A CD40L-Fc fusion protein, gene fragment, recombinant plasmid, recombinant cell, and preparation method and use thereof

Abstract

The present invention belongs to the field of genetic engineering, and specifically relates to a CD40L-Fc fusion protein, a gene fragment, a recombinant plasmid, a recombinant cell, and a preparation method and use thereof. By means of the fusion protein technology, the present invention prepares a murine IgG1 subtype CD40L-Fc fusion protein; further, the Fc segment of IgG1 is subjected to mutagenesis and modification to obtain a mutated CD40L-Fc fusion protein. The IgG1 subtype CD40-Fc fusion protein and the mutated fusion protein of the present invention can significantly enhance the activation of B cells, have the effects of immune activation and anti-tumor, and have broad application prospects in drug research and development and disease treatment.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a CD40L-Fc fusion protein, a gene fragment, a recombinant plasmid, a recombinant cell, and a preparation method and use thereof. Background Art

[0002] According to statistics of the World Health Organization, cancer is one of the main causes leading to human death, shortening life expectancy, and threatening human life and health. Affected by various objective factors such as changes in lifestyle, environmental deterioration, and increasing social pressure, the number of newly added tumors globally has been continuously increasing in recent years. In recent years, immunotherapy for cancer has shown unique advantages. Immunotherapy can identify and eliminate tumor cells by activating or enhancing the patient's own immune system, and has achieved remarkable therapeutic effects in various cancer types, such as lung cancer, melanoma, renal cancer, etc.; compared with traditional treatment methods, immunotherapy can bring lasting survival benefits to patients and even achieve long-term survival of some cancer patients; the application of immunotherapy is gradually advancing from advanced tumor patients to early patients, and some patients with inoperable tumors have achieved tumor downstaging after receiving immunotherapy, thereby prolonging the survival period. However, some patients may develop drug resistance after initial treatment, resulting in a decline in the treatment effect, or triggering immune-related adverse reactions, etc. Currently, the causes of common immune-related adverse reactions are T cell infiltration, cytokines, and autoantibodies. With the continuous in-depth research and understanding of how tolerance, immunity, and immunosuppression regulate the anti-tumor immune response, active immunotherapy is considered a way for cancer patients to obtain a lasting response, and new immunotherapy drugs are needed to improve the above problems.

[0003] CD40 is a type I transmembrane protein, which is widely expressed in platelets, B cells, myeloid cells, etc. The homologous ligand of CD40 is CD154, also known as CD40L, which is a type II transmembrane protein and is usually expressed in platelets, granulocytes, activated T cells, activated B cells, NK cells, and some tumor cells, etc. The binding of CD40 / CD40L activates a variety of innate immune cells, including dendritic cells, macrophages, NK cells, and B cells, resulting in the stimulation of CD8+ and CD4+ T cells, and further leading to immune activation and tumor apoptosis. At the same time, the binding of CD40 / CD40L can expand the immune response at the front end to kill tumor cells. The CD40 / CD40L plays a key role in immune activation and tumor treatment. It has been reported in the literature that CD40L has obvious anti-tumor activity and immune activity. Different from previous studies on traditional antibodies, agonist antibodies targeting co-stimulatory molecules do not need to continuously improve their affinity, and a balanced low affinity can mediate a stronger therapeutic effect. The CD40L-Fc fusion protein based on natural binding and affinity has more potential and advantages.

[0004] B cells are a type of immune cells that play important roles in immune system regulation and antibody production. B cells recognize antigens through B cell receptors (BCRs) on their surfaces. Activated B cells differentiate into plasma cells and memory B cells, promoting inflammatory conditions and activating immune cells, etc. In terms of immune regulation, B cells play an important role in the activation and function of T cells. By expressing antigen-presenting molecules, B cells can present the absorbed antigens to T cells, promoting the activation and expansion of T cells. B cells can also secrete cytokines to regulate immune responses, such as secreting interferon and cytokines, etc. The activation of B cells is important for immune responses, and activated B cells can achieve immune activation. In anti-tumor aspects, by activating B cells, the antigen-presenting and antibody-secreting abilities of B cells are enhanced. The antigen-presenting ability of B cells enables tumor antigens to be presented to T cells, promoting the activation and proliferation of T cells and enhancing cellular immune responses; specific antibodies can recognize and bind to antigens on the surface of tumor cells, marking the tumor cells, and then activating other cells in the immune system (such as macrophages, natural killer cells, etc.) to attack the tumor cells. Therefore, activating B cells can achieve the effects of immune activation or anti-tumor.

[0005] The binding of CD40 / CD40L activates a variety of innate immune cells, and the CD40L-Fc fusion protein has more potential and advantages in activating B cells. Currently, there is only a human-derived CD40L-Fc fusion protein, and it is of the IgG4 subtype. There is no murine CD40L-Fc fusion protein, and there are no CD40L-Fc fusion proteins of other subtypes either. The murine CD40L-Fc fusion protein can avoid the immune responses that may be triggered by human proteins in mouse models, which is more conducive to basic research and early drug development; the development of CD40L-Fc fusion proteins of other subtypes provides multiple possibilities for the drug research and development of CD40L-Fc fusion proteins, which is beneficial to promoting the development of CD40L-Fc fusion proteins in immune activation and anti-tumor drugs.

[0006] Therefore, it is very important to explore a murine CD40L-Fc fusion protein of other subtypes that has a strong activating effect on B cells. Summary of the Invention

[0007] Aiming at the defects of the prior art, the present invention provides a murine IgG1 subtype CD40L-Fc fusion protein, aiming to more strongly activate B cells.

[0008] The present invention provides a CD40L-Fc fusion protein, and the CD40L-Fc fusion protein comprises CD40L and the Fc segment of IgG1; the amino acid sequence of the Fc segment of IgG1 comprises the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0009] Preferably, the amino acid sequence of the CD40L-Fc fusion protein comprises the sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0010] The present invention provides a gene fragment encoding the CD40L-Fc fusion protein described in any one of the above.

[0011] Preferably, the gene fragment comprises the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0012] The present invention provides a recombinant plasmid comprising the gene fragment described in any one of the above.

[0013] Preferably, the recombinant plasmid is a plasmid obtained by inserting the gene fragment described in any one of the above into a vector; the vector is selected from at least one of pcDNA and pCMV.

[0014] The present invention provides a recombinant cell containing the recombinant plasmid described in any one of the above.

[0015] Preferably, the recombinant cell is selected from at least one of 293T cells, HEK293 cells, 293S cells, and CHO cells.

[0016] The preparation method of the CD40L-Fc fusion protein described in any one of the above comprises:

[0017] Inserting the gene fragment encoding the CD40L-Fc fusion protein into a plasmid, then transforming it into a recombinant cell for expression, and separating and purifying to obtain the product.

[0018] Use of the CD40L-Fc fusion protein described in any one of the above, the gene fragment described in any one of the above, the recombinant plasmid described in any one of the above, and the recombinant cell described in any one of the above in the preparation of B cell activation-dependent immune activation and / or anti-tumor drugs, or for constructing B cell-specific research tools.

[0019] The present invention prepares a murine IgG1 subtype CD40L-Fc fusion protein through a fusion protein technology; further, the Fc segment of IgG1 is mutated and modified to prepare a mutated CD40L-Fc fusion protein. Experiments have found that the murine IgG1 subtype CD40-Fc fusion protein of the present invention has a significantly enhanced effect on B cell activation, does not trigger other immune responses in a mouse model, and is beneficial to basic research and early drug development; further, compared with the unmutated CD40L-Fc fusion protein, the mutated fusion protein has a similar CD40 binding ability and a significantly enhanced B cell activation effect, and has a better application prospect both as a drug itself and as a drug development reagent. The CD40L-Fc fusion protein prepared by the present invention has a broad application prospect in immune activation and anti-tumor aspects.

[0020] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0021] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0022] Figure 1 It is the SDS-PAGE protein denaturing non-reducing gel electrophoresis diagram of the purified CD40L-Fc fusion proteins mIgG1 and MUT1; among them, the G1 lane refers to the lane of the fusion protein mIgG1, and MUT1 refers to the lane of the mutant MUT1.

[0023] Figure 2 It is the result diagram of the binding ability of the fusion proteins mIgG1 and MUT1 to CD40.

[0024] Figure 3 It is the experimental result diagram of the activation of B cells by the fusion proteins mIgG1 and MUT1; among them, A is the result diagram of the influence of the fusion protein on the number of B cells; among them, B is the result diagram of the influence of the fusion protein on the number of B cells expressing CD54; among them, C is the result diagram of the influence of the fusion protein on the number of B cells expressing CD83; among them, D is the result diagram of the influence of the fusion protein on the number of B cells expressing CD86; among them, E is the result diagram of the influence of the fusion protein on the number of B cells expressing IgM.

[0025] Figure 4Results of the competitive activation of B cells by the fusion proteins mIgG1 and MUT1 with free CD40L; among which, A is the result of the influence of the fusion protein on the number of B cells; among which, B is the result of the influence of the fusion protein on the number of B cells expressing CD54; among which, C is the result of the influence of the fusion protein on the number of B cells expressing CD83; among which, D is the result of the influence of the fusion protein on the number of B cells expressing CD86; among which, E is the result of the influence of the fusion protein on the number of B cells expressing IgM. Detailed implementation mode

[0026] In the following examples and experimental examples, reagents and materials not specifically stated are all commercially available products.

[0027] Example 1 Construction of the CD40-Fc fusion protein of the present invention

[0028] I. Construction of the plasmid of the CD40L-Fc fusion protein

[0029] 1. Construction of the plasmid of the Fc fusion protein based on natural IgG1

[0030] (1). Synthesize the CD40L-Fc(G1) DNA fragment formed by connecting CD40L and the mouse IgG1 Fc fragment, and add homologous arm sequences at both ends of this fragment (the homologous arm sequences include two segments. The nucleic acid sequence of the first homologous arm sequence is as shown in SEQ ID NO. 3, added to the 5' end of the aforementioned fragment, and the nucleic acid sequence of the second homologous arm sequence is as shown in SEQ ID NO. 4, added to the 3' end of the aforementioned fragment), to obtain the G1 fragment with homologous arms, named homologous arm-G1.

[0031] (2). Using pcDNA3.4 as the vector, linearize the vector pcDNA3.4 by double digestion at the cloning sites Eco53k and SacI, and perform homologous recombination on the synthesized G1 fragment with homologous arms and the linearized vector, and name the obtained recombinant plasmid as the pcDNA3.4-1-G1 vector.

[0032] (3). After the recombination reaction is completed, it can be directly transformed or stored at -20°C for later use.

[0033] (4). Transform the recombinant product into competent cells (such as DH5α), and obtain monoclonal colonies through steps such as heat shock, resuscitation, and plating. Screen positive clones through colony PCR and restriction enzyme digestion identification after plasmid extraction.

[0034] (5). Send the bacterial solution of the suspected positive clone to a sequencing company for bidirectional sequencing, and compare whether the sequencing result is consistent with the designed sequence. Perform subsequent experiments on the clones with correct sequencing results.

[0035] 2. Construction of Plasmid of Fc Fusion Protein Based on Mutated IgG1

[0036] (1). To generate the construction of the mutated mouse fusion protein plasmid, first synthesize the DNA fragment of IgG1-MUT1 with site-directed mutation, and then synthesize the CD40-Fc(G1-MUT1) DNA fragment formed by ligating the CD40L and IgG1-MUT1 fragments.

[0037] (2). Use the same construction method as the plasmid of the Fc fusion protein based on natural IgG1 to construct the plasmid of the mutated fusion protein. The obtained G1-MUT1 fragment with homologous arms is named homologous arm-G1-MUT1, and the obtained recombinant plasmid is named pcDNA3.4-1-G1-MUT1 vector.

[0038] In this example, the nucleotide sequences of the DNA fragment encoding CD40L, the DNA fragment encoding mouse IgG1 Fc, the CD40L-Fc(G1) DNA fragment, the DNA fragment encoding IgG1-MUT1, the CD40-Fc(G1-MUT1) DNA fragment, and the homologous arm sequence are shown in Table 1:

[0039] Table 1 Nucleotide Sequences of Gene Fragments in Example 1

[0040]

[0041] Note: The single underline " " represents the nucleotide sequence of the DNA fragment encoding CD40L; the single wavy line " " represents the nucleotide sequence of the DNA fragment encoding mouse IgG1Fc; the double underline " " represents the nucleotide sequence of the DNA fragment encoding IgG1-MUT1.

[0042] II. Expression of CD40L-Fc Fusion Protein

[0043] The fusion protein of this example is expressed by 293T cells, and the method is as follows:

[0044] 1. Start expressing the protein after the 293T cells in 7 dishes grow well;

[0045] 2. Prepare serum-free (SFM) medium: Add 1% GlutaMAX additive and 1% PS (penicillin / streptomycin) to the SFM medium;

[0046] 3. Prepare the DNA solution: Add 0.41 mL of MEM medium and 25 μg of the pcDNA3.4-1-G1 vector plasmid or pcDNA3.4-1-G1-MUT1 vector plasmid prepared in this example to each dish;

[0047] 4. Prepare the PEI solution: Add 0.4 mL of MEM medium and 50 μg of PEI (molecular weight: average M n ~10,000 by GPC, average M w ~25,000 by LS, brand: merck (sigma), product number: 408727) to each dish;

[0048] 5. Mix the DNA solution and the PEI solution, slowly invert 10 times, and incubate in the dark for 15 min;

[0049] 6. Add the mixed solution from step 5 to the prepared SFM medium and mix well to obtain the culture solution;

[0050] 7. Take out the cells, aspirate the supernatant, add 10 mL of PBS to wash the cells and then aspirate;

[0051] 8. Add 20 mL of the culture solution to each cell dish and culture in an incubator at 37 °C and 5% CO 2 incubator;

[0052] 9. After culturing for four days, collect the supernatant of the culture solution, centrifuge at 400×g for 5 min, filter using a vacuum filtration system, add 0.03% - 0.05% Proclin300, and store at 4 °C.

[0053] III. Purification of CD40L-Fc fusion protein

[0054] 1. Slowly shake the affinity chromatography medium protein A, take 1.2 mL and put it into a test tube, add 10 mL of PBS and mix well, centrifuge at 2900 rpm for 3 min, discard the supernatant, add 10 mL of PBS again, mix well and centrifuge, discard the supernatant, add 10 mL of PBS again, mix well and centrifuge, and evenly add the washed protein A to the supernatant of the fusion protein obtained in step 2 of this example, gently invert for 15 min;

[0055] 2. Assemble the filter beads. First, pre-wet the filter cake with 1 mL of PBS. After the PBS flows out, add the mixed solution of the protein supernatant and protein A. After all are filtered, add 5 mL of PBS to wash the tube wall and let it flow out, then add 5 mL of PBS to wash the tube wall and let it flow out;

[0056] 3. Add 150 μL of Tris-HCl to three centrifuge tubes in advance. Add 1 mL of glycine to the filter cake and let it flow into the first centrifuge tube, then add another 1 mL of glycine and let it flow into the second centrifuge tube, and finally add 1 mL of glycine and let it flow into the third centrifuge tube to obtain the effluent of the purified fusion protein sample in three tubes. Note that the filter cake should not dry out during the process.

[0057] 4. Prepare 10×PBS solution. Weigh the reagents according to the formula in Table 2 below, add 1 L of double distilled water, autoclave (121 °C, 20 min), and after cooling to room temperature, take 10×PBS solution and dilute it with double distilled water to obtain 4 L of 1×PBS solution, pour it into a 5 L beaker, and place it in a 4 °C refrigerator.

[0058] Table 2 Formula of 10×PBS solution

[0059]

[0060] 5. Inject 3 mL of the effluent of the purified fusion protein sample into the dialysis membrane, place it in a 4 L beaker of 1×PBS for rotary dialysis, keep the whole at 4 °C, change the 1×PBS liquid after 4 h, and after 4 h, suck out the dialyzed protein to obtain the purified fusion protein, which is stored in a 4 °C refrigerator.

[0061] According to the same purification method, two fusion proteins, mIgG1 and MUT1, can be purified respectively. mIgG1 is a CD40-Fc fusion protein based on the Fc of natural IgG1, and MUT1 is a CD40-Fc fusion protein based on the Fc of mutant IgG1.

[0062] The amino acid sequences of the Fc segments of natural / mutant IgG1 and the amino acid sequences of the fusion proteins mIgG1 and MUT1 are shown in Table 3:

[0063] Table 3

[0064]

[0065] IV. Detection of Fusion Proteins

[0066] 1. Experimental Method

[0067] The molecular weights of the fusion proteins mIgG1 or MUT1 were determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) under denaturing and non-reducing conditions.

[0068] (1) Pour the separating gel

[0069] ① Assemble the gel mold.

[0070] ②Mix different volumes of 30% Acr - Bis (29:1), SDS - PAGE Separating Gel Buffer, and pure water in a small beaker or test tube.

[0071] ③Add 10% ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), and gently stir to mix evenly, avoiding the generation of bubbles.

[0072] ④Pour 4 / 5 of the separating gel solution into the gel mold, and then gently cover the surface of the separating gel solution with a 1 - cm layer of water to keep the gel surface flat.

[0073] ⑤Let it stand for 30 - 60 minutes. After a clear interface appears between the separating gel and the water layer, the surface gel has polymerized.

[0074] (2)Pour the stacking gel

[0075] ①Remove the water layer covering the separating gel.

[0076] ②Mix different volumes of 30% Acr - Bis (29:1), SDS - PAGE Stacking Gel Buffer, and pure water in a small beaker or test tube.

[0077] ③Add 10% ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), and gently stir to mix evenly, avoiding the generation of bubbles.

[0078] ④Add the stacking gel solution on top of the separating gel until the gel solution reaches the top of the front glass plate

[0079] ⑤Insert the comb into the gel, avoiding the generation of bubbles.

[0080] ⑥Let it stand for 10 - 20 minutes and wait for the stacking gel to polymerize.

[0081] ⑦After the gel has polymerized, carefully pull out the comb to avoid damaging the sample wells.

[0082] (3)Electrophoresis

[0083] ①Prepare the loading buffer. Mix the protein sample (mIgG1 or MUT1) and 5×loading buffer (containing SDS), then heat in a 100℃ water bath for 5 minutes and cool to room temperature.

[0084] ②Place the gel in the electrophoresis chamber, add an appropriate amount of gel buffer, and slowly add the sample mixture and Marker into the sample wells.

[0085] ③ Close the lid of the electrophoresis tank and connect the electrode plugs. Adjust the voltage and current. The voltage is 80 V. When the indicator band reaches the edge of the separating gel and the stacking gel, change the electrophoresis voltage to 120 V and continue electrophoresis. When the indicator band moves to about 1 cm from the gel front edge, turn off the power supply and stop electrophoresis.

[0086] ④ Carefully remove the gel from the electrophoresis tank with a scraper.

[0087] (4)Staining and decolorization

[0088] Place the gel in a petri dish and add Coomassie Brilliant Blue protein gel staining solution. The staining solution should just cover the gel. Shake well on a horizontal shaker for 4 h. Then decolorize the gel with double-distilled water and continue decolorization on the shaker, changing the double-distilled water every half hour. Take a photo when the bands are clear.

[0089] 2. Experimental results

[0090] The results of SDS-PAGE are as Figure 1 shown: For the fusion proteins mIgG1 and MUT1, the bands were clear and single after purification, with the correct molecular weight and high purity, and they can be used for activity research and analysis. The experimental results prove that two target CD40-Fc fusion proteins were prepared in the present invention: the fusion protein mIgG1 and the fusion protein MUT1.

[0091] The technical solution of the present invention will be further described through experiments below. The fusion protein mIgG1 and the fusion protein MUT1 in the following experimental examples were both prepared by the method of Example 1 above:

[0092] Experimental Example 1 Characterization of the binding ability of the CD40-Fc fusion protein of the present invention to CD40

[0093] I. Experimental method

[0094] 1. Experimental grouping

[0095] This experimental example was divided into three groups: mIgG1, MUT1, and Ctrl IgG groups. The mIgG1 group used the fusion protein mIgG1, the MUT1 group used the mutant fusion protein MUT1, and the Ctrl IgG group used the protein natural non-specific mouse IgG (brand: Jackson ImmunoResearch, product number: 015-000-003). The concentration of each group of proteins was 2.5 μg / mL.

[0096] 2. Characterization of the binding ability of the fusion protein to CD40

[0097] (1)Prepare a 0.1 μg / ml CD40 protein solution by dissolving CD40 protein in 1×PBS.

[0098] (2) Coat the CD40 protein (100 μl per well) in a 96-well plate and incubate overnight at 4°C.

[0099] (3) Dissolve it with 1×PBS to prepare the blocking solution (5% skim milk powder).

[0100] (4) Take out the 96-well plate, pat it dry, add 200 μl of the blocking solution to each well, and let it stand at room temperature for 2 h.

[0101] (5) Prepare the washing solution PBST: Add 0.5 ml of TWEEN 80 to 1×PBS.

[0102] (6) After discarding the liquid, add 200 μl of PBST, discard it again, add 200 μl of PBST, pat it dry, and add 100 μl of antibodies and fusion proteins with different concentration gradients (concentration range: 0.0316 μg / ml - 31.6 μg / ml), and let it stand at room temperature for one hour.

[0103] (7) Dilute mouse Fc-HPR 10,000 times with 1xPBS.

[0104] (8) After discarding the liquid, add 200 μl of PBST, discard it again, add 200 μl of PBST, pat it dry, and add 100 μl of the secondary antibody mouse Fc HPR, and let it stand at room temperature for one hour.

[0105] (9) After discarding the liquid, add 200 μl of PBST, discard it again, add 200 μl of PBST, pat it dry, and add 100 μl of the chromogenic solution to each well, let it stand for 15 min, and wait for color development.

[0106] (10) Detect the absorbance at 620 nm, terminate the color development with 1 M sulfuric acid after detection, and detect the absorbance value at 450 nm.

[0107] II. Experimental Results

[0108] The experimental results of the binding ability are as Figure 2 shown: The CD40L-Fc fusion proteins mIgG1 and MUT1 of the IgG1 subtype have similar binding abilities to CD40 and can be used for activity research and analysis.

[0109] Experimental Example 2 Study on the Effect of CD40-Fc Fusion Protein in Activating B Cells

[0110] I. Experimental Methods

[0111] 1. Experimental Grouping

[0112] This experimental example was divided into three groups, namely the mIgG1, MUT1, and Ctrl IgG groups. The mIgG1 group used the fusion protein mIgG1, the MUT1 group used the mutant fusion protein MUT1, and the Ctrl IgG group used the protein natural non-specific mouse IgG (brand: Jackson ImmunoResearch, product number: 015-000-003). The concentration of each group of protein was 2.5 μg / mL.

[0113] 2. Extraction and treatment of splenocytes

[0114] In this experimental example, mouse splenocytes were used to study the activation effect of the fusion protein on B cells. The whole process of extracting splenocytes was carried out on ice, and the method was as follows:

[0115] (1) Prepare the culture medium (RPMIm): RPMI + 10% FBS + 100×PS (penicillin / streptomycin);

[0116] (2) Prepare the Facs solution: 20 mL PBS + 100 μL FBS;

[0117] (3) Take 10 mL of the Facs solution into a culture dish and place a filter;

[0118] (4) Grind the spleen of C57 mice (8 weeks old, female), aspirate the solution, and centrifuge (400×g, 5 min);

[0119] (5) Discard the supernatant, add 5 mL of filtered erythrocyte lysate (previously filtered and sterilized through a filter membrane), incubate on ice for 5 - 8 min, add 9 mL of Facs solution, mix well, and centrifuge (400×g, 5 min);

[0120] (6) Discard the supernatant, add 4 mL of the prepared RPMIm culture medium, and count the cells;

[0121] (7) According to the cell counting results, dilute the cell solution to prepare a cell solution of 2×10 6 cells / ml.

[0122] 3. B cell activation experiment

[0123] According to the grouping situation, different proteins were added to the cell solution in step 2 to prepare a suspension. Place it in a 96-well cell culture plate and perform staining analysis after culturing in an incubator at 37°C and 5% CO 2 for two days.

[0124] 4. Competitive B cell activation experiment with CD40L

[0125] According to the grouping situation, different proteins and free CD40L protein were added to the cell lysate in step 2 to prepare a suspension. The suspension was placed in a 96-well cell culture plate and cultured in an incubator at 37°C and 5% CO 2 After culturing for two days, staining analysis was performed.

[0126] The concentration of free CD40L added to each group was 0.25 μg / mL. The effects of mIgG1 and MUT1 fusion proteins on B cell activation were analyzed under the competition of free CD40L.

[0127] 5. Staining experiment

[0128] Staining (operate on ice throughout the process):

[0129] (1) Take out the cells in the well plate, centrifuge (400×g, 5 min), discard the supernatant, and remove the cell culture medium;

[0130] (2) Add 200 μL of 1×PBS, fully suspend the cells, then centrifuge (400×g, 5 min), and discard the supernatant;

[0131] (3) Add 500 μL of antibody dilution solution (the antibody staining solution is diluted 400 times with PBS), mix well, incubate in the dark for 15 min, add 150 μL of 1×PBS, mix well, then centrifuge (400×g, 5 min), and discard the supernatant;

[0132] (4) Add 200 μL of 1×PBS, suspend the cells, centrifuge (400×g, 5 min), and discard the supernatant;

[0133] (5) Add 100 μL of 7-AAD (7-aminoactinomycin D) solution diluted with PBS and stain in the dark for 10 - 20 min;

[0134] (6) Analyze on the machine.

[0135] II. Experimental results

[0136] 1. Results of B cell activation

[0137] The results of the B cell activation experiment are as Figure 3 shown: It can be seen from the figure that the CD40L-Fc fusion protein of the IgG1 subtype can activate a variety of immune molecules, significantly increasing the number of B cells expressing CD54, CD83, CD8, and IgM; in particular, the effect of the mutant MUT1 on activating B cells is significantly stronger than that of mIgG1.

[0138] 2. Results of competitive B cell activation with CD40L

[0139] The results of competitive B cell activation with CD40L are as Figure 4Shown: In the presence of free CD40L, the CD40L-Fc fusion protein of IgG1 subtype has a significant effect of competitively activating B cells, indicating that the CD40L-Fc fusion protein of IgG1 subtype may achieve stronger immune activation by competitively binding to CD40; in particular, the effect of the mutant MUT1 on competitive B cell activation is significantly stronger than that of mIgG1.

[0140] The above results indicate that, whether free CD40L is present or not, the CD40-Fc fusion protein of IgG1 subtype has a significantly enhanced effect on B cell activation, and the mutant MUT1 has a stronger effect on B cell activation than mIgG1.

[0141] The results of the above experimental examples indicate that the CD40L-Fc fusion protein of IgG1 subtype has an obvious effect of activating B cells.

[0142] On the one hand, activating B cells can enhance the antigen presentation and antibody secretion abilities of B cells. The antigen presentation ability of B cells presents tumor antigens to T cells, promotes the activation and proliferation of T cells, and enhances the cellular immune response; specific antibodies can recognize and bind to antigens on the surface of tumor cells, label tumor cells, and then activate other cells in the immune system (such as macrophages, natural killer cells, etc.) to attack tumor cells. Therefore, the CD40L-Fc fusion protein of IgG1 subtype has a better anti-tumor effect. Activated B cells can also affect the cell composition and cytokine levels in the immune microenvironment, promote the activation and function of immune cells, and enhance the immune response. Therefore, the CD40L-Fc fusion protein of IgG1 subtype has an immune activation effect. The two CD40L-Fc fusion proteins of IgG1 subtype of the present invention can be used as immune activation drugs or anti-tumor drugs respectively, especially the mutant MUT1 has greater potential for development as immune activation and anti-tumor drugs.

[0143] Furthermore, an anti-tumor experiment was also carried out on the CD40L-Fc fusion protein of the present invention using the MC38 murine colon cancer model. The experimental method is as follows:

[0144] 1. Culture MC38 cells in vitro (RPMI, 10% FBS, 1% Pen / Strep, 1 mM sodium pyruvate, 10 mM HEPES, 50 μM 2-mercaptoethanol). When the cells are in the logarithmic phase and in good condition, inoculate C57BL / 6 mice subcutaneously at a dose of 2×10 6 cells / mouse.

[0145] 2. After inoculation, measure the tumor size of the inoculated mice once every 3 days, and weigh the mice at the same time. Use the formula (L1 2The volume of the tumor was calculated as (L1 × L2) / 2, where L1 is the smallest tumor diameter and L2 is the largest tumor diameter.

[0146] 3. Six days later, the mice were randomly grouped according to the tumor volume, and the mice in different experimental groups were marked with characteristics and injected intraperitoneally with drugs. The drugs were different CD40L-Fc fusion proteins (mIgG1 and MUT1 prepared in the present invention) or control IgG (5 - 50 μg / mouse). The drug was administered 3 times, on day 0, day 2, and day 4 respectively.

[0147] 4. After drug administration (until the tumor of the mice grew to a diameter of 15 - 20 mm), the volume of the mice's tumors was measured and recorded every 3 days, and the status of the mice was observed. After the measurement was completed, the anti-tumor effect was statistically analyzed.

[0148] On the other hand, the present invention provides two murine IgG1 subtype CD40L-Fc fusion proteins. The activation effects of the two proteins on B cells are different, which can be used for comparative studies on the different immune changes in the body caused by different degrees of B cell activation, and can be used for basic research related to B cell activation and the early R & D of drugs for various immune-related diseases.

[0149] In immunological research, B cells are an important part of the adaptive immune system. To deeply understand their functions, various tools and models have been developed in the prior art, including Mb1-Cre tool mice, CD40-activated B cell models, etc. The two murine IgG1 subtype CD40L-Fc fusion proteins provided by the present invention can cause the activation of murine B cells. In particular, the activation effect of the mutant MUT1 on B cells is particularly obvious, and it can also be used as an important tool for specific research on B cells or for studying the proliferation degree of B cells and the immune changes caused by them in the body.

[0150] From the above examples and experimental examples, it can be seen that the present invention prepares a murine IgG1 subtype CD40L-Fc fusion protein through fusion protein technology; further mutates and modifies the Fc segment of IgG1 to prepare a mutant CD40L-Fc fusion protein. Experiments have found that the murine IgG1 subtype CD40-Fc fusion protein of the present invention has a significantly enhanced effect on B cell activation and does not cause other immune reactions in the mouse model, which is beneficial to basic research and early drug development; further compared with the unmutated CD40L-Fc fusion protein, the mutated fusion protein has a similar CD40 binding ability and a significantly enhanced B cell activation effect, and has a better application prospect both as a drug itself or as a drug development reagent. The CD40L-Fc fusion protein prepared by the present invention has a broad application prospect in immune activation and anti-tumor aspects.

Claims

1. A CD40L-Fc fusion protein, characterized in that: The amino acid sequence of the CD40L-Fc fusion protein is the sequence shown in SEQ ID NO.

8.

2. A gene, characterized in that: The gene encodes the CD40L-Fc fusion protein of claim 1.

3. The gene according to claim 2, characterized in that: The gene comprises the sequence shown in SEQ ID NO.

2.

4. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the gene according to claim 2 or 3.

5. The recombinant plasmid according to claim 4, characterized in that: The recombinant plasmid is a plasmid obtained by inserting the gene described in claim 2 or 3 into a vector; the vector is selected from at least one of pcDNA and pCMV.

6. A recombinant cell, characterized in that: The recombinant cell contains the recombinant plasmid according to claim 4 or 5.

7. The recombinant cell according to claim 6, characterized in that: The recombinant cells are selected from at least one of 293T cells, HEK293 cells, 293S cells and CHO cells.

8. The method for preparing the CD40L-Fc fusion protein according to claim 1, characterized in that: It includes: The gene encoding the CD40L-Fc fusion protein is inserted into a plasmid, which is then transformed into a recombinant cell for expression, and separated and purified to obtain the fusion protein.

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