Recombinant vectors for preparing hla / beta2m complex and methods of making and using the same
By introducing FOS and JUN leucine zippers into the original vector, a recombinant vector was constructed, which solved the problem of low expression level of HLA-β2M complex, achieved efficient expression and refolding, and supported the development of biopharmaceuticals targeting intracellular antigens.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- PUJIAN BIOLOGICAL (WUHAN) TECH CO LTD
- Filing Date
- 2022-11-28
- Publication Date
- 2026-04-21
AI Technical Summary
Existing technologies struggle to efficiently express and refold the HLA-β2M complex, resulting in low expression levels and low refolding rates, which hinders the development of biopharmaceuticals targeting intracellular antigens.
By introducing FOS and JUN leucine zippers into the original vector to form a stable heterodimer, a recombinant vector was constructed to achieve efficient expression of the HLA/β2M complex. The specific steps included inserting the corresponding gene sequence at specific restriction sites and expressing it in CHO cells.
We achieved high expression levels of the HLA/β2M complex in CHO cells, up to 92.45 mg/L, which significantly improved expression level and refolding rate, supporting the development of biopharmaceuticals targeting intracellular antigens.
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Figure CN116004718B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of antibody preparation technology for MHC-peptide complexes, and particularly to recombinant vectors for preparing HLA / β2M complexes, their preparation methods, and applications. Background Technology
[0002] In the cutting-edge research and development of biopharmaceuticals, targeting tumor-related intracellular proteins is gradually coming into focus. Due to the limitations of antibody molecular weight, it is difficult for antibody molecules to directly target intracellular antigens through the cell membrane. Currently, most common methods of targeting intracellular antigens are indirect, such as introducing the target antibody into cells for expression through viral vectors, nanospheres, liposomes, etc., or fusing the target antibody with peptides to mediate internalization.
[0003] Targeting intracellular antigens can also be achieved by utilizing cellular immune surveillance mechanisms. Generally, intracellular antigens are degraded into specific short peptides by intracellular hydrolases. These peptides are then presented by the cell as MHC1 peptide complexes on the cell surface. CD8+ T cells can recognize these tumor- or virus-associated MHC1 peptide complexes via TCRs (antigen receptors) and kill malignant tumor cells or virus-infected cells. Therefore, finding antibodies (TCRm Abs) or TCRs targeting tumor-associated MHC1 peptide complexes allows for targeting intracellular antigens and achieving therapeutic goals. Among biotechnology companies targeting intracellular protein antigens in the form of MHC1 peptide complexes, Adaptimmune / Immunecore's TCR-T cell therapy and Eureka's CAR-T cell therapy are representative. Adaptimmune / Immunecore are sister companies; both companies use their SPEAR display platform to screen for high-affinity mutant TCRs targeting MHC1 peptide complexes. Eureka also targets MHC1 peptide complexes, using its E-ALPHA display platform to screen for high-affinity antibodies. Cell therapy targeting intracellular protein antigens and targeting MHCI peptide complexes on the cell surface is widely used in the treatment of solid tumors such as non-small cell lung cancer, gastric cancer, and ovarian cancer. Furthermore, leading companies in the cell therapy field, such as Juno and Kite, have also made significant investments in this technology.
[0004] Targeting intracellular protein antigens, particularly targeting tumor antigen-related MHC I peptide complexes, will represent a growth market for new biologics. In the field of cell therapy, cell therapy for hematologic malignancies has shown significant efficacy, with two drugs already on the market; however, drug development for solid tumors has progressed slowly. The National Cancer Institute (NCI) has compiled and scored cancer antigens, providing valuable tumor-related antigens. Targeting tumor-related intracellular protein antigens in the form of MHC I peptide complexes will offer more options for cell therapy of solid tumors. Targeting intracellular antigens to locate tumor cells also provides new target options for bispecific antibodies, antibody-drug conjugates, and other fields.
[0005] In the field of precision medicine, high-throughput gene sequencing technology can be combined to detect tumor mutational antigens and neoantigens. New tumor vaccines can be developed in the form of MHCII peptide complexes or MHCCI peptide complexes, or the body's immune cells can be activated in vitro. The U.S. Food and Drug Administration (FDA) has granted Fast Track designation to the investigational drug tebentafusp (IMCgp100) for the treatment of previously untreated (treatment-naïve) HLA-A*020-positive metastatic uveal melanoma (UM). Previously, the FDA had granted tebentafusp orphan drug designation for the treatment of UM. These examples demonstrate the immense research and development potential of these technologies.
[0006] Obtaining recombinantly expressed HLA-β2M complexes is extremely difficult. The HLA-β2M complex undergoes numerous post-translational modifications, resulting in very low expression levels in eukaryotic expression systems. In E. coli expression systems, only inclusion bodies are expressed, leading to extremely low refolding recovery rates. Currently, the most common method involves purifying HLA and β2M inclusion bodies separately using recombinant expression in E. coli, denaturing them with denaturing agents (such as high-concentration urea or guanidine hydrochloride), and finally refolding them. It is well known that protein refolding is very difficult, with no fixed formula, often requiring a single method for each protein; and the refolding yield is very low, typically around tens of μg / L. Even when refolded complexes are obtained, their activity is relatively low. Summary of the Invention
[0007] To address the shortcomings of existing technologies, this invention provides a recombinant vector for preparing the HLA / β2M complex, its preparation method, and its applications. In MHCI class molecules, HLA molecules interact with β2M molecules, and β2M can stabilize the structure of HLA. This invention modifies the original vector by introducing a FOS / JUN leucine zipper, allowing the proto-oncogene FOS and transcription factor JUN to form a very strong heterodimer through the leucine zipper, thus achieving efficient expression of the HLA / β2M complex. This is specifically achieved through the following techniques.
[0008] A recombinant vector for preparing the HLA / β2M complex is provided by inserting a FOS leucine zipper bZIP motif sequence between the NheI / AvrII restriction sites of the original vector, a JUN leucine zipper bZIP motif sequence between the BsiWI / BlpI restriction sites, and a β2M gene sequence between the AgeI / NheI restriction sites.
[0009] The FOS leucine zipper bZIP motif sequence is shown in SEQ ID NO.1, the JUN leucine zipper bZIP motif sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the β2M gene is shown in SEQ ID NO.3.
[0010] Preferably, a corresponding HLA sequence is also inserted between the PmeI / BsiWI restriction sites of the recombinant vector.
[0011] It should be noted that the restriction enzyme sites for inserting HLA gene sequences can be adjusted according to actual needs, and therefore do not require special specification; for example, PmeI / BsiWI double restriction sites can be used. Depending on the amino acid sequence of the HLA protein, there are also corresponding different gene sequences encoding HLA proteins; therefore, different HLA protein gene sequences can be inserted according to actual needs.
[0012] Preferably, the original vector is the pTRIOZ-hIgG1K vector.
[0013] The present invention also provides a method for preparing the above-mentioned recombinant vector, comprising the following steps:
[0014] S1. The original vector was double-digested with restriction endonucleases NheI and AvrII, and the vector was recovered. PCR amplification was performed using the FOS leucine zipper bZIP motif plasmid as a template, and the 100bp fragment was recovered. The FOS leucine zipper bZIP motif sequence was inserted between the NheI / AvrII restriction sites to obtain the first recombinant vector. The nucleotide sequence of the first recombinant vector is shown in SEQ ID NO.4.
[0015] S2. Take the first recombinant vector obtained in step S1, and double-digest it with restriction endonucleases BsiWI and BlpI, then recover the vector; use the JUN leucine zipper bZIP motif plasmid as a template for PCR amplification, recover the 100bp fragment, and insert the JUN leucine zipper bZIP motif sequence between the BsiWI / BlpI restriction sites to obtain the second recombinant vector; the nucleotide sequence of the second recombinant vector is shown in SEQ ID NO. 5;
[0016] S3. Take the second recombinant vector obtained in step S2, and double-digest it with restriction endonucleases AgeI and NheI. Recover the vector. Perform PCR amplification using the β2M gene sequence as a template, recover the 400bp fragment, and insert it between the AgeI / NheI restriction sites to finally obtain the recombinant vector for preparing the HLA / β2M complex. The nucleotide sequence of the recombinant vector for preparing the HLA / β2M complex is shown in SEQ ID NO. 6.
[0017] Preferably, in step S1 of the above method, the forward primer sequence for PCR amplification is shown in SEQ ID NO.7, and the reverse primer sequence is shown in SEQ ID NO.8.
[0018] Preferably, in step S2 of the above method, the forward primer sequence for PCR amplification is shown in SEQ ID NO.9, and the reverse primer sequence is shown in SEQ ID NO.10.
[0019] Preferably, in step S3 of the above method, the forward primer sequence for PCR amplification is shown in SEQ ID NO.11, and the reverse primer sequence is shown in SEQ ID NO.12.
[0020] Preferably, step S4 of the above method is as follows:
[0021] The present invention also provides the application of the HLA / β2M complex prepared by the above method in the preparation of reagents for diagnosing cancer or in the preparation of drugs for treating cancer.
[0022] The present invention also provides a cancer diagnostic test kit containing the HLA / β2M complex prepared by the above method.
[0023] The HLA / β2M complex provided by this invention can bind to different peptides based on the differences in HLA proteins. Therefore, the HLA / β2M complex provided by this invention can be used as a tool in various technical fields where it is necessary to specifically bind to a certain peptide. Based on current research results, if it binds to a specific peptide of cancer cells, the HLA / β2M / peptide complex can effectively stimulate T cells, thereby killing cancer cells and playing a role in cancer treatment.
[0024] Compared with existing technologies, the advantages of this invention are as follows: By introducing a FOS / JUN leucine zipper into the original vector, the proto-oncogene FOS and transcription factor JUN proteins form a very strong heterodimer through the leucine zipper. This heterodimer structure is stable, thereby achieving efficient expression of the HLA / β2M complex. By utilizing nearly 60 different HLA sequences to construct corresponding recombinant vectors and employing methods such as transfection into CHJO cells for expression, the final HLA / β2M complex expression levels were all very high, exceeding 1 mg / L; the highest expression level reached 92.45 mg / L. Attached Figure Description
[0025] Figure 1 This is an electrophoresis image of the 400bp fragment (i.e., pATX-β2M-FOS-JUN plasmid) recovered by electrophoresis in step S3 of Example 1.
[0026] Figure 2 This is a schematic diagram of the structure of the pATX-β2M-FOS-HLA-A*02:01-JUN plasmid prepared in Example 2;
[0027] Figure 3 This is an SDS-PAGE electrophoresis image of the HLA-A*02:01 / β2M complex recovered in Example 2;
[0028] Figure 4 This is an SDS-PAGE electrophoresis image of the HLA-A*03:01:01:01 / β2M complex recovered in Example 3;
[0029] Figure 5 This is an SDS-PAGE electrophoresis image of the HLA-G / β2M complex recovered in Example 4;
[0030] Figure 6 This is an SDS-PAGE electrophoresis image of the HLA-A*11:01 / β2M complex recovered in Example 5. Detailed Implementation
[0031] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0032] In the following examples, all PCR systems used for PCR amplification are shown in Table 1 below.
[0033] Table 1 PCR amplification system
[0034] project volume template 1μL upstream primer 1μL Downstream primer 1μL 2xMix 25μL Ultrapure water 22μL
[0035] The PCR amplification program was as follows: first, react at 95℃ for 5 min; then react at 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 30 s, for 30 cycles; then extend at 72℃ for 5 min; and finally incubate at 12℃ until the sample is removed.
[0036] Example 1: Preparation of a recombinant vector for preparing the HLA / β2M complex
[0037] The recombinant vector for preparing the HLA / β2M complex provided in this embodiment was prepared by the following method.
[0038] S1. Take the pTRIOZ-hIgG1K plasmid (purchased from Invivogen, i.e. the original vector), double digest it with restriction endonucleases NheI and AvrII, and recover the 7kb fragment using 1% agarose gel.
[0039] Then, PCR amplification was performed using the FOS leucine zipper bZIP motif plasmid (as shown in SEQ ID NO.13) as a template to insert the FOS leucine zipper bZIP motif sequence (as shown in SEQ ID NO.1) between the NheI / AvrII restriction sites; the sequences of the amplification primer pairs are as follows.
[0040] Forward primer (as shown in SEQ ID NO.7):
[0041] 5'-gtagatatcacgtcatgaaagctagcggcggcggcggcggcctgac-3';
[0042] Reverse primer (as shown in SEQ ID NO.8):
[0043] 5'-gtcattggggaaacctgctcctaggtcagtggtggtggtggtggtggctgccgcc-3';
[0044] The PCR amplification program was as follows: first, react at 95℃ for 5 min; then react at 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, and repeat 30 times; then extend at 72℃ for 5 min; and incubate at 12℃ until the sample is removed.
[0045] The 100bp fragment was recovered using a 1% agarose gel, and the first recombinant vector, pTRIOZ-hIgG1K-FOS plasmid, was obtained by sequencing verification; the nucleotide sequence of pTRIOZ-hIgG1K-FOS plasmid is shown in SEQ ID NO.4.
[0046] S2. Take the first recombinant vector pTRIOZ-hIgG1K-FOS obtained in step S1, and double-digest it with restriction endonucleases BsiWI and BlpI. Recover the 6.7kb fragment using a 1% agarose gel. Perform PCR amplification using the JUN leucine zipper bZIP motif plasmid (as shown in SEQ ID NO.14) as a template to insert the JUN leucine zipper bZIP motif sequence (as shown in SEQ ID NO.2) between the BsiWI / BlpI restriction sites. The sequences of the amplification primer pair are as follows.
[0047] Forward primer (as shown in SEQ ID NO.9):
[0048] 5'-caagtttaaacaccatggaacgtacgggcggcggcggcggccgcat-3';
[0049] Reverse primer (as shown in SEQ ID NO.10):
[0050] 5'-atgtctggccagctaggtccctctacttctcgaactgggggtggct-3';
[0051] The PCR amplification program was as follows: first, react at 95℃ for 5 min; then react at 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, and repeat 30 times; then extend at 72℃ for 5 min; and incubate at 12℃ until the sample is removed.
[0052] The 100bp fragment was recovered using a 1% agarose gel, and the second recombinant vector, pATX-FOS-JUN plasmid, was obtained by sequencing verification. The nucleotide sequence of pATX-FOS-JUN plasmid is shown in SEQ ID NO.5 below.
[0053] S3. Take the second recombinant vector pATX-FOS-JUN obtained in step S2, and double-digest it with restriction endonucleases AgeI and NheI. Recover the 7.3kb fragment using a 1% agarose gel. Using the synthesized β2M gene sequence (as shown in SEQ ID NO. 3) as a template, insert it between the AgeI / NheI restriction sites. The sequences of the amplification primer pair are as follows.
[0054] Forward primer (as shown in SEQ ID NO.11):
[0055] 5'-aaccaccgctaattcaaagcaaccggtgccgccaccatgagcagaagcgtggcc-3';
[0056] Reverse primer (as shown in SEQ ID NO.12):
[0057] 5'-ggtcaggccgccgccgccgcccatgtctcgatcccacttaacg-3';
[0058] The PCR amplification program was as follows: first, react at 95℃ for 5 min; then react at 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, and repeat 30 times; then extend at 72℃ for 5 min; and incubate at 12℃ until the sample is removed.
[0059] The 400bp fragment was recovered using 1% agarose gel electrophoresis, such as... Figure 1 As shown, the recombinant vector for preparing the HLA / β2M complex, namely the pATX-β2M-FOS-JUN plasmid, was obtained after sequencing verification; the nucleotide sequence of the pATX-β2M-FOS-JUN plasmid is shown in SEQ ID NO.6.
[0060] In subsequent Examples 2-5, for different types of HLA proteins, the nucleotide sequence of the corresponding HLA protein was inserted between the PmeI / BsiWI sites of the above-mentioned pATX-β2M-FOS-JUN plasmid vector to prepare pATX-β2M-FOS-HLA-JUN plasmid that can be directly used to prepare HLA / β2M complex.
[0061] Example 2: Preparation of HLA-A*02:01 / β2M complex
[0062] The amino acid sequence of the HLA-A*02:01 protein is shown in SEQ ID NO.15, and the corresponding nucleotide sequence of HLA-A*02:01 is shown in SEQ ID NO.16. Using a self-synthesized plasmid containing the HLA-A*02:01 nucleotide sequence (SEQ ID NO.17) as a template, amplification was performed, and a 1 kb fragment was recovered using a 1% agarose gel. The nucleotide sequence shown in SEQ ID NO.16 was then inserted between the PmeI / BsiWI sites of the vector. After sequencing verification, the pATX-β2M-FOS-HLA-A*02:01-JUN plasmid was prepared. The plasmid structure is shown below. Figure 2 As shown; the sequences of the amplification primer pairs are as follows.
[0063] Forward primer (as shown in SEQ ID NO.18):
[0064] 5'-ccaccggcgaggcgcgccaagtttaaacgccgccaccatggccgtgatgg-3';
[0065] Reverse primer (as shown in SEQ ID NO.19):
[0066] 5'-cgatgcggccgccgccgccgcccacgatggcgatggtgggctgg-3';
[0067] The PCR amplification program was as follows: first, react at 95℃ for 5 min; then react at 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, and repeat 30 times; then extend at 72℃ for 5 min; and incubate at 12℃ until the sample is removed.
[0068] The HLA-A*02:01 / β2M complex was prepared using the above plasmid. The specific preparation method is as follows:
[0069] 1. Plasmid extraction: Using ThermoFisher PureLink TM HiPure Plasmid Extraction Kit (Catalog No.: K210007), extract plasmids according to the instructions;
[0070] 2. Transfection: Transfection is performed using FreeStyle. TM MAX Transfection reagent (ThermoFisher, catalog number: 16447100) was used for cell culture and transfection according to the instructions. 300 mL of CHO cells were transfected, and the culture supernatant was collected and purified on the 6th day after transfection.
[0071] 3. The target HLA / β2M complex was purified from the collected culture supernatant using Ni-TED resin (brand: Roche, catalog number: 5893801001), and a total of 9 ml of eluent was collected.
[0072] 4. The protein concentration of the eluent was determined using the Bradford protein assay (ThermoFisher, catalog number 23200). The protein concentration of the eluent was found to be 0.715 mg / mL. The SDS-PAGE electrophoresis image of the purified HLA-A*02:01 / β2M complex is shown below. Figure 3 As shown.
[0073] After final calculation, the plasmid constructed above was transfected into CHO cells, and the expression level reached 21.45 mg / L.
[0074] Example 3: Preparation of HLA-A*03:01:01:01 / β2M complex
[0075] The amino acid sequence of the HLA-A*03:01:01:01 protein is shown in SEQ ID NO.20, and the corresponding nucleotide sequence of the HLA-A*03:01:01:01 protein is shown in SEQ ID NO.21. Using a plasmid containing the HLA-A*03:01:01:01 nucleotide sequence synthesized by the applicant (as shown in SEQ ID NO.22) as a template, amplification was performed, and a 1 kb fragment was recovered using a 1% agarose gel. The nucleotide sequence of the HLA-A*03:01:01:01 protein was then inserted between the PmeI / BsiWI sites of the vector. After sequencing verification, the pATX-β2M-FOS-HLA-A*03:01:01:01-JUN plasmid was prepared. The HLA-A*03:01:01:01 / β2M complex was prepared using the above plasmid, following the same method as in Example 2. The sequences of the amplification primer pairs are as follows.
[0076] Forward primer (as shown in SEQ ID NO.23):
[0077] 5'-ccaccggcgaggcgcgccaagtttaaacgccgccaccatggctgtgatggca-3';
[0078] Reverse primer (as shown in SEQ ID NO.24):
[0079] 5'-cgatgcggccgccgccgccgcccacgatggggattgtgggctgg-3';
[0080] The SDS-PAGE electrophoresis image of the purified HLA-A*03:01:01:01 / β2M complex is shown below. Figure 4 As shown in the figure. After final calculation, the expression level of the HLA-A*03:01:01:01 / β2M complex was finally obtained by transfecting the constructed plasmid into CHO cells, reaching 54.30 mg / L.
[0081] Example 4: Preparation of HLA-G / β2M complex
[0082] The amino acid sequence of the HLA-G protein is shown in SEQ ID NO.25, and the corresponding nucleotide sequence of the HLA-G protein is shown in SEQ ID NO.26. Using a plasmid containing the above-mentioned nucleotide sequence of the HLA-G protein (as shown in SEQ ID NO.27), synthesized by the applicant, as a template, amplification was performed. A 1kb fragment was recovered using a 1% agarose gel. The nucleotide sequence of the HLA-G protein was then inserted between the PmeI / BsiWI sites of the vector. After sequencing verification, the pATX-β2M-FOS-HLA-G-JUN plasmid was prepared. The HLA-G / β2M complex was prepared using the above plasmid, following the same method as in Example 2. The sequences of the amplification primer pairs are as follows.
[0083] Forward primer (as shown in SEQ ID NO.28):
[0084] 5'-gaggcgcgccaagtttaaacgccgccaccatggctgtgatggccccccgga-3';
[0085] Reverse primer (as shown in SEQ ID NO.29):
[0086] 5'-cgatgcggccgccgccgccgcccacgattgggatagttggcagg-3'.
[0087] The SDS-PAGE electrophoresis image of the purified HLA-G / β2M complex is shown below. Figure 5 As shown in the figure. After final calculation, the expression level of the finally prepared HLA-G / β2M complex reached 1 mg / L.
[0088] Example 5: Preparation of HLA-A*11:01 / β2M complex
[0089] The amino acid sequence of the HLA-A*11:01 protein is shown in SEQ ID NO.30, and the corresponding nucleotide sequence of the HLA-A*11:01 protein is shown in SEQ ID NO.31. Using a plasmid containing the above-mentioned nucleotide sequence of the HLA-A*11:01 protein (as shown in SEQ ID NO.32), synthesized by the applicant, as a template for amplification, a 1 kbp fragment was recovered using a 1% agarose gel. The above nucleotide sequence was inserted between the PmeI / BsiWI sites of the vector, and after sequencing verification, the pATX-β2M-FOS-HLA-A*11:01-JUN plasmid was prepared. The HLA-A*11:01 / β2M complex was prepared using the above plasmid, and the specific preparation method was the same as that in Example 2. The sequences of the amplification primer pairs are as follows.
[0090] Forward primer (as shown in SEQ ID NO.33):
[0091] 5'-gaggcgcgccaagtttaaacgccgccaccatggctgtgatggcaccccgg-3';
[0092] Reverse primer (as shown in SEQ ID NO.34):
[0093] 5'-cgatgcggccgccgccgccgcccacgatggggattgtgggcaag-3';.
[0094] The SDS-PAGE electrophoresis image of the purified HLA-A*11:01 / β2M complex is shown below. Figure 6 As shown in the figure. After final calculation, the expression level of the finally prepared HLA-A*11:01 / β2M complex reached 92.45 mg / L.
[0095] Ultimately, the applicant of this invention inserted a total of 57 HLA nucleotide sequences into the pATX-β2M-FOS-JUN plasmid to construct corresponding recombinant vectors. These vectors were then transfected into CHO cells for recombinant expression. Testing showed that the expression levels of the corresponding complexes in these recombinant vectors all exceeded 1 mg / L, with the highest reaching 92.45 mg / L, indicating very high expression levels. This demonstrates that this invention, by modifying the vector and introducing the FOS / JUN leucine zipper, achieved high expression of the HLA / β2M complex in CHO cells. Compared to conventional complex expression without the FOS / JUN leucine zipper, the expression level was significantly increased. Compared to the expression of the HLA-Fc / β2M complex fused with the human IgG1 Fc tag (obtained by the applicant in their own experiments, with an expression level of 0.12 mg / L), the expression level also achieved an order-of-magnitude increase.
[0096] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. A recombinant vector for preparing an HLA / β2M complex, characterized in that, A FOS leucine zipper bZIP motif sequence was inserted between the NheI / AvrII restriction sites of the pTRIOZ-hIgG1K vector, a JUN leucine zipper bZIP motif sequence was inserted between the BsiWI / BlpI restriction sites, a β2M gene sequence was inserted between the AgeI / NheI restriction sites, and the corresponding HLA sequence was inserted between the PmeI / BsiWI restriction sites of the recombinant vector. The FOS leucine zipper bZIP motif sequence is shown in SEQ ID NO.1, the JUN leucine zipper bZIP motif sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the β2M gene is shown in SEQ ID NO.3; the HLA sequence is the gene expression sequence of HLA-A*02:01 protein, HLA-A*03:01:01:01, or HLA-A*11:01; the amino acid sequence of the HLA-A*02:01 protein is shown in SEQ ID NO.15, the amino acid sequence of the HLA-A*03:01:01:01 protein is shown in SEQ ID NO.20, and the amino acid sequence of the HLA-A*11:01 protein is shown in SEQ ID NO.
30.
2. A method of producing the recombinant vector of claim 1, characterized by, Includes the following steps: S1. The original vector was double-digested with restriction endonucleases NheI and AvrII, and the vector was recovered. PCR amplification was performed using the FOS leucine zipper bZIP motif plasmid as a template, and the 100bp fragment was recovered. The FOS leucine zipper bZIP motif sequence was inserted between the NheI / AvrII restriction sites to obtain the first recombinant vector. The nucleotide sequence of the first recombinant vector is shown in SEQ ID NO.
4. S2. Take the first recombinant vector obtained in step S1, and double-digest it with restriction endonucleases BsiWI and BlpI, then recover the vector; use the JUN leucine zipper bZIP motif plasmid as a template for PCR amplification, recover the 100bp fragment, and insert the JUN leucine zipper bZIP motif sequence between the BsiWI / BlpI restriction sites to obtain the second recombinant vector; the nucleotide sequence of the second recombinant vector is shown in SEQ ID NO.5; S3. Take the second recombinant vector obtained in step S2, and double-digest it with restriction endonucleases AgeI and NheI. Recover the vector. Perform PCR amplification using the β2M gene sequence as a template, recover the 400bp fragment, and insert it between the AgeI / NheI restriction sites to finally obtain the recombinant vector for preparing the HLA / β2M complex. The nucleotide sequence of the recombinant vector for preparing the HLA / β2M complex is shown in SEQ ID NO.
6.
3. The method of claim 2, wherein the recombinant vector is prepared by the steps of: In step S1, the forward primer sequence for PCR amplification is shown in SEQ ID NO.7, and the reverse primer sequence is shown in SEQ ID NO.
8.
4. The method of claim 2, wherein the recombinant vector is prepared by the steps of: In step S2, the sequence of the forward primer for PCR amplification is shown as SEQ ID NO. 9, and the sequence of the reverse primer is shown as SEQ ID NO.
10.
5. The method of claim 2, wherein the recombinant vector is prepared by the steps of: In step S3, the sequence of the forward primer for PCR amplification is shown as SEQ ID NO. 11, and the sequence of the reverse primer is shown as SEQ ID NO.
12.
6. An HLA / β2M complex prepared by using the recombinant vector of claim 1.