fourth and fifth α helices at the carboxyl terminus, and then connecting the mutated gene sequence to a vector, transforming, expressing, separating and purifying, and obtaining recombinant human light chain ferritin with His-tag and SUMO-tag (shown in SEQ ID No.1).

[0065] Specifically, in this step, His-tag and SUMO-tag are inserted into the amino terminus of the natural human light chain ferritin monomer amino acid sequence, and VHP functional peptide is inserted into the fourth and fifth α-helical disordered structure regions of the carboxyl terminus. Based on this human light chain ferritin mutant sequence, and on the premise of being familiar with the mutant amino acid sequence and the cDNA complete gene sequence of the natural human light chain ferritin monomer, those skilled in the art can easily obtain and realize the construction of the recombinant vector. Subsequently, the constructed recombinant expression vector is transferred into E. coli (DE3) competent cells were used for picking, culture and expression of recombinant proteins. After centrifugation, fragmentation, affinity chromatography and filtration, fusion proteins with His-tag and SUMO-tag were obtained.

[0066] Step E2: His-tag and SUMO-tag removal: The tagged protein obtained in step E1 and SUMO protease are placed in a tris (hydroxymethyl) aminomethane (Tris), sodium chloride, β-mercaptoethanol buffer, and digested at 4°C for 12-20 hours. Centrifugation, filtration, affinity chromatography, and dialysis purification are performed in sequence to obtain the target fusion protein.

[0067] Step E3: Preparation of functionalized human light chain ferritin: Use polyethylene glycol 20000 (PEG 20000) for dialysis concentration and ultrafiltration concentration, use a nucleic acid protein quantifier and a BCA protein concentration detection kit to determine the protein concentration, and sterilize and filter again to obtain a higher concentration of functionalized human light chain ferritin.

[0068] In a seventh aspect, the present invention provides a pharmaceutical composition, the active ingredients of which include the anti-inflammatory functionalized human light chain ferritin described in the first aspect.

[0069] Preferably, the anti-inflammatory functionalized modified human light chain ferritin is administered by intravenous injection.

[0070] In an eighth aspect, the present invention provides the use of the functionally modified human light chain ferritin described in the first aspect in drugs for vascular inflammation and chronic inflammation-related diseases.

[0071] The concentrations of reagents involved in the above steps, such as human light chain ferritin, SUMO protease, and the specific concentrations of reagents used in the purification step, are all selected by those skilled in the art under the guidance of the above preparation method according to the specific circumstances, and are all within the scope of protection of the present invention.

[0072] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0073] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0074] The functionally modified human light chain ferritin mentioned in the following specific examples refers to a mutant formed by inserting His-tag and SUMO-tag into the amino terminus of the amino acid sequence of the human light chain ferritin monomer and inserting the VHP functional peptide sequence into the disordered structure region of the fourth and fifth α helices at the carboxyl terminus, i.e., the modified monomer. The recombinant expression vector pET30a(+)-Histag-SUMO-LFn-VHP mentioned refers to the one formed by connecting the coding gene of Histag-SUMO-LFn-VHP to the plasmid vector pET30a(+), wherein the sequence of the pET30a(+) plasmid is shown in SEQ ID No.6.

[0075] The unit "mM" mentioned in the following specific examples represents "mmol / L" (millimole per liter).

[0076] Example 1 Construction, prokaryotic expression and purification of functional protein expression plasmids with His-tag and SUMO-tag

[0077] A1: Based on the cDNA nucleotide sequence of the wild-type LFn monomer, it was transferred into the pET30a(+) vector using molecular biological methods, and the recombinant vector pET30a(+)-Histag-SUMO-LFn-VHP was successfully constructed.

[0078] A2: DNA sequencing confirmed that the mutant gene sequence in the vector was completely correct. The recombinant expression vector pET30a(+)-Histag-SUMO-LFn-VHP was then transferred into E. coliIn BL21 (DE3) competent cells, spread the culture on LB plates in a sterile environment. After culturing overnight at 37°C, pick a single colony from the plate and inoculate it into 5mL LB test tube culture medium, add kanamycin sulfate (final concentration 100 μg / mL), and culture at 37°C and 220 rpm for 12-20 hours. According to the inoculation volume of 1%, transfer 5 mL of bacterial solution into a 500 mL LB culture medium flask, add kanamycin sulfate (final concentration 100 μg / mL), and culture at 37°C and 200 rpm for about 2-4 hours (OD 600 Between 0.4-0.6), add isopropylthiogalactoside inducer (final concentration of 0.3 mM IPTG), and continue shaking induction culture at 18°C ​​for 12-20 hours. Collect the cells by centrifugation at 8000 rpm for 5 minutes; wash the collected cells once with tris(hydroxymethyl)aminomethane buffer, resuspend in 40 mL lysis buffer (10 mM Tris, 10 mM β-mercaptoethanol, pH 8.0), and perform ultrasonic disruption in an ice water bath for 60 minutes. Centrifuge the disrupted solution at 12000 rpm for 30 minutes to collect the supernatant.

[0079] A3: Purification of functional protein with tag: The supernatant obtained in step A2 was dialyzed into chromatography loading buffer (20mM Tris, 20mM imidazole, 10mM β-mercaptoethanol, pH 8.0), and the liquid was changed at low temperature for 2-3 times overnight. The precipitate was removed by centrifugation at 12000rpm for 20 min, and the supernatant was sterilized and filtered at 0.22 μm. 5mL HisTrap HP pre-packed column was loaded into the AKTA Pure 25 system, and the column was first equilibrated with loading buffer (20mM Tris, 20mM imidazole, pH 8.0) for 3 column volumes, and then the system was loaded, and the equilibrated solution was used to wash the column after loading. The isocratic gradient elution was adopted, and the elution gradient was 50mM, 100mM, 200mM, 500mM, 1M imidazole, respectively. The chromatography flow rate was 5mL / min, and the sample elution peak was collected. The target protein was detected by SDS-PAGE. The sample eluted with 500 mM imidazole was dialyzed into 20 mM Tris, 10 mM β-mercaptoethanol, pH 8.0 buffer to remove imidazole. The sample was dialyzed 2-3 times at 4°C overnight.

[0080] The protein Histag-SUMO-LFn-VHP obtained in this example is a fusion protein with His-tag and SUMO-tag after mutation, with His-tag and SUMO-tag inserted at the amino terminus of the amino acid sequence, and VHP peptide inserted at the fourth and fifth α-helical disordered structure regions at the carboxyl terminus, and its amino acid sequence is shown in SEQ ID No.1.

[0081] like Figure 1 The SDS-PAGE image of the functionally modified human light chain ferritin with a tag obtained in this example is shown, wherein lane M is a protein molecular weight standard band, lane 1 is a supernatant after fragmentation and a chromatographic loading sample band, lane 2 is a flow-through band of the first cycle chromatography, lane 3 is a flow-through band of the second cycle chromatography, lane 4 is an eluent band of 100 mM imidazole, lane 5 is an eluent band of 200 mM imidazole, and lane 6 is an eluent band of 500 mM imidazole. As can be seen from the figure, a tagged fusion protein (i.e., recombinant human light chain ferritin) that meets the theoretical value is obtained by purification means.

[0082] Example 2 Fusion protein tag removal and separation and purification

[0083] SUMO protease (Biyuntian, P2312M) was slowly added to the tagged fusion protein and mixed evenly, placed in enzyme digestion buffer (20 mM Tris, 150 mM NaCl, 10 mM β-mercaptoethanol, pH 8.0), and digested at 4°C for 12-20 hours. After digestion, the sample was centrifuged at 12000 rpm for 20 min to remove impurities and sterilized by 0.22 μm syringe filter. Affinity chromatography was performed on the AKTA Pure25 chromatography system using a 5 mL HisTrap HP pre-packed column, and the sample was collected. The collected sample was used to detect the target protein using SDS-PAGE, and the sample was dialyzed into 20 mM Tris, 10 mM β-mercaptoethanol, pH 8.0 buffer to remove imidazole and obtain functionalized human light chain ferritin.

[0084] like Figure 2 As shown, it is the SDS-PAGE image of the functionalized human light chain ferritin obtained in this example, lane M expresses the protein molecular weight standard, lane 1 is SUMO protease, lane 2 is the functionalized human light chain ferritin with His-tag and SUMO-tag before protease cleavage, and lane 3 is the functionalized human light chain ferritin after the tag is removed by enzyme cleavage. Figure 2 It can be seen that the LFn-VHP obtained by purification after enzyme digestion is consistent with the theoretical value.

[0085] like Figure 3This is a transmission electron microscope image of the functionalized modified human light chain ferritin obtained in this example. Human light chain ferritin is a protein assembly of a caged protein structure formed by self-assembly, and the protein assembly has a cavity with an inner diameter of about 8 nm. The functionalized modified human light chain ferritin shown in the figure has an assembly structure with a size of about 15-20 nm, and the central part of the protein appears black under an electron microscope, indicating that it has a complete protein cage central cavity.

[0086] like Figure 4 The mass spectrum of the functionalized human light chain ferritin obtained in this example is shown in FIG. Figure 4 It can be seen that the molecular weight of LFn-VHP after enzyme digestion is consistent with the theoretical value.

[0087] Example 3 Preparation of functionalized human light chain ferritin

[0088] The fusion protein obtained in Example 2 was qualitatively analyzed by SDS-PAGE, and then dialysis with PEG 20000 for concentration reduction and ultrafiltration concentration. The protein concentration was determined using a nucleic acid protein quantifier and a BCA protein concentration determination kit, and then sterilized and filtered through a 0.22 μm syringe filter to obtain a high concentration of functionally modified LFn-VHP protein.

[0089] Example 4 Experiment on targeting inflammatory cells with functional human light chain ferritin

[0090] Inflammation induction experiment was performed on human umbilical vein endothelial cells (HUVEC) using 1 μg / mL LPS: After LPS stimulated HUVEC cells in 48-well plates for 24 h, VCAM-1 was highly expressed, and then wild-type ferritin and functional ferritin labeled with FITC were used for fluorescent labeling. FITC was coupled to the protein at a molar ratio of 20:1, and ultrafiltration was performed to remove free FITC. The protein concentration of coupled FITC was then determined, and the protein was diluted to a final concentration of 25 μg / mL using serum-free Gibco RPMI Medium 1640 cell culture medium for cell incubation for 2 h. The number of FITC-labeled cells was detected using a flow cytometer. FITC-labeled cells under the same conditions were washed 2-3 times with PBS and fixed with methanol. After the cells were fixed, the cell nuclei were stained with Hoechst dye, and fluorescence microscopy was performed after the staining was completed.

[0091] like Figure 5 As shown, it is the flow cytometry diagram of the functionally modified human light chain ferritin obtained in this example. Figure 6As shown, it is the cell fluorescence image of the functionalized human light chain ferritin obtained in this example. Figure 5 and 6 It can be seen that the LFn-VHP protein after enzyme cleavage can successfully target inflammatory cells.

[0092] In summary, the present invention constructs the Histag-SUMO-LFn-VHP functional protein through the above technical scheme, utilizes the SUMO-tag at the amino terminus of the functional protein to promote the expression of the insoluble modified human ferritin, uses the His-tag to purify the protein, and utilizes the fourth and fifth α-helical disordered structure regions at the carboxyl terminus to insert the VHP functional peptide to target inflammatory cells and perform inflammation inhibition treatment, providing a new treatment strategy for vascular inflammation.

[0093] The preparation process provided in the above embodiments of the present invention has simple steps and mild conditions, which solves the technical problems that human light chain ferritin is difficult to transform and express; and the obtained functional protein has good dispersibility, stability and biosafety, and has potential learning and application value in the treatment of vascular inflammation and functional modification of human ferritin.

[0094] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0095] The various aspects, embodiments, features and examples of the present invention should be considered as illustrative in all aspects and are not intended to limit the present invention, the scope of the present invention is defined only by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0096] The use of headings in the present invention is not meant to limit the present invention; both the Summary and Detailed Description sections may apply to any aspect, embodiment, or feature of the present invention.

[0097] Throughout this disclosure, where a composition is described as having, containing, or comprising particular components, or where a process is described as having, containing, or comprising particular process steps, it is contemplated that the compositions taught by the present invention also consist essentially of, or consist of, the recited components, and that the processes taught by the present invention also consist essentially of, or consist of, the recited process steps.

[0098] Unless specifically stated otherwise, use of the terms "include," "includes," "including," "have," "has," or "having" should generally be construed as open ended and non-limiting.

[0099] It should be understood that the order of the various steps or the order in which a particular action is performed is not very important, as long as the teachings of the present invention remain operable. In addition, two or more steps or actions may be performed simultaneously.

[0100] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or capable of achieving the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. A recombinant human light chain ferritin, comprising a functional tag combination inserted at the N-terminus, a self-assembly, and a functional domain inserted into the disordered amino acid sequence region between the fourth and fifth α helices of a subunit of the self-assembly; wherein, The functional tag combination consists of a functional domain for promoting expression inserted at the N-terminus and a functional domain for promoting purification inserted at the N-terminus; The functional domain of the disordered amino acid sequence region is a polypeptide having a targeted anti-inflammatory effect; The self-assembly body is a homologous twenty-tetrameric cage protein formed by self-assembly of self-assembly monomers.

2. The recombinant human light chain ferritin according to claim 1, characterized in that The functional domain of the disordered amino acid sequence region is selected from any one of a VHP peptide targeting vascular cell adhesion molecule-1, a GHK peptide, an RGD peptide targeting multiple integrin receptors, or an NGR peptide targeting aminopeptidase N (APN / CD13); The human light chain ferritin self-assembling monomer is a natural human light chain ferritin subunit.

3. The recombinant human light chain ferritin according to claim 1 or 2, characterized in that: The amino acid sequence of the functionally modified human light chain ferritin is as shown in SEQ ID No. 1; And / or, the functional domain promoting purification is a His-tag; And / or, the functional domain promoting expression is a SUMO-tag; and / or, the amino acid sequence of His-tag is shown in SEQ ID No.4; and / or, the amino acid sequence of SUMO-tag is shown in SEQ ID No.5; and / or, the amino acid sequence of a natural human light chain ferritin subunit is as shown in SEQ ID No. 3; and / or, the amino acid sequence of the functional domain in the disordered amino acid sequence region is as shown in SEQ ID No. 2; Preferably, the recombinant human light chain ferritin further comprises a fluorescent substance connected to the functionalized modified human light chain ferritin by chemical modification.

4. A nucleic acid molecule encoding the recombinant human light chain ferritin according to any one of claims 1 to 3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector is inserted with the nucleic acid molecule according to claim 4, and the expression vector can, after transforming or transfecting the host cell, cause the transformed or transfected host cell to express the functionally modified human light chain ferritin according to any one of claims 1 to 3.

6. A method for preparing recombinant human light chain ferritin, characterized in that: The specific steps include: Using the natural human light chain ferritin gene sequence as a template, constructing the recombinant expression vector as claimed in claim 5; The recombinant expression vector is transferred into a host cell for expression to obtain recombinant human light chain ferritin.

7. A functionally modified human light chain ferritin, characterized in that: The recombinant human streptavidin as claimed in claim 1 is obtained by enzymatically removing the functional tags.

8. A method for preparing the functionalized modified human light chain ferritin according to claim 7, characterized in that: The specific steps include: Using the natural human light chain ferritin gene sequence as a template, constructing the recombinant expression vector as claimed in claim 5; The recombinant expression vector is transferred into a host cell for expression to obtain recombinant human light chain ferritin; Using SUMO protease to cut off the His-tag domain and the SUMO-tag domain at the N-terminus of the recombinant human light chain ferritin; The functionally modified human light chain ferritin is obtained.

9. A pharmaceutical composition, characterized in that The active ingredient of the pharmaceutical composition includes the functionally modified human light chain ferritin as claimed in claim 7; Preferably, the functionally modified light chain ferritin is administered by intravenous injection.

10. Use of the functionally modified human light chain ferritin according to any one of claim 7 in the preparation of drugs for preventing vascular inflammation and / or chronic inflammation-related diseases.

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