Human beta2-glycoprotein 1 recombinant protein and its use

Abstract

The application relates to the technical field of protein engineering, in particular to a human beta2-glycoprotein 1 recombinant protein and application thereof. The human beta2-glycoprotein 1 recombinant protein has an amino acid sequence as shown in any one of SEQ ID NO:1 to SEQ ID NO:6. The human beta2-glycoprotein 1 recombinant protein provided by the application is high-efficiency expressed in insect cells, can be used for human beta2-GP1 antibody detection, and has consistent reactivity with nature. In the clinical detection process, the recombinant beta2-GP1 antigen shows excellent detection capability by comparison with multiple manufacturers' kits.

Description

Technical Field

[0001] This application relates to the field of protein engineering technology, and more specifically, to the expression and application of recombinant human β2-glycoprotein 1. Background Technology

[0002] β2-glycoprotein 1 (β2-GP1, also known as apolipoprotein H) is a key plasma phospholipid-binding protein in the human body, mainly synthesized by the liver. It is widely involved in physiological processes such as coagulation, complement regulation, and vascular endothelial homeostasis, and is an important functional protein for maintaining the balance between coagulation and anticoagulation. Since researchers confirmed in the 1990s that the core recognition target of antiphospholipid antibodies is β2-GP1 rather than cardiolipin, the clinical detection value of this protein has been gradually clarified. It has now become a core diagnostic marker for autoimmune diseases such as antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE), and has been formally included in the international classification diagnostic criteria for APS. It is a key indicator for clinical thrombosis risk assessment, prediction of pathological pregnancy complications, and monitoring of autoimmune diseases.

[0003] In clinical immunoassay systems, recombinant β2-GP1 protein is a core antigenic raw material for preparing various in vitro diagnostic products such as enzyme-linked immunosorbent assay (ELISA) reagents, colloidal gold assays, and chemiluminescence assays. Its protein purity, spatial conformation, biological activity, and antigen specificity directly determine the accuracy, sensitivity, and specificity of anti-β2-GP1 antibody detection results. High-quality, highly active, and conformationally intact recombinant β2-GP1 protein is a crucial prerequisite for achieving accurate detection of anti-β2-GP1 antibodies and reducing the rate of missed and misdiagnosed cases in clinical practice. It has vital clinical significance for the early screening, disease grading, and prognostic assessment of autoimmune diseases.

[0004] Currently, the preparation methods for β2-GP1 antigen protein used in diagnostic reagents in the industry are mainly divided into two categories: natural protein extraction and prokaryotic expression recombinant preparation. Natural human β2-GP1 protein is isolated and purified from human plasma. Although it possesses the natural conformation and biological activity, it suffers from drawbacks such as limited raw material sources, large batch-to-batch variations in plasma, complex purification processes, and high costs. Furthermore, human plasma carries the risk of viral contamination, making it difficult to meet the safety requirements for large-scale, standardized production of in vitro diagnostic reagents and thus unsuitable for large-scale clinical testing. Existing prokaryotic expression systems (E. coli expression systems) for preparing recombinant β2-GP1 protein offer advantages such as rapid propagation, low cost, and high yield, but lack a eukaryotic protein post-translational modification system, making it impossible to complete the glycosylation modification, spatial folding, and conformational remodeling of the β2-GP1 protein. The protein's spatial conformation differs significantly from that of natural human β2-GP1, and the antigenic epitope exposure is incomplete. This makes it impossible to accurately identify specific anti-β2-GP1 antibodies in serum, leading to false negatives and false positives. The detection sensitivity and specificity are poor, severely limiting the detection performance and application scenarios of β2-GP1-related in vitro diagnostic products. While truncated expression using insect expression systems has been used for clinical testing, the detection rate in disease groups is less than 80%, indicating a low detection rate.

[0005] To address the numerous shortcomings of existing technologies, the industry urgently needs a technology for preparing recombinant β2-GP1 protein that can be efficiently prepared, possesses a natural spatial conformation, complete post-translational modifications, and high antigenic activity. Summary of the Invention

[0006] This invention utilizes an insect expression system to specifically prepare recombinant full-length β2-GP1 protein for detection, which can be used for antibody detection in clinical serum samples. This application provides a recombinant human β2-glycoprotein 1 protein and its applications.

[0007] In a first aspect, this application provides a recombinant human β2-glycoprotein 1 protein, employing the following technical solution: Human β2-glycoprotein 1 recombinant protein, which has the following characteristics: (1) An amino acid sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:6; or (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

[0008] In the above technical solution, SEQ ID The sequence of NO:1 is: MISPVLILFSSFLCHVAIAGRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGGMRKFICPLTGLWPINTLKCTPRVCPFAGILENGAVRYTTFEYPNTISFSCNTGFYLNGADSAKCTEEGKWSPELPVCAPIICPPPSIPTFATLRVYKPSAGNNSLYRDTAV FECLPQHAMFGNDTITCTTHGNWTKLPECREVKCPFPSRPDNGFVNYPAKPTLYYKDKATFGCHDGYSLDGPEEIECTKLGNWSAMPSCKASCKLPVKKATVVYQGERVKIQEKFKNGMLHGDKVSFFCKNKEKKCSYTEDAQCIDGTIEVPKCFKEHSSLAFWKTDASDVKPCHHHHHH.

[0009] In the above technical solution, the sequence of SEQ ID NO:2 is: MISPVLILFSSFLCHVAIAGRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGGMRKFICPLTGLWPINTLKCTPRVHHHHHH.

[0010] In the above technical solution, the sequence of SEQ ID NO:3 is: MISPVLILFSSFLCHVAIAGRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGGMRKFICPLTGLWPINTLKCTPRVCPFAGILENGAVRYTTFEYPNTISFSCNTGFYLNGADSAKCTEEGKWSPELPVCAPIICHHHHHH.

[0011] In the above technical solution, the sequence of SEQ ID NO:4 is: MISPVLILFSSFLCHVAIAGRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGGMRKFICPLTGLWPINTLKCTPRVCPFAGILENGAVRYTTFEYPNTISFSCNTGFYLNGADSAKCTEEGKWSPELPVCAPIICPPPSIPTFATLRVYKPSAGNNSLYRDTAVFECLPQHAMFGNDTITCTTHGNWTKLPECREVKCHHHHHH.

[0012] In the above technical solution, SEQ ID The sequence of NO:5 is: MISPVLILFSSFLCHVAIAGRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGGMRKFICPLTGLWPINTLKCTPRVCPFAGILENGAVRYTTFEYPNTISFSCNTGFYLNGADSAKCTEEGKW SPELPVCAPIICPPPSIPTFATLRVYKPSAGNNSLYRDTAVFECLPQHAMFGNDTITCTTHGNWTKLPECREVKCPFPSRPDDNGFVNYPAKPTLYYKDKATFGCHDGYSLDGPEEIECTKLGNWSAMPSCKAHHHHHH.

[0013] In the above technical solution, SEQ ID The sequence of NO:6 is: MRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGGMRKFICPLTGLWPINTLKCTPRVCPFAGILENGAVRYTTFEYPNTISFSCNTGFYLNGADSAKCTEEGKWSPELPVCAPIICPPPSIPTFATLRVYKPSAGNNSLYRDTAVFECLPQHAMF GNDTITCTTHGNWTKLPECREVKCPFPSRPDNGFVNYPAKPTLYYKDKATFGCHDGYSLDGPEEIECTKLGNWSAMPSCKASCKVPVKKATVVYQGERVKIQEKFKNGMLHGDKVSFFCKNKEKKCSYTEDAQCIDGTIEVPKCFKEHSSLAFWKTDASDVKPCHHHHHH.

[0014] In the above technical solution, the recombinant human β2-glycoprotein 1 protein has the following characteristics: (4) The amino acid sequence as shown in SEQ ID NO:1 and / or SEQ ID NO:6; or (5) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (4), and which has the same or similar function as the amino acid sequence shown in (4); or (6) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (4) or (5).

[0015] Preferably, the recombinant human β2-glycoprotein 1 protein has the amino acid sequence shown in SEQ ID NO:6.

[0016] Preferably, the sequence of the recombinant human β2-glycoprotein 1 is as shown in SEQ ID NO:6.

[0017] Secondly, this application provides a nucleic acid molecule encoding the aforementioned recombinant human β2-glycoprotein 1 protein.

[0018] In the above technical solution, the nucleic acid molecule has: (7) A nucleotide sequence as shown in any of SEQ ID NO:7 to SEQ ID NO:12; or (8) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (7), and whose function is the same as or similar to that of (7); or (9) A nucleotide sequence that is at least 70% homologous to the nucleotide sequence shown in (7) or (8).

[0019]

[0020] In the above technical solution, the sequence of SEQ ID NO: 8 is: ATGATCAGCCCGGTTCTGATCCTCTTTAGTTCTTTCCTGTGCCATGTGGCTATTGCGGGAAGAACTTGTCCCAAGCCGGACGACCTGCCTTTTTCCACGGTCGTCCCTCTCAAGACGTTTTATGAGCCTGGCGAAGAGATAACCTACAGTTGCAAACCCGGTTACGTAAGCAGAGGCGGAATGAGAAAATTCATATGCCCTCTGACTGGACTTTGGCCGATTAATACACTCAAGTGCACCCCAAGAGTGCATCATCATCATCATCACTAA (expressing fragment 1-83).

[0021] In the above technical solution, the sequence of SEQ ID NO: 9 is: ATGATCAGCCCGGTTCTGATCCTCTTTAGTTCTTTCCTGTGCCATGTGGCTATTGCGGGAAGAACTTGTCCCAAGCCGGACGACCTGCCTTTTTCCACGGTCGTCCCTCTCAAGACGTTTTATGAGCCTGGCGAAGAGATAACCTACAGTTGCAAACCCGGTTACGTAAGCAGAGGCGGAATGAGAAAATTCATATGCCCTCTGACTGGACTTTGGCCGATTAATACACTCAAGTGCACCCCAAGAGTGTGCCCTTTTGCTGGCATCTTGGAGAATGGCGCCGTGCGCTACACAACGTTCGAATACCCGAATACGATCTCTTTCTCGTGCAACACCGGCTTCTACTTGAACGGCGCCGATTCTGCCAAATGTACTGAAGAGGGCAAGTGGAGCCCCGAGTTGCCTGTGTGCGCACCTATCATCTGCCATCATCATCATCATCACTAA (expressing fragment 1-142).

[0022] In the above technical solution, the sequence of SEQ ID NO: 10 is: ATGATCAGCCCGGTTCTGATCCTCTTTAGTTCTTTCCTGTGCCATGTGGCTATTGCGGGAAGAACTTGTCCCAAGCCGGACGACCTGCCTTTTTCCACGGTCGTCCCTCTCAAGACGTTTTATGAGCCTGGCGAAGAGATAACCTACAGTTGCAAACCCGGTTACGTAAGCAGAGGCGGAATGAGAAAATTCATATGCCCTCTGACTGGACTTTGGCCGATTAATACACTCAAGTGCACCCCAAGAGTGTGCCCTTTTGCTGGCATCTTGGAGAATGGCGCCGTGCGCTACACAACGTTCGAATACCCGAATACGATCTCTTTCTCGTGCAACACCGGCTTCTACTTGAACGGCGCCGATTCTGCCAAATGTACTGAAGAGGGCAAGTGGAGCCCCGAGTTGCCTGTGTGCGCACCTATCATCTGCCCACCCCCTTCTATCCCCACGTTCGCCACGCTTAGGGTATACAAGCCAAGCGCAGGCAACAATTCCCTGTACCGTGATACGGCGGTGTTCGAGTGCTTGCCACAGCACGCTATGTTTGGCAACGACACCATTACATGCACAACCCATGGTAACTGGACAAAGCTCCCCGAATGTCGTGAAGTTAAGTGCCATCATCATCATCATCACTAA (expressing fragment 1-205).

[0023] In the above technical solution, the sequence of SEQ ID NO:11 is: ATGATCAGCCCGGTTCTGATCCTCTTTAGTTCTTTCCTGTGCCATGTGGCTATTGCGGGAAGAACTTGTCCCAAGCCGGACGACCTGCCTTTTTCCACGGTCGTCCCTCTCAAGACGTTTTATGAGCCTGGCGAAGAGATAACCTACAGTTGCAAACCCGGTTACGTAAGCAGAGGCGGAATGAGAAAATTCATATGCCCTCTGACTGGACTTTGGCCGATTAATACACTCAAGTGCACCCCAAGAGTGTGCCCTTTTGCTGGCATCTTGGAGAATGGCGCCGTGCGCTACACAACGTTCGAATACCCGAATACGATCTCTTTCTCGTGCAACACCGGCTTCTACTTGAACGGCGCCGATTCTGCCAAATGTACTGAAGAGGGCAAGTGGAGCCCCGAGTTGCCTGTGTGCGCACCTATCATCTGCCCACCCCCTTCTATCCCCACGTTCGCCACGCTTAGGGTATACAAGCCAAGCGCAGGCAACAATTCCCTGTACCGTGATACGGCGGTGTTCGAGTGCTTGCCACAGCACGCTATGTTTGGCAACGACACCATTACATGCACAACCCATGGTAACTGGACAAAGCTCCCCGAATGTCGTGAAGTTAAGTGCCCATTTCCCTCACGCCCTGACAATGGTTTTGTGAACTACCCTGCTAAGCCCACTCTGTACTACAAGGATAAGGCAACCTTTGGATGCCACGACGGTTACTCTCTCGATGGTCCTGAAGAGATCGAATGCACCAAGCTTGGCAACTGGTCCGCAATGCCTTCCTGCAAGGCCCATCATCATCATCATCACTAA (expressed fragment 1-262).

[0024]

[0025] Thirdly, this application provides a recombinant vector containing the aforementioned nucleic acid molecules.

[0026] In the above technical solution, the backbone plasmid in the recombinant vector is selected from pFastBac1 vector, pFastBacDUAL vector, pFastBac HT series vector, pFastBac EGT series vector, pFastBac ET vector or pBlueBacHis2 series vector.

[0027] Preferably, the backbone plasmid is the pFastBac1 vector.

[0028] The aforementioned vectors all contain a strong promoter for the baculovirus polyhedrosis protein gene and can recombine with the baculovirus shuttle plasmid in *E. coli* DH10Bac via the Tn7 transposition principle, thereby rapidly and efficiently obtaining recombinant baculovirus. Those skilled in the art should understand that any transfer vector possessing the above-mentioned functions, compatible with the Bac-to-Bac system, and capable of expressing the target gene in insect cells can be used as the backbone plasmid of this invention and also falls within the scope of protection of this invention.

[0029] Fourthly, this application provides a host cell characterized by being transformed and / or transfected with the aforementioned recombinant vector.

[0030] In the above technical solution, the transformed and / or transfected chassis cells in the host cells include insect cells.

[0031] In the above technical solution, the host cell is selected from: the grassland noctuid moth ( Spodoptera frugiperda ) Ovarian cell lines Sf9 and Sf21, *Spodoptera litura* ( Trichoplusia ni Embryonic cell line BTI-Tn5B1-4 (trade names High Five™ or Hi5), and fruit flies ( Drosophila melanogaster One or more of the following cell lines: S2, etc. All of the above cell lines can be cultured in suspension or adherent form, making them suitable for large-scale recombinant protein production.

[0032] Preferably, the insect cells are Sf9 cells.

[0033] Fifthly, this application provides a method for preparing recombinant human β2-glycoprotein 1 protein, wherein the host cells are cultured, expressed, and purified to obtain the recombinant human β2-glycoprotein 1 protein.

[0034] Sixthly, this application provides the use of the recombinant human β2-glycoprotein 1 protein, the nucleic acid molecule, the recombinant vector, the host cell, and / or the recombinant human β2-glycoprotein 1 protein obtained by the preparation method in the preparation of a product for detecting anti-β2-GP1 antibodies.

[0035] In a seventh aspect, this application provides a detection product comprising: the recombinant human β2-glycoprotein 1 protein, the nucleic acid molecule, the recombinant vector, the host cell, and / or the recombinant human β2-glycoprotein 1 protein obtained by the preparation method.

[0036] In the above technical solution, the detection product is used to detect the presence and / or content of human β2-glycoprotein 1 and / or anti-human β2-glycoprotein 1 antibody in biological samples. The biological sample is selected from one or more of serum, plasma, cell culture supernatant, urine, and tissue homogenate.

[0037] In the above technical solution, the detection product can be in the form of a reagent kit. The types of reagent kits include, but are not limited to, one or more of the following: enzyme-linked immunosorbent assay (ELISA) kits, chemiluminescent immunoassay kits, magnetic microparticle chemiluminescent immunoassay kits, fluorescent immunoassay kits, and immunochromatographic test strips.

[0038] In the above technical solution, the detection product further includes one or more of the following auxiliary components: a solid-phase carrier coated with recombinant β2-glycoprotein 1 protein or its antibody; detection antibody or detection antigen and its marker; calibrators and / or quality control products; sample diluent, washing solution, substrate chromogenic solution and / or stop solution; and instructions for use.

[0039] Those skilled in the art should understand that any detection product that uses the recombinant human β2-glycoprotein 1 protein of the present invention as the core raw material for qualitative or quantitative detection of β2-glycoprotein 1 or its antibody falls within the protection scope of the present invention.

[0040] In summary, this application has the following beneficial effects: The recombinant human β2 glycoprotein 1 protein provided by this invention is efficiently expressed in insect cells and can be used for the detection of human β2-GP1 antibodies, exhibiting reactivity consistent with the natural protein. In clinical testing, comparisons with kits from multiple manufacturers showed that the recombinant β2-GP1 antigen demonstrated excellent detection capabilities. Attached Figure Description

[0041] Figure 1 The results of SDS-PAGE analysis of the recombinant human β2 glycoprotein 1 protein implemented in this application. Detailed Implementation

[0042] The amino acid sequence of the recombinant human β2-GP1 protein provided by this invention is shown in any of SEQ ID NO:1 to SEQ ID NO:6.

[0043] The present invention also provides an expression vector comprising the polynucleotide described herein. That is, the present invention provides an expression vector encoding the recombinant protein described herein. In some embodiments, the backbone vector of the expression vector is selected from the pFastBac1 vector.

[0044] The expression vector of the present invention is a baculovirus, and the cell line containing the polynucleotide or the expression vector is an insect cell capable of transfecting baculovirus and expressing protein.

[0045] In this invention, the host cell is a eukaryotic insect cell containing an expression vector for the polynucleotide or expressed within the insect cell. In some embodiments, the host insect cell is an sf9 or High Five cell line.

[0046] The expression vector of the present invention is a baculovirus, and the cell line containing the polynucleotide or the expression vector described herein is an insect cell capable of transfecting baculovirus and expressing protein.

[0047] The present invention also provides a method for preparing the recombinant protein, comprising: culturing the host cells, transfecting the host cells with baculovirus and expressing the protein.

[0048] In some embodiments, the method for preparing the recombinant protein further includes steps of disrupting, extracting, and purifying the expressed cells. The disruption is performed using high-pressure homogenization. The extraction and purification are performed using Ni metal chelation chromatography and CM ion exchange chromatography.

[0049] The application of the recombinant protein, the polynucleotide, the expression vector, the cell line, the host cell, or the product prepared by the method described in this invention in the detection of human β2-GP1 antibody.

[0050] The amino acid sequence of recombinant human β2 glycoprotein 1 is shown in any of SEQ ID NO:1 to SEQ ID NO:6. Recombinant human β2 glycoprotein 1 is efficiently expressed in insect cells and can be used for the detection of human β2-GP1 antibodies, exhibiting reactivity consistent with the natural protein. In clinical testing, we compared the results with kits from multiple manufacturers, and the recombinant β2-GP1 antigen demonstrated excellent detection capabilities.

[0051] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0052] The present application will be further described in detail below with reference to the embodiments.

[0053] Unless otherwise specified, the raw materials used in the embodiments and comparative examples of this application are all commercially available.

[0054] Example 1: Gene Synthesis The gene containing the β2-GP1 recombinant protein (NCBI accession number: X58100.1) has a 6-terminus appended to its C-terminus. The his tags were synthesized after codon optimization (SEQ ID NO:7 to SEQ ID NO:12). The plasmid preparation, expression and purification described below take the β2-GP1 recombinant protein gene of SEQ ID NO:7 as an example. The plasmid preparation, expression and purification methods for the other β2-GP1 polynucleotides are the same.

[0055] Example 2 Preparation of pFastbac-β2-GP1 plasmid Take 1 μg of β2-GP1 polynucleotide aqueous solution and 2 μg of pFastbac1 plasmid, and add 100 units of BamHI and 100 units of HindIII restriction endonuclease, respectively. React at 37℃ for 30 minutes. After the reaction, remove excess enzyme using a purification kit. Mix the digested β2-GP1 and pFastBac1 plasmids and add 100 units of T4 ligase. Incubate at room temperature for 2 hours, then remove excess T4 enzyme using a purification kit. The purified pFastBac1... The β2-GP1 plasmid was transformed into electrocompetent *E. coli* DH10Bac™ using a Gene Pulser Xcell electroporator. After incubation at 37°C for 1 hour in 1 mL of LSOC convalescent medium, the plasmid was plated onto LB agarose gels containing 100 μg / mL ampicillin and 70 μg / mL gentamicin. Positive clones were selected after overnight incubation and inoculated into SB medium containing 100 μg / mL ampicillin and 70 μg / mL gentamicin. The bacteria were cultured to the logarithmic growth phase, and the purified pFastBac-β2-GP1 plasmid was obtained using the QIAGEN Maxi Prep plasmid extraction kit.

[0056] Example 3: Expression of recombinant human β2-GP1 protein SF9 insect cells were cultured in Grace insect culture medium supplemented with 10% fetal bovine serum until the logarithmic growth phase (1.5~2.5×10⁻⁶). 6 Cells were added per milliliter of culture medium with a survival rate higher than 95%. 2 ml of LGrace insect culture medium was added to each well of a 6-well plate, and 8 × 10⁸ g of [unspecified antibiotic] ​​was added to each well without additional antibiotics. 5 sf9 cells.

[0057] Add 8 μL of Cellfectin II (purchased from Thermo Fisher Scientific) to 100 μL of additive-free Grace insect culture medium, and dilute 1 μg of pFastBac-β2-GP1 to 100 μL of additive-free Grace insect culture medium. Mix the diluted Cellfectin II and pFastBac-β2-GP1 plasmid, and incubate at 25℃±5℃ for 15-30 minutes.

[0058] The Cellfectin II and pFastBac-β2-GP1 plasmid mixture was added dropwise to sf9 cells cultured in 6-well cell culture plates and incubated at 27°C for 3–5 hours. Afterward, the transfection mixture was removed, and 2 mL of Grace insect culture medium containing 10% fetal bovine serum was added to each well. Additionally, 100 μg / mL ampicillin and 70 μg / mL gentamicin were added to the culture medium.

[0059] After culturing sf9 cells in culture medium for 72 hours (late stage of infection) until a large number of cells can be observed floating, the cells are resuspended, centrifuged at 500g for 5 minutes to remove cells and debris, the supernatant is collected, and centrifuged again at 5000×g for 5 minutes to obtain P1 generation virus after removing other residues.

[0060] Passage the P1 generation virus and prepare 6-well cell culture plates, adding 2 × 10⁻⁶ cells / well to each well. 6 1 sf9 insect cell was incubated at room temperature for 1 hour to allow the cells to adhere to the culture plate.

[0061] An appropriate amount of P1 generation virus was added to each culture well, with the amount of virus added adjusted to maintain the infection copy number (MOI) between 0.05 and 0.1 as needed. Cells and virus were incubated at 27°C under humid conditions for 48 hours.

[0062] Forty-eight hours later, 2 mL of culture medium was aspirated from each well. After mixing all the culture media, the mixture was centrifuged at 5000×g for 5 minutes, and the supernatant was collected. The collected virus is the P2 generation, and its viral load must reach 1×10⁻⁶. 8 Only when the concentration of pfu / mL is sufficient can it be used to reinfect cells and express proteins.

[0063] Add an appropriate amount of P2 generation virus to each culture flask, adjusting the infection copy number (MOI) to be between 0.05 and 0.1 as needed. Incubate the cells and virus at 27°C under humid conditions for 48 hours.

[0064] Forty-eight hours later, all cell culture media were transferred to a 1000 mL centrifuge cup and centrifuged at 8000 rpm and 4°C for 20 min. The culture supernatant containing recombinant human β2-GP1 protein was collected.

[0065] Example 4: Purification and preparation of recombinant human β2-GP1 protein (1) Centrifugation: 2L of cultured cells and supernatant were placed into a 1L centrifuge cup, balanced, and centrifuged under the following conditions: 4℃, 5000rpm, 30min. Immediately after centrifugation, the supernatant was poured into a clean beaker.

[0066] (2) Filtration: Use an ultrafiltration cup with a pore size of 0.45 μm to filter the filtrate into another clean beaker.

[0067] (3) Ni metal chelation chromatography was performed using AKTA pure. Washing: After loading and rebalancing, impurities were washed away with 20 mM PBS + 1 mM DTT pH 7.4. The trend of UV curve changes was observed. When the UV absorbance value increased, the waste tube was placed into a clean beaker to collect the eluent. The collection was stopped when the UV value reached the level, and then the waste tube was put back into the waste container.

[0068] (4) Dissociation of target protein: The target protein was eluted with 20mM PBS + 1mM DTT + 1M imidazole pH 7.4. The trend of UV curve change was observed. When the UV value increased, the sample was collected. When the UV value was at a certain level, the collection was stopped and the waste liquid tube was put back into the waste liquid bucket.

[0069] (5) Change the buffer: Use a dialysis bag with a molecular cutoff of 14KD to change the buffer. The buffer solution is 20 mM PB + 1 mM MTT pH 7.4. Dialyze twice, each time for no less than 3 hours.

[0070] (6) CM ion exchange chromatography was performed using AKTA pure. Washing: After loading and rebalancing, impurities were washed away with 20 mM PB + 100 mM NaCl + 1 mM DTT at pH 7.4. The trend of UV curve changes was observed. When the UV absorbance value increased, the waste liquid tube was placed into a clean beaker to collect the eluent. The collection was stopped when the UV value reached a certain level, and then the waste liquid tube was put back into the waste liquid container.

[0071] (7) Dissociation of target protein: Use 20 mM PB + 200 mM NaCl + 1 mM DTT pH 7.4 to elute the target protein, observe the trend of UV curve changes, start collecting samples when the UV value rises, stop collecting when the UV value is at a certain level, and then put the waste liquid tube back into the waste liquid bucket.

[0072] (8) Treatment of the target protein: The medium was changed using a dialysis bag with a molecular cutoff of 14 kDa. The storage buffer was 20 mM PBS + 1 mM DTT + 10% glycerol, pH 7.5. The protein was concentrated using a 10 kDa ultrafiltration tube. The concentration was measured using a G250 during the process. When the concentration reached 1 mg / mL, it was stored at -20℃.

[0073] Example 5: Screening for Recombinant Human β2-GP1 Protein Expression To screen for reactive β2-GP1 recombinant proteins, we expressed truncated versions of the full-length protein at different lengths. Different lengths of the β2-GP1 polypeptide sequence (SEQ ID NO:2:1-83, SEQ ID NO:3:1-142, SEQ ID NO:4:1-205, SEQ ID NO:5:1-262, SEQ ID NO:1:1-345, SEQ ID NO:6:21-345) were expressed, and reactive fragments were screened using an indirect ELISA method. SEQ ID NO:6 was reactive and highly expressed, and was therefore preferred for expression and clinical testing.

[0074] Table 1

[0075] Example 6: Application of recombinant human β2-GP1 protein in serum sample detection Using the purified antigen (SEQ ID NO:6) described above, an indirect method was used to detect anti-human β2-GP1 antibodies in clinical samples. Positive samples were detected, and the kit could distinguish between clinically positive and negative samples. The results are shown in Table 2. The detection rate of the antigen preparation kit developed in this patent is consistent with that of products A, B, C, and D on the market. Of the 51 samples containing both negative and positive samples, 23 were positive, while E and F only detected 16 and 19, respectively. Furthermore, product B explicitly states that it uses naturally extracted β2-GP1, and the antigen detection is completely consistent with it, indicating that the recombinant full-length β2-GP1 expressed in insect cells has the same clinical reactivity as the natural protein.

[0076] Table 2

[0077] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Recombinant human β2-glycoprotein 1, characterized in that, It has the following characteristics: (1) An amino acid sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:6; or (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

2. The recombinant human β2-glycoprotein 1 protein as described in claim 1, characterized in that, The recombinant human β2-glycoprotein 1 protein has the following characteristics: (4) The amino acid sequence as shown in SEQ ID NO:1 and / or SEQ ID NO:6; or (5) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (4), and which has the same or similar function as the amino acid sequence shown in (4); or (6) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (4) or (5).

3. A nucleic acid molecule encoding the recombinant human β2-glycoprotein 1 protein as described in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The nucleic acid molecule has: (7) A nucleotide sequence as shown in any of SEQ ID NO:7 to SEQ ID NO:12; or (8) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (7), and whose function is the same as or similar to that of (7); or (9) A nucleotide sequence that is at least 80% homologous to the nucleotide sequence shown in (7) or (8).

5. A recombinant vector, characterized in that, It contains the nucleic acid molecule as described in claim 3 or 4.

6. A host cell, characterized in that, Transformation and / or transfection with the recombinant vector as described in claim 5.

7. The host cell as described in claim 6, characterized in that, The transformed and / or transfected chassis cells include insect cells.

8. A method for preparing recombinant human β2-glycoprotein 1 protein, characterized in that, The host cells as described in claim 6 are cultured, and after expression and purification, the recombinant human β2-glycoprotein 1 protein is obtained.

9. Any of the following applications in the preparation of products for detecting anti-β2-GP1 antibodies: (1) The recombinant human β2-glycoprotein 1 protein as described in claim 1 or 2; and / or (2) The nucleic acid molecule as described in claim 3 or 4; and / or (3) The recombinant vector as described in claim 5; and / or (4) The host cell as described in claim 6 or 7; and / or (5) The recombinant human β2-glycoprotein 1 obtained by the preparation method described in claim 8.

10. The product being tested, characterized in that, Includes any of the following: (1) The recombinant human β2-glycoprotein 1 protein as described in claim 1 or 2; and / or (2) The nucleic acid molecule as described in claim 3 or 4; and / or (3) The recombinant vector as described in claim 5; and / or (4) The host cell as described in claim 6 or 7; and / or (5) The recombinant human β2-glycoprotein 1 obtained by the preparation method described in claim 8.

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