CD3-HLA-B27 recombinant protein, encoding gene thereof, preparation method and application thereof

Abstract

The application provides a CD3-HLA-B27 recombinant protein and a coding gene, a preparation method and an application thereof, and belongs to the technical field of genetic engineering. The CD3-HLA-B27 recombinant protein is obtained by fusion expression of an extracellular segment of a CD3 epsilon chain or a mutant thereof and HLA-B27 or a mutant thereof. A flexible connecting peptide is further included between the extracellular segment of the CD3 epsilon chain or the mutant thereof and the HLA-B27 or the mutant thereof, and the flexible connecting peptide is (GGGGS) n wherein n=1~5. In the application, the extracellular segment of the CD3 epsilon chain or the mutant thereof and the HLA-B27 or the mutant thereof are fused and expressed, so that the cell can express the extracellular segment of the CD3 epsilon chain or the mutant thereof and the HLA-B27 or the mutant thereof at a ratio of 1:1, and can be applied to a scenario of simultaneously detecting CD3 and HLA-B27.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and in particular to a CD3-HLA-B27 recombinant protein, its encoding gene, preparation method, and application. Background Technology

[0002] Methods for detecting HLA-B27 include microlymphocytotoxicity assay (MLCT), molecular biology methods (PCR), and flow cytometry (FC). Among these, MLCT may produce false positives due to changes in the number or structure of B27 molecules on the cell membrane surface caused by factors such as infection. PCR has high sensitivity, but its false positive rate is also relatively high, limiting its clinical use. Flow cytometry is a commonly used method for clinical detection of HLA-B27, with relatively good specificity and sensitivity.

[0003] CD3 is a surface marker of T lymphocytes. Detecting HLA-B27 in peripheral blood CD3+ cells can exclude interference from other cells and improve specificity.

[0004] To ensure the reliability of test results, quality control materials are needed during the testing process. However, the proportion of HLA-B27 positive cases in the population is low, and qualified sample donors are very limited. Laboratories struggle to obtain a sufficient quantity and quality of patient-derived samples for batch quality control or long-term storage, resulting in an unstable supply of quality control materials and affecting the standardization of testing.

[0005] Therefore, there is an urgent need in this field for a quality control product for the combined detection of HLA-B27 and CD3. Summary of the Invention

[0006] To solve at least one of the above-mentioned technical problems, the technical solution adopted in this application is as follows.

[0007] According to the first aspect of this application, compared with the prior art, this application has the following beneficial effects: This application provides a recombinant CD3-HLA-B27 protein, obtained by fusing an extracellular segment of the CD3ε chain or a mutant thereof with HLA-B27 or a mutant thereof. The amino acid sequence of the extracellular segment of the CD3ε chain is shown in SEQ ID No. 25, and the amino acid sequence of the HLA-B27 is shown in SEQ ID No. 1. A flexible linker peptide, (GGGGS), is further included between the extracellular segment of the CD3ε chain or its mutant and the HLA-B27 or its mutant. nWhere n=1~5, the mutant of the extracellular segment of the CD3ε chain is based on the amino acid sequence shown in SEQ ID No. 25 with 1~2 mutations, the mutant of HLA-B27 is based on the amino acid sequence shown in SEQ ID No. 1 with 1~2 mutations, and the fusion expression is selected from one of the following: (1) No mutations were found in the extracellular segment of the CD3ε chain, and no mutations were found in HLA-B27; (2) The extracellular segment of the CD3ε chain has L200I and V204A mutations, and HLA-B27 has M6L mutations; (3) No mutations were found in the extracellular segment of the CD3ε chain, but S5G and F9R mutations were found in HLA-B27; (4) No mutation was found in the extracellular segment of the CD3ε chain, but the HLA-B27 underwent the Y8W mutation; (5) The extracellular segment of the CD3ε chain underwent a V204A mutation, and HLA-B27 underwent an S5G mutation; (6) No mutations were found in the extracellular segment of the CD3ε chain, but G2S and M6L mutations were found in HLA-B27; (7) The extracellular segment of the CD3ε chain underwent the L200I mutation, and HLA-B27 underwent the H10W mutation.

[0008] The second aspect of this application provides a gene encoding the CD3-HLA-B27 recombinant protein described in the first aspect of this application, comprising a nucleotide sequence encoding an extracellular segment of the CD3ε chain or a mutant thereof, and a nucleotide sequence encoding HLA-B27 or a mutant thereof.

[0009] In some embodiments of this application, the CD3-HLA-B27 recombinant protein is obtained by fusing an extracellular segment of the CD3ε chain with HLA-B27, the nucleotide sequence encoding the extracellular segment of the CD3ε chain is shown in SEQ ID No. 26, and the nucleotide sequence encoding HLA-B27 is shown in SEQ ID No. 2.

[0010] In some other embodiments of this application, the CD3-HLA-B27 recombinant protein is obtained by fusing a mutant of the extracellular segment of the CD3ε chain with a mutant of HLA-B27. The amino acid sequence of the mutant of the extracellular segment of the CD3ε chain is shown in SEQ ID No. 31, the amino acid sequence of the mutant of HLA-B27 is shown in SEQ ID No. 33, the nucleotide sequence encoding the mutant of the extracellular segment of the CD3ε chain is shown in SEQ ID No. 32, and the nucleotide sequence encoding the mutant of HLA-B27 is shown in SEQ ID No. 34.

[0011] A third aspect of this application provides an expression vector comprising any of the genes described in the second aspect of this application.

[0012] In some embodiments of this application, the expression vector further includes a nucleotide sequence encoding B2M following the nucleotide sequence encoding HLA-B27 or a mutant thereof.

[0013] In some embodiments of this application, the expression vector further includes a nucleotide sequence for an internal ribosome entry site after the nucleotide sequence encoding HLA-B27 or a mutant thereof and before the nucleotide sequence encoding B2M.

[0014] In some embodiments of this application, the expression vector further includes a nucleotide sequence encoding a first signal peptide before the nucleotide sequence encoding the extracellular segment of the CD3ε chain or a mutant thereof, and a nucleotide sequence encoding a second signal peptide before the nucleotide sequence encoding B2M.

[0015] The fourth aspect of this application provides a host cell containing any of the expression vectors described in the third aspect of this application.

[0016] The fifth aspect of this application provides a method for preparing the CD3-HLA-B27 recombinant protein described in the first aspect of this application, including the step of inducing the host cells described in the fourth aspect of this application to express the protein.

[0017] The sixth aspect of this application provides the use of the host cells described in the fourth aspect of this application in the preparation of a kit for detecting HLA-B27 expression in biological samples, wherein the host cells serve as a positive control.

[0018] The seventh aspect of this application provides a kit for detecting HLA-B27 in biological samples, comprising the host cells described in the fourth aspect of this application, wherein the host cells serve as a positive control.

[0019] Compared with the prior art, this application has the following effective technical effects: This application fuses the extracellular segment of the CD3ε chain or its mutant with HLA-B27 or its mutant, enabling cells to express the extracellular segment of the CD3ε chain or its mutant and HLA-B27 or its mutant in a 1:1 ratio, which is suitable for scenarios that simultaneously detect CD3 and HLA-B27.

[0020] This application further incorporates a designed signal peptide that enables the extracellular segment of the CD3ε chain or its mutant and recombinant proteins of HLA-B27 or its mutant to cross the membrane and reside on the cell membrane, and can be used as a positive control for the detection of HLA-B27.

[0021] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0022] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which: Figure 1 The following is a flow cytometry scatter plot of the monoclonal cell line obtained by double plasmid transfection in Example 1 of this application: A: HLA-B27 detection result of cells co-transfected with double plasmids; B: CD3 detection result of cells co-transfected with double plasmids; C: HLA-B27 detection result of untransfected cells; D: CD3 detection result of untransfected cells. Figure 2 The following is a flow cytometry scatter plot of the monoclonal cell line obtained by three-plasmid transfection in Example 2 of this application: A: HLA-B27 detection result of cells co-transfected with three plasmids; B: CD3 detection result of cells co-transfected with three plasmids; C: HLA-B27 detection result of untransfected cells; D: CD3 detection result of untransfected cells. Figure 3 A schematic diagram of the CD3-HLA-B27 plasmid structure constructed in Example 3 of this application is shown; Figure 4 The following is a flow cytometry scatter plot of the monoclonal cell line obtained by transfection with CD3-HLA-B27 plasmid in Example 3 of this application: A: HLA-B27 detection results of cells co-transfected with single plasmid; B: CD3 detection results of cells co-transfected with single plasmid; C: HLA-B27 detection results of untransfected cells; D: CD3 detection results of untransfected cells. Figure 5 The following are the results of flow cytometry detection of HLA-B27 in monoclonal cell lines obtained by transfection with plasmids containing the original or mutated sequences in Example 4 of this application: A: Detection results of monoclonal cell lines obtained by transfection with plasmids containing the original sequence; B: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 1 sequence; C: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 4 sequence; D: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 6 sequence; E: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 7 sequence; F: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 8 sequence; G: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 10 sequence; H: Detection results of the pool of cells without positive results. Figure 6The following diagram illustrates the flow cytometry results of CD3 detection in monoclonal cell lines obtained by transfection with plasmids containing either the original or mutated sequences, as described in Example 4 of this application: A: Detection results of monoclonal cell lines obtained by transfection with plasmids containing the original sequence; B: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 1; C: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 4; D: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 6; E: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 7; F: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 8; G: Detection results of monoclonal cell lines obtained by transfection with plasmids containing mutation 10; H: Detection results of the pool of cells without positive results. Detailed Implementation

[0023] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0024] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0025] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0027] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0029] Example 1: Preparation of double-positive cell lines by co-transfection with two plasmids To simultaneously express HLA-B27 and CD3, this embodiment co-transfects a plasmid containing the HLA-B27 gene nucleotide sequence and a protein sequence including the full-length CD3ε chain (NCBI ID 1SY6_A, CD3E_HUMAN) into one host cell, thereby enabling simultaneous expression of HLA-B27 and the CD3ε chain. Specifically: 1. Construction of plasmids containing the HLA-B27 gene The amino acid sequence of HLA-B27 is as follows: GGSHSMRYFHTSVSRPGRGEPRFITVGYVDDTLFVRFDSDAASPREEPRAPWIEQEGPEYWDRETQICKAKAQTDREDLRTLLRYYNQSEAGSHTLQNMYGCDVGPDGRLLRGYHQHAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAARVAEQLRAYLEGECVEWLRR YLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQSTVPIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSYSQAACSDSAQGSDVSLTA (SEQ ID No. 1) After codon optimization, the nucleotide sequence of HLA-B27 is as follows: In this embodiment, a plasmid expressing the HLA-B27 fusion protein was constructed. The structure of the HLA-B27 fusion protein sequentially includes: signal peptide 1, HLA-B27, signal peptide 2, and B2M, wherein: The amino acid sequence of signal peptide 1 is as follows: METDTLLLWVLLLWVPGSTG (SEQ ID No. 3) The nucleotide sequence of signal peptide 1 is as follows: ATGGAAACCGATACTCTTCTTCTCTGGGTACTGCTGCTGTGGGTACCTGGCTCCACCGGG (SEQ ID No. 4) The amino acid sequence of signal peptide 2 is the same as that of signal peptide 1. The nucleotide sequence of signal peptide 2 is as follows: ATGGAAACCGATACACTTCTTCTCTGGGTATTGCTCTTGTGGGTGCCTGGATCTACAGGA (SEQ ID No. 5) The amino acid sequence of B2M is as follows: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID No. 6) The nucleotide sequence of B2M is as follows: ATTCAGAGGACACCAAAGATCCAGGTTTTCAAGACATCCTGCCGAGAATGGGAAATCCAATTTCCTGAACTGCTACGTCAGCGGTTTTCACCCCTCAGACATAGAAGTCGATCTCCTGAAAAATGGGGAGCGTATCGAAAAGGTCGAG CATTCAGACCTCAGCTTCTCTAAGGATTGGTCCTTTTATCTGCTGTACTATACCGAGTTCACCCCAACTGAAAAGGACGAGTACCGCTTGCAGAGTTAACCATGTTACCCTGTCCCAACCTAAAATCGTGAAGTGGGACAGAGACATG (SEQ ID No. 7) When constructing the recombinant expression vector, a stop codon (TAATGA) is included after both the nucleotide sequences of HLA-B27 and B2M. Furthermore, after the nucleotide sequence of HLA-B27 and before the nucleotide sequence of signal peptide 2, the nucleotide sequence of the internal ribosome entry site (IRES) is also included. (SEQ ID No. 8) The nucleotide sequence of the HLA-B27 fusion protein is thus obtained as follows: The nucleotide sequence shown in SEQ ID No. 9 was synthesized and cloned into the eukaryotic expression vector pGXC-WPRE containing the glutamine synthase (GS) gene to construct a recombinant plasmid, named HLA-B27 plasmid.

[0030] 2. Construction of plasmids containing the full-length CD3ε chain sequence The UniProt ID for the full-length CD3ε protein sequence is: P07766 (CD3E_HUMAN), as follows: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID No. 10) After codon optimization, the full-length nucleotide sequence of the CD3ε chain gene is as follows: (SEQ ID No. 11) Adding a stop codon (TGA) to the end of the full-length nucleotide sequence of the CD3ε chain gene, and then linking it to the nucleotide sequence of signal peptide 1, we get: (SEQ ID NO: 12) The nucleotide sequence shown in SEQ ID No. 12 was synthesized and cloned into a eukaryotic expression vector containing the GS gene to construct a recombinant plasmid, named the full-length CD3ε plasmid.

[0031] 3. Transfection of CHO cells and GS pressure selection Cell resuscitation: CHO-K1 cells were cultured in suspension in CHO cell culture medium containing 4 mM L-glutamine.

[0032] Electroporation: Logarithmically growing cells were harvested, and 15 μg of HLA-B27 plasmid and 15 μg of full-length CD3ε plasmid were added. Transfection was performed using an electroporator. The electroporation parameters were: electric field strength 700 V / cm, pulse duration 15 ms.

[0033] Methionine sulfoxide (MSX) screening: 24 hours after transfection, the medium was replaced with L-glutamine-free selective medium.

[0034] MSX was added for pressure selection (to inhibit the expression of endogenous glutamine synthase in cells), with concentration gradients of 25 μM, 50 μM, and 100 μM. Cells were cultured for approximately 2-3 weeks until resistant cells formed.

[0035] 4. Flow cytometry identification and monoclonalization of double-positive cell lines Resistant cell pools were collected and subjected to limiting dilution for monoclonalization, yielding monoclonal cell lines after approximately 4-5 weeks. Flow cytometry was then performed to select cell lines with good cell populations and high HLA-B27 and CD3 fluorescence signals. Fluorescently labeled antibodies were used for staining: FITC-HLA-B27 (Thermo Fisher Scientific (China) Co., Ltd., clone number HLA-ABC-m3, catalog number MA1-82540) and PE-CD3 (Thermo Fisher Scientific (China) Co., Ltd., clone number UCHT1, catalog number 12-0038-41). Flow cytometry was performed, and a gating strategy was used to separately delineate HLA-B27-positive and CD3-positive cell populations. The results are shown in Table 1. Figure 1 As shown.

[0036] Table 1: Flow cytometry results of double plasmid co-transfection .

[0037] As shown in Table 1, the HLA-B27 positive cell population accounted for 99.71%, with good cell differentiation and high fluorescence signal intensity. The CD3 cell population was similar to that of the untransfected cells, indicating that CD3 signal could not be detected on the cell membrane.

[0038] Example 2: Preparation of double-positive cell lines by co-transfection of three plasmids The inventors analyzed that the human CD3ε chain containing the antigen recognition epitope cannot be expressed independently and located on the cell membrane surface. In this embodiment, the inventors attempted to express the complete T cell receptor complex (composed of TCRα / β, CD3γ / ε, CD3δ / ε, and CD3ζ / ζ).

[0039] To this end, the inventors first obtained the HLA-B27 plasmid according to the method in Example 1, and then prepared the TCRα-TCRβ-CD3γ plasmid and the CD3ε-CD3δ-CD3ζ plasmid, specifically: 1. Construction of TCRα-TCRβ-CD3γ plasmid The UniProt sequence number of TCRα is P01848 (TRAC_HUMAN), as follows: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQID No. 13) After codon optimization, its nucleotide sequence is as follows: ATTCAGAATCCTGATCCAGCCGTATATCAGCTGAGAGATTCTAAGTCCTCAGATAAGTCTGTTTTGCCTCTTCACCGACTTCGATTCCCAGACAAACGTATCCCAGAGCAAAGACAGCGACGTATACATCACCGATAAAACTGTCCTTGATATGAGAAGCATGGACTTCAAAAGCAATAGTGCCGTGGCTTGGTCAAATAAGTCCGATTTCGC TTGCGCCAATGCCTTCAACACAGTATCATCCCCGAAGATACGTTTCTTCCCTAGCCCCGAGAGCAGTTGCGACGTCAAATTGGTGGAAAAGAGCTTTGAAACCGATACTAACCTGAATTTCCAGAACTTGAGCGTGATTGGCTTCCGCATTCTGCTGCTGAAAGTGGCTGGCTTCAATCTTCTGATGACACTGAGGCTGTGGAGTAGT (SEQ ID No. 14) The UniProt sequence number of TCRβ is P01850 (TRBC1_HUMAN), as follows: DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID No. 15) After codon optimization, its nucleotide sequence is as follows: GATTTGAATAAGGTTTTTCCTCCCGAGGTTGCAGTGTTCGAGCCGTCCGAAGCAGAGATTTCTCACACACAGAAGGCTACTCTCGTGTGTCTCGCCACAGGCTTCTTTCCTGATCACGTGGAGTTGTCCTGGTGGGTGAATGGTAAAGAGGTTCATTCCGGCGTGAGCACAGACCCACAGCCCCTTAAGGAGCAGCCTGCCTTGAACGATTCCCGCTATTGCCTGAGTTCACGCCTCAGAGTGTCTGCCACATTTTGGCAGAATCCTAGGAACCACTTCCGGTGTCAGGTGCAGTTTTACGGTCTCAGCGAGAACGATGAGTGGACCCAGGATAGAGCAAAGCCAGTCACCCAAATCGTTTCCGCCGAGGCCTGGGGCCGGGCCGATTGTGGCTTCACCAGCGTTTCCTACCAACAGGGAGTGCTGAGCGCCACCATCCTGTACGAGATTCTCTTGGGGAAGGCTACTTTGTATGCCGTGCTGGTCTCTGCCCTCGTACTCATGGCCATGGTGAAGAGGAAGGACTTC (SEQ ID No. 16) The UniProt accession number of the protein sequence of CD3γ is: P09693 (CD3G_HUMAN), which is specifically as follows: QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID No. 17) After codon optimization, the nucleotide sequence thereof is as follows: CAAAGTATCAAAGGTAATCACCTGGTCAAGGTGTACGACTACCAGGAGGACGGAAGTGTTTTGCTTACATGTGATGCTGAGGCTAAGAATATCACCTGGTTTAAAGACGGTAAGATGATCGGCTTCCTCACAGAGGACAAGAAGAAGTGGAACCTGGGAAGTAACGCAAAAGACCCCCGGGGAATGTACCAGTGCAAAGGGTCTCAGAATAAGAGTAAGCCTCTGCAGGTGTACTACAGGAT GTGTCAGAACTGCATCGAGTTGAATGCAGCTACTATTAGCGGGTTTCTCTCGCAGAGATAGTCTCAATCTTCGTCCTGGCCGTGGGTGTGTATTTCATAGCCGGGCAGGACGGTGTGAGG CAGAGCCGCGCCTCCGATAAGCAGACTCTGCTCCCAAACGATCAGCTCTACCAACCCTGAAGGACCGAGAGGATGATCAGTACTCTCACCTGCAGGGTAACCAGCTCAGAAGGAAC (SEQ ID No. 18) A TCRα-TCRβ-CD3γ fusion gene was constructed using the nucleotide sequences of TCRα, TCRβ, and CD3γ. Adjacent genes were linked by an IRES sequence (SEQ ID No. 8). An additional nucleotide sequence of signal peptide 1 (SEQ ID No. 4) was added before each gene. Specifically, the nucleotide sequences of signal peptide 1, TCRα, IRES, signal peptide 1, TCRβ, IRES, signal peptide 1, and CD3γ were linked together. Furthermore, stop codons (TGAs) were added after the nucleotide sequences of TCRα, TCRβ, and CD3γ. The resulting nucleotide sequence of the TCRα-TCRβ-CD3γ fusion gene is as follows: The nucleotide sequence shown in SEQ ID No. 19 was synthesized and cloned into a eukaryotic expression vector containing the GS gene to construct a recombinant plasmid, named TCRα-TCRβ-CD3γ plasmid.

[0040] 2. Construction of CD3ε-CD3δ-CD3ζ plasmid The full-length amino acid and nucleotide sequences of the CD3ε chain are shown in SEQ ID No. 10 and SEQ ID No. 11 in Example 1, respectively.

[0041] The UniProt ID for the full-length CD3δ protein sequence is: P04234 (CD3D_HUMAN), as follows: FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID No. 20) After codon optimization, the full-length nucleotide sequence of the CD3δ chain gene is as follows: TTCAAAATCCCAATCGAGGAACTCGAAGACAGGGTGTTTGTTAATTGTAATACCAGCATCACATGGGTGGAGGGTACTGTCGGGACCCTCCTGAGCGACATCACCAGGCTTGATTTGGGCAAAAGAATCCTCGATCCCAGAGGTATATACCGGTGCAACGGCACCGATATCTACAAGGACAAAGAATCCACAGTCCAGGTTCACTACAGGATGTGCCAGTCATGTGT CGAGCTGGATCCAGCTACTGTCGCAGGCATTGTAACAGACGTGATTGCGACGCTCCTCTTGGCTTTGGGAGTGTTCTGCTTCGCCGGGCATGAAACTGGCAGACTGTCTGGAGCAGCGGATACTCAGGCTCTCCTTAGAAATGACCAGGTGTACCAGCCCCTGCGAGATCGGGATGACGCCCAGTATAGCCACCTGGGGGGAAATTGGGCAAGGAACAAG (SEQ ID No. 21) The UniProt ID for the full-length CD3ζ protein sequence is: P20963 (CD3Z_HUMAN), as follows: QSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No. 22) After codon optimization, the full-length nucleotide sequence of the CD3ζ chain gene is as follows: CAGTCATTCGGGCTGCTCGACCCAAAACTGTGCTACTTGCTTGACGGCATCCTGTTTATCTACGGCGTGATCCTGACGGCTCTTTTTCTCTCGGGTCAAATTCTCCAGGTCCGCTGACGCCCCTGCATATCAACAAGGGCAGAATCAGCTTTACAATGAGCTGAACCTGGGCAGACGCGAAGAATACGACGTTCTGGACAAGCGCCGCGTCGAGAT CCAGAGATGGGCGGCAAACCCCAGCGCAGGAAGAACCCGCAGGAGGGGTTGTACAATGAGCTGCAGAAAGACAAGATGGCCGAAGCCTACTCCGAGATTGGTATGAAAGGCGAAAGGCGGAGAGGCAAAGGCCATGACGGGCTTTACCAAGGGTTGTCCACCGCTACTAAGGACACATACGATGCCCTTCACATGCAGGCTCTGCCACCTAGA (SEQ ID No. 23) A fusion gene was constructed using the nucleotide sequences of CD3ε, CD3δ, and CD3ζ. Adjacent genes were linked by an IRES sequence (SEQ ID No. 8). An additional nucleotide sequence of signal peptide 1 (SEQ ID No. 4) was added before each gene. Specifically, the nucleotide sequences of signal peptide 1, CD3ε, IRES, signal peptide 1, CD3δ, IRES, signal peptide 1, and CD3ζ were linked together. Furthermore, stop codons (TGAs) were added after the nucleotide sequences of CD3ε, CD3δ, and CD3ζ. The resulting nucleotide sequence of the CD3ε-CD3δ-CD3ζ fusion gene is as follows: The nucleotide sequence shown in SEQ ID No. 24 was synthesized and cloned into a eukaryotic expression vector containing the GS gene to construct a recombinant plasmid, named CD3ε-CD3δ-CD3ζ plasmid.

[0042] 3. Transfection of CHO cells and GS pressure selection Cell resuscitation: CHO-K1 cells were cultured in suspension in CHO cell culture medium containing 4 mM L-glutamine.

[0043] Electroporation: Logarithmically growing cells were collected and 10 μg of HLA-B27 plasmid, 10 μg of TCRα-TCRβ-CD3γ plasmid, and 10 μg of CD3ε-CD3δ-CD3ζ plasmid were added. Electroporation was performed using an electroporator. The electroporation parameters were: electric field strength 700 V / cm, pulse duration 15 ms.

[0044] Methionine sulfoxide (MSX) screening: 24 hours after transfection, the medium was replaced with L-glutamine-free selective medium.

[0045] MSX was added for pressure selection (to inhibit the expression of endogenous glutamine synthase in cells), with concentration gradients of 25 μM, 50 μM, and 100 μM. Cells were cultured for approximately 2-3 weeks until resistant cells formed.

[0046] 4. Flow cytometry identification and monoclonalization of double-positive cell lines Resistant cell pools were collected and subjected to limiting dilution for monoclonalization, yielding monoclonal cell lines after approximately 4-5 weeks. Flow cytometry was then performed to select cell lines with good cell populations and high HLA-B27 and CD3 fluorescence signals. Fluorescently labeled antibodies, FITC-HLA-B27 and PE-CD3, were used for staining. Flow cytometry was performed, and a gating strategy was used to separately delineate HLA-B27-positive and CD3-positive cell populations. The results are shown in Table 2. Figure 2 As shown.

[0047] Table 2: Flow cytometry results of triple plasmid co-transfection .

[0048] Table 2 shows that HLA-B27 positive cells accounted for 91.48%, with good cell differentiation and high fluorescence signal intensity. CD3 positive cells accounted for 73.29%, with poor cell differentiation and low fluorescence signal intensity. Furthermore, to express the complete T cell receptor complex, co-transfection with plasmids encoding six polypeptide chains is required, making it difficult to quickly obtain cell lines with high CD3 expression.

[0049] Example 3: Construction of CD3-HLA-B27 recombinant expression vector As demonstrated in Examples 1 and 2, when HLA-B27 and CD3 are transfected into cell lines separately, the two proteins cannot simultaneously achieve high signal values. Therefore, the inventors attempted to express CD3 and HLA-B27 as a fusion protein.

[0050] 1. Construction of CD3-HLA-B27 plasmid First, according to Example 1, the CD3 positivity rate was 0, indicating that CD3 was not effectively expressed on the cell membrane. Therefore, when constructing the recombinant expression vector for the fusion protein, the extracellular segment of the CD3ε chain (NCBI ID 1SY6_A) was selected.

[0051] The amino acid sequence of the extracellular segment of the CD3ε chain is as follows: MQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMGSADDAKKDAAKKDDAKKDDAK KDGSDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARV (SEQ ID No. 25) The nucleotide sequence of the extracellular segment of the CD3ε chain is as follows: (SEQ ID No. 26) To facilitate the folding of the fusion protein, the inventors introduced a linker peptide between CD3 and HLA-B27. In this embodiment, the linker peptide is (GGGGS)3, and its amino acid sequence is as follows: GGGGSGGGGSGGGGS (SEQ ID No. 27) The nucleotide sequence of the linker peptide is as follows: GGCGGCGGAGGATCTGGCGGCGGAGGTTCTGGCGGCGGAGGCTCT (SEQ ID No. 28) In constructing the recombinant expression vector, the nucleotide sequence of B2M is included after the nucleotide sequence of HLA-B27. Both the nucleotide sequences of HLA-B27 and B2M include a stop codon (TAATGA). Furthermore, the nucleotide sequence of signal peptide 1 (SEQ ID No. 4) is included before the nucleotide sequence of the extracellular segment of the CD3ε chain, and the nucleotide sequence of signal peptide 2 (SEQ ID No. 5) is included before the nucleotide sequence of B2M. Following the nucleotide sequence of HLA-B27 and preceding the nucleotide sequence of signal peptide 2, the IRES sequence (SEQ ID No. 8) is also included. The resulting nucleotide sequence of the recombinant gene expressing the CD3-HLA-B27 fusion protein is as follows: The nucleotide sequence shown in SEQ ID No. 29 was synthesized and cloned into a eukaryotic expression vector containing the GS gene to construct a recombinant plasmid, named CD3-HLA-B27 plasmid, with the structure shown below. Figure 3 As shown.

[0052] 2. Transfection of CHO cells and GS pressure selection Cell resuscitation: CHO-K1 cells were cultured in suspension in CHO cell culture medium containing 4 mM L-glutamine.

[0053] Electroporation: Logarithmically growing cells were taken and 30 μg of CD3-HLA-B27 plasmid was added. Transfection was performed using an electroporator. The electroporation parameters were: electric field strength 700 V / cm, pulse duration 15 ms.

[0054] Methionine sulfoxide (MSX) screening: 24 hours after transfection, the medium was replaced with L-glutamine-free selective medium.

[0055] MSX was added for pressure selection (to inhibit the expression of endogenous glutamine synthase in cells), with concentration gradients of 25 μM, 50 μM, and 100 μM. Cells were cultured for approximately 2-3 weeks until resistant cells formed.

[0056] 3. Flow cytometry identification and monoclonalization of double-positive cell lines Resistant cell pools were collected and subjected to limiting dilution for monoclonalization, yielding monoclonal cell lines after approximately 4-5 weeks. Flow cytometry was then performed to select cell lines with good cell populations and high HLA-B27 and CD3 fluorescence signals. Fluorescently labeled antibodies, FITC-HLA-B27 and PE-CD3, were used for staining. Flow cytometry was performed, and a gating strategy was used to separately delineate HLA-B27-positive and CD3-positive cell populations. The results are shown in Table 3. Figure 4 As shown.

[0057] Table 3: Flow cytometry results of single plasmid transfection (fusion expression) .

[0058] As shown in Table 3, the proportion of HLA-B27 positive cells was 98.73%, the proportion of CD3 positive cells was as high as 99.35%, and the proportion of CD3 and HLA-B27 double positive cells was 99.58%, with high fluorescence signal intensity.

[0059] Example 4: Sequence Optimization Design The amino acid sequence of the fusion protein formed by the extracellular segment of the CD3ε chain (SEQ ID No. 25) and HLA-B27 (SEQ ID No. 1) via a flexible linker peptide (SEQ ID No. 27) is as follows: (SEQ ID No. 30) Because the extracellular segment of the CD3ε chain contains a flexible linker peptide after linking with HLA-B27, the structure of the adjacent region (±10 amino acids from both ends of the flexible linker peptide) is incorrect, ultimately leading to reduced expression of the CD3-HLA-B27 fusion protein or decreased antigen detection activity. Therefore, the inventors optimized the sequence of the fusion protein.

[0060] 1. AlphaFold3 program predicts protein structure The AlphaFold3 program reads the following main parameters from a JSON file: (1) Amino acid sequence of the fusion protein: SEQ ID No. 30; (2) Random number seed: seed=42; (3) Number of random number seeds = 2048.

[0061] The AlphaFold3 program outputs the CIF file of the protein prediction model with the highest ranking score. The confidence scores of amino acid residues (pLDDT) are greater than 90 points at positions ±10 amino acids at both ends of the flexible linker peptide (SEQ ID No. 27), indicating that the protein structure model has a high confidence level.

[0062] 2. PyMol program for format conversion The PyMol program reads the aforementioned CIF file and re-outputs it as a PDB file that can be processed in subsequent steps.

[0063] 3. ProteinMPNN program predicts mutant amino acids Read the aforementioned CIF file, generate 1024 sample sequences under the following parameters, and save them to a FASTA file: (1) Exclude the amino acid "Cys"; (2) Random number seed: seed=42; (3) Temperature: temperature = 0.2; (4) Only mutate ±10 amino acids at both ends of the flexible linker peptide; (5) Structural noise 0.200 angstroms.

[0064] 4. AF3Score Program Evaluation The AF3Score program reads the 1024 sampled sequences generated in the previous steps, outputs scores, and selects the top 10 sequences, as shown in Table 4.

[0065] Table 4: Mutation information of 10 mutant sequences .

[0066] Wherein, V(-1)A indicates that the first amino acid of the flexible linker peptide is mutated from V to A, corresponding to SEQ ID No. 25, and the mutation is V204A; M(+6)L indicates that the sixth amino acid of the flexible linker peptide is mutated from M to L, corresponding to SEQ ID No. 1, and the mutation is M6L, and so on for the remaining mutations.

[0067] Nucleotide sequences of 10 mutant sequences were obtained, and recombinant vectors were constructed according to the method in Example 3. These vectors were transfected into CHO cells and subjected to GS pressure selection to collect resistant cell pools. Fluorescently labeled antibodies FITC-HLA-B27 and PE-CD3 were used for staining to detect fluorescence signals in the cell pools. The results showed that cell pools transfected with recombinant vectors constructed using mutants 2, 3, 5, and 9 showed no fluorescence signal. Cell pools exhibiting fluorescence signals were subjected to limiting dilution and monoclonalization. Monoclonal cell lines were obtained approximately 4-5 weeks later. Flow cytometry was then performed to select cell lines with good cell clustering and the highest HLA-B27 and CD3 fluorescence signals. The detection results are shown in Table 5. Figure 5 , Figure 6 As shown.

[0068] Table 5: Flow cytometry results of cells transfected with mutant sequences .

[0069] From Table 5, Figure 5 and Figure 6 It can be seen that the fluorescence signal of most of the 10 mutated sequences is lower than that of the original sequence. This may be because the mutation causes the protein to be unable to be expressed or the expression level to be reduced, or the mutation causes the protein structure to be incorrect and lose the original antibody binding site, resulting in a low detection signal.

[0070] For mutation 1, the amino acid sequence of the extracellular segment of the CD3ε chain becomes: MQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMGSADDAKKDAAKKDDAKKDDAK KDGSDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYIRARA (SEQ ID No. 31) Its nucleotide sequence becomes: ATGGAAACCGATACTCTTCTTCTCTGGGTACTGCTGCTGTGGGTACCTGGCTCCACCGGGATGCAAAGTATCAAGGGTAATCATCTAGTGAAAGTCTACGACTACCAAGAGGACGGCTCCGTGCTGCTGACCTGCGACGCCGAGGCCAAGAACATCACCTGGTTCAAGGACGGCAAGATGATCGGCTTTCTGACCGAGGATAAGAAGAAGTGGAACCTGGGCTCCAACGCCAAGGATCCTAGAGGCATGTACCAGTGCAAGGGCAGCCAGAACAAGTCCAAGCCTCTGCAGGTGTACTATCGGATGGGCTCTGCCGACGACGCCAAGAAGGACGCTGCTAAAAAGGACGACGCTAAGAAGGACGATGCTAAGAAAGATGGCTCCGACGGCAACGAGGAAATGGGCGGCATCACCCAGACACCTTACAAGGTGTCCATCTCCGGCACCACCGTGATCCTCACCTGTCCTCAGTACCCCGGCTCTGAGATCCTGTGGCAGCACAACGACAAGAATATCGGAGGCGACGAAGATGACAAGAACATCGGCTCTGACGAGGACCACCTGTCCCTGAAAGAGTTCTCCGAGCTGGAACAGAGCGGCTACTACGTGTGCTACCCACGGGGCAGCAAGCCCGAAGATGCCAACTTCTACCTGTACATCAGAGCCAGAGCC (SEQ ID No. 32) The amino acid sequence of HLA-B27 becomes: GGSHSLRYFHTSVSRPGRGEPRFITVGYVDDTLFVRFDSDAASPREEPRAPWIEQEGPEYWDRETQICKAKAQTDREDLRTLLRYYNQSEAGSHTLQNMYGCDVGPDGRLLRGYHQHAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAARVAEQLRAYLEGECVEWLRRYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQSTVPIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSYSQAACSDSAQGSDVSLTA (SEQ ID No. 33) Its nucleotide sequence becomes: Example 5: Cell passage stability and application testing Genetic stability: Cell lines obtained from the mutant 1 sequence were passaged for 38 consecutive generations, and the MFI value was detected by flow cytometry.

[0071] One vial of cells stored in liquid nitrogen was revived and cultured to approximately 30 passages. Then, another vial of cells from the same batch was revived and cultured for a few more days until the viability of the second-revised cells reached over 98%. At this point, the first-revised cells had reached 38 passages, and the second-revised cells had reached 7 passages.

[0072] In this application, the culture passage number refers to the number of cell divisions, not the number of cell passages. The culture passage number is defined using the following formula:

[0073] The test results are shown in Table 6.

[0074] Table 6: Stability analysis of cell lines after 38 passages (changes in CD3 and B27 fluorescence intensity) .

[0075] As shown in Table 6, when the cell line culture passage number increased from 7 to 38, the proportion of positive cells did not change significantly, but the MFI of CD3 fluorescence signal decreased by 10.9%, and the MFI of HLA-B27 fluorescence signal decreased by 15.2%. Since the sensitivities of CD3 antibody and HLA-B27 antibody are not the same, the decrease in MFI of fluorescence signal is not 1:1.

[0076] This shows that the cell line can still meet the detection requirements after 38 passages, indicating that the stability of the cell line can meet the needs of long-term application and will not produce significant batch-to-batch differences as the number of culture passages increases.

[0077] Example 6: Simulated Clinical Testing After the cell line obtained by the mutant 1 sequence was fixed, it was used as a quality control and run on a clinical flow cytometer. The results showed that it could be correctly identified by the instrument as "HLA-B27 positive population in T cell population".

[0078] Furthermore, it should be understood that after reading the foregoing content of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A recombinant CD3-HLA-B27 protein, characterized in that, It is obtained by fusing a mutant of the extracellular segment of the CD3ε chain with a mutant of HLA-B27. Between the mutant of the extracellular segment of the CD3ε chain and the mutant of HLA-B27, there is also a flexible linker peptide, which is (GGGGS). n Where n=1~5, the mutant of the extracellular segment of the CD3ε chain is based on the amino acid sequence shown in SEQ ID No. 25 with L200I and V204A mutations, and the mutant of HLA-B27 is based on the amino acid sequence shown in SEQ ID No. 1 with M6L mutation.

2. A gene encoding the CD3-HLA-B27 recombinant protein of claim 1, characterized in that, This includes the nucleotide sequences of mutants encoding the extracellular segment of the CD3ε chain, and the nucleotide sequences of mutants encoding HLA-B27.

3. An expression carrier, characterized in that, Includes the gene described in claim 2.

4. The expression vector according to claim 3, characterized in that, The expression vector further includes a nucleotide sequence encoding B2M following the nucleotide sequence encoding the HLA-B27 mutant.

5. The expression vector according to claim 4, characterized in that, Following the nucleotide sequence encoding the HLA-B27 mutant and preceding the nucleotide sequence encoding B2M, there is also a nucleotide sequence for the internal ribosome entry site.

6. The expression vector according to claim 4 or 5, characterized in that, The mutant sequence encoding the extracellular segment of the CD3ε chain is preceded by a nucleotide sequence encoding a first signal peptide, and the mutant sequence encoding B2M is preceded by a nucleotide sequence encoding a second signal peptide.

7. A host cell, characterized in that, It contains the expression vector as described in any one of claims 3 to 6.

8. The method for preparing the CD3-HLA-B27 recombinant protein according to claim 1, characterized in that, Includes the step of inducing the host cell of claim 7 to express a protein.

9. The use of the host cell according to claim 7 in the preparation of a kit for detecting HLA-B27 expression in biological samples, characterized in that, The host cell was used as a positive control.

10. A kit for detecting HLA-B27 in biological samples, characterized in that, Includes the host cell as described in claim 7, wherein the host cell serves as a positive control.

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