Method for producing high-titer antiserum based on combinatorial immunity

Abstract

The invention discloses a method for producing high-titer antiserum based on combinatorial immunity, and belongs to the technical field of biological medicine. According to the invention, the TLR agonist and the cell factor are added as immunologic adjuvants, and the TLR agonist activates innate immunity and provides a good immune microenvironment for mRNA-induced antigen-specific immune reaction; the cytokines regulate functions of immune cells and promote recognition and response of the immune cells to mRNA expression antigens. The TLR agonist and the cell factor have a synergistic effect, so that the functions of immune cells can be effectively regulated, proliferation and differentiation of T cells can be promoted, Th1 type immune response can be enhanced, and the immune function of cells can be improved; meanwhile, B cells are promoted to generate high-titer antiserum, and the humoral immune function is enhanced. Compared with pure mRNA immunization, the combined immunization method disclosed by the invention can be used for obtaining antiserum with remarkably improved titer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for generating high-titer antiserum based on combined immunization. Background Technology

[0002] mRNA vaccines, with their advantages of rapid development and efficient antigen expression, have shown great potential in the field of disease prevention and treatment. However, the current mRNA-based immunization strategy still has certain limitations. On the one hand, the immunogenicity of mRNA in the body is limited, making it difficult to fully stimulate a strong and lasting immune response; on the other hand, the activation and functional regulation of immune cells are insufficient, resulting in inadequate strength and breadth of the immune response.

[0003] Toll-like receptor (TLR) agonists and cytokines play important roles in immune regulation. TLR agonists can activate innate immune cells and initiate immune responses, while cytokines can regulate the proliferation, differentiation, and function of immune cells.

[0004] However, there are currently no research reports on combining mRNA, TLR agonists, and cytokines for immunization to produce high-titer antiserum. Summary of the Invention

[0005] The purpose of this invention is to provide a method for generating high-titer antiserum based on combined immunization. This addresses the problem of low immunogenicity in existing single-mRNA immunization methods.

[0006] In a first aspect, the present invention provides a method for generating high-titer antiserum based on combined immunization, comprising the following steps: providing a complex of lipid nanoparticles encapsulating mRNA, a TLR agonist, and cytokines respectively; immunizing animals with a mixture of the lipid nanoparticle-encapsulated mRNA complex, the TLR agonist, and the cytokines, and performing a second immunization two weeks later, then collecting serum to obtain high-titer antiserum; wherein the lipid nanoparticle-encapsulated mRNA complex is obtained by encapsulating 1,2-dioleoyl-3-trimethylammonium propane, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, cholesterol, and mRNA using a thin-film hydration method.

[0007] In this invention, the inventors discovered that by adding TLR agonists and cytokines as immune adjuvants during mRNA immunization, the TLR agonists activate innate immunity, providing a favorable immune microenvironment for mRNA-induced antigen-specific immune responses; cytokines regulate immune cell function, promoting the recognition and response of immune cells to mRNA-expressed antigens. Through synergistic effects, the TLR agonists and cytokines effectively regulate immune cell function, promote T cell proliferation and differentiation, enhance Th1 immune responses, and improve cellular immune function; simultaneously, they promote the production of high-titer antiserum by B cells, enhancing humoral immunity; furthermore, they enhance the killing activity of innate immune cells such as NK cells, comprehensively improving the body's immune defense capabilities. Compared to mRNA immunization alone, the combined immunization method of this invention yields antiserum with significantly improved titers.

[0008] In some embodiments, in the provided lipid nanoparticle-encapsulated mRNA complex, the molar ratio of 1,2-dioleoyl-3-trimethylammonium propane, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, and cholesterol is (45-55):(25-35):(15-25).

[0009] In some embodiments, in the provided lipid nanoparticle-encapsulated mRNA complex, the concentration of mRNA is 0.5-1.5 mg / ml, and the mRNA includes at least one of CLDN6 mRNA and BTNL10 mRNA; wherein the nucleotide sequence of CLDN6 mRNA is shown in SEQ ID NO: 1, and the nucleotide sequence of BTNL10 mRNA is shown in SEQ ID NO: 2.

[0010] In some embodiments, the concentration of the lipid nanoparticle-encapsulated mRNA complex is 0.5-1.5 mg / ml.

[0011] In some implementations, the concentration of the TLR agonist is 0.005-0.015 mg / ml, and the TLR agonist includes Poly I:C.

[0012] In some embodiments, the concentration of the cytokine is 0.005-0.015 mg / ml, and the cytokine includes IL-21.

[0013] In some implementations, in the step of immunizing animals with a mixture of lipid nanoparticle-encapsulated mRNA complex, TLR agonist, and cytokine, the mass ratio of the mixture of lipid nanoparticle-encapsulated mRNA complex, TLR agonist, and cytokine is 50:(0.5-1):(0.05-0.1).

[0014] In some implementations, the mass ratio of the lipid nanoparticle-encapsulated mRNA complex, TLR agonist, and cytokine mixture is 50:1:0.05.

[0015] In some implementations, the animals include mice.

[0016] In some implementation schemes, immunizing animals specifically includes immunizing animals by intramuscular injection.

[0017] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention incorporates TLR agonists and cytokines as immune adjuvants. The TLR agonist activates innate immunity, providing a favorable immune microenvironment for mRNA-induced antigen-specific immune responses; the cytokines regulate immune cell function, promoting the recognition and response of immune cells to mRNA-expressed antigens. Through synergistic action, the TLR agonist and cytokines effectively regulate immune cell function, promote T cell proliferation and differentiation, enhance Th1-type immune responses, and improve cellular immune function. Simultaneously, they promote the production of high-titer antiserum by B cells, enhancing humoral immunity. Furthermore, they enhance the killing activity of innate immune cells such as NK cells, comprehensively improving the body's immune defense capabilities. Compared to simple mRNA immunization, the combined immunization method of this invention yields antiserum with significantly improved titers. Attached Figure Description

[0018] Figure 1 This is a flowchart of the method for generating high-titer antiserum based on combined immunization in this invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0021] Currently, existing single-mRNA immunization methods suffer from low immunogenicity.

[0022] To address the problem of low immune titers in existing single-mRNA immunization, this invention provides a method for generating high-titer antiserum based on combined immunization.

[0023] In a first aspect, the present invention provides a method for generating high-titer antiserum based on combined immunization, comprising the following steps: providing a complex of lipid nanoparticles encapsulating mRNA, a TLR agonist, and cytokines respectively; immunizing animals with a mixture of the lipid nanoparticle-encapsulated mRNA complex, the TLR agonist, and the cytokines, and performing a second immunization two weeks later, then collecting serum to obtain high-titer antiserum; wherein the lipid nanoparticle-encapsulated mRNA complex is obtained by encapsulating 1,2-dioleoyl-3-trimethylammonium propane, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, cholesterol, and mRNA using a thin-film hydration method.

[0024] The method for generating high-titer antiserum based on combined immunization provided by this invention involves adding a TLR agonist and cytokines as immune adjuvants during mRNA immunization. The TLR agonist activates innate immunity, providing a favorable immune microenvironment for mRNA-induced antigen-specific immune responses; cytokines regulate immune cell function, promoting the recognition and response of immune cells to mRNA-expressed antigens. Through synergistic action, the TLR agonist and cytokines effectively regulate immune cell function, promote T cell proliferation and differentiation, enhance Th1 immune responses, and improve cellular immune function. Simultaneously, they promote the production of high-titer antiserum by B cells, enhancing humoral immunity. Furthermore, they enhance the killing activity of innate immune cells such as NK cells, comprehensively improving the body's immune defense capabilities. Compared to simple mRNA immunization, the combined immunization method of this invention yields antiserum with significantly higher titers.

[0025] In some embodiments, in the provided lipid nanoparticle-encapsulated mRNA complex, the molar ratio of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and cholesterol (Chol) is (45-55):(25-35):(15-25), preferably 50:30:20.

[0026] In this invention, by controlling the molar ratio of DOTAP, DOPE and Chol within a specific range, the encapsulation, delivery efficiency and stability of the prepared lipid nanoparticles for mRNA can be improved.

[0027] In some embodiments, in the provided lipid nanoparticle-encapsulated mRNA complex, the concentration of mRNA is 0.5-1.5 mg / ml, preferably 1 mg / ml; and the mRNA includes at least one of CLDN6 mRNA and BTNL10 mRNA; wherein the nucleotide sequence of CLDN6 mRNA is shown in SEQ ID NO: 1, and the nucleotide sequence of BTNL10 mRNA is shown in SEQ ID NO: 2.

[0028] In this invention, by optimizing the nucleotide sequences of CLDN6 mRNA and BTNL10 mRNA with codons, the immune response of mRNA can be enhanced and the titer of antiserum can be increased.

[0029] Understandably, the type of mRNA can be selected from conventional mRNAs in the prior art according to actual needs, as long as it produces the corresponding high-titer antiserum. For example, in this invention, the mRNA preferably includes at least one of CLDN6 mRNA and BTNL10 mRNA.

[0030] In some embodiments, the concentration of the lipid nanoparticle-encapsulated mRNA complex is 0.5-1.5 mg / ml, preferably 1 mg / ml.

[0031] In this invention, by controlling the concentration of the complex of lipid nanoparticles encapsulating mRNA within a specific range, the immune response of mRNA can be activated, resulting in high-titer antiserum.

[0032] In some embodiments, the concentration of the TLR agonist is 0.005-0.015 mg / ml, preferably 1 mg / ml; and the TLR agonist includes Poly I:C.

[0033] In this invention, by controlling the concentration of TLR agonists within a specific range, immunity can be activated, resulting in antiserum with increased potency.

[0034] In some embodiments, the concentration of the cytokine is 0.005-0.015 mg / ml, preferably 1 mg / ml; and the cytokine includes IL-21.

[0035] In this invention, by controlling the concentration of cytokines within a specific range, immune cells can recognize and respond to mRNA-expressed antigens, resulting in antiserum with improved potency.

[0036] In some implementations, in the step of immunizing animals with a mixture of lipid nanoparticle-encapsulated mRNA complex, TLR agonist, and cytokine, the mass ratio of the mixture of lipid nanoparticle-encapsulated mRNA complex, TLR agonist, and cytokine is 50:(0.5-1):(0.05-0.1).

[0037] In this invention, by controlling the mass ratio of the complex of lipid nanoparticles encapsulating mRNA, TLR agonist, and cytokine mixture within a specific range, the three components work synergistically to obtain an antiserum with further enhanced potency.

[0038] In some implementations, the mass ratio of the lipid nanoparticle-encapsulated mRNA complex, TLR agonist, and cytokine mixture is 50:1:0.05.

[0039] In this invention, by controlling the optimal mass ratio of the lipid nanoparticle-encapsulated mRNA complex, TLR agonist, and cytokine mixture, the antiserum with the highest potency can be obtained.

[0040] In some implementations, the animals include mice.

[0041] Understandably, the species of animal can be conventionally selected based on actual needs, as long as it can mount an immune response and produce high-titer antiserum. For example, in this invention, the preferred animal species include mice.

[0042] In some implementation schemes, immunizing animals specifically includes immunizing animals by intramuscular injection.

[0043] Understandably, the method of immunizing animals can be conventionally selected according to actual needs, as long as it ensures efficient and stable delivery of the immunizing agent to the animals. For example, in this invention, intramuscular injection is preferred as the method of immunizing animals.

[0044] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0045] Please see Figure 1 This is a flowchart of the method for generating high-titer antiserum based on combined immunization in this invention. Specifically, the method includes the following steps: providing a complex of lipid nanoparticles encapsulating mRNA, a TLR agonist, and cytokines; immunizing animals with the mixture of the lipid nanoparticle-encapsulated mRNA complex, TLR agonist, and cytokines, followed by a second immunization two weeks later, and then collecting the serum to obtain high-titer antiserum.

[0046] Example 1 This embodiment provides a method for generating high-titer antiserum based on combined immunization, wherein the mRNA in the complex of lipid nanoparticles encapsulating mRNA is CLDN6 mRNA, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0047] Specifically, it includes the following steps: S1. First, prepare a complex of lipid nanoparticles encapsulating mRNA (LNP-mRNA), specifically including the following steps: Preparation of lipid membrane: Weigh the raw materials according to the molar ratio of DOTAP, DOPE, and Chol of 50:30:20 and add them to a glass round-bottom flask; add 10 mL of chloroform to the flask and vortex to completely dissolve the lipids and form a homogeneous lipid film; connect the round-bottom flask to a rotary evaporator, with a water bath temperature of 35℃, a rotation speed of 80 rpm, and a vacuum degree gradually increased to 0.09 MPa; rotary evaporate for 15-30 minutes until the organic solvent is completely evaporated and a homogeneous, transparent lipid film (without visible droplets) forms on the inner wall of the flask; after rotary evaporation, keep the flask on the rotary evaporator, open the nitrogen valve, and purge the inner wall of the flask with a slow airflow for 10-15 minutes to thoroughly remove residual organic solvent.

[0048] Hydration and mRNA encapsulation: Dilute the mRNA solution to 1 mg / mL with RNase-free hydration buffer, mix gently, avoiding vigorous vortexing, to obtain mRNA hydration solution; add the pre-cooled mRNA hydration solution to a flask containing a lipid membrane, ensuring that the liquid completely covers the lipid membrane on the inner wall of the flask; place in a 37°C water bath and gently shake for 30-60 minutes to fully hydrate the lipid membrane and form an LNP suspension encapsulating mRNA.

[0049] Homogenization: Transfer the above suspension to an RNase-free centrifuge tube and sonicate in a water bath for 10-20 minutes until the solution is clear or semi-transparent (Note: the sonication intensity should not be too high to avoid mRNA degradation or lipid membrane rupture); then squeeze through a polycarbonate membrane (e.g., 100nm pore size) 3-10 times to obtain an LNP-mRNA suspension.

[0050] Purification and collection: Transfer the above LNP-mRNA suspension to an RNase-free centrifuge tube and centrifuge at 3000 rpm for 10 minutes at 4°C to remove any possible large particle aggregates; take the supernatant to obtain an LNP-mRNA complex with a concentration of 1 mg / ml, transfer it to a sterile cryovial, and store temporarily at 4°C or long-term at -80°C (avoid repeated freeze-thaw cycles).

[0051] Poly I:C was then dissolved in physiological saline to prepare a TLR agonist with a concentration of 0.01 mg / ml; IL-21 was dissolved in PBS to prepare a cytokine with a concentration of 0.01 mg / ml.

[0052] S2. Take 50 μg of the lipid nanoparticle-encapsulated mRNA complex obtained in step S1, 0.5 μg of the TLR agonist obtained in step S1, and 0.05 μg of the cytokine obtained in step S1, and immunize mice by intramuscular injection. Two weeks later, a second immunization was performed. The experiment was repeated twice. An equal amount of Tris-HCl (20 mM, pH 7.4) was injected as a negative control. One week after the second immunization, serum was collected to obtain high-titer antiserum.

[0053] The method for determining the high titer of antiserum is as follows: 1) Dilute the antigen with CB to 2 μg / ml, and coat each well with 100 μl of the antigen into a 96-well plate. Incubate overnight at 4°C. 2) Pat the plate dry, seal each well with 200μl of 4% skim milk powder, and incubate at 37°C for 2 hours; 3) Wash the plate 5 times with TBS for 1 minute each time; dilute the antiserum with PBS at dilution ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, and 1:1024000, add 100 μl to each well, and incubate at 37°C for 1 hour; 4) Wash the plate 3 times with TBS, 1 minute each time; after washing, add 100 μl of goat anti-mouse secondary antibody (1:5000) to each well; 5) After 40 minutes, wash the plate 5 times with TBS for 1 minute each time, add 50 μl of TBM to each well, and incubate at 37°C for 15 minutes.

[0054] 6) Readings from the ELISA reader.

[0055] The measurement results are shown in Table 1 below.

[0056] Table 1. Results of mouse serum detection

[0057] As can be seen from Table 1, high-titer antiserum can be obtained after immunization with a combination of LNP-CLDN6 mRNA, Poly I:C and IL-21.

[0058] Example 2 The method for generating high-titer antiserum based on combined immunization provided in this embodiment is basically the same as that in Embodiment 1, except that in step S2, the injection volume of cytokines is 0.1 μg.

[0059] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 2 below.

[0060] Table 2 Results of mouse serum detection

[0061] As can be seen from Table 2, immunization with a combination of LNP-CLDN6 mRNA, Poly I:C and IL-21 can produce high-titer antiserum, and the titer of the antiserum can be further increased with the increase of IL-21 dosage.

[0062] Example 3 The method for generating high-titer antiserum based on combined immunization provided in this embodiment is basically the same as that in Embodiment 1, except that in step S2, the injection volume of TLR agonist is 1 μg.

[0063] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 3 below.

[0064] Table 3. Results of mouse serum detection

[0065] As can be seen from Table 3, immunization with a combination of LNP-CLDN6 mRNA, Poly I:C and IL-21 can produce high-titer antiserum, and the titer of the antiserum can be further increased with the increase of Poly I:C dosage.

[0066] Example 4 The method for generating high-titer antiserum based on combined immunization provided in this embodiment is basically the same as that in Embodiment 1, except that in step S2, the injection amount of TLR agonist is 1 μg and the injection amount of cytokine is 0.1 μg.

[0067] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 4 below.

[0068] Table 4. Results of mouse serum detection

[0069] As shown in Table 4, immunization with the combination of LNP-CLDN6 mRNA, Poly I:C, and IL-21 yielded high-titer antiserum. Furthermore, the titer of the antiserum was further increased with the simultaneous increase of the dosage of Poly I:C and IL-21, but it was still lower than the titer of the antiserum in Example 3. The results indicate that increasing the dosage of Poly I:C and continuing to increase the dosage of IL-21 will decrease the titer of the antiserum.

[0070] Example 5 The method for generating high-titer antiserum based on combined immunization provided in this embodiment is basically the same as that in Embodiment 1, except that in step S1, the mRNA is BTNL10 mRNA.

[0071] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 5 below.

[0072] Table 5. Results of mouse serum detection

[0073] As can be seen from Table 5, immunization with the combination of LNP-BTNL10 mRNA, Poly I:C and IL-21 can produce high-titer antiserum, and the titer of the antiserum can be further increased with the increase of IL-21 dosage.

[0074] Example 6 The method for generating high-titer antiserum based on combined immunization provided in this embodiment is basically the same as that in embodiment 5, except that in step S2, the injection volume of cytokines is 0.1 μg.

[0075] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 6 below.

[0076] Table 6. Results of mouse serum detection

[0077] As can be seen from Table 6, immunization with the combination of LNP-BTNL10 mRNA, Poly I:C and IL-21 can produce high-titer antiserum, and the titer of the antiserum can be further increased with the increase of IL-21 dosage.

[0078] Example 7 The method for generating high-titer antiserum based on combined immunization provided in this embodiment is basically the same as that in embodiment 5, except that in step S2, the injection volume of TLR agonist is 1 μg.

[0079] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 7 below.

[0080] Table 7 Results of mouse serum detection

[0081] As can be seen from Table 7, high-titer antiserum can be obtained after immunization with the combination of LNP-BTNL10 mRNA, Poly I:C and IL-21, and the titer of the antiserum can be further improved with the increase of Poly I:C dosage.

[0082] Example 8 The method for generating high-titer antiserum based on combined immunization provided in this embodiment is basically the same as that in embodiment 5, except that in step S2, the injection amount of TLR agonist is 1 μg and the injection amount of cytokine is 0.1 μg.

[0083] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 8 below.

[0084] Table 8 Results of mouse serum detection

[0085] As shown in Table 8, immunization with the combination of LNP-BTNL10 mRNA, Poly I:C, and IL-21 yielded high-titer antiserum. Furthermore, the titer of the antiserum was further increased with the simultaneous increase of the dosage of Poly I:C and IL-21, but it was still lower than the titer of the antiserum in Example 7. The results indicate that increasing the dosage of Poly I:C and continuing to increase the dosage of IL-21 will decrease the titer of the antiserum.

[0086] Comparative Example 1 The method for generating high-titer antiserum based on combined immunization provided in this comparative example is basically the same as that in Example 1, except that in step S2, the injection volume of TLR agonist and cytokine is 0 μg.

[0087] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 9 below.

[0088] Table 9. Results of mouse serum detection

[0089] As can be seen from Table 9, the titer of the antiserum was significantly reduced after immunization with only LNP-CLDN6 mRNA.

[0090] Comparative Example 2 The method for generating high-titer antiserum based on combined immunization provided in this comparative example is basically the same as that in Example 1, except that the injection volume of cytokines in step S2 is 0 μg.

[0091] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 10 below.

[0092] Table 10 Results of mouse serum detection

[0093] As can be seen from Table 10, the titer of the antiserum decreased to some extent after immunization with only LNP-CLDN6 mRNA and Poly I:C.

[0094] Comparative Example 3 The method for generating high-titer antiserum based on combined immunization provided in this comparative example is basically the same as that in Example 1, except that in step S2, the injection volume of TLR agonist is 1 μg and the injection volume of cytokines is 0 μg.

[0095] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 11 below.

[0096] Table 11 Results of mouse serum detection

[0097] As can be seen from Table 11, after immunization with LNP-CLDN6 mRNA and Poly I:C, the titer of the antiserum was consistent with that in Example 1; however, the amount of Poly I:C used was significantly increased. The use of a small amount of Poly I:C and cytokines, with their synergistic effect, can improve the titer of the antiserum.

[0098] Comparative Example 4 The method for generating high-titer antiserum based on combined immunization provided in this comparative example is basically the same as that in Example 5, except that in step S2, the injection volume of TLR agonist and cytokine is 0 μg.

[0099] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 12 below.

[0100] Table 12 Results of mouse serum detection

[0101] As can be seen from Table 12, the titer of the antiserum was significantly reduced after immunization with LNP-BTNL10 mRNA alone.

[0102] Comparative Example 5 The method for generating high-titer antiserum based on combined immunization provided in this comparative example is basically the same as that in Example 5, except that the injection volume of cytokines in step S2 is 0 μg.

[0103] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 13 below.

[0104] Table 13 Results of mouse serum detection

[0105] As can be seen from Table 13, the titer of the antiserum decreased to some extent after immunization with only LNP-BTNL10 mRNA and Poly I:C.

[0106] Comparative Example 6 The method for generating high-titer antiserum based on combined immunization provided in this comparative example is basically the same as that in Example 5, except that in step S2, the injection volume of TLR agonist is 1 μg and the injection volume of cytokines is 0 μg.

[0107] The high-titer antiserum was determined using the method described in Example 1, and the results are shown in Table 14 below.

[0108] Table 14 Results of mouse serum detection

[0109] As can be seen from Table 14, the titer of the antiserum after immunization with LNP-BTNL10 mRNA and Poly I:C was consistent with that in Example 5; however, the amount of Poly I:C used was significantly increased. The use of a small amount of Poly I:C and cytokines, with their synergistic effect, can improve the titer of the antiserum.

[0110] In summary, the combined immunization method of the present invention can yield antiserum with significantly improved titer.

[0111] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0112] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for generating high-titer antiserum based on combined immunization, characterized in that, Includes the following steps: The formulations include a complex of lipid nanoparticles encapsulating mRNA, a TLR agonist, and a cytokine. The complex of the lipid nanoparticles encapsulating mRNA, the TLR agonist, and the cytokines were mixed and then used to immunize animals. A second immunization was performed two weeks later, and then the serum was collected to obtain high-titer antiserum. The lipid nanoparticle-encapsulated mRNA complex is obtained by encapsulating 1,2-dioleoyl-3-trimethylammonium propane, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, cholesterol, and mRNA using a thin-film hydration method.

2. The method for generating high-titer antiserum based on combined immunization according to claim 1, characterized in that, In the provided lipid nanoparticle-encapsulated mRNA complex, the molar ratio of 1,2-dioleoyl-3-trimethylammonium propane, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, and cholesterol is (45-55):(25-35):(15-25).

3. The method for generating high-titer antiserum based on combined immunization according to claim 1, characterized in that, In the provided lipid nanoparticle-encapsulated mRNA complex, the concentration of the mRNA is 0.5-1.5 mg / ml, and the mRNA includes at least one of CLDN6 mRNA and BTNL10 mRNA; The nucleotide sequence of the CLDN6 mRNA is shown in SEQ ID NO: 1, and the nucleotide sequence of the BTNL10 mRNA is shown in SEQ ID NO:

2.

4. The method for generating high-titer antiserum based on combined immunization according to claim 1, characterized in that, In the provided lipid nanoparticle-encapsulated mRNA complex, the concentration of the lipid nanoparticle-encapsulated mRNA complex is 0.5-1.5 mg / ml.

5. The method for generating high-titer antiserum based on combined immunization according to claim 1, characterized in that, The concentration of the TLR agonist is 0.005-0.015 mg / ml, and the TLR agonist includes Poly I:C.

6. The method for generating high-titer antiserum based on combined immunization according to claim 1, characterized in that, The concentration of the cytokine is 0.005-0.015 mg / ml, and the cytokine includes IL-21.

7. The method for generating high-titer antiserum based on combined immunization according to claim 1, characterized in that, In the step of immunizing animals with a mixture of the lipid nanoparticle-encapsulated mRNA complex, the TLR agonist, and the cytokines, the mass ratio of the mixture of the lipid nanoparticle-encapsulated mRNA complex, the TLR agonist, and the cytokines is 50:(0.5-1):(0.05-0.1).

8. The method for generating high-titer antiserum based on combined immunization according to claim 7, characterized in that, The mass ratio of the lipid nanoparticle-encapsulated mRNA complex, the TLR agonist, and the cytokine mixture is 50:1:0.

05.

9. The method for generating high-titer antiserum based on combined immunization according to claim 1, characterized in that, The animals mentioned include mice.

10. The method for generating high-titer antiserum based on combined immunization according to claim 1, characterized in that, The immunized animals specifically include animals immunized by intramuscular injection.

Images