Human endogenous retrovirus envelope protein mutants, truncates and their uses

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to human endogenous retrovirus envelope protein mutants, truncates and their uses. Based on HERVs, the envelope protein mutants and truncates designed by the inventors of the present invention can induce a strong immune response in the immune system, and can be used not only in various therapeutic and prophylactic vaccines such as mRNA vaccines, protein subunit vaccines, polypeptide vaccines, etc., the design of therapeutic and diagnostic antibodies, the design of specific therapeutic cell products such as CAR-T cells, specific cytotoxic T cells, CAR-NK cells, specific NK cells, etc., for the diagnosis, prevention and treatment of diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to human endogenous retrovirus envelope protein mutants, truncates, and their uses. Background Art

[0002] Human endogenous retroviruses (HERVs) are remnants of retroviruses that infected humans millions of years ago and integrated into the human genome, and have been inherited to date in a Mendelian manner, accounting for about 8% of the human genome. Among them, most HERVs have lost their coding ability due to mutations, deletions, and the presence of stop codons in the coding sequences, and were previously considered to be junk DNA. However, in recent years, with the in-depth study of the biological functions of HERVs, it has been found that some HERVs not only have important physiological functions in some specific tissues and organs, but may also be closely related to the occurrence and development of blood and solid tumors, autoimmune diseases, neurodegenerative diseases, etc. It is known that the C-terminal transmembrane region (TM) of the HERVs envelope protein (Env) has multiple immunosuppressive domains, such as the fusion peptide region (FP), the immunosuppressive domains (ISD), and the transmembrane domain (TMD), which have a powerful immunosuppressive function and are an important cause of tumor immune tolerance. The N-terminal (SU) of the envelope protein is involved in various inflammatory responses.

[0003] Therefore, studying whether the HERVs envelope protein has important physiological functions that can be utilized and modified by humans, so as to apply it to the diagnosis, prevention, and treatment of diseases is the pain point and hotspot of current industry research. Summary of the Invention

[0004] The present invention relates to mutants and truncates of HERV-K, HERV-H, and HERV-F envelope proteins and their uses in the preparation of drugs for disease diagnosis and / or treatment, wherein the mutation sites of the envelope protein mutants and the truncated domains of the truncates are conserved regions not affected by different subtypes (HERV-K, HERV-H, and HERV-F).

[0005] A human endogenous retrovirus envelope protein mutant or truncate, wherein one or more amino acids in the immunosuppressive domain of the mutant or truncate are mutated.

[0006] Preferably, the immunosuppressive domain is the immunosuppressive region.

[0007] Preferably, the human endogenous retrovirus is any one of HERV-K, HERV-H, or HERV-F.

[0008] Preferably, the HERV-K is any one of HERV-K108 or HERV-K102, and the amino acid sequence corresponding to each type of human endogenous retrovirus typing is as follows:

[0009] The sequence of the HERV-K108 envelope protein mutant is shown in SEQ ID NO: 1,

[0010] The sequence of the truncated HERV-K108 envelope protein is shown in SEQ ID NO: 5,

[0011] The sequence of the HERV-K102 envelope protein mutant is shown in SEQ ID NO: 3,

[0012] The sequence of the truncated HERV-K102 envelope protein is shown in SEQ ID NO: 7,

[0013] The sequence of the HERV-F envelope protein mutant is shown in SEQ ID NO: 9,

[0014] The sequence of the truncated HERV-F envelope protein is shown in SEQ ID NO: 11,

[0015] The sequence of the HERV-H envelope protein mutant is shown in SEQ ID NO: 13,

[0016] The sequence of the truncated HERV-H envelope protein is shown in SEQ ID NO: 15.

[0017] Preferably, the mutant or truncated body is a transmembrane protein formed by the hydrolysis of the human endogenous retrovirus envelope protein by furin protease, and the corresponding amino acid sequence is as follows:

[0018] The sequence of the HERV-K108 transmembrane protein mutant is shown in SEQ ID NO: 2,

[0019] The sequence of the truncated HERV-K108 transmembrane protein is shown in SEQ ID NO: 6,

[0020] The sequence of the HERV-K102 transmembrane protein mutant is shown in SEQ ID NO: 4,

[0021] The sequence of the truncated HERV-K102 transmembrane protein is shown in SEQ ID NO: 8,

[0022] The sequence of the HERV-F transmembrane protein mutant is shown in SEQ ID NO: 10,

[0023] The sequence of the truncated HERV-F transmembrane protein is shown in SEQ ID NO: 12,

[0024] The sequence of the HERV-H transmembrane protein mutant is shown in SEQ ID NO: 14.

[0025] The sequence of the truncated HERV-H transmembrane protein is shown in SEQ ID NO: 16.

[0026] Use of the envelope protein mutant or truncated body, including any of the human endogenous retrovirus envelope protein mutants or truncated bodies described above, in the preparation of a drug for inducing a strong immune response in the immune system for diagnosis and / or treatment.

[0027] Preferably, the drug for inducing a strong immune response in the immune system for diagnosis and / or treatment is any one of mRNA vaccines, protein subunit vaccines, recombinant gene vaccines, animal-derived antibodies, and envelope protein-specific cells.

[0028] Preferably, the recombinant gene vaccine is any one of plasmid vector vaccines, viral vector vaccines, or live cell vector vaccines.

[0029] Preferably, the animal-derived antibody is any one of mouse, rat, rabbit, or alpaca animal-derived antibodies.

[0030] Preferably, the alpaca animal-derived antibody is an alpaca nanobody.

[0031] Preferably, the envelope protein-specific cell is any one of cytotoxic T cells, NK cells, plasma cells, and B cells.

[0032] Preferably, the cytotoxic T cell is any one of CD8+ or CD4+.

[0033] Use of the antibody prepared from the envelope protein mutant or truncated body antigen described in any of the above, in the preparation of a specific cell drug.

[0034] Preferably, the specific cell drug is any one of CAR-T, CAR-NK, or CAR-VAC cell drugs.

[0035] The beneficial effects of the present invention mainly lie in:

[0036] The envelope protein mutants and truncated bodies designed by the inventors based on HERVs can induce a strong immune response in the immune system, and can be used not only for various therapeutic and prophylactic vaccines such as mRNA vaccines, protein subunit vaccines, polypeptide vaccines, etc., the design of therapeutic and diagnostic antibodies, and the design of specific therapeutic cell products such as CAR-T cells, specific cytotoxic T cells, CAR-NK cells, specific NK cells, etc., for the diagnosis, prevention, and treatment of diseases. Description of the Drawings

[0037] Figure 1 It is the plasmid map of pCW-Cas9 vector;

[0038] Figure 2 It is the plasmid map of pcDNA3.1(+) vector;

[0039] Figure 3 It is the expression of HERV-K transmembrane protein K-TM in different hematological malignancies and normal blood cells;

[0040] Figure 4 It is the expression of HERV-W transmembrane protein W-TM in different hematological malignancies and normal blood cells;

[0041] Figure 5 It is the expression of ERVFRD envelope protein FRD-Env in different tumors and normal blood cells;

[0042] Figure 6 It is the effect of K-TM and W-TM on the immune escape of tumor cells;

[0043] Figure 7 It is the interaction between K-TM and T cell CD3 molecule;

[0044] Figure 8 It is the effect of HERVs envelope protein mutants, truncates and wild types on the immune response of mice. Detailed implementation methods

[0045] 1. Glossary:

[0046] Human endogenous retroviruses (HERVs);

[0047] Envelope protein (envelope, Env);

[0048] C-terminal transmembrane region (TM), TM is the transmembrane protein formed after Env is hydrolyzed by furin protease;

[0049] Fusion peptide region (Fusion Peptide, FP);

[0050] Immunosuppressive Domains (ISD);

[0051] Transmembrane Domain (TMD);

[0052] HERV-W-Env (W-Env);

[0053] HERV-F-Env (FRD-Env);

[0054] HERV-K transmembrane protein (K-TM);

[0055] HERV-W transmembrane protein (W-TM);

[0056] Envelope protein N-terminus (SU).

[0057] 2. Main instruments and equipment

[0058]

[0059] 3. Main reagents

[0060]

[0061]

[0062] 4. Cell culture

[0063] Cell lines such as Kasumi-1, KM3, NB4, Raji, Jurkat, K562, K562 / adr, KG1, MV4-11, Mia, Panc-1, Hunh7, HepG2, SW620, U2OS, L02, Nalm6, LY3, Jeko1, THP-1, HL-60, MOLM13, ARP-1, 8226, MM1.S, U266, H9, A549, H1975, IMR-32, U87, HT-29 used in this patent are all purchased from the ATCC cell bank and stored in the cell bank of the Institute of Oncology, Zhejiang University. The cells are cultured using RPMI 1640 complete medium or DMEM complete medium. During the culture process, the cells are placed in an incubator at 37°C with 5% CO2, and are regularly detected to exclude contamination by bacteria, fungi, mycoplasma, etc. According to the experimental plan and cell status, the cell culture medium is changed and the cells are passaged every 2 - 3 days to maintain the cells in the logarithmic growth phase.

[0064] 5. Isolation and culture of mononuclear cells from primary sample peripheral blood

[0065] (1) Draw the patient's peripheral blood, gently shake it in an EDTA anticoagulation tube to prevent blood coagulation, and bring it back to the laboratory in an ice box. (2) Operate in a laminar flow hood: First, transfer the sample into a clean 15 mL centrifuge tube, and gently pipette and mix it. Then add an equal volume of pre-cooled 1×PBS buffer to the sample and gently mix and dilute. Subsequently, slowly add 2 volumes of human lymphocyte separation medium to the sample volume. Centrifuge at 2500 rpm horizontally for 20 minutes at room temperature, and adjust the centrifuge to increase the speed by 2 and decrease the speed by 1. (3) Aspirate the middle white mononuclear cell layer and wash it once with pre-cooled 1×PBS buffer. Centrifuge at 1500 rpm for 5 minutes at room temperature.

[0066] (4) Discard the supernatant, add 2 mL of red blood cell lysate, incubate at room temperature for 10 minutes, and then centrifuge at 1000 rpm for 5 minutes. Wash once with

[0067] 1×PBS buffer.

[0068] (5) Collect the cells, add protein lysate, and extract the total protein of primary sample PBMC cells for subsequent western blot experiments.

[0069] Example 1 Expression of HERV-K transmembrane protein K-TM in different hematological tumors and normal blood cells

[0070] Detect the expression of HERV-K transmembrane protein K-TM in hematological tumor cell lines, primary tumor cell samples, normal blood cell samples, and bone marrow samples of a leukemia patient before treatment (UT) and after treatment remission (CR) by western blot technology.

[0071] The specific experimental steps are as follows:

[0072] (1) Cell protein extraction and quantification

[0073] a Collect the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend with 1 mL of pre-cooled PBS, and transfer to a 1.5 mL EP

[0074] tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0075] b Prepare the protein lysate in advance, and prepare it according to the ratio of adding 10 μL of protease inhibitor mixture and 10 μL of EDTA to every 1 mL of M-PER lysate. Add an appropriate amount of protein lysate to the cell pellet and lyse on ice for 30 min. c After centrifuging at 13000 rcf at 4°C for 15 min, aspirate the supernatant and place it in a 0.6 mL centrifuge tube. Take 5 μL and quantify the protein concentration with a BCA kit according to the instructions. Add the remaining supernatant to

[0076] 5×Loading Buffer at a ratio of supernatant:loading 4:1, vortex and mix well, heat in a 100°C metal bath for 10 min, and place on ice for standby or store in a -80°C refrigerator for long-term preservation.

[0077] (2) Western blot experiment

[0078] a Prepare SDS-PAGE gel: According to the molecular weight of the target protein, prepare a separating gel with a concentration of 10%. Add successively to 50 mL

[0079] Add to a centrifuge tube: 4.0 mL of ddH2O, 3.3 mL of 30% acrylamide, 2.5 mL of 1.5 M Tris-HCl (pH 8.8), 0.1 mL of 10% SDS, 0.1 mL of 10% AP, and 0.004 mL of TEMED. After quickly mixing, add it to the glass plate, and immediately add 0.2 mL of isopropanol to flatten the gel surface. Let it stand at room temperature for 30 min, and wait for the separating gel to completely solidify. Wash off the isopropanol and let it stand to dry. Meanwhile, prepare the stacking gel. Add to a 50 mL centrifuge tube in sequence: 1.72 mL of ddH2O, 0.5 mL of 30% acrylamide, 0.76 mL of 0.5 M Tris-HCl (pH 6.8), 0.03 mL of 10% SDS, 0.03 mL of 10% AP, and 0.003 mL of TEMED. A total of 10 mL. After mixing, add it to the separating gel and immediately insert the comb. Let it stand at room temperature for 30 min

[0080] After that, the stacking gel completely solidifies. Store it in a 4°C refrigerator for future experimental use.

[0081] b SDS-PAGE gel electrophoresis: Assemble the electrophoresis equipment and add the pre-prepared 1× electrophoresis buffer. Pull out the comb, add equal amounts of protein samples to the lanes, and at the same time load 5 μL of protein prestained marker on both sides of the lanes. When running the stacking gel,

[0082] Adjust the voltage to 80 V. When electrophoresis reaches the separating gel, then adjust the voltage to 130 V. Stop electrophoresis when the bromophenol blue runs to the bottom of the separating gel.

[0083] c Transfer membrane: Take out the SDS-PAGE gel from the electrophoresis device and soak it in the transfer buffer. Take out the PVDF membrane and soak it in methanol for 10 s. Install the transfer device. From bottom to top in sequence: the positive electrode of the transfer clip, the sponge pad, double-layer filter paper, PVDF membrane, gel, double-layer filter paper, sponge pad, the negative electrode of the transfer clip. At the same time, drain the air bubbles between the gel and the PVDF membrane. Fix the transfer device, add the transfer buffer, perform ice bath, and transfer the membrane at a constant current of 250 mA for 100 min.

[0084] d Blocking: Take out the PVDF membrane and cut a corner at the upper right corner to mark the front side. Put the membrane into the blocking solution and block it at room temperature for 1 h. After that, wash the membrane with 1×TBST, slowly shake the membrane on a shaker at room temperature for 10 min, and repeat 3 times.

[0085] e Incubation with primary antibody: Put the PVDF membrane into the pre-prepared primary antibody and incubate it on a shaker at 4°C overnight.

[0086] f Incubation with secondary antibody: The next day, take out the PVDF membrane, first wash the membrane with 1×TBST, wash it 3 times for 10 min each. Then put the PVDF membrane into the prepared secondary antibody and incubate it on a shaker at room temperature for 1 h.

[0087] g Color development: After the secondary antibody incubation is completed, wash the membrane with 1×TBST, wash 3 times for 10 minutes. At the same time, prepare developing solution A and solution B in a 1:1 ratio, mix them well, cover the PVDF membrane, and perform color development in a developing instrument.

[0088] Liquid and solution B, mix them well and cover the PVDF membrane, and perform color development in a developing instrument.

[0089] Example 2 Expression of HERV-W transmembrane protein W-TM in different hematological malignancies and normal blood cells

[0090] Detect the expression of HERV-W transmembrane protein W-TM in hematological malignancy cell lines, primary leukemia tumor cell samples, and normal blood cell samples by western blot technique.

[0091] The specific experimental steps are the same as those in Example 1.

[0092] Example 3 Expression of HERV-FRD envelope protein FRD-Env in different tumors and normal blood cells

[0093] Detect the expression of HERV-FRD transmembrane protein FRD-Env in different hematological malignancy cell lines, solid tumor cell lines, primary leukemia tumor cell samples, and normal blood cell samples by western blot technique.

[0094] The specific experimental steps are the same as those in Example 1.

[0095] Example 4 Effects of HERV-K-TM and W-TM on the immune escape of tumor cells

[0096] (1) Construction of lentivirus overexpression plasmids pCW-K-TM-HA and pCW-W-TM-HA

[0097] The full-length K-TM amino acid sequence is derived from the transmembrane region 466-699aa of the human retrovirus K family member 6 Env protein (ENK-6, UniProt, Q69384). At the same time, a signal peptide (Signal Peptide, UniProt, Q69384, 1-89aa) is fused to the amino terminus of the sequence, and an HA tag is fused to the carboxyl terminus.

[0098] The full-length W-TM amino acid sequence is derived from the transmembrane region 291-538aa of the human retrovirus W family member Syncytin-1 protein (ERVW-1, UniProt, Q9UQF0). At the same time, a signal peptide (Signal Peptide, UniProt, Q9UQF0, 1-20aa) is fused to the amino terminus of the sequence, and an HA tag is fused to the carboxyl terminus.

[0099] The amino acid sequence was optimized for human codons using the OptimumGene codon optimization technology, and the resulting nucleic acid sequence was inserted between the EcoRⅠ and BamHⅠ restriction sites of the pCW-Cas9 vector (plasmid map as shown in Figure 1 ), to construct plasmids pCW-K-TM-HA and pCW-W-TM-HA.

[0100] (2) Construction of MCA205 cell lines stably overexpressing K-TM and W-TM

[0101] a Package the constructed pCW-K-TM-HA and pCW-W-TM-HA plasmids into virus solutions.

[0102] b Resuspend MCA205 cells with 1 mL of complete 1640 medium and add them to a 12-well plate. The plate needs to be seeded one day in advance to allow

[0103] MCA205 cells to adhere.

[0104] c Add 250 μL of virus solution and 10 μg / mL Polybrene to the culture plate and infect the cells for 24 h.

[0105] d Discard the virus supernatant, replace with fresh medium, transfer the cells to a culture flask, and add puromycin at a working concentration of 2 μg / mL to screen for positive cells.

[0106] e When the cells reach a certain growth density, take a small amount of cells and verify the expression of K-TM-HA and W-TM-HA by western blot. The remaining cells continue to be screened with puromycin for 14 days, thus constructing MCA205-K-TM-HA,

[0107] MCA205-W-TM-HA stable overexpressing cell lines.

[0108] (3) Construction of MCA205 allogeneic tumor model

[0109] a BALB / c mice (female, 6 weeks old, body weight about 20 g) provided by Shanghai SLAC Laboratory Animal Co., Ltd. were used for animal experiments. The experimental animals were housed in the SPF clean environment of the Animal Experiment Center of Zhejiang Chinese Medical University. The animal experiment protocol and operation process were approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine.

[0110] b After MCA205 cells overexpressing K-TM protein and W-TM protein were induced with 2 μg / mL Dox for 48 h, the cells were collected, washed once with PBS, and then resuspended with PBS to make the cell suspension density 5×10 6 / mL.

[0111] c BALB / c mice were randomly divided into 2 groups, with 5 mice in each group. 0.2 mL of MCA205-K-TM-HA and MCA205-W-TM-HA stable overexpression cell lines were inoculated into the left axilla of the mice respectively. After inoculating the tumor fluid, the two groups of mice were given Dox at a dose of 100 mg / kg to induce the expression of K-TM and W-TM proteins, and administered by gavage once a day for 20 consecutive days.

[0112] d The status of the mice and the growth of subcutaneous tumors were monitored twice a week. After the experiment, the mice were euthanized, the subcutaneous tumors were dissected and photographed.

[0113] Example 5 Interaction between HERV-K-TM and T cell CD3 molecule

[0114] (1) Construction of Jurkat stable overexpression cell line with inducible overexpression of K-TM

[0115] a The constructed pCW-K-TM-HA plasmid and pCW empty plasmid were packaged into virus solutions.

[0116] b Jurkat cells were resuspended in 1 mL of complete 1640 medium and added to a 12-well plate.

[0117] c 250 μL of virus solution and 10 μg / mL Polybrene were added to the culture plate to infect the cells for 24 h.

[0118] d The virus supernatant was discarded, and after replacing the fresh medium, the cells were transferred to a culture flask and positive cells were screened with puromycin at a working concentration of 2 μg / mL.

[0119] e When the cells reached a certain growth density, a small amount of cells were taken to verify the expression of K-TM-HA by western blot. The remaining cells were continuously screened with puromycin for 14 days, and the Jurkat-K-TM-OE stable overexpression cell line was thus constructed.

[0120] (2) Immunofluorescence co-localization experiment of K-TM and CD3ε

[0121] a Jurkat-K-TM-OE cells were collected, washed once with 1×PBS, and then resuspended in fetal bovine serum to an appropriate density.

[0122] b The cells were smeared on a glass slide, fixed with 4% paraformaldehyde, and blocked with 10% rabbit antiserum.

[0123] c CD3ε primary antibody was added and incubated at 4°C overnight.

[0124] On the next day, the slides were taken out of the refrigerator, washed three times with 1×PBS, and then incubated with an anti-mouse secondary antibody labeled with Alexa Fluor 594 fluorescence in the dark at room temperature for 1 h.

[0125] e The cell nuclei were stained with DAPI in the dark at room temperature for 10 min. Photographs were taken and observed using a Zeiss confocal laser scanning microscope, and image acquisition and analysis were performed using ZEN software.

[0126] Example 6 Effects of HERVs envelope protein mutants, truncates and wild type on mouse immune responses

[0127] (1) Antigen preparation: The inventors prepared full-length eukaryotic overexpression plasmids of H-Env envelope protein including wild type, mutant and truncate, pcDNA3.1(+)-H-Env, pcDNA3.1(+)-Mutant-H-Env, pcDNA3.1(+)

[0128] -Truncated-H-Env. The full-length H-Env amino acid sequence is derived from the 1-584 aa of the Env protein (ENH1, UniProt, Q9N2K0) of a member of the human retrovirus H (abbreviated as HERV-H) family. The mutant H-Env amino acid sequence refers to the Appendix No7-1-XUHMV sequence, and the truncated H-Env amino acid sequence refers to the Appendix No8-1-XUHMV sequence. The OptimumGene codon optimization technology was used to optimize the amino acid sequence with human codons, and the obtained nucleic acid sequence was inserted between the EcoRⅠ and BamHⅠ restriction sites of the pcDNA3.1(+) vector (the plasmid map is as Figure 2 shown), and the plasmids pcDNA3.1(+)-H-Env, pcDNA3.1(+)-Mutant-H-Env and pcDNA3.1(+)

[0129] -Truncated-H-Env were constructed.

[0130] (2) Immunization and serum evaluation: According to the conventional mouse immunization procedure, the above plasmids were used to immunize 6-week-old Balb / c mice respectively. The titers of antibodies in mouse sera were analyzed by titration. The OD450nm was >1.0 at a dilution of 1:8000, and at the same time the titer was >72900

[0131] (P / N>2.1). After the titers were qualified, stable transfected cell lines, human tumor cell lines, etc. were used for flow cytometry verification, and finally mice with good immune effects were selected for fusion.

[0132] (3) Cell fusion: According to the results of immunoserum titers and experimental verifications such as flow cytometry and western blot, after evaluation and confirmation, the optimal mouse was selected and arranged for fusion.

[0133] (4) Monoclonalization of positive cells: After cell fusion, through pre-screening detection, the culture medium was replaced to reduce the detection background. One week later, ELISA primary screening was carried out, ELISA positive clones were selected, and subcloning was carried out by two-step limited dilution method. 5 - 10 positive clones detected by ELISA were expanded in culture, verified by flow cytometry with stable transfected cell lines, human tumor cell lines, etc., and the finally selected positive clones were expanded in culture and cryopreserved.

[0134] (5) Ascites preparation and antibody purification: Ascites was prepared from the positive clones, the titer of antibodies in the ascites was analyzed by indirect ELISA, and the subtype was also analyzed at the same time. And affinity purification of the antibodies in the ascites was carried out.

[0135] (6) Detection of antibody titer by western blot technique: The specific experimental steps were the same as those in Example 1.

[0136] Experimental results

[0137] The results of Example 1 showed: A: Expression of K-TM in different hematological tumor cell lines; B: Expression of K-TM in different primary leukemia cells. Jurkat was used as a positive control; C: Expression of K-TM in normal blood cell samples, K562 was used as a positive control; D: Expression of K-TM in bone marrow samples before treatment (UT) and after treatment remission (CR) in a leukemia patient.

[0138] The results of Example 2 showed: A: Expression of W-TM in different tumor cell lines; B: Expression of W-TM and PD-L1 in different primary leukemia cells. KG1 was used as a positive control; C: Expression of W-TM and PD-L1 in normal blood cell samples.

[0139] The results of Example 3 showed: A: Expression of FRD-Env in different tumor cell lines; B: Expression of FRD-Env in different primary leukemia cells. THP1 was used as a positive control; C: Expression of FRD-Env in normal blood cell samples. THP1 was used as a positive control.

[0140] The results of Example 4 showed: When MCA-205 cells expressing K-TM protein were inoculated into BALB / C mice, rapid growth of tumor cells was visible, while MCA-205 cells expressing W-TM could not grow in BALB / c mice.

[0141] The results of Example 5 showed: A: K-TM protein binds to the CD3 molecule on the surface of T cells; B: Schematic diagram of K-TM inhibiting T cell activation. K-TM binds to the CD3 molecule on the surface of T cells, blocking its binding to the TCR receptor, thereby inhibiting T cell activation.

[0142] The results of Example 6 showed that: A: Six different types of antibodies could be produced after immunizing mice with the mutant envelope protein plasmid, as indicated by the black arrows; B: Two specific antibodies could be produced after immunizing mice with the truncated envelope protein plasmid, as indicated by the black arrows; C: Only one specific antibody could be produced after immunizing mice with the wild-type envelope protein plasmid, as indicated by the black arrows.

[0143] Results and Analysis

[0144] The inventor of the present invention used Western blot technology to detect the HERV-K transmembrane protein (K-TM) in multiple different tumor cell lines, primary tumor cell samples from leukemia patients, and normal blood cell samples. The results showed that most tumor cell lines and primary tumor cell samples highly expressed K-TM, while normal peripheral blood cell samples had low or no expression of K-TM (Example 1, Figure 3 A-C). Notably, in some leukemia patients with high expression of K-TM, K-TM also significantly decreased after achieving complete remission after treatment (Example 1, Figure 3 D).

[0145] HERV-W is an endogenous retrovirus in humans that is considered to have important physiological functions during placental development. Recently, some literature reports have shown that severe T cell function exhaustion also occurs in some COVID-19 patients. In these patients, peripheral blood T lymphocytes abnormally highly expressed HERV-W-Env (W-Env), and its expression level was not only significantly positively correlated with the levels of the T cell exhaustion marker PD-1 and the inflammatory factors IL-6 and IL-10, but also closely related to the disease severity. These results suggest that abnormal activation of W-Env may be closely related to T cell function exhaustion in COVID-19 patients. The inventor of the present invention used Western blot to detect the expression of the W-TM protein encoded by the HERV-W-Env gene in different tumor cell lines, primary tumor cell samples, and normal blood cell samples. The results showed that the W-TM protein was highly expressed in most tumor cell lines (Example 2, Figure 4 A) and primary tumor cell samples (Example 2, Figure 4 B), while normal blood cell samples had low or no expression (Example 2, Figure 4 C). Notably, some tumor samples with high expression of the W-TM protein also expressed the cell-derived immune checkpoint PD-L1 (Example 2, Figure 4 B). These results suggest that W-TM may directly or indirectly participate in tumor immune escape.

[0146] HERV-F is another human endogenous retrovirus that is normally only expressed in placental tissue. The envelope protein sample FRD-Env encoded by it (the C-terminus is the TM protein) has significant immunosuppressive functions. At present, little is known about the role of the sample FRD-Env and TM (F-TM) in tumors. Since there is currently no antibody against the F-TM protein, the inventor of the present invention used Western blot to detect the expression of the FRD-Env protein of HERV-F in different tumor cell lines, primary tumor cell samples, and normal blood cell samples. The results showed that there was varying degrees of expression of the sample FRD-Env in 21 different tumor cell lines (Example 3, Figure 5 A), 9 out of 20 primary tumor cell samples from leukemia patients had FRD-Env expression (45%) (Example 3, Figure 5 B), while FRD-Env was not expressed or was weakly expressed in 20 normal human blood cell samples (Example 3, Figure 5 C).

[0147] To verify whether K-TM and W-TM have the function of helping tumor cells escape immunity, the inventor of the present invention used MCA-205 murine fibrosarcoma cells (derived from C57BL / 6 mice) to construct cells with high expression of K-TM and W-TM respectively. Then, they were inoculated into wild-type BALB / c mice with normal immune function to observe the effect of these two endogenous retroviral proteins on the growth of MCA-205 cells in BALB / c mice. For BALB / c mice, MCA-205 cells are allogeneic cells, and when inoculated into BALB / c mice, they will be cleared by their immune cells and cannot grow in BALB / c mice. However, if MCA-205 cells carry an immunosuppressive functional protein that inhibits the function of murine immune cells, they can grow in mice. The experimental results showed that MCA-205 cells expressing W-TM could not grow in BALB / c mice, while MCA-205 cells expressing K-TM could grow rapidly in BALB / c mice (Example 4, Figure 6 ). These results indicate that K-TM has a strong immunosuppressive function and can endow MCA-205 tumor cells with the function of immune escape.

[0148] Similar to most human tumors, some tumors caused by retroviral infections are also often accompanied by high expression of cell-derived ICIs such as PD1 / PDL1 and CTLA-4 on T cells, resulting in T cell immune function exhaustion and tumor immune escape phenomena, such as murine leukemia, bovine leukemia, and HTLV-1 adult T cell leukemia. Although its exact etiology and molecular mechanism are not yet fully understood, the viral envelope protein Env and the TM protein may be the most critical regulatory molecules. The retroviral TM is a transmembrane protein formed by the hydrolysis of Env by furin protease, containing multiple immunosuppressive functional domains such as the fusion peptide FP, ISD, and TMD, among which ISD is highly conserved among different retroviruses. The mature and functional TM protein is mainly distributed on the cell membrane and has a powerful immunosuppressive function, which is a powerful weapon for retroviruses to escape the attack of the host immune system. It should be particularly noted that the envelope proteins and their transmembrane proteins TM of some endogenous retroviruses contain multiple immunosuppressive domains. In HTLV-1 gp21 and HIV-gp41 TM, FP, ISD, and TMD have been shown to interfere with the binding of CD3 to the TCR receptor and inhibit T cell function. It is known that K-TM can significantly inhibit T cell growth and upregulate the immunosuppressive inflammatory factors IL-6 and IL-10. We used co-IP and immunofluorescence co-localization experiments and found that K-TM can bind to the T cell CD3 molecule (Example 5, Figure 7 A). These preliminary results suggest that K-TM may directly inhibit T cell function, and its mechanism of action may prevent CD3 from activating T cells (Example 5, Figure 7 B).

[0149] To verify whether the envelope proteins of HERVs have immunosuppressive functions, the inventors designed different types of envelope protein mutants and truncations, and then injected plasmids expressing these mutants, truncations, and wild types into mice through plasmid gene immunization technology, immunizing once every 1 week for a total of 3 times. After immunizing the mice, ascites were obtained to prepare antibodies, and the titers of the prepared antibodies were detected by western blot experiments. Then, the immune response effects of the mutants, truncations, and wild-type plasmids on the mice were observed and compared respectively. The results showed that through three immunizations, the types and titers of specific antibodies produced by immunization with mutant envelope proteins and truncated envelope proteins were significantly higher than those of wild-type envelope proteins. In particular, the mutant envelope protein could produce 6 different types of antibodies (Example 6, Figure 8 A), the truncation could produce 2 specific antibodies (Example 6, Figure 8 B), while in the mice immunized with the wild-type envelope protein plasmid, the production of specific antibodies was not only small in number (only 1 type) but also had a very low titer (Example 6, Figure 8C). This result indicates that the envelope protein mutants and truncations designed by the present inventors can induce a strong immune response in the immune system and can be used not only in various therapeutic and prophylactic vaccines such as mRNA vaccines, protein subunit vaccines, polypeptide vaccines, etc., but also in the design of therapeutic and diagnostic antibodies and the design of specific therapeutic cell products such as CAR-T cells, specific cytotoxic T cells, CAR-NK cells, specific NK cells, etc., for the diagnosis, prevention, and treatment of diseases.

[0150] Design of mutants and truncations of human endogenous retrovirus (HERVs) envelope proteins

[0151] The HERV-K envelope protein (Env) mutant refers to the mutant of Env generated by the present inventors by mutating one or more amino acids of the conserved region of the immunosuppressive domain ISD of Env, namely L(A)A(G)NQINDLRQTVIW(R)MGD ) L 522A (or other amino acids), A 523G (or other amino acids), W 535R (or other amino acids). The preferred mutation sites are amino acids L522A, A523G, and W535R, and more preferably the C-terminal transmembrane protein TM of the envelope protein mutant.

[0152] The HERV-K envelope protein truncation refers to the truncation of the envelope protein generated by the present inventors by deleting one or more of the fusion peptide region (FP) FIFTLIAVIMGLIAVTATAAV and / or the immunosuppressive domain (ISD)

[0153] LANQINDLRQTVIWMGD and / or the transmembrane domain (TMD)

[0154] IGSTTIINLILILVCLFCLLL. The preferred one is the C-terminal transmembrane protein TM of the envelope protein truncation.

[0155] The HERV-H envelope protein mutant refers to the mutant of the envelope protein generated by the present inventors by mutating one or more amino acids of the conserved region of the immunosuppressive domain ISD of the envelope protein (L(A)Q(A)NRRGLDLLTAEK(R)GGL), namely L454A (or other amino acids), Q455A (or other amino acids), K467R (or other amino acids). The preferred mutation sites are amino acids L454A, Q455A, and K467R, and more preferably the C-terminal transmembrane protein TM of the envelope protein mutant.

[0156] The truncated HERV-H envelope protein refers to the truncated envelope protein generated by the inventors through deletion mutations of one or more of the fusion peptide region (FP) VIPLIPLMVGLGLSASTVALG and / or immunosuppressive domains (ISD)

[0157] LQNRRGLDLLTAEKGGLCIF and / or transmembrane domain (TMD)

[0158] LPIVSPLIPIFLLLLFGPCIF. Preferably, it is the C-terminal transmembrane protein TM of the truncated envelope protein.

[0159] The HERV-F envelope protein mutant refers to the envelope protein mutant generated by the inventors through mutations of one or more of the amino acids L(A)Q(A)NRRGLDMLTAA Q(R) GGICLA(F) at positions L414A (or other amino acids), Q415A (or other amino acids), Q427R (or other amino acids), A433F (or other amino acids). The preferred mutation sites are amino acids L414A, Q415A, Q427R, and A433F. More preferably, it is the C-terminal transmembrane protein TM of the envelope protein mutant.

[0160] The truncated HERV-F envelope protein refers to the truncated envelope protein generated by the inventors through deletion mutations of one or more of the fusion peptide region (FP) AIHFIPLLAGLGILAGTGTGIAGITK and / or immunosuppressive domains (ISD) LQNRRGLDMLTAAQGGI and / or transmembrane domain (TMD)

[0161] WFSWVLPLTGPLVSLLLLLLF. Preferably, it is the C-terminal transmembrane protein TM of the truncated envelope protein.

[0162] The specific amino acid sequences are as follows:

[0163] SEQ ID NO:1No1-1-XUKMV(mutant)(K108-Env Mutant, K108 envelope protein mutant)

[0164] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLTQLATKY

[0165] LENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVPFPPLIRAVTWMDNPTEVYVNDS

[0166] VWVPGPIDDRCPAKPEEEGMMINISIGYHYPPICLGRAPGCLMPAVQNWLVEVPTVSPICRFTYHMVSGM

[0167] SLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQF

[0168] YHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIVSPVSGPEHPELWRL

[0169] TVASHHIRIWSGNQTLETRDRKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCID

[0170] STFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIAVIMGLIAVTATAAVA

[0171] GVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQK AGNQINDLRQTVIRMGD RLMSLEHRFQLQCDWNT

[0172] SDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNP

[0173] VTWVKTIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVT

[0174] VSV(699aa).

[0175] SEQ ID NO:2 No1-2-XUKMV (mutant) (K108-TM Mutant, K108 transmembrane protein mutant) FIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQK AGNQINDLRQTVIRMGD RLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV (234aa).

[0176] SEQ ID NO:3 No2-1-XUKMV (mutant) (K102-Env Mutant, K102 envelope protein mutant) MVTPVTWMDNPIEIYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIVSPVSGPEHPELWRLTVASHHIRIWSGNQTLETRDCKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQK AGNQINDLRQTVIRMGD RLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV (588aa).

[0177] SEQ ID NO:4 No2-2-XUKMV (mutant) (K102-TM Mutant, K102 transmembrane protein mutant) FIFTLIAVIMGLIAVTATAAVAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQK AGNQINDLRQTVIRMGD RLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV (234aa)

[0178] SEQ ID NO:5 No3-1-XUKTV (truncated) (K108-Env Truncation, K108 envelope protein truncation) MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPTWAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAYVPFPPLIRAVTWMDNPTEVYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYHYPPICLGRAPGCLMPAVQNWLVEVPTVSPICRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSAVILQNNEFGTIIDWAPRG

[0179] QFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGISTPRPKIVSPVSGPEHPEL

[0180] WRLTVASHHIRIWSGNQTLETRDRKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLL

[0181] TCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRAGVALHSSVQSVNFV

[0182] NDWQKNSTRLWNSQSSIDQKRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNL

[0183] TLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTVCRCTQQLRRDSDHRERAMMTM

[0184] AVLSKRKGGNVGKSKRDQIVTVSV(640aa)

[0185] SEQ ID NO:6No3-2-XUKTV(truncated)(K108-TM Truncation,K108 transmembrane protein truncation)

[0186] AGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQKRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHH

[0187] WDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTVCRCTQQ

[0188] LRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV(175aa)

[0189] SEQ ID NO:7No4-1-XUKMV(truncated)(K102-Env Truncation,K102 envelope protein truncation)

[0190] MVTPVTWMDNPIEIYVNDSVWVPGPIDDRCPAKPEEEGMMINISIGYRYPPICLGRAPGCLMPAVQNWLV

[0191] EVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPKEIPKESKNTEVLVWEECVANSA

[0192] VILQNNEFGTIIDWAPRGQFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYPWEWGEKGIS

[0193] TPRPKIVSPVSGPEHPELWRLTVASHHIRIWSGNQTLETRDCKPFYTIDLNSSLTVPLQSCVKPPYMLVVG

[0194] NIVIKPDSQTITCENCRLLTCIDSTFNWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRS

[0195] KRAGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQKRLMSLEHRFQLQCDWNTSDFCITPQIYNESEH

[0196] HWDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTVCRCTQ

[0197] QLRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV(529aa)

[0198] SEQ ID NO:8No4-2-XUKMV(truncated)(K102-TM Truncation,K102 transmembrane protein truncation)

[0199] AGVALHSSVQSVNFVNDWQKNSTRLWNSQSSIDQKRLMSLEHRFQLQCDWNTSDFCITPQIYNESEHH

[0200] WDMVRRHLQGREDNLTLDISKLKEQIFEASKAHLNLVPGTEAIAGVADGLANLNPVTWVKTVCRCTQQ

[0201] LRRDSDHRERAMMTMAVLSKRKGGNVGKSKRDQIVTVSV(175aa)

[0202] SEQ ID NO:9No5-1-XUFMV(F-Env Mutant,F envelope protein mutant)

[0203] MGLLLLVLILTPSLAAYRHPDFPLLEKAQQLLQSTGSPYSTNCWLCTSSSTETPGTAYPASPREWTSIEAEL

[0204] HISYRWDPNLKGLMRPANSLLSTVKQDFPDIRQKPPIFGPIFTNINLMGIAPICVMAKRKNGTNVGTLPST

[0205] VCNVTFTVDSNQQTYQTYTHNQFRHQPRFPKPPNITFPQGTLLDKSSRFCQGRPSSCSTRNFWFRPADYN

[0206] QCLQISNLSSTAEWVLLDQTRNSLFWENKTKGANQSQTPCVQVLAGMTIATSYLGISAVSEFFGTSLTPLF

[0207] HFHISTCLKTQGAFYICGQSIHQCLPSNWTGTCTIGYVTPDIFIAPGNLSLPIPIYGNSPLPRVRRAIHFIPLL

[0208] AGLGILAGTGTGIAGITKASLTYSQLSKEIANNIDTMAKALTTMQEQIDSLAAVVAANRRGLDMLTAARG

[0209] GICLFLDEKCCFWVNQSGKVQDNIRQLLNQASSLRERATQGWLNWEGTWKWFSWVLPLTGPLVSLLLL

[0210] LLFGPCLLNLITQFVSSRLQAIKLQTNLSAGRHPRNIQESPF(538aa)

[0211] SEQ ID NO:10No5-2-XUFMV(F-TM Mutant,F transmembrane protein mutant)

[0212] AIHFIPLLAGLGILAGTGTGIAGITKASLTYSQLSKEIANNIDTMAKALTTMQEQIDSLAAVVAANRRGLD

[0213] MLTAARGGICLFLDEKCCFWVNQSGKVQDNIRQLLNQASSLRERATQGWLNWEGTWKWFSWVLPLTG

[0214] PLVSLLLLLLFGPCLLNLITQFVSSRLQAIKLQTNLSAGRHPRNIQESPF(188aa)

[0215] SEQ ID NO:11 No6-1-XUFTV (F-Env Truncation, F envelope protein truncated form)

[0216] MGLLLLVLILTPSLAAYRHPDFPLLEKAQQLLQSTGSPYSTNCWLCTSSSTETPGTAYPASPREWTSIEAEL

[0217] HISYRWDPNLKGLMRPANSLLSTVKQDFPDIRQKPPIFGPIFTNINLMGIAPICVMAKRKNGTNVGTLPST

[0218] VCNVTFTVDSNQQTYQTYTHNQFRHQPRFPKPPNITFPQGTLLDKSSRFCQGRPSSCSTRNFWFRPADYN

[0219] QCLQISNLSSTAEWVLLDQTRNSLFWENKTKGANQSQTPCVQVLAGMTIATSYLGISAVSEFFGTSLTPLF

[0220] HFHISTCLKTQGAFYICGQSIHQCLPSNWTGTCTIGYVTPDIFIAPGNLSLPIPIYGNSPLPRVRRASLTYSQ

[0221] LSKEIANNIDTMAKALTTMQEQIDSLAAVVCLALDEKCCFWVNQSGKVQDNIRQLLNQASSLRERATQG

[0222] WLNWEGTWKGPCLLNLITQFVSSRLQAIKLQTNLSAGRHPRNIQESPF (474aa)

[0223] SEQ ID NO:12 No6-2-XUFTV (F-TM Truncation, F transmembrane protein truncated form)

[0224] ASLTYSQLSKEIANNIDTMAKALTTMQEQIDSLAAVVCLALDEKCCFWVNQSGKVQDNIRQLLNQASSLRERATQGWLNWEGTWKGPCLLNLITQFVSSRLQAIKLQTNLSAGRHPRNIQESPF (124aa)

[0225] SEQ ID NO:13 No7-1-XUHMV (H-Env Mutant, H envelope protein mutant)

[0226] MIFAGKAPSNTSTLMKFYSLLLYSLLFSFPFLCHPLPLPSYLHHTINLTHSLLAASNPSLVNNCWLCISLSSS

[0227] AYTAVPAVQTDWATSPISLHLRTSFNSPHLYPPEELIYFLDRSSKTSPDISHQQAAALLRTYLKNLSPYINST

[0228] PPIFGPLTTQTTIPVAAPLCISWQRPTGIPLGNLSPSRCSFTLHLRSPTTNINETIGAFQLHITDKPSINTDKLK

[0229] NISSNYCLGRHLPCISLHPWLSSPCSSDSPPRPSSCLLIPSPENNSERLLVDTRRFLIHHENRTFPSTQLPHQS

[0230] PLQPLTAAALAGSLGVWVQDTPFSTPSHLFTLHLQFCLAQGLFFLCGSSTYMCLPANWTGTCTLVFLTPKI

[0231] QFANGTEELPVPLMTPTQQKRVIPLIPLMVGLGLSASTVALGTGIAGISTSVMTFRSLSNDFSASITDISQT

[0232] LSVLQAQVDSLAAVVAANRRGLDLLTAERGGLCIFLNEECCFYLNQSGLVYDNIKKLKDRAQKLANQAS

[0233] NYAEPPWALSNWMSWVLPIVSPLIPIFLLLLFGPCIFRLVSQFIQNRIQAITNHSIRQMFLLTSPQYHPLPQDLPSA(584aa)

[0234] SEQ ID NO:14 No7-2-XUHMV (H-TM Mutant, H transmembrane protein mutant)

[0235] VIPLIPLMVGLGLSASTVALGTGIAGISTSVMTFRSLSNDFSASITDISQTLSVLQAQVDSLAAVVAANRRG

[0236] LDLLTAERGGLCIFLNEECCFYLNQSGLVYDNIKKLKDRAQKLANQASNYAEPPWALSNWMSWVLPIVSPLIPIFLLLLFGPCIFRLVSQFIQNRIQAITNHSIRQMFLLTSPQYHPLPQDLPSA(197aa)

[0237] SEQ ID NO:15No8-1-XUHTV(H-Env Truncation,H包膜蛋白截短体)

[0238] MIFAGKAPSNTSTLMKFYSLLLYSLLFSFPFLCHPLPLPSYLHHTINLTHSLLAASNPSLVNNCWLCISLSSS

[0239] AYTAVPAVQTDWATSPISLHLRTSFNSPHLYPPEELIYFLDRSSKTSPDISHQQAAALLRTYLKNLSPYINST

[0240] PPIFGPLTTQTTIPVAAPLCISWQRPTGIPLGNLSPSRCSFTLHLRSPTTNINETIGAFQLHITDKPSINTDKLK

[0241] NISSNYCLGRHLPCISLHPWLSSPCSSDSPPRPSSCLLIPSPENNSERLLVDTRRFLIHHENRTFPSTQLPHQS

[0242] PLQPLTAAALAGSLGVWVQDTPFSTPSHLFTLHLQFCLAQGLFFLCGSSTYMCLPANWTGTCTLVFLTPKI

[0243] QFANGTEELPVPLMTPTQQKRTGIAGISTSVMTFRSLSNDFSASITDISQTLSVLQAQVDSLAAVVLNEEC

[0244] CFYLNQSGLVYDNIKKLKDRAQKLANQASNYAEPPWALSNWMSWVRLVSQFIQNRIQAITNHSIRQMFLLTSPQYHPLPQDLPSA(522aa)

[0245] SEQ ID NO:16No8-2-XUHTV (H-TM Truncation, Truncated form of H transmembrane protein)

[0246] RTGIAGISTSVMTFRSLSNDFSASITDISQTLSVLQAQVDSLAAVVLNEECCFYLNQSGLVYDNIKKLKDR AQKLANQASNYAEPPWALSNWMSWVRLVSQFIQNRIQAITNHSIRQMFLLTSPQYHPLPQDLPSA(136aa)

[0247] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited in this application for reference as if each document was separately cited for reference.

Claims

1. A human endogenous retrovirus envelope protein mutant or truncation, characterized in that, Mutate one or more amino acids in the mutant or truncated immunosuppressive domain described above; The immunosuppressive domain described above is an immunosuppressive region; The human endogenous retrovirus is HERV-H; The corresponding amino acid sequence is: The sequence of the HERV-H envelope protein mutant is shown in SEQ ID NO: 13, The sequence of the HERV-H envelope protein truncated body is shown in SEQ ID NO:

15.

2. A human endogenous retrovirus envelope protein mutant or truncation, characterized in that, The mutant or truncated body described above is a transmembrane protein formed after the human endogenous retrovirus envelope protein is hydrolyzed by furin protease, and its corresponding amino acid sequence is: The sequence of the HERV-H transmembrane protein mutant is shown in SEQ ID NO: 14, The sequence of the HERV-H transmembrane protein truncated body is shown in SEQ ID NO:

16.

3. A drug, characterized in that, The drug described above includes the human endogenous retrovirus envelope protein mutant or truncated body according to any one of claims 1 to 2.

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