Construction method and application of BCMA full-length receptor in nano phospholipid plate

Abstract

The invention relates to the technical field of biology, and discloses a construction method and application of a BCMA full-length receptor in a nano phospholipid plate. The construction method of the BCMA full-length receptor comprises the following steps: reacting a BCMA extracellular domain, a BCMA transmembrane region and an intracellular domain containing site-specific mutagenesis, CaCl2 and sortase A, after the reaction is finished, purifying by using a nickel affinity column, collecting an imidazole elution component, and enriching to obtain the BCMA full-length receptor in the nano phospholipid plate. According to the construction method, the extracellular domain of the BCMA and the BCMA transmembrane region and the intracellular domain in the nano phospholipid plate are connected through Sortase A enzyme, the BCMA full-length receptor is obtained in an in-vitro near-physiological environment, the method is simple, protein is easy to obtain, and the defect that the transmembrane region is poor in solubility is overcome; meanwhile, the structure-function of the BCMA can be studied in vitro by marking the functional structure domain of the BCMA at a fixed point.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing and applying a full-length BCMA receptor in a nanophospholipid disc. Background Technology

[0002] B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily and is one of the receptors for a proliferation-inducing ligand (APRIL) and a B-cell activating factor (BAFF). Diseases associated with BCMA include multiple myeloma, systemic lupus erythematosus, and chronic lymphocytic leukemia. Currently, various therapies have been developed in this field, including antibody-drug conjugates, bispecific antibodies, and CAR-T therapy, and the phenomena and mechanisms of these diseases have been comprehensively studied, involving genetic, cellular, animal, and clinical levels. However, in the research process, the main methods are the expression of the full-length BCMA on the cell surface or the expression of the extracellular soluble portion of BCMA in vitro, and research on the structure and function of the full-length BCMA under membrane anchoring is very limited. To solve the above technical problems, this invention provides a method for constructing the full-length BCMA receptor in a nano-phospholipid disc. Summary of the Invention

[0003] This invention provides a method for constructing a full-length BCMA receptor, enabling visualization studies of the structure and function of the full-length BCMA under in vitro membrane anchoring.

[0004] The purpose of this invention is to provide a method for constructing and applying a full-length BCMA receptor in a nanophospholipid disc. The extracellular domain of BCMA and the transmembrane and intracellular domains of BCMA in the nanophospholipid disc are linked using Sortase A enzyme, resulting in a full-length BCMA receptor in a near-physiological environment in vitro. The method is simple, the protein is easy to obtain, and it overcomes the defect of poor solubility of the transmembrane domain. At the same time, it can be used to study the structure-function of BCMA in vitro by site-specific labeling of its functional domains.

[0005] To achieve the above objectives, this invention provides a method for constructing a BCMA transmembrane region and intracellular domain containing site-directed mutagenesis, comprising the following steps:

[0006] Step 1: A cysteine ​​mutation was introduced at a specific site into the gene sequence encoding the transmembrane region and intracellular domain of BCMA, and a downstream sequence GGG linked by Sortase A was inserted at the amino terminus.

[0007] Step 2: Optimize the gene sequence according to the codon preference of E. coli and insert it between the Hind III and BamHI restriction sites of pMM-LR6 to enable efficient expression of the protein in E. coli, resulting in the BCMA transmembrane region and intracellular domain containing site-directed mutations.

[0008] To achieve the above objectives, the present invention also provides a BCMA transmembrane region and intracellular domain containing site-directed mutations, prepared by the above preparation method.

[0009] To achieve the above objectives, the present invention also provides a method for constructing nano-phospholipid disks, the steps of which are as follows:

[0010] The lyophilized sample powder containing the site-directed mutagenesis-containing BCMA transmembrane region and intracellular domain was dissolved in equal volumes of hexafluoroisopropanol and isopropanol to obtain a protein organic solution.

[0011] Long-chain phospholipids and short-chain phospholipids were added sequentially to a protein organic solution, dried under nitrogen, and then freeze-dried overnight to obtain a freeze-dried sample.

[0012] The lyophilized sample was dissolved in urea solution and dialyzed with HEPES solution to obtain a twin-cell solution.

[0013] Membrane scaffold protein MSP was added to a twin-cell solution and incubated. The resulting nanophospholipid discs containing the transmembrane region and intracellular domain of BCMA after site-directed mutation were obtained by dialysis with HEPES solution.

[0014] Furthermore, the ratio of BCMA transmembrane region and intracellular domain containing site-directed mutations is: long-chain phospholipid DMPC: short-chain phospholipid DHPC = 1:200:800 (molar ratio); the ratio of BCMA transmembrane region and intracellular domain containing site-directed mutations is: long-chain phospholipid DMPC: membrane scaffold protein MSP = 1:200:2 (molar ratio).

[0015] Furthermore, the preparation method of the membrane scaffold protein MSP is as follows: a strep tag sequence WSHPQFEK (SEQ ID NO.1) is introduced into the carboxyl terminus of the amino acid sequence of the membrane scaffold protein MSP, and the coding gene sequence of the membrane scaffold protein MSP is optimized with codons preferred by E. coli and then inserted into the vector pET-28b(+) to enable the protein to be expressed efficiently in E. coli, thus obtaining the membrane scaffold protein MSP.

[0016] To achieve the above objectives, the present invention also provides a nano-phospholipid disk, which is prepared by the above construction method.

[0017] To achieve the above objectives, the present invention also provides a method for constructing a full-length BCMA receptor in a nano-phospholipid disc, the steps of which are as follows:

[0018] The extracellular domain of BCMA and the aforementioned nanophospholipid discs were ligated using Sortase A. After the reaction, purification was performed using a nickel affinity column, and the imidazole eluent was collected and enriched to obtain the nanophospholipid discs.

[0019] BCMA full-length receptor.

[0020] Among them, the extracellular domain of BCMA: the nanophospholipid disc contains the site-directed mutated transmembrane region and intracellular domain of BCMA: CaCl2: Sortase A = 0.6mM: 0.6mM: 0.1mM: 2.5μM.

[0021] Furthermore, the preparation method of the BCMA extracellular domain is as follows:

[0022] Step 1: A histidine tag HHHHHH (SEQ ID NO. 2) is introduced at the amino terminus of the extracellular domain amino acid sequence of BCMA, and a Sortase A enzyme ligation upstream site sequence LPETGG (SEQ ID NO. 3) is introduced at the carboxyl terminus.

[0023] Step 2: The coding DNA sequence of the BCMA extracellular domain was optimized with codons preferred by E. coli. The optimized gene was constructed between the BamHI and SalI restriction sites of the vector pGEX-6p-1. The original HRV3C restriction site LEVLFQGP (SEQ ID NO.4) on the vector was mutated to the TEV restriction site ENLYFQS (SEQ ID NO.5) to enable efficient expression of the protein in E. coli and obtain the BCMA extracellular domain.

[0024] To achieve the above objectives, the present invention also provides a full-length BCMA receptor in a nanophospholipid disc, which is prepared by the above preparation method.

[0025] To achieve the above objectives, the present invention also provides the application of the above-mentioned BCMA transmembrane region and intracellular domain containing site-directed mutations, the above-mentioned nanophospholipid discs, or the full-length BCMA receptor in the above-mentioned nanophospholipid discs in in vitro studies of BCMA structure and function.

[0026] The advantages and positive effects of the method for constructing and applying the full-length BCMA receptor in the nanophospholipid discs described in this invention are as follows:

[0027] This invention connects the extracellular domain of BCMA and the transmembrane region of BCMA in nanophospholipid discs to the intracellular domain using Sortase A enzyme, obtaining the full-length BCMA receptor in a near-physiological environment in vitro. The method is simple, the protein is easy to obtain, and it overcomes the defect of poor solubility of the transmembrane region. At the same time, it can conduct structure-function studies of BCMA in vitro by site-specific labeling of its functional domains.

[0028] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0029] Figure 1 This is a flowchart and schematic diagram illustrating the construction process and principle of the full-length BCMA receptor in the nanophospholipid disc of the present invention.

[0030] Figure 2 The HPLC purification results of TMCT in Example 1 of this invention;

[0031] Figure 3 The mass spectrometry results of TMCT in Example 1 of this invention;

[0032] Figure 4 The FPLC results of the ECD in Embodiment 2 of the present invention;

[0033] Figure 5 The mass spectrometry results of ECD in Example 2 of this invention;

[0034] Figure 6 This is a gel image of the purified membrane scaffold protein in Example 3 of the present invention;

[0035] Figure 7 The image shows the gel image of the nanophospholipid disc in Example 4 of this invention;

[0036] Figure 8 This is a gel image of the full-length BCMA receptor in Example 5 of the present invention. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0038] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0039] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0040] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0041] The plasmid vectors and culture media used in this invention, such as pMM-LR6, pGEX-6p-1, pET-28b(+), LB medium, etc., were all available for purchase.

[0042] The construction process and principle of the full-length BCMA receptor in the nanophospholipid disc in this invention are as follows: Figure 1 As shown.

[0043] Example 1: Preparation and characterization of the transmembrane region and intracellular domain (TMCT) of BCMA

[0044] A method for preparing BCMA transmembrane and intracellular domains containing site-directed mutations includes the following steps:

[0045] Step 1: A cysteine ​​mutation was introduced at a specific site into the gene sequence encoding the transmembrane region and intracellular domain of BCMA, and a downstream sequence GGG linked by Sortase A was inserted at the amino terminus.

[0046] Step 2: Optimize the gene sequence according to the codon preference of E. coli and insert it between the Hind III and BamHI restriction sites of pMM-LR6 to enable efficient expression of the protein in E. coli, resulting in the BCMA transmembrane region and intracellular domain containing site-directed mutations.

[0047] Among them, after optimization, the gene sequence encoding the BCMA transmembrane region and intracellular domain is GGTGGTGG TATCTTGTGGACCTTTCTGGGTCTGAGCCTGATCATCTCTCTGGCTGTGTTCGTTCTGAGCTTTCTGCTGCGTAAGATCAACTCTGAACCGCTGAAAGACGAATTTAAGAACACCGGTTCTGGTCTGCTGGGTAAAGCGAACATCGACCTGGAGAAGAGCCGTACCGGTGACGAAATCATTCTGCCGCGTGGTCTGGAA TACACCGTTGAAGAAGCTACCGCTGAAGATGCGATCAAATCTAAACCGAAAGTTGACTCTGACCACGCGTTTCCGCTGCCGGCACTGGAAGAAGGTTGCACCACCTAGACCAACGACTACTCCAAATCTCTGCCGGCTGCTCTGTCTGCGACCGAAATCGAGAAGTCCATCTCTGCGCGTTAA(SEQ ID NO.6).

[0048] The amino acid sequences of the transmembrane region and intracellular domain of BCMA are: GGGILWTFLGLSLIISLAVFVL SFLLRKINSEPLKDEFKNTGSGLLGKANIDLEKSRTGDEIILPRGLEYTVEEA TAEDAIKSKPKVDSDHAFPLPALEEGCTILVTTKTNDYSKSLPAALSATEIE KSISAR (SEQ ID NO.7).

[0049] This invention does not impose any particular limitation on the protein expression method; any method well-known to those skilled in the art can be used, such as prokaryotic expression or eukaryotic expression. In this embodiment, the optimized coding gene fragment is subcloned into a plasmid for expression; the preferred expression method is prokaryotic expression; the preferred host is *Escherichia coli*, and the preferred plasmid is pMM-LR6.

[0050] The expression and purification methods are described in detail below:

[0051] Transform 1 μL of the expression vector containing the transmembrane and intracellular domains of BCMA into E. coli host cells.

[0052] In BL21(DE3), transformed single colonies were cultured overnight on antibiotic plates (containing 100 μg / mL Kana). Well-grown single colonies were then transferred to LB medium and cultured at 37°C until OD... 600 When the bacterial cell concentration was 0.6–0.8, IPTG was added as an inducer, and expression was induced overnight at 18°C. The bacterial cell pellet was collected by centrifugation and resuspended in 20 mM Tris + 150 mM NaCl (pH 7.4), followed by sonication for lysis. After lysis, the pellet was centrifuged at 12,000 rpm for 30 min, the supernatant was discarded, and the lysed pellet was resuspended in guanidine hydrochloride buffer (6 M guanidine hydrochloride + 50 mM Tris + 200 mM NaCl (pH 8.0)) and stirred overnight. The next day, the pellet was centrifuged at 12,000 rpm for 90 min, and the supernatant was collected. A certain volume of nickel packing material was equilibrated using guanidine hydrochloride buffer (6M guanidine hydrochloride, 50mM Tris, 200mM NaCl, pH 8.0). After equilibration, the buffer was discarded, and the supernatant from the previous centrifugation step was poured into the column for incubation. After incubation, flow-through was performed. Impurities were then washed away using guanidine hydrochloride buffer (6M guanidine hydrochloride, 50mM Tris, 200mM NaCl, pH 8.0), urea (8M), and deionized water. Finally, the target protein was eluted with formic acid. Cyanogen bromide solid was added and vortexed to dissolve the protein. The reaction was carried out under nitrogen gas, and after the reaction, the sample was dialyzed against deionized water. The sample was lyophilized overnight. The lyophilized sample was dissolved in 9 mL of deionized water, 9 mL of HPLC organic phase solution (50% IPA, 50% ACN, 0.1% TFA), and 2 mL of formic acid. The sample was then subjected to HPLC twice, and the target protein was collected based on the elution peaks. The sample was lyophilized overnight to obtain the sample powder.

[0053] HPLC purification results are as follows Figure 2 As shown, the mass spectrometry results are as follows: Figure 3 As shown. Figure 2 The middle arrow points to the isolated BCMA transmembrane region and intracellular domain proteins. From Figure 3 The mass spectrometry results show that its molecular weight is almost consistent with the theoretical molecular weight, which is the target product of this embodiment.

[0054] Example 2: Construction and characterization of extracellular domain ECD

[0055] The method for preparing the extracellular domain of BCMA is as follows:

[0056] Step 1: A histidine tag HHHHHH (SEQ ID NO. 2) is introduced at the amino terminus of the extracellular domain amino acid sequence of BCMA, and a Sortase A enzyme ligation upstream site sequence LPETGG (SEQ ID NO. 3) is introduced at the carboxyl terminus.

[0057] Step 2: The coding DNA sequence of the BCMA extracellular domain was optimized with codons preferred by E. coli. The optimized gene was constructed between the BamHI and SalI restriction sites of the vector pGEX-6p-1. The original HRV3C restriction site LEVLFQGP (SEQ ID NO.4) on the vector was mutated to the TEV restriction site ENLYFQS (SEQ ID NO.5) to enable efficient expression of the protein in E. coli and obtain the BCMA extracellular domain.

[0058] The optimized gene sequence encoding the extracellular domain of BCMA is: catcatcatcatcatcacCTGG GATCCATGCTGCAGATGGCTGGTCAATGCTCTCAGAACGAATACTTCGACTCTCTGCTGCACGCATGCATCCCGTGCCAGCTGCGTTGCAGCTCCAACACTCCACCGCTGACTTGCCAGCGTTACTGCAACGCGTCCGTGACTAACTCTGTGAAAGGTACCAACGCGCTGCCAGAAACTGGCGGTTAA (SEQ ID NO. 8).

[0059] The amino acid sequence of the extracellular domain of BCMA is HHHHHHLGSMLQMAGQCSQNEYFDSL LHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNALPETGG (SEQ ID NO.9).

[0060] This invention does not impose any particular limitation on the protein expression method; any method well-known to those skilled in the art can be used, such as prokaryotic expression or eukaryotic expression. In this embodiment, the optimized coding gene fragment is subcloned into a plasmid for expression; the preferred expression method is prokaryotic expression; the preferred host is *Escherichia coli*, and the preferred plasmid is pGEX-6p-1.

[0061] The expression and purification methods are described in detail below:

[0062] Transform 1 μL of the BCMA extracellular domain expression vector into E. coli host BL21(DE3) and culture the transformed single colonies overnight on antibiotic plates (containing 100 μg / mL Amp). Select well-grown single colonies and culture them in LB medium at 37°C until OD... 600 When the protein concentration was 0.6–0.8, IPTG was added as an inducer, and expression was induced at 37°C for 4 hours. The bacterial pellet was collected by centrifugation and resuspended in 50 mM Tris and 300 mM NaCl (pH 8), followed by sonication for lysis. The supernatant was collected after lysis, loaded onto a column, and washed with Tris buffer (50 mM Tris, 300 mM NaCl, pH 8) to remove impurities. The target protein was eluted with 20 mM GSH. After enrichment using ultrafiltration concentrators, the protein was digested with TEV. The digested protein was purified by rapid protein liquid chromatography (FPLC), and the enriched protein was verified by gel electrophoresis.

[0063] FPLC purification results are as follows Figure 4 As shown, the mass spectrometry results are as follows: Figure 5 As shown. Figure 4 The middle arrow points to the BCMA extracellular domain protein. From Figure 5 The mass spectrometry results show that its molecular weight is almost consistent with the theoretical molecular weight, which is the target product of this embodiment.

[0064] Example 3: Preparation and characterization of membrane scaffold protein MSP

[0065] The preparation method of membrane scaffold protein MSP is as follows: a strep tag sequence WSHPQFEK (SEQ ID NO.1) is introduced into the carboxyl terminus of the amino acid sequence of membrane scaffold protein MSP, and the coding gene sequence of membrane scaffold protein MSP is optimized with codons preferred by E. coli and then inserted into the vector pET-28b(+) to enable the protein to be expressed efficiently in E. coli, thus obtaining membrane scaffold protein MSP.

[0066] Among them, after optimization, the coding gene sequence of the membrane scaffold protein MSP is ATGGGCTCCTCTA CCTTTTCCAAACTGCGTGAACAGCTGGGTCCGGTTACGCAGGAATTCTGGGATAACCTGGAAAAAGAAACGGAGGGCCTGCGTCAGGAAATGAGCAAAGATCTGGAAGAAGTGAAGGCTAAAGTTCAGCCGTACCTGGATGACTTCCAGAAAAAGTGGCAGGAAGAGATGGAACTGTACCGTCAGAAGGTTGAACCGCTGCGTGCAGAACTGCAGGAAGGCGCACGTCAGAAACTGCATGAACTGCAGGAAAAGCTGTCTCCGCTGGGTGAAGAAATGCGTGACCGTGCACGTGCTCATGTCGACGCGCTGCGCACGCACCTGGCGCCGTACTCTGATGAACTGCGTCAGCGTCTGGCTGCTCGCCTGGAAGCACTGAAGGAAAACGGTGGCGCACGTCTGGCTGAATACCACGCGAAAGCAACCGAGCACCTGTCTACCCTGAGCGAGAAAGCGAAGCCGGCGCTGGAGGACCTGCGCCAGGGCCTGCTGCCAGTACTGGAGTCTTTCAAGGTTAGCTTCCTGTCTGCTCTGGAGGAATACACCAAAAAGCTGAACACCCAGGGCGGCGGTGGTTCCAGCTCTGCGTGGTCTCACCCGCAGTTCGAAAAA(SEQ ID NO.10).

[0067] The amino acid sequence of the membrane scaffold protein MSP is MGSSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGGGGSSSAWSHPQFEK(SEQ ID NO.11).

[0068] This invention does not specifically limit the protein expression method; any method well-known to those skilled in the art, such as prokaryotic or eukaryotic expression, can be used. In this embodiment, the optimized coding gene fragment is subcloned into a plasmid for expression; the preferred expression method is prokaryotic expression; the preferred host is *Escherichia coli*, and the preferred plasmid is pET-28b(+).

[0069] The expression and purification methods are described in detail below:

[0070] Transform 1 μL of the expression vector from MSP into *E. coli* host BL21(DE3) and culture the transformed single colonies overnight on antibiotic plates (containing 100 μg / mL Kana). Select well-grown single colonies and incubate at 37°C in LB medium until OD (Organic Growth Rate) is reached. 600 When the concentration was 0.6–0.8, IPTG was added as an inducer, and expression was induced at 37°C for 4 hours. The bacterial pellet was collected by centrifugation and resuspended in 50 mM Tris-500 mM NaCl (pH 8), followed by sonication for lysis. The supernatant was collected after lysis, loaded onto a column, and washed with Tris buffer (50 mM Tris-500 mM NaCl, pH 8) and HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) to remove impurities. The target protein was eluted with 2.5 mM desthiobiotin. Enrichment was performed using ultrafiltration concentrators, followed by gel electrophoresis. The results are shown below. Figure 6 As shown.

[0071] Example 4: Construction and Characterization of Nanophospholipid Discs

[0072] A method for constructing nanophospholipid disks, comprising the following steps:

[0073] The lyophilized protein sample powder containing the transmembrane and intracellular domains of BCMA was dissolved in equal volumes of hexafluoroisopropanol and isopropanol, and the protein concentration was measured to obtain a protein organic solution. The amount of phospholipid used was calculated according to a molar ratio of BCMA transmembrane and intracellular domains containing site-directed mutagenesis: long-chain phospholipid DMPC: short-chain phospholipid DHPC of 1:200:800. The phospholipid was then added to the protein organic solution, dried under nitrogen, and lyophilized overnight. The next day, the lyophilized sample was dissolved in urea solution (8M urea in 20mM HEPES 75mM NaCl pH 7.4), and dialysis was performed using HEPES solution (20mM HEPES 75mM NaCl pH 7.4) to construct twins. After construction, the molar ratio of BCMA transmembrane and intracellular domains containing site-directed mutations to long-chain phospholipid DMPCs to membrane scaffold protein MSPs was 1:200:2. The amount of MSP was calculated, and then the MSPs were added to the bicellular solution. After incubation for half an hour, the mixture was dialyzed with HEPES solution (20 mM HEPES, 75 mM NaCl, pH 7.4) to obtain nanophospholipid discs containing the intracellular and transmembrane domains of BCMA. The gel electrophoresis results are as follows. Figure 7 As shown, by Figure 7 It can be concluded that a nanophospholipid disk sample containing the BCMA transmembrane region and intracellular domain was successfully obtained.

[0074] Example 5: Construction and characterization of the full-length BCMA receptor

[0075] The extracellular domain of BCMA and the transmembrane region of BCMA in nanophospholipid discs were ligated to the intracellular domain using Sortase A. The reaction was carried out at a ratio of BCMA extracellular domain: nanophospholipid disc containing the site-directed mutagenic BCMA transmembrane region and intracellular domain: CaCl2: Sortase A = 0.6 mM: 0.6 mM: 0.1 mM: 2.5 μM. After 5 h, the reaction was completed, and purification was performed using a nickel affinity column. The elution fractions from 30 mM imidazole to 250 mM imidazole were collected and enriched, and validated by gel electrophoresis. The results are as follows: Figure 8 As shown, by Figure 8 It can be concluded that a nanophospholipid disk sample containing the full-length BCMA receptor was successfully constructed.

[0076] Therefore, this invention employs the above-mentioned method for constructing and applying the full-length BCMA receptor in a nanophospholipid disc. The extracellular domain of BCMA and the transmembrane region and intracellular domain of BCMA in the nanophospholipid disc are linked using Sortase A enzyme, resulting in the full-length BCMA receptor in a near-physiological environment in vitro. The method is simple, the protein is easy to obtain, and it overcomes the defect of poor solubility of the transmembrane region. At the same time, it can conduct structure-function studies of BCMA in vitro by site-specific labeling of its functional domains.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing BCMA transmembrane region and intracellular domain containing site-directed mutagenesis, characterized in that, Includes the following steps: Step 1: A cysteine ​​mutation was introduced at a specific site into the gene sequence encoding the transmembrane region and intracellular domain of BCMA, and a downstream sequence GGG linked by Sortase A was inserted at the amino terminus. Step 2: Optimize the gene sequence according to the codon preference of E. coli and insert it between the HindIII and BamHI restriction sites of pMM-LR6 to enable efficient expression of the protein in E. coli, resulting in the BCMA transmembrane region and intracellular domain containing site-directed mutations.

2. A BCMA transmembrane region and intracellular domain containing site-directed mutagenesis, characterized in that: It is prepared by the preparation method described in claim 1.

3. A method for constructing nanophospholipid discs, characterized in that, The steps are as follows: The lyophilized sample powder containing the site-directed mutagenesis-mediated BCMA transmembrane region and intracellular domain as described in claim 2 was dissolved in equal volumes of hexafluoroisopropanol and isopropanol to obtain a protein organic solution. Long-chain phospholipids and short-chain phospholipids were added sequentially to a protein organic solution, dried under nitrogen, and then freeze-dried overnight to obtain a freeze-dried sample. The lyophilized sample was dissolved in urea solution and dialyzed with HEPES solution to obtain a twin-cell solution. Membrane scaffold protein MSP was added to a twin-cell solution and incubated. The resulting nanophospholipid discs containing the transmembrane region and intracellular domain of BCMA after site-directed mutation were obtained by dialysis with HEPES solution.

4. The construction method according to claim 3, characterized in that, The molar ratio of BCMA transmembrane region and intracellular domain containing site-directed mutations: long-chain phospholipid DMPC: short-chain phospholipid DHPC = 1:200:800; the molar ratio of BCMA transmembrane region and intracellular domain containing site-directed mutations: long-chain phospholipid DMPC: membrane scaffold protein MSP = 1:200:

2.

5. The construction method according to claim 4, characterized in that, The preparation method of membrane scaffold protein MSP is as follows: a strep tag sequence WSHPQFEK is introduced into the carboxyl terminus of the amino acid sequence of membrane scaffold protein MSP, and the coding gene sequence of membrane scaffold protein MSP is optimized with codons preferred by E. coli and then inserted into the vector pET-28b(+) to enable the protein to be expressed efficiently in E. coli and obtain membrane scaffold protein MSP.

6. A nano-phospholipid disc, characterized in that, It is prepared by the construction method according to any one of claims 3-5.

7. A method for constructing a full-length BCMA receptor in a nanophospholipid disc, characterized in that, The steps are as follows: The extracellular domain of BCMA and the nanophospholipid disc described in claim 6 were linked by Sortase A enzyme. After the reaction was completed, the mixture was purified using a nickel affinity column, and the imidazole elution fraction was collected and enriched to obtain the full-length BCMA receptor in the nanophospholipid disc.

8. The construction method according to claim 7, characterized in that, The method for preparing the extracellular domain of BCMA is as follows: Step 1: A histidine tag HHHHHH is introduced at the amino terminus of the extracellular domain amino acid sequence of BCMA, and a Sortase A enzyme ligation upstream site sequence LPETGG is introduced at the carboxyl terminus. Step 2: The coding DNA sequence of the BCMA extracellular domain was optimized with codons preferred by E. coli. The optimized gene was constructed between the BamHI and SalI restriction sites of the vector pGEX-6p-1, and the original HRV3C restriction site LEVLFQGP on the vector was mutated to the TEV restriction site ENLYFQS, so that the protein was efficiently expressed in E. coli and the BCMA extracellular domain was obtained.

9. A full-length BCMA receptor in a nanophospholipid disc, characterized in that, It is prepared by the construction method according to any one of claims 7-8.

10. The application of the BCMA transmembrane region and intracellular domain containing the site-directed mutation of claim 2, or the full-length BCMA receptor in the nanophospholipid disc of claim 6, or the nanophospholipid disc of claim 9, in in vitro studies of BCMA structure and function.

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