A nucleic acid immunization formulation, its preparation method and use

Abstract

This invention discloses a nucleic acid immunoassay agent, its preparation method, and its application. The nucleic acid immunoassay agent comprises a single or multiple vector sequence DNA strands, wherein the vector sequence DNA strand is obtained by circularization and enzymatic amplification of the vector sequence and ligation primers; wherein the vector sequence has a sequence as shown in any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; and the ligation primer has a sequence as shown in SEQ ID NO.4. The nucleic acid immunoassay agent of this invention is synthesized using enzymatic amplification, which greatly simplifies the synthesis steps and reaction substrates, significantly reducing costs; simultaneously, the prepared nucleic acid immunoassay agent can, to a certain extent, delay the degradation time of nucleases, prolong its survival time in vivo, and the DNA base ligation method is natural, resulting in a wider applicable dosage window with less toxicity to organisms.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nucleic acid immunomodulator, its preparation method, and its application. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease that predominantly affects women. Its pathogenesis is complex and not yet fully understood. It is generally believed that in susceptible individuals, environmental or infectious factors induce abnormal immune function, activation of T and B cells, production of autoantibodies, and deposition of immune complexes in tissues and organs, leading to autoimmune diseases.

[0003] The above process is triggered by infection and abnormal activation of immune regulation. Toll-like receptors (TLRs), as crucial nucleic acid sensors, activate cellular functions by recognizing immune complexes containing DNA or RNA fragments. Exogenous factors such as bacterial infection bind to Toll-like receptors like TLR7 / 9 via CpG motifs and are considered pathological factors in SLE. TLR7 and TLR9 share the downstream MyD88 pathway, promoting B cell activation and the production of large amounts of autoantibodies by inducing type I IFN gene transcription.

[0004] Studies have shown that by modulating the CpG motif or the oligodeoxynucleotide CpG ODN backbone, TLR signaling can be inhibited, thereby downregulating the inflammatory response and even altering the secretion of inflammatory cytokines and the proportion of Th cells in vivo. Currently, some fully thiolated nucleic acid sequences have been found to regulate TLR9 signaling and inhibit immune cell activation. For example, the fully thiolated nucleic acid immunosuppressive sequence IRS 954 (5'-TGCTCCTGGAGGGGTTGT-3') has been shown in mice to reduce the level of autoanti-nucleic acid-related antibodies in the blood, inhibit a series of immune responses, and thus alleviate the symptoms of lupus erythematosus. However, this nucleic acid immunosuppressive sequence requires full thiolation of the nucleotide phosphate bonds to resist degradation by nucleases in vivo. The sequence is fully synthesized using a DNA synthesizer and subsequently purified by high-performance liquid chromatography. Therefore, the synthesis cost is high, and the error rate of artificial synthesis increases sharply with the length of the fragment, while the yield of the purified product decreases, making it difficult to synthesize longer DNA fragments (e.g., Eur. J. Immunol. 2007. 37: 3582-3586. Journal of Leukocyte Biology Volume 73, June 2003). Current nucleic acid immunosuppressive sequences are mostly fully thiolated oligonucleotides, several tens of nucleotides in length. The oxygen atom with a double bond on the phosphate group of the nucleotide is replaced by a sulfur atom, thus resisting degradation by nucleases and prolonging its duration of action in vivo. However, because these oligonucleotides are non-natural, their production relies entirely on artificial chemical synthesis. Furthermore, the error rate of artificially synthesized fragments increases with the length, leading to high manufacturing costs and limiting the synthetic length of nucleic acid immunosuppressive sequences.

[0005] To prevent rapid degradation of nucleic acid sequences by nucleases in vivo, current technologies mostly employ fully thiolated modification of nucleotide phosphate bonds, followed by chemical synthesis of the DNA sequence using a DNA synthesizer, and then separation and purification of the target fully thiolated DNA using high-performance liquid chromatography. The entire synthesis process is complex and involves numerous steps, resulting in high costs and the inability to synthesize relatively long nucleic acid fragments. Furthermore, because fully thiolated nucleotides cannot be degraded by nucleases, their toxicity in vivo is significant, limiting the dosage that can be used in vivo. Summary of the Invention

[0006] The main objective of this invention is to provide a nucleic acid immunoassay agent, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0008] This invention provides a nucleic acid immunoassay agent, which includes a single vector sequence DNA strand or multiple vector sequence DNA strands, wherein the vector sequence DNA strand is prepared by circularization of a vector sequence and a linker primer, followed by enzymatic amplification.

[0009] The vector sequence has a sequence as shown in any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; the ligation primer has a sequence as shown in SEQ ID NO.4.

[0010] This invention also provides a method for preparing the aforementioned nucleic acid immunomodulator, comprising:

[0011] The vector sequence is hybridized and circulated with the linker primer to obtain a circular vector sequence;

[0012] Furthermore, the circular vector sequence is enzymatically amplified with DNA polymerase to prepare a nucleic acid immunoassay agent.

[0013] This invention also provides the use of the aforementioned nucleic acid immunomodulators in the preparation of drugs that regulate immune function.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] (1) The nucleic acid immunomodulator in this invention is made by folding and compressing an ultra-long carrier sequence DNA chain into micro-nano spherical particles, which can delay the degradation time of nucleases to a certain extent and prolong the retention time in the body. The DNA base linkage is natural and can be completely degraded by nucleases in the end. The toxic side effects on organisms can be used within a larger dosage range window.

[0016] (2) The nucleic acid immunoassay agent in this invention is synthesized by enzymatic amplification. By synthesizing a small amount of primers and circular nucleic acids, a large amount of ultra-long immunosuppressive nucleic acid sequences can be produced under the catalysis of DNA polymerase. It does not require DNA synthesis for DNA modification and total synthesis, and there is no need for complex purification steps such as high performance liquid chromatography. The cost is greatly reduced and the steps are greatly simplified. It can be synthesized in large quantities using simple reaction containers. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1This is a schematic diagram illustrating the preparation principle of nucleic acid immunosuppressants in a typical embodiment of the present invention;

[0019] Figures 2a-2c These are scanning electron microscope images of the nucleic acid immunosuppressant vector sequences 151, 154, and 869 prepared in Example 1 of this invention. Detailed Implementation

[0020] In view of the shortcomings of existing technologies, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention. This invention employs an enzymatic method to amplify nucleic acid immunoassay units via rolling circle amplification, synthesizing ultralong DNA with immunosuppressive sequences and dispersing them into nanoparticle structures. These nanoparticles of DNA can delay the degradation by nucleases, thereby prolonging their duration of action in vivo. Furthermore, the base linkage of this DNA is natural and can ultimately be completely degraded, resulting in minimal toxicity to the organism and thus a wider applicable dosage window. Most importantly, this nucleic acid immunosuppressant can be synthesized enzymatically, significantly reducing manufacturing costs and allowing for the synthesis of longer sequences.

[0021] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Specifically, as one aspect of the technical solution of the present invention, the nucleic acid immunoassay agent involved includes a single vector sequence DNA strand or multiple vector sequence DNA strands, wherein the vector sequence DNA strand is prepared by circularization and enzymatic amplification of the vector sequence and the connecting primer;

[0023] The vector sequence has a sequence as shown in any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; the ligation primer has a sequence as shown in SEQ ID NO.4.

[0024] In some preferred embodiments, when the nucleic acid immunoassay agent is a single vector sequence DNA strand, the single vector sequence DNA strand forms a spherical nucleic acid immunoassay agent by self-entanglement.

[0025] In some preferred embodiments, when the nucleic acid immunoassay agent is a plurality of vector sequence DNA strands, the plurality of vector sequence DNA strands are cross-linked and intertwined to form a spherical nucleic acid immunoassay agent.

[0026] In some preferred embodiments, the nucleic acid immunoassay agent is a micro- or nano-sized spherical particle.

[0027] Furthermore, the particle size of the nucleic acid immunoassay agent is 100 nm to 10 μm.

[0028] In some preferred embodiments, the length of the vector sequence DNA strand is 40–8000 bp.

[0029] Furthermore, the length of the vector sequence DNA strand is 40–5000 bp.

[0030] Furthermore, the length of the vector sequence DNA strand is 40–100 bp.

[0031] In some preferred embodiments, the 5' end of the carrier sequence is phosphorylated with a phosphate group.

[0032] In some preferred embodiments, the 3' end of the vector sequence is modified with a hydroxyl group.

[0033] Another aspect of the present invention provides a method for preparing the aforementioned nucleic acid immunoassay agent, comprising:

[0034] The vector sequence is hybridized and circulated with the linker primer to obtain a circular vector sequence;

[0035] Furthermore, the circular vector sequence is enzymatically amplified with DNA polymerase to prepare a nucleic acid immunoassay agent.

[0036] In some preferred embodiments, the preparation method specifically includes: mixing the vector sequence, ligation primers, and hybridization system and incubating at 95–65°C for 5 min, then cooling to 37–4°C and adding ligase and ATP for hybridization circularization to obtain the circular vector sequence; wherein the hybridization system includes 0.05–150 mM Tris-HCl, 2–20 mM Mg salt, and 0–50 mM dithiothreitol, and the pH of the hybridization system is 5.0–9.0.

[0037] Furthermore, the Mg salt includes, but is not limited to, MgCl2.

[0038] In some more specific embodiments, the preparation method specifically includes: mixing the vector sequence, ligation primers and hybridization system and incubating at 95°C for 5 min, then cooling to 4°C and adding ligase and ATP for hybridization and circularization to obtain the circular vector sequence; wherein the hybridization system includes 50 mM Tris-HCl, 10 mM MgCl2 and 10 mM dithiothreitol, and the pH value of the hybridization system is 7.5.

[0039] Furthermore, the ligase includes any one or a combination of two or more of T4 ligase, T3 ligase, T7 ligase, and single-stranded DNA circularization ligase, and is not limited thereto.

[0040] In some more specific embodiments, the preparation method specifically includes: mixing the circular vector sequence with the rolling circle amplification reaction system and reacting at 20–45°C for 30 min–36 h, followed by inactivation treatment at 65°C for 10 min, and then post-processing to obtain the nucleic acid immunoassay agent; wherein the rolling circle amplification reaction system includes pH buffer and / or reaction buffer, dNTPs, magnesium salt, dithiothreitol and DNA polymerase.

[0041] Furthermore, the rolling circle amplification reaction system includes pH buffer 5.0–9.0, dNTP 1–50 mM, magnesium salt 1–20 mM, dithiothreitol 0–50 mM, and DNA polymerase 0.1–500 U.

[0042] In some more specific embodiments, the preparation method specifically includes: mixing the circular vector sequence with the rolling circle amplification reaction system and reacting at 37°C for 8–24 h, followed by inactivation treatment at 65°C for 10 min, and then post-processing to obtain the nucleic acid immunoassay agent; wherein the rolling circle amplification reaction system includes 1× reaction buffer, 10 mM dNTP, 8 mM MgCl2, 1 mM dithiothreitol and 10 U DNA polymerase.

[0043] In some preferred embodiments, the DNA polymerase includes any one or more of phi29 DNA polymerase, Bst polymerase, and large fragments of Bst polymerase, and is not limited thereto.

[0044] In this invention, the vector sequence having the sequence shown in SEQ ID NO.1 is designated as vector sequence 154; the vector sequence having the sequence shown in SEQ ID NO.2 is designated as vector sequence 151; and the vector sequence having the sequence shown in SEQ ID NO.1 is designated as vector sequence 154, as shown in Table 1 below:

[0045] Table 1. Specific sequences of the vector sequence and ligation primers in this invention.

[0046]

[0047] In Table 1 of this invention, the underlined portion of the vector sequence is complementary to half of the ligation primer. Both sequences hybridize with the ligation primer, and the vector sequences can be joined end-to-end to form a circular structure with the assistance of the ligation primer and DNA ligase. Subsequently, rolling circle amplification is performed under the action of phi29 DNA polymerase to synthesize a nucleic acid immunosuppressant. The principle is as follows: Figure 1 As shown.

[0048] In some more specific embodiments, the preparation method of the nucleic acid immunosuppressant includes: the 5' end of the above-mentioned vector sequence is a phosphate group, and the 3' end is a hydroxyl group. A ligation primer (5'-TCACCATAGTCCTTGTCATCAC-3') is used, which is complementary to half of the underlined portion of the vector sequence and half of the ligation primer. Hybridization is performed at both ends of the vector sequence during ligation. Specifically, 1 μM of ligation primer and different vector sequences are added to the hybridization system (50 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol, pH 7.5), incubated at 95°C for 5 min, then cooled to 4°C according to a -1°C / min program, and then 20 U / μL T4 ligase and 1 mM ATP are added. The mixture is incubated overnight at 4°C to obtain a circular vector sequence. Then, 50 μL of the reaction mixture is added to a 400 μL rolling circle amplification reaction system (1× reaction buffer, 10 mM dNTP, 8 mM... MgCl2, 1mM dithiothreitol, 10U phi29 DNA polymerase were reacted at 37℃ for 8-24 hours. After inactivating the DNA polymerase at 65℃ for 10 minutes, the reaction solution was thoroughly dispersed, washed with sterile water, and centrifuged at 20000g to separate micro-nano-sized spherical particles, which were then used to prepare nucleic acid immunosuppressants. The solutions were stored at 4℃ for later use.

[0049] The particle size of the nucleic acid immunosuppressants prepared above ranges from hundreds of nanometers to several micrometers. They are composed of a large number of carrier sequence DNA strands that are entangled and cross-linked. The length of a single carrier sequence DNA strand can reach more than 70kb, which is much longer than that of fully thiolated DNA (tens of bp in length).

[0050] The nucleic acid immunosuppressant sequences of this invention are synthesized using enzymatic amplification. By synthesizing a small amount of primers and circular nucleic acids, a large number of ultra-long immunosuppressive nucleic acid sequences can be produced under the catalysis of DNA polymerase. The synthesis steps and reaction substrates are greatly simplified, and the synthesis cost is significantly reduced. These ultra-long immunosuppressive nucleic acid sequences are folded and compressed into micro- and nano-sized spherical particles, which can delay the degradation time of nucleases to a certain extent and prolong their survival time in vivo. This DNA base linkage method is natural and can ultimately be completely degraded by nucleases, resulting in a wider applicable dosage window for toxic side effects on organisms.

[0051] This invention constructs three types of nucleic acid sequences containing immunosuppressive properties and corresponding linking primers, and then prepares them into micro- and nano-sized nucleic acid immunosuppressants through nucleic acid linking circularization and enzymatic amplification.

[0052] Another aspect of the present invention provides the use of the aforementioned nucleic acid immunomodulators in the preparation of immunomodulatory drugs.

[0053] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0054] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0055] Example 1

[0056] 1 μM ligation primers and different vector sequences (vector sequence 151, vector sequence 154, vector sequence 869) were added to the hybridization system (50 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol, pH 7.5), incubated at 95 °C for 5 min, then cooled to 4 °C at a rate of -1 °C / min, and 20 U / μL T4 ligase and 1 mM ATP were added. The mixture was incubated overnight at 4 °C to obtain circular vector sequences. Then, 50 μL of the reaction mixture was added to a 400 μL rolling circle amplification reaction system (1× reaction buffer, 10 mM dNTP, 8 mM MgCl2, 1 mM dithiothreitol, 10 U phi29). DNA polymerase was reacted at 37°C for 8-24 hours, and then inactivated at 65°C for 10 minutes. The reaction solution was then thoroughly dispersed, washed with sterile water, and centrifuged at 20,000g to separate micro- and nano-sized spherical particles, which were then used to prepare nucleic acid immunosuppressants (denoted as vector sequence group 151, vector sequence group 154, and vector sequence group 869, respectively). These were stored at 4°C for later use. Scanning electron microscope images of the nucleic acid immunosuppressant vector sequences 151, 154, and 869 prepared in this embodiment are shown below. Figures 2a-2c As shown.

[0057] Verification of immunosuppressive effect:

[0058] After 5 days of acclimatization, all mice were intraperitoneally injected with 0.5 ml of pritane per mouse to establish a lupus erythematosus (SLE) model. Antinuclear antibody (ANA) levels were measured in mice 3 months later, and intervention was initiated 100 days after modeling. The intervention group consisted of six animals per group: model group, vector sequence group 151, vector sequence group 154, and vector sequence group 869. The intervention was administered subcutaneously every Tuesday and Friday, with each group receiving a 10 μg / 250 μL / mouse / injection of the corresponding nucleic acid immunosuppressant (twice a week) for 14 weeks. Animals were euthanized on the second day after the 14-week intervention period, and hair loss was observed. Animal serum was diluted 1:100 with ANA detection kit diluent, and standards and test samples were added sequentially into microplates, with two replicates per well. After incubation with the antibody, the mice were washed three times, and the absorbance was measured at 450 nm after adding the stop solution. The ANA levels in the serum of each group of animals were obtained according to the standard curve (as shown in Table 2). The results showed that all model mice treated with vector sequences 151, 154, and 869 survived, and their blood ANA levels were significantly lower than those in the model group, with no significant hair loss. In contrast, the model group mice showed significant hair loss on their heads and backs, exhibiting typical signs of lupus erythematosus.

[0059] Table 2. Results of ANA content in animal serum from different groups.

[0060]

[0061] Note: Compared with the Model group, ** means P < 0.01, and * means P < 0.05.

[0062] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0063] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention. sequence list <110> Suzhou University <120> A nucleic acid immunomodulator, its preparation method and application <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 45 <212> DNA <213> Artificial sequence (artificial sequence) <400> 1 gactatggtg actcacccct caagcttgag gactgtgatg acaag 45 <210> 2 <211> 52 <212> DNA <213> Artificial sequence (artificial sequence) <400> 2 gactatggtg accaacccta accctaaccc taaccctaac agtgatgaca ag 52 <210> 3 <211> 46 <212> DNA <213> Artificial sequence (artificial sequence) <400> 3 gactatggtg actcacaacc cctccaggag caactgtgat gacaag 46 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence (artificial sequence) <400> 4 tcaccatagt ccttgtcatc ac 22

Claims

1. A nucleic acid immunomodulator, characterized in that: The nucleic acid immunoassay agent comprises a single vector sequence DNA strand or multiple vector sequence DNA strands, wherein the vector sequence DNA strand is prepared by circularization and enzymatic amplification of a vector sequence and a connecting primer; the nucleic acid immunoassay agent is a micro / nano-sized spherical particle; the particle size of the nucleic acid immunoassay agent is 100 nm to 10 μm; when the nucleic acid immunoassay agent is a single vector sequence DNA strand, the single vector sequence DNA strand forms a spherical nucleic acid immunoassay agent through self-entanglement; when the nucleic acid immunoassay agent is multiple vector sequence DNA strands, the multiple vector sequence DNA strands form a spherical nucleic acid immunoassay agent through mutual entanglement and cross-linking; The vector sequence is any one of SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3; the ligation primer is a sequence as shown in SEQ ID NO.

4.

2. The nucleic acid immunomodulator according to claim 1, characterized in that: The 5' end of the carrier sequence is modified with a phosphate group by phosphorylation.

3. The nucleic acid immunomodulator according to claim 1, characterized in that: The 3' end of the vector sequence is modified with a hydroxyl group.

4. A method for preparing the nucleic acid immunomodulator according to any one of claims 1-3, characterized in that, include: The vector sequence is hybridized and circulated with the linker primer to obtain a circular vector sequence; Furthermore, the circular vector sequence is enzymatically amplified with DNA polymerase to prepare a nucleic acid immunoassay agent.

5. The preparation method according to claim 4, characterized in that, Specifically, it includes: The vector sequence, ligation primers, and hybridization system were mixed and incubated at 95–65°C for 5 min. The mixture was then cooled to 37–4°C, and ligase and ATP were added for hybridization and circularization to obtain the circular vector sequence. The hybridization system comprised 0.05–150 mM Tris-HCl, 2–20 mM Mg salt, and 0–50 mM dithiothreitol, with a pH of 5.0–9.

0. The ligase includes any one or a combination of two or more of T4 ligase, T3 ligase, T7 ligase, and single-stranded DNA circularization ligase.

6. The preparation method according to claim 4, characterized in that, Specifically, it includes: The circular vector sequence is mixed with the rolling circle amplification reaction system and reacted at 20-45°C for 30-36 hours, followed by inactivation treatment at 65°C for 10 minutes, and then post-processed to obtain the nucleic acid immunoassay agent; wherein the rolling circle amplification reaction system includes pH buffer and / or reaction buffer, dNTP, magnesium salt, dithiothreitol and DNA polymerase.

7. The preparation method according to claim 6, characterized in that: The DNA polymerase includes any one or a combination of two or more of the following: phi29 DNA polymerase, Bst polymerase, and large fragments of Bst polymerase.

8. Use of the nucleic acid immunomodulator according to any one of claims 1-3 in the preparation of a medicament for treating systemic lupus erythematosus.

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