CD40L-Fc fusion protein, gene segment, recombinant plasmid, recombinant cell, preparation method and application

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a CD40L-Fc fusion protein, a gene segment, a recombinant plasmid, a recombinant cell, a preparation method and application. According to the invention, through a fusion protein technology, a CD40L-Fc fusion protein of a mouse IgG1 subtype is prepared; the Fc segment of the IgG1 is further subjected to mutation modification, and the mutant CD40L-Fc fusion protein is prepared. The IgG1 subtype CD40-Fc fusion protein and the mutated fusion protein can significantly enhance the activation of B cells, have immune activation and anti-tumor effects, and have wide application prospects in the aspects of drug research and development and disease treatment.

Description

Technical Field

[0001] 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. Background Art

[0002] According to statistics from the World Health Organization, cancer is one of the main causes of human death, shortened life expectancy, and threatened human life and health. Affected by various objective factors such as changes in lifestyle, environmental degradation, and increased social pressure, the number of new tumors in the world has continued to grow in recent years. In recent years, immunotherapy for cancer has shown unique advantages. Immunotherapy activates or enhances the patient's own immune system to identify and eliminate tumor cells, and has achieved significant therapeutic effects in many types of cancer, such as lung cancer, melanoma, and kidney cancer. Compared with traditional treatment methods, immunotherapy can bring patients lasting survival benefits and even achieve long-term survival for some cancer patients. The application of immunotherapy has gradually advanced from patients with advanced tumors to patients with early tumors. Some patients with unresectable tumors have achieved tumor downstaging after receiving immunotherapy, thereby prolonging their survival. However, some patients may develop drug resistance after initial treatment, resulting in a decrease in treatment efficacy or immune-related adverse reactions. At present, the causes of common adverse reactions related to immunotherapy are T cell infiltration, cytokines, and autoantibodies. As people's research and understanding of how tolerance, immunity, and immunosuppression regulate anti-tumor immune responses continue to deepen, active immunotherapy is considered to be a way for cancer patients to obtain lasting responses, and new immunotherapy drugs are needed to improve the above problems.

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

[0004] B cells are a type of immune cell that plays an important role in regulating the immune system and producing antibodies. B cells recognize antigens through the B cell receptor BCR on their surface. Activated B cells differentiate into plasma cells and memory B cells, promote inflammatory conditions, activate 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 absorbed antigens to T cells, promote the activation and proliferation of T cells, and B cells can also secrete cytokines to regulate immune responses, such as secreting interferons and cytokines. The activation of B cells plays an important role in immune responses, and activated B cells can achieve immune activation. In terms of anti-tumor, by activating B cells, the antigen presentation and antibody secretion capabilities of B cells are enhanced. The antigen presentation ability of B cells enables tumor antigens to be presented to T cells, promotes the activation and proliferation of T cells, and enhances cellular immune responses; specific antibodies can recognize and bind to antigens on the surface of tumor cells, mark tumor cells, and then activate other cells in the immune system (such as macrophages, natural killer cells, etc.) to attack tumor cells. Therefore, activating B cells can achieve immune activation or anti-tumor effects.

[0005] CD40 / CD40L binding activates a variety of innate immune cells, and CD40L-Fc fusion protein has more potential and advantages in activating B cells. Currently, there is only human CD40L-Fc fusion protein, and it is of the IgG4 subtype. There is no mouse CD40L-Fc fusion protein, and there are no other subtypes of CD40L-Fc fusion protein. The mouse CD40L-Fc fusion protein can avoid the immune response that human proteins may induce in mouse models, which is more conducive to basic research and early drug development; the development of other subtypes of CD40L-Fc fusion proteins provides multiple possibilities for the drug development of CD40L-Fc fusion proteins, which is conducive 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 mouse CD40L-Fc fusion protein of other subtypes that has a strong activation effect on B cells. Summary of the invention

[0007] In view of the defects of the prior art, the present invention provides a mouse-derived IgG1 subtype CD40L-Fc fusion protein, which aims to more strongly activate B cells.

[0008] The present invention provides a CD40L-Fc fusion protein, wherein 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, wherein the gene fragment encodes any one of the above-mentioned CD40L-Fc fusion proteins.

[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, which comprises any one of the gene fragments described above.

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

[0014] The present invention provides a recombinant cell, wherein the recombinant cell contains any of the recombinant plasmids described above.

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

[0016] The method for preparing any of the above CD40L-Fc fusion proteins comprises: The gene fragment 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.

[0017] Use of any of the above-mentioned CD40L-Fc fusion proteins, any of the above-mentioned gene fragments, any of the above-mentioned recombinant plasmids, and any of the above-mentioned recombinant cells in the preparation of B cell activation-dependent immune activation and / or anti-tumor drugs, or use in constructing B cell-specific research tools.

[0018] The present invention prepares a mouse IgG1 subtype CD40L-Fc fusion protein through fusion protein technology; further mutates the Fc segment of IgG1 to prepare a mutated CD40L-Fc fusion protein. Experiments have found that the mouse IgG1 subtype CD40-Fc fusion protein of the present invention has a significantly enhanced B cell activation effect, does not induce other immune responses in mouse models, and is beneficial to basic research and early drug development; further compared with the unmutated CD40L-Fc fusion protein, the mutated fusion protein has similar CD40 binding ability, has a significantly enhanced B cell activation effect, and has better application prospects as a drug itself or a drug development reagent. The CD40L-Fc fusion protein prepared by the present invention has broad application prospects in immune activation and anti-tumor.

[0019] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0020] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is the SDS-PAGE protein denaturing non-reducing gel electrophoresis diagram of the purified CD40L-Fc fusion protein mIgG1 and MUT1; wherein the G1 lane refers to the lane of the fusion protein mIgG1, and the MUT1 lane refers to the lane of the mutant MUT1.

[0022] Figure 2 This is a graph showing the binding ability of the fusion proteins mIgG1 and MUT1 to CD40.

[0023] Figure 3 The figures are the experimental results of the fusion protein mIgG1 and MUT1 on B cell activation; A is the result of the effect of the fusion protein on the number of B cells; B is the result of the effect of the fusion protein on the number of B cells expressing CD54; C is the result of the effect of the fusion protein on the number of B cells expressing CD83; D is the result of the effect of the fusion protein on the number of B cells expressing CD86; E is the result of the effect of the fusion protein on the number of B cells expressing IgM.

[0024] Figure 4The figures are the results of competitive activation of B cells by fusion proteins mIgG1 and MUT1 with free CD40L respectively; A is the result of the effect of fusion protein on the number of B cells; B is the result of the effect of fusion protein on the number of B cells expressing CD54; C is the result of the effect of fusion protein on the number of B cells expressing CD83; D is the result of the effect of fusion protein on the number of B cells expressing CD86; E is the result of the effect of fusion protein on the number of B cells expressing IgM. DETAILED DESCRIPTION

[0025] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0026] Example 1 Construction of the CD40-Fc fusion protein of the present invention 1. Construction of CD40L-Fc fusion protein plasmid 1. Construction of plasmids for Fc fusion protein based on natural IgG1 (1) A CD40L-Fc (G1) DNA fragment formed by connecting CD40L and mouse IgG1 Fc fragment was synthesized, and homology arm sequences were added to both ends of the fragment (the homology arm sequence includes two segments, the nucleic acid sequence of the first homology arm sequence is shown in SEQ ID NO. 3, added to the 5' end of the aforementioned fragment, and the nucleic acid sequence of the second homology arm sequence is shown in SEQ ID NO. 4, added to the 3' end of the aforementioned fragment), to obtain a G1 fragment with homology arms, named homology arm-G1. (2) Using pcDNA3.4 as the vector, the vector pcDNA3.4 was linearized by double restriction digestion at the cloning sites Eco53k and SacI. The synthesized G1 fragment with homology arms and the linearized vector were homologously recombined. The resulting recombinant plasmid was named pcDNA3.4-1-G1 vector.

[0027] (3) After the recombination reaction is completed, it can be directly transformed or stored at -20℃ for future use.

[0028] (4) Transform the recombinant product into competent cells (such as DH5α), and obtain monoclonal colonies after heat shock, recovery, and plating. Screen positive clones through colony PCR and enzyme digestion identification after plasmid extraction.

[0029] (5) Send the bacterial solution of suspected positive clones to a sequencing company for bidirectional sequencing and compare the sequencing results with the designed sequence to see if they are consistent. Only those with correct sequencing results can proceed to subsequent experiments.

[0030] 2. Construction of plasmids based on mutant IgG1 Fc fusion protein (1) To generate mutant mouse fusion protein plasmids, first synthesize a DNA fragment of IgG1-MUT1 with site-directed mutations, and then synthesize a CD40-Fc (G1-MUT1) DNA fragment consisting of CD40L and IgG1-MUT1 fragments.

[0031] (2) The plasmid of the mutant fusion protein was constructed using the same construction method as the plasmid of the Fc fusion protein based on natural IgG1. The obtained G1-MUT1 fragment with homology arms was named homology arm-G1-MUT1, and the obtained recombinant plasmid was named pcDNA3.4-1-G1-MUT1 vector.

[0032] 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 homology arm sequence are shown in Table 1: Table 1 Nucleotide sequences of gene fragments in Example 1 Note: Single underscore " " 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.

[0033] 2. Expression of CD40L-Fc fusion protein The fusion protein of this example was expressed by 293T cells in the following manner: 1. After 7 dishes of 293T cells have grown well, start expressing proteins; 2. Prepare serum-free (SFM) medium: add 1% GlutaMAX supplement and 1% PS (penicillin / streptomycin) to SFM medium; 3. Prepare DNA solution: add 0.41 mL of MEM medium and 25 μg of pcDNA3.4-1-G1 vector plasmid or pcDNA3.4-1-G1-MUT1 vector plasmid prepared in this example to each dish; 4. Prepare PEI solution: Add 0.4 mL of MEM medium and 50 μg PEI (molecular weight: average M n~10,000 by GPC, average M w ~25,000 by LS, Brand: merck (sigma) Product No.: 408727); 5. Mix the DNA solution and PEI solution, slowly flip 10 times, and protect from light for 15 minutes; 6. Add the mixed solution in step 5 to the prepared SFM medium and mix well to obtain culture solution; 7. Take out the cells, remove the supernatant, add 10mL PBS to wash the cells and then remove it; 8. Add 20 mL of culture medium to each cell plate and incubate at 37°C and 5% CO 2 Culture in an incubator; 9. After four days of cultivation, collect the culture supernatant, centrifuge at 400×g for 5 minutes, filter using a vacuum filtration system, add 0.03%-0.05% Proclin300, and store at 4°C.

[0034] 3. Purification of CD40L-Fc fusion protein 1. Slowly shake the affinity chromatography medium protein A and take 1.2 mL into a test tube, add 10 mL PBS to mix, centrifuge at 2900 rpm for 3 min, remove the supernatant, add 10 mL PBS to mix and centrifuge, remove the supernatant, add 10 mL PBS to mix and centrifuge, add the washed protein A evenly to the supernatant of the fusion protein obtained in step 2 of this example, and gently flip for 15 min; 2. Assemble the filter beads, first add 1mL PBS to moisten the filter cake, and after the PBS flows out, add the mixture of protein supernatant and protein A. After all are filtered, add 5mL PBS to wash the tube wall, and then add 5mL PBS to wash the tube wall; 3. Add 150 μL Tris-HCl to three centrifuge tubes respectively in advance, add 1 mL of glycine to the filter cake and flow it into the first centrifuge tube, then add 1 mL of glycine to the second centrifuge tube, and finally add 1 mL of glycine to the third centrifuge tube to obtain three tubes of purified fusion protein sample effluent. Note that the filter cake cannot dry out during the process; 4. Prepare 10×PBS solution. Weigh the reagents according to the formula in Table 2 below, add 1L of double-pure water, sterilize under high pressure (121°C, 20min), cool to room temperature, take 10×PBS solution and prepare 4L of 1×PBS solution with double-pure water, pour into a 5L beaker, and place in a 4°C refrigerator; Table 2 10×PBS solution formula 5. Inject 3 mL of the purified fusion protein sample effluent into the dialysis membrane, place it in a 4L 1×PBS beaker for rotational dialyzation, and place the whole at 4°C. Replace the 1×PBS liquid after 4 hours. After 4 hours, aspirate the dialyzed protein to obtain the purified fusion protein, and store it in a 4°C refrigerator.

[0035] According to the same purification method, two fusion proteins, mIgG1 and MUT1, can be purified separately. 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.

[0036] The amino acid sequences of the Fc segment of natural / mutated IgG1 and the amino acid sequences of the fusion proteins mIgG1 and MUT1 are shown in Table 3: Table 3 4. Detection of fusion proteins 1. Experimental methods The molecular weight of the fusion protein mIgG1 or MUT1 was determined by SDS-PAGE protein denaturing non-reducing gel electrophoresis experiment.

[0037] (1) Pouring separation gel ①Assemble the gel mold.

[0038] ② Mix different volumes of 30% Acr-Bis (29:1), SDS-PAGE Separating GelBufer and pure water in a small beaker or test tube.

[0039] ③ Add 10% ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), and stir gently to mix them evenly to avoid generating bubbles.

[0040] ④ Pour 4 / 5 of the separation gel solution into the gel mold, and then gently cover the separation gel solution with a 1 cm layer of water to keep the gel surface flat.

[0041] ⑤ Let it stand for 30-60 minutes until a clear interface appears between the separation gel and the water layer, and the surface gel has polymerized.

[0042] (2) Pouring concentrated gel ① Remove the water layer covering the separation gel.

[0043] ② Mix different volumes of 30% Acr-Bis (29:1), SDS-PAGE Stacking Gel Buffer and pure water in a small beaker or test tube.

[0044] ③ Add 10% ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), and stir gently to mix them evenly to avoid generating bubbles.

[0045] ④ Add the concentrated gel solution to the separation gel until the gel solution reaches the top of the front glass plate ⑤Insert the comb into the gel to avoid creating bubbles.

[0046] ⑥Let it stand for 10 to 20 minutes to allow the concentrated gel to polymerize.

[0047] ⑦ After the gel is polymerized, carefully pull out the comb to avoid damaging the sample wells.

[0048] (3) Electrophoresis ① Prepare loading buffer, mix the protein sample (mIgG1 or MUT1) and 5× loading buffer (containing SDS), incubate in a 100℃ water bath for 5 minutes, and cool to room temperature. ② Place the gel in the electrophoresis box, add an appropriate amount of gel buffer, and slowly add the sample mixture and marker into the sample well.

[0049] ③ Close the electrophoresis tank cover and connect the electrode plug. Adjust the voltage and current to 80V. When the indicator strip reaches the edge of the separation gel and the concentrated gel, change the electrophoresis voltage to 120V and continue electrophoresis. When the indicator strip moves to about 1cm from the front edge of the gel, turn off the power and stop electrophoresis.

[0050] ④ Carefully remove the gel from the electrophoresis tank using a scraper.

[0051] (4) Dyeing and bleaching Place the gel in a culture dish and add Coomassie Brilliant Blue protein gel staining solution until the staining solution covers the gel. Shake the gel on a horizontal shaker for 4 hours, decolorize the gel with double-pure water, and continue to decolorize on the shaker, changing the double-pure water every half an hour. Take pictures when the bands are clear.

[0052] 2. Experimental results The results of SDS-PAGE experiments are shown in Figure 1 As shown: the fusion proteins mIgG1 and MUT1 have clear and single bands after purification, correct molecular weight, high purity, and can be used for activity research and analysis. The experimental results show that the present invention has prepared two target CD40-Fc fusion proteins: fusion protein mIgG1 and fusion protein MUT1.

[0053] The technical solution of the present invention is further described below through experiments. The fusion protein mIgG1 and the fusion protein MUT1 in the following experimental examples are both prepared by the method of the aforementioned Example 1: Experimental Example 1 Characterization of the binding ability of the CD40-Fc fusion protein of the present invention to CD40 1. Experimental Methods 1. Experimental Grouping This experiment was divided into three groups, namely mIgG1, MUT1 and Ctrl IgG groups. The mIgG1 group used the fusion protein mIgG1, the MUT1 group used the mutated fusion protein MUT1, and the Ctrl IgG group used natural non-specific mouse IgG (brand: Jackson ImmunoResearch, product number: 015-000-003). The protein concentration of each group was 2.5μg / mL.

[0054] 2. Characterization of the binding ability of fusion protein to CD40 (1) Prepare a 0.1 μg / ml CD40 protein solution using 1× PBS.

[0055] (2) Coat the CD40 protein in a 96-well plate (100 μl per well) at 4°C overnight.

[0056] (3) Dissolve in 1× PBS and prepare blocking solution (5% skimmed milk powder).

[0057] (4) Take out the 96-well plate and pat it dry. Add 200 μl of blocking solution to each well and let it stand at room temperature for 2 h.

[0058] (5) Prepare the cleaning solution PBST: add 0.5 ml TWEEN 80 to 1× PBS.

[0059] (6) After shaking out the liquid, add 200 μl PBST, discard it and add another 200 μl PBST. Pat dry and add 100 μl of antibodies and fusion proteins with different concentration gradients (concentration range is 0.0316 μg / ml-31.6 μg / ml), and let it stand at room temperature for one hour.

[0060] (7) Dilute mouse Fc-HPR 10,000-fold with 1x PBS.

[0061] (8) After shaking out the liquid, add 200 μl PBST, discard it and add another 200 μl PBST. Pat dry and add 100 μl mouse Fc HPR secondary antibody. Let it stand at room temperature for one hour.

[0062] (9) After shaking out the liquid, add 200 μl PBST, discard it and add another 200 μl PBST. Pat dry and add color developing solution, 100 μl per well. Let it stand for 15 minutes and wait for color development.

[0063] (10) Detect the absorbance at 620 nm. After detection, use 1 M sulfuric acid to stop the color development and detect the absorbance value at 450 nm.

[0064] 2. Experimental Results The experimental results of binding ability are as follows Figure 2 As shown: The IgG1 subtype CD40L-Fc fusion proteins mIgG1 and MUT1 have similar binding abilities to CD40 and can be used for activity research and analysis.

[0065] Experimental Example 2 Study on the effect of CD40-Fc fusion protein on activating B cells 1. Experimental Methods 1. Experimental Grouping This experiment was divided into three groups, namely mIgG1, MUT1 and Ctrl IgG groups. The mIgG1 group used the fusion protein mIgG1, the MUT1 group used the mutated fusion protein MUT1, and the Ctrl IgG group used natural non-specific mouse IgG (brand: Jackson ImmunoResearch, product number: 015-000-003). The protein concentration of each group was 2.5μg / mL.

[0066] 2. Extraction and processing of spleen cells In this experiment, mouse spleen cells were used to study the activation effect of fusion protein on B cells. The spleen cell extraction was performed on ice as follows: (1) Prepare the culture medium (RPMIm): RPMI + 10% FBS + 100×PS (penicillin / streptomycin); (2) Prepare FACS solution: 20 mL PBS + 100 μL FBS; (3) Take 10 mL of Facs solution into a culture dish and put it into a filter; (4) Grind the spleen of C57 mice (8 weeks, female), aspirate the solution and centrifuge (400 × g, 5 min); (5) Remove the supernatant and add 5 mL of filtered red blood cell lysate (sterilized by filtration through a membrane in advance). Keep on ice for 5-8 minutes, add 9 mL of Facs solution, mix well and centrifuge (400 × g, 5 minutes). (6) Remove the supernatant, add 4 mL of RPMI culture medium, and count the cells; (7) According to the cell counting results, dilute the cell solution to prepare 2x10 6 / ml of cell fluid.

[0067] 3. B cell activation experiment According to the grouping situation, different proteins were added to the cell solution in step 2 to prepare a suspension. The suspension was placed in a 96-well cell culture plate and incubated at 37°C and 5% CO 2 After two days of culture in an incubator, staining analysis was performed.

[0068] 4. Competitive B cell activation experiment with CD40L According to the grouping situation, different proteins and free CD40L protein were added to the cell solution in step 2 to prepare a suspension. The suspension was placed in a 96-well cell culture plate and incubated at 37°C and 5% CO 2 After two days of culture in an incubator, staining analysis was performed.

[0069] The concentration of free CD40L added to each group was 0.25 μg / mL, and the effects of mIgG1 and MUT1 fusion proteins on B cell activation under the competition of free CD40L were analyzed.

[0070] 5. Staining experiment Staining (operate on ice throughout the process): (1) Remove the cells from the well plate, centrifuge (400 × g, 5 min), discard the supernatant, and remove the cell culture medium; (2) Add 200 μL 1× PBS, fully suspend the cells, centrifuge (400×g, 5 min), and discard the supernatant; (3) Add 500 μL of antibody diluent (antibody staining solution diluted 400 times with PBS), mix well, protect from light for 15 min, add 150 μL of 1× PBS, mix well, centrifuge (400×g, 5 min), and discard the supernatant; (4) Add 200 μL 1x PBS, suspend the cells, centrifuge (400 × g, 5 min), and discard the supernatant; (5) Add 100 μL of 7-AAD (7-aminoactinomycin D) diluted in PBS and stain for 10-20 minutes in the dark; (6) On-computer analysis.

[0071] 2. Experimental Results 1. Results of B cell activation The results of B cell activation experiments were Figure 3 As shown in the figure, it can be seen that the IgG1 subtype CD40L-Fc fusion protein has an activating effect on a variety of immune molecules, significantly increasing the number of B cells expressing CD54, CD83, CD8 and IgM; in particular, the effect of mutant MUT1 in activating B cells is significantly stronger than mIgG1.

[0072] 2. Competitive B cell activation with CD40L Competitive B cell activation with CD40L results in Figure 4As shown: In the presence of free CD40L, the IgG1 subtype CD40L-Fc fusion protein has a significant competitive activation effect on B cells, indicating that the IgG1 subtype CD40L-Fc fusion protein may achieve stronger immune activation by competitively binding to CD40; in particular, the competitive B cell activation effect of mutant MUT1 is significantly stronger than that of mIgG1.

[0073] The above results indicate that, in the presence or absence of free CD40L, the IgG1 subtype CD40-Fc fusion protein has a significantly enhanced B cell activation effect, and the mutant MUT1 has a stronger B cell activation effect than mIgG1.

[0074] The results of the above experimental examples show that the IgG1 subtype CD40L-Fc fusion protein has a significant effect on activating B cells.

[0075] On the one hand, activating B cells can enhance the antigen presentation and antibody secretion capabilities of B cells. The antigen presentation capability of B cells enables tumor antigens to be presented to T cells, promotes the activation and proliferation of T cells, and enhances cellular immune responses; specific antibodies can recognize and bind to antigens on the surface of tumor cells, mark tumor cells, and then activate other cells in the immune system (such as macrophages, natural killer cells, etc.) to attack tumor cells, so the IgG1 subtype CD40L-Fc fusion protein 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 immune responses, so the IgG1 subtype CD40L-Fc fusion protein has an immune activation effect. The two IgG1 subtype CD40L-Fc fusion proteins 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.

[0076] Furthermore, the MC38 mouse colon cancer model was also used to conduct an anti-tumor experiment on the CD40L-Fc fusion protein of the present invention. The experimental method is as follows: 1. MC38 cells were cultured in vitro (RPMI, 10% FBS, 1% Pen / Strep, 1mM sodium pyruvate, 10mM HEPES, 50μM 2-mercaptoethanol), and cells in good logarithmic phase were taken and cultured at 2×10 6 C57BL / 6 mice were subcutaneously inoculated with a dose of 1 cell / mouse.

[0077] 2. After inoculation, measure the tumor size of the inoculated mice every 3 days and weigh the mice at the same time. Use the formula (L1 2× L2) / 2, where L1 is the smallest tumor diameter and L2 is the largest tumor diameter.

[0078] 3. After 6 days, the mice were randomly divided into groups according to the tumor size, and the mice in different experimental groups were marked with characteristics and intraperitoneally injected with drugs. The drugs were different CD40L-Fc fusion proteins (mIgG1, MUT1 prepared by the present invention) or control IgG (5-50 μg / mouse). The number of administrations was 3 times, on day 0, day 2, and day 4, respectively.

[0079] 4. After administration (until the mouse tumor grows to a diameter of 15-20 mm), continue to measure and record the size of the mouse tumor every 3 days, and observe the condition of the mouse. After the measurement, statistically analyze its anti-tumor effect.

[0080] On the other hand, the present invention provides two mouse IgG1 subtype CD40L-Fc fusion proteins, which have different activation effects on B cells. They can be used to compare and study the different immune changes in the body caused by different degrees of B cell activation. They can be used for basic research related to B cell activation and early development of drugs for various immune-related diseases.

[0081] In immunology research, B cells are an important part of the adaptive immune system. In order to gain a deeper understanding of their functions, the prior art has developed a variety of tools and models, including Mb1-Cre tool mice, CD40-activated B cell models, and the like. The present invention provides two mouse-derived IgG1 subtype CD40L-Fc fusion proteins that can cause mouse B cell activation, especially the B cell activation effect caused by mutant MUT1 is particularly significant, and can also be used as a tool for B cell specificity research, or an important tool for studying the degree of B cell proliferation and the immune changes caused by it in the body.

[0082] Through the above embodiments and experimental examples, it can be seen that the present invention prepares a mouse-derived IgG1 subtype CD40L-Fc fusion protein through fusion protein technology; further mutates the Fc segment of IgG1 to prepare a mutated CD40L-Fc fusion protein. Experiments have found that the mouse-derived IgG1 subtype CD40-Fc fusion protein of the present invention has a significantly enhanced B cell activation effect, does not induce other immune responses in the 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 similar CD40 binding ability and significantly enhanced B cell activation, and has better application prospects as a drug itself or a drug development reagent. The CD40L-Fc fusion protein prepared by the present invention has broad application prospects in immune activation and anti-tumor.

Claims

1. A CD40L-Fc fusion protein, characterized in that: 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.

2. The CD40L-Fc fusion protein according to claim 1, characterized in that: The amino acid sequence of the CD40L-Fc fusion protein comprises the sequence shown in SEQ ID NO. 7 or SEQ ID NO.

8.

3. A gene fragment, characterized in that: The gene fragment encodes the CD40L-Fc fusion protein according to claim 1 or 2.

4. The gene fragment according to claim 3, characterized in that: The gene fragment comprises the sequence shown in SEQ ID NO. 1 or SEQ ID NO.

2.

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

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

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

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

9. The method for preparing the CD40L-Fc fusion protein according to claim 1 or 2, characterized in that: It includes: The gene fragment 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.

10. Use of the CD40L-Fc fusion protein according to claim 1 or 2, the gene fragment according to claim 3 or 4, the recombinant plasmid according to claim 5 or 6, or the recombinant cell according to claim 7 or 8 in the preparation of B cell activation-dependent immune activation and / or anti-tumor drugs, or in the construction of B cell-specific research tools.

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