CN113509542A - Medicine for expressing interleukin 12 and aiming at tumor based on mRNA and preparation method thereof - Google Patents
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Abstract
The invention discloses a vaccine for expressing interleukin 12 and aiming at tumor based on mRNA and a preparation method thereof, wherein the vaccine comprises the following components: a liposome complex of self-replicating mRNA expressing interleukin-12; the self-replicating mRNA expressing interleukin-12 is mRNA-IL 12; the liposome complex of the self-replicating mRNA expressing interleukin-12 is mRNA-LNP; mRNA-IL12 comprises: an mRNA replicon designed based on an alphavirus genome and containing an interleukin 12 protein gene, the interleukin 12 protein gene; according to the invention, the mRNA sequence is designed according to the genome of the alphavirus family, and the obtained vaccine has a good effect of mobilizing an immune system, can achieve the immune effect of killing tumors, and has a good application prospect; and the self-replicating mRNA technology is matched, so that the inoculation dose is small, and the process is simple.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a medicine for expressing interleukin 12 and aiming at tumor based on mRNA and a preparation method thereof.
Background
Interleukin 12(IL-12) is a key regulator in the cellular immune response and has important biological activity. IL-12 is expressed primarily by modulating receptors on the surface of dendritic cells, natural killer cells (NK); activating NK and T cells, and inducing macrophages in a tumor microenvironment to differentiate towards M1 type; enhancing the expression of tumor cell autophagy-related protein and inducing autophagy; affecting the secretion of IL-10 or gamma interferon, and regulating down vascular endothelial growth factor, programmed death ligand 1, etc. to affect the growth and metastasis of tumor. Therefore, IL-12 is considered to be the most effective antitumor cytokine, using IL-12 for antitumor therapy gradually become the research of the hot spot. However, direct injection of IL-12 protein has strong toxic and side effects, and direct application of IL-12 has systemic adverse effects, as do other cytokines. Therefore, gene therapy or combination therapy of tumors using a virus or gene encoding IL-12 is often used. The current research on IL-12 anti-tumor is mainly based on experiments, and the reasonable application of IL-12 in clinic will become the future research direction.
Only a few patients can benefit fully from conventional tumor regression therapy. Successful anti-tumor immunity is thought to be associated with the induction of a self-sustaining "cancer immune cycle". In this cycle, the immunogen can destroy tumor cells and activate dendritic cells to initiate an anti-tumor response of T cells in the lymph nodes. These T cells then enter the disease site, along with other immune cells, promoting sustained killing of tumor cells and remodeling of the Tumor Microenvironment (TME). However, initiation of this cycle requires initiation of a series of interrelated events including the induction of ICDs, activation of dendritic cells, recruitment of immune cells to the tumor site, return to possible immunosuppression in the TME, and activation of immunoinflammatory factors, among others. The therapeutic approach that can cause all of these changes in the tumor remains a significant challenge.
Since BMS and PD1 drugs have been marketed, the development of immunotherapeutic drugs has progressed rapidly, new immunotherapeutic technologies such as monoclonal antibodies, diabodies, ADC drugs, CAR-T, oncolytic viruses have emerged, and a number of drugs (or therapies) have been approved for marketing. The mRNA technology applied to the development of tumor immunotherapy and infectious disease vaccines has been developed for more than 10 years, and has attracted much attention due to the excellent protective efficacy and the characteristic of rapid development and production amplification since two new mRNA vaccines come into the market in the last year. Therefore, the application of mRNA technology to tumor immunotherapy would be highly expected to make products available on the market as early as possible.
The traditional inactivated vaccine and recombinant protein vaccine have long production period and complex process and cannot meet the large-scale inoculation requirement of the sudden large-scale epidemic. mRNA is a rapid response vaccine development platform for outbreak epidemic situations. Recently, mRNA (messenger ribonucleic acid) vaccines developed by companies have initiated clinical trials, providing prophase data on the safety and efficacy of mRNA vaccines. The mRNA vaccine is synthesized by taking linearized plasmid DNA as a template and performing enzyme transcription reaction in vitro, and the synthesis strategy avoids the problems of living cell culture production mode, safety, complex production process and the like which need to be considered. The mRNA vaccine platform has the characteristics of safety, effectiveness, short production period and simple process, thereby being particularly suitable for treating tumors.
The mRNA technology route has a distinction between self-replicating and non-self-replicating. Compared with non-replicating mRNA, the self-replicating mRNA has a replication process in vivo due to the use of a replication vector, has lower initial administration dosage, better safety and lower side effect, has strong immune agonist effect when being replicated in vivo, can trigger strong innate immune response of an organism, and is very suitable for the research and development of tumor immunotherapy drugs according to a target gene required to be expressed according to specific application codes; the application of the technology to the preparation of tumor vaccines has a plurality of technical problems to be overcome, the market needs to realize the application of the technology to the preparation of the tumor vaccines, and the obtained vaccines need to have good effect of mobilizing the immune system, so that the immune effect of killing tumors can be achieved; the present invention solves such problems.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a medicine for expressing interleukin 12 and aiming at tumors based on mRNA and a preparation method thereof, the mRNA sequence is designed according to the genome of an alphavirus family, and the obtained medicine has good effect of mobilizing the immune system and can achieve the immune effect of killing tumors; and the self-replicating mRNA technology is matched, so that the inoculation dose is small, and the process is simple.
In order to achieve the above object, the present invention adopts the following technical solutions:
a drug for expressing interleukin 12 against tumors based on self-replicating mRNA, comprising: a liposome complex of a self-replicating mRNA that expresses interleukin-12; the self-replicating mRNA expressing interleukin-12 is mRNA-IL 12; the liposome complex of the self-replicating mRNA expressing interleukin-12 is mRNA-LNP; mRNA-IL12 comprises: an mRNA replicon designed based on an alphavirus genome and containing an interleukin 12 protein gene, the interleukin 12 protein gene; the sequence of the mRNA replicon contains a gene encoding an alphavirus autonomous replication function, and lacks a gene that produces a structural protein of an infectious alphavirus particle.
The mRNA-based medicine for expressing interleukin 12 to the tumor comprises the following lipids: DMG-PEG2000, cholesterol, distearoylphosphatidylcholine or cationic lipids.
The mRNA-based drug for expressing interleukin 12 to tumor is provided, and the alphavirus genome comprises: a venezuelan encephalomyelitis virus genome, a sindbis virus genome, a chikungunya virus genome, an eastern equine encephalomyelitis virus genome, a western equine encephalomyelitis virus genome, a maja virus genome, a sheng likissenlin virus genome, or a venezuelan equine encephalomyelitis virus genome.
In the mRNA-based drug for expressing interleukin 12 to a tumor, mRNA-IL12 was synthesized by an enzymatic transcription reaction in vitro using linearized plasmid DNA as a template.
In the mRNA-based drug for expressing interleukin 12 to tumor, the plasmid DNA contains kanamycin resistance gene, T7 promoter gene, nsp gene, IL12 gene and polyA sequence; the gene sequence is shown in SEQ ID NO. 3.
A preparation method of a medicine for expressing interleukin 12 and aiming at tumors based on mRNA comprises the following steps:
step one, synthesizing an IL12 gene fragment: inquiring an IL12 amino acid sequence, carrying out codon optimization according to a human preference codon table to obtain an optimized sequence, adding a promoter and an ApaI enzyme digestion site GGGCCC at the upstream of the optimized sequence, adding a NotI enzyme digestion site GCGGCCGC at the downstream of the optimized sequence, and synthesizing the designed sequence to obtain the optimized sequence;
step two, preparing and obtaining mRNA-IL12, wherein the mRNA-IL12 is self-replicating mRNA for expressing interleukin-12;
step three, mixing mRNA-IL12 with lipid, wrapping mRNA-IL12 into LNP to obtain mRNA-LNP, and concentrating and changing liquid to a preparation solution; mRNA-LNP is a liposomal complex of self-replicating mrnas that express interleukin-12.
In the second step, the specific steps for preparing the mRNA-IL12 of the mRNA-based drug for expressing interleukin 12 against tumors include:
step a, constructing a TC-83 self-replicating vector: designing a self-replicating mRNA sequence containing an interleukin 12 protein gene based on an alpha virus genome, taking a plasmid constructed by the self-replicating mRNA sequence as a template for amplification, and synthesizing the designed sequence to obtain the self-replicating mRNA sequence; the sequence of the mRNA replicon includes a gene encoding an alphavirus autonomous replication function and lacks a gene that produces a structural protein of an alphavirus particle having infectivity; step b, preparing a recombinant plasmid JCXH-203: inserting an IL12 gene carrying ApaI at the upstream and NotI at the downstream into the enzyme cutting sites of ApaI and NotI of a TC-83 replication vector to obtain a recombinant plasmid JCXH-203; step c, linearizing the JCXH-203 plasmid by using restriction enzyme digestion; and d, firstly carrying out in-vitro transcription reaction on the linearized JCXH-203 plasmid, then degrading the template DNA, and finally adding the 7-methylated guanylic acid cap structure to the 5' end of the transcribed mRNA to obtain the in-vitro transcribed mRNA-IL 12.
In the mRNA-based drug for expressing interleukin 12 against tumors, the restriction enzyme in step c is BspQI enzyme.
In the step d, the linearized JCXH-203 plasmid is subjected to in vitro transcription reaction by using T7 RNA polymerase, the Turbo DNase enzyme is used for degrading the template DNA, and the capping enzyme is used for adding the 7-methylated guanylic acid cap structure to the 5' end of the transcribed mRNA to obtain the in vitro transcribed mRNA-IL 12.
In the third step, the mRNA-IL12 and the lipid are mixed by a Nanolasemblr mixer.
The invention has the advantages that:
according to the invention, an mRNA sequence is designed according to the genome of an alphavirus family, so that the mRNA can express IL12 for treating tumors, and the obtained medicine has a good effect of mobilizing an immune system and can achieve the immune effect of killing the tumors;
the invention has strong therapeutic effect in animal experiments based on the self-replicating mRNA medicament, can replicate self sequence by taking the self as a template, so compared with the conventional mRNA vaccine, the invention has less required inoculation dose, and can induce stronger immune response by adjuvant effect formed by immune response induced during self replication, thereby further enhancing humoral and cellular immune response;
the invention uses self-replicating mRNA and takes linearized plasmid DNA as a template to carry out a strategy of enzyme transcription reaction synthesis in vitro, and the synthesis strategy avoids a living cell culture production mode and avoids the problems of considering safety, complex production process and the like;
the invention utilizes self-replicating mRNA to code IL12, and expresses IL12 in vivo to enhance the retention of cell factors in a tumor microenvironment TME, thereby avoiding toxicity; and then the strong synergistic effect of cell death ICD induced by immunoreaction promoted by gene expression and innate immune stimulation is replicated by matching with lipid-mediated tumor cell transfection, so that the immune effect is further improved.
The invention has simple process, and only needs in vitro transcription and liposome encapsulation to prepare the mRNA medicament.
Drawings
FIG. 1 is a plasmid map of mRNA-IL12 of the present invention, which contains kanamycin resistance gene (Kan), T7 promoter gene, nsp gene, IL12 gene, polyA gene;
FIG. 2 is a diagram showing the results of electrophoresis after in vitro transcription of mRNA-IL12 of the present invention; m is RNA ladder, A is mRNA-IL12 size after in vitro transcription;
FIG. 3 is a Western test result, an expression diagram of IL12 protein, Non is a negative control of untransfected BHK cells, and IL12 is an expression diagram of IL12 protein after BHK cells are transfected in vitro by mRNA-IL 12;
FIG. 4 is a graph showing the results of the evaluation of the therapeutic effect (mean tumor volume) of the tumor model by the treatment of model mice with the mRNA-IL12 drug of the present invention (Group01 Vehicle: saline control Group one, Group02 LNP-srRNA (IL 12): inoculated with drug Group two of the present invention, Group03 keytruda: inoculated with drug Group III of the market, and Group04 keytruda + LNP-srRNA (IL12) inoculated with drug Group keytruda + drug Group IV of the present invention).
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
An mRNA-based agent for expressing interleukin 12 against a tumor, comprising: the method comprises the following steps: a liposome complex of a self-replicating mRNA that expresses interleukin-12; the self-replicating mRNA expressing interleukin-12 is mRNA-IL 12; the liposome complex of the self-replicating mRNA expressing interleukin-12 is mRNA-LNP; mRNA-IL12 comprises: an mRNA replicon designed based on an alphavirus genome and containing an interleukin 12 protein gene, the interleukin 12 protein gene; the sequence of the mRNA replicon contains a gene encoding an alphavirus autonomous replication function, and lacks a gene that produces a structural protein of an infectious alphavirus particle.
As an example, lipids include: DMG-PEG2000 (1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000), cholesterol, Distearoylphosphatidylcholine (DSPC), cationic lipid DLinDMA. The term "comprising" is intended to mean either one or a mixture of several of them, or other lipids, and is not intended to be exhaustive.
As an example, the alphavirus genome is derived from: venezuelan Equine encephalomyelitis Virus (TC83 Venezuelan equi Encephalitis Virus, VEEV), sindbis Virus (Sin-dbis Virus), Chikungunya Virus (Chikungunya Virus), Eastern Equine encephalomyelitis Virus (Eastern Equine encphalis-tis Virus), Western Equine encephalomyelitis Virus (Western equiencephalitis Virus), mayaru Virus (Mayarovirus), shenglisen forest Virus (semlikriest Virus), Venezuelan Equine encephalomyelitis Virus (Venezuelan equi Encephalitis Virus), and the like; preferably, Venezuelan encephalomyelitis Virus (TC83 Venezuelan Equisene encphalitis Virus, VEEV) is used. It should be noted that: the alphavirus family of viruses herein is not intended to be exhaustive, and any virus whose mRNA is designed to express the interleukin 12 protein gene is within the scope of the present invention.
The mRNA includes: conventional mRNA or self-replicating mRNA. There are two major routes to mRNA vaccine platform technology, one based on conventional mRNA and the other based on self-replicating mRNA; both routes can be applied to the present invention. After the self-replicating mRNA vaccine is inoculated into an animal body, a self sequence can be replicated by taking the self as a template, so that compared with the conventional mRNA vaccine, the self-replicating mRNA vaccine requires less inoculation dosage, and an adjuvant effect formed by an immune response induced during self replication can induce stronger immune response and further enhance humoral and cellular immune responses; thus, as a preferred option, self-replicating mRNA is synthesized in vitro by enzymatic transcription using linearized plasmid DNA as a template.
A preparation method of a vaccine for expressing interleukin 12 to aim at tumors based on mRNA comprises the following steps:
step one, synthesizing an IL12 gene fragment:
an IL12 amino acid sequence is inquired, codon optimization is carried out according to a human preference codon table to obtain an optimized sequence, a promoter and an ApaI enzyme digestion site (GGGCCC) are added at the upstream of the optimized sequence, a NotI enzyme digestion site (GCGGCCGC) is added at the downstream of the optimized sequence, the designed sequence is directly obtained through synthesis, and the gene sequence is shown as SEQ ID NO.1 in a sequence table;
step two, preparing and obtaining mRNA-IL 12;
the method comprises the following specific steps:
step a, constructing a TC-83 self-replicating vector:
designing a self-replicating mRNA sequence containing an interleukin 12 protein gene based on an alpha virus genome, taking a plasmid constructed by the self-replicating mRNA sequence as a template for amplification, and synthesizing the designed sequence to obtain the self-replicating mRNA sequence; the sequence of the mRNA replicon comprises a gene encoding the self-replication function of alphavirus, lacking a gene that produces a structural protein of an infectious alphavirus particle;
preferably, the self-replicating mRNA is designed based on the genome of Venezuelan Equine encephalomyelitis Virus (TC83 Venezuelan equi enchaitis Virus, VEEV) in alphavirus family, contains a gene encoding an alphavirus self-replicating module, but lacks a structural protein encoding an alphavirus particle having infectivity, and the constructed plasmid is amplified as a template, and the designed sequence is directly obtained by synthesis, and the gene sequence is represented by SEQ ID No.2 in the sequence table.
Step b, preparing a recombinant plasmid JCXH-203:
inserting an IL12 gene carrying ApaI at the upstream and NotI at the downstream into the enzyme cutting sites of ApaI and NotI of a TC-83 replication vector to obtain a recombinant plasmid JCXH-203;
preferably, an IL12 gene carrying ApaI at the upstream and NotI at the downstream is inserted between ApaI and NotI enzyme cutting sites of a TC-83 replication vector to obtain a recombinant plasmid JCXH-203 (shown in figure 1), wherein the plasmid DNA contains a kanamycin resistance gene, a T7 promoter gene, an nsp gene, an IL12 gene and a polyA gene; the gene sequence is shown as SEQ ID NO.3 in the sequence table.
Step c, linearizing the JCXH-203 plasmid by using restriction enzyme digestion; as an example, the restriction enzyme is BspQI enzyme.
D, firstly carrying out in-vitro transcription reaction on the linearized JCXH-203 plasmid, then degrading the template DNA, and finally adding the 7-methylated guanylic acid cap structure to the 5' end of the transcribed mRNA to obtain the in-vitro transcribed mRNA-IL 12; as a preferred example, the linearized JCXH-203 plasmid in vitro transcription reaction (IVT) is performed using T7 RNA polymerase, the degradation of template DNA is performed using Turbo DNase, and a 7-methylated guanylate Cap structure (referred to as Cap 0) is added to the 5' end of the transcribed mRNA using a capping enzyme (Vaccidia capping enzyme). The in vitro transcription reaction (IVT) of the linearized JCXH-203 plasmid can also be performed by degrading the template DNA using SP6 RNA polymerase using DNase I enzyme, adding a 7-methylated guanylate Cap structure (referred to as Cap 0) to the 5 ' end of the transcribed mRNA using a capping enzyme (Vaccinia capping enzyme), and adding a methyl group to the 5 ' end of the RNA at the 2 ' -O position of the first nucleotide of the Cap structure immediately adjacent to the 5 ' end of the RNA using Cap2 oxymethyltransferase (Cap 2 ' -O-Methyltransferase, referred to as Cap 1).
It should be noted that: the examples herein are not exhaustive and any enzymes capable of achieving in vitro transcription, degradation, and capping may be used in the present invention (see FIG. 2).
It should be noted that the mRNA may also be a conventional mRNA, and the specific steps include: first, a conventional mRNA template was prepared, including the T7 promoter, 5 'UTR, Kozak sequence, IL12, 3' UTR, polyA to obtain a template plasmid by synthesis. The template was obtained after linearization with BspQI enzyme, the regular mRNA was obtained by plasmid in vitro transcription reaction (IVT), and after removal of the template by DNase I enzyme, the 5' end was capped by the corresponding capping enzyme.
Step three, rapidly mixing mRNA-IL12 and lipid through a Nanolasembler mixer so as to cause precipitation of the lipid and effect of charge, wrapping mRNA-IL12 into LNP to obtain an mRNA-LNP compound, and concentrating and changing the solution into a preparation solution; the preparation solution comprises: tris, NaCl, sucrose, reagent composition and dosage of the preparation solution are not limited.
The technical effects of the invention are verified through experiments as follows:
experiment I, preparation of vaccine mRNA-IL12 and verification experiment of expression condition in cells;
1, IL12 Gene fragment Synthesis
The method comprises the steps of inquiring an IL12 protein gene from NCBI, carrying out corresponding optimization according to human codons, adding Apa I enzyme cutting site and a promoter at the upstream, adding a Not I enzyme cutting site at the downstream, and finally directly obtaining a DNA sequence (obtained in a form of a clone plasmid pUC57-IL12 provided by a company) through synthesis (the sequence is shown as a sequence table SEQ ID NO. 1).
2, construction of TC-83 self-replicating vector
The self-replicating mRNA is designed according to the genome of Venezuelan Equine encephalomyelitis Virus (TC83 Venezuelan Escherichia Virus, VEEV) in alphavirus family, contains genes capable of coding alphavirus self-replicating components, lacks structural proteins for manufacturing infectious alphavirus particles, is used as a template for amplification in constructed plasmids, and is directly obtained by synthesis (the sequence is shown as SEQ ID NO.2 in a sequence table).
3, the preparation method of the recombinant plasmid JCXH-203 is as follows: pUC57-IL12 and TC-83 self-replicating vectors were double digested with Apa I and Not I, 20. mu.L: pUC57-IL12 or TC-83 self-replicating vectors < 1. mu.g, ApaI 1. mu.L, NotI 1. mu.L, 10 × CutSmart Buffer 2. mu.L, ddH2O make up the system to 20. mu.L. Carrying out enzyme digestion on the plasmid in 25 ℃ water bath for 1h, then carrying out enzyme digestion on the plasmid in 37 ℃ water bath for 1h, adding 0.5 mu L CIP into the vector fragment, and carrying out dephosphorylation in 37 ℃ water bath for 30 min. The digested mixture was mixed with 6 × Loading Buffer, electrophoresed (1% agarose, 94V) and gel recovered fragments of the corresponding length (pUC57-IL12 recovered fragment IL12 gene fragment 1698bp in length; TC-83 recovered fragment from the replica vector, about 9.5Kb in length), eluted with 30 μ L of Elution Buffer.
Taking an IL12 gene fragment and a TC-83 self-replicating vector fragment respectively according to a connection system: vector 50ng, insert moles: vector fragment molar number 5:1, T4 Ligase 1. mu.L, 10 XLigase Buffer 5. mu.L, ddH2O make up the system to 10. mu.l, and ligate at 22 ℃ for 1 h. Gently mixing the ligation product with competent cells of Escherichia coli DH5 alpha at a volume of 1:10, ice-cooling for 30min, heat shock at 42 deg.C for 45s, ice-cooling for 3min, adding preheated SOB broth 500. mu.L, mixing, culturing at 37 deg.C and 180rpm for 1h, and spreading on LK plates (LB-Kan plates: containing 50. mu.L of culture medium)g/mL LB plate of Kan), cultured at 37 ℃ for 16-20 h.
And (3) PCR screening: amplifying a target band IL12 by using bacterial plasmids extracted by a boiling method as a template, wherein an upstream primer F: 5'-TATGGCCATGACTACTCTAGCTA-3', downstream primer R: 5'-GGGAAACGCCTGGTATCTTT-3', the reaction circulation conditions are as follows: 94 ℃ 3min → (94 ℃ 1min, 47 ℃ 30s, 72 ℃ 3min) × 30 cycles → 72 ℃ 10min → 4 ℃; electrophoretic Observation of PCR results (electrophoresis conditions: 1% agarose gel; 90V, loading: 5. mu.l PCR product), expected results: the IL12 fragment was approximately 2101 bp.
Enzyme digestion verification: plasmids were extracted from positive bacteria screened and verified by PCR, and the digestion was carried out according to the above digestion system using Apa I and Not I, and the digestion mixture was mixed with 6 Xloading Buffer for electrophoresis (1% agarose, 94V). Sequencing and verifying: and extracting the plasmid which is expected by PCR and enzyme digestion verification, and sending the plasmid to a sequencing company for sequencing. The coliform bacteria carrying the positive plasmid, which were completely correct after sequencing verification, were stored at-80 ℃.
4, the method for linearization and recovery of the JCXH-203 plasmid by BspQI enzyme digestion is as follows: JCXH-203 plasmid 10. mu.g, BspQI 1. mu.L, 10 XNEBuffer 3.15. mu.L, ddH2O, the system is complemented to 50 mu L, and the plasmid is cut by enzyme in water bath at 50 ℃ for 1 h. The cleavage mixture was mixed with 6 Xloading Buffer, electrophoresed (1% agarose, 94V) and the corresponding length of the fragment recovered in gel (JCXH-203 plasmid used, length of about 13Kb), 30. mu.L of Elution Buffer Elution.
After linearization the JCXH-203 plasmid was used to initiate an in vitro transcription reaction (IVT) using T7 RNA polymerase, Turbo DNase enzyme to degrade the template DNA, and capping enzyme (Vaccidia capping enzyme) to add 7-methylated guanylate Cap structure (called Cap 0) to the 5' end of the transcribed mRNA as follows.
10 Xreaction Buffer 2. mu.L, NTP 0.5mM each, linearized JCXH-2031. mu.g, T7 RNA Polymerase 2. mu.L, water supplemented to 20. mu.L, reacted at 30 ℃ for 1 hour and added, 1. mu.L of TURBOTMDNase, 4. mu.L of 10X clamping Buffer, 2. mu.L of GTP (10mM), 2. mu.L of SAM (2mM), 2. mu.L of Vaccinia clamping Enzyme, total volume supplemented with water to a total volume of 40. mu.L, and reaction at 30 ℃ for 1 hour. Adding water to 200 μ L, adding 7.5M lithium chloride 120 μ L, and mixingAfter the mixture was homogenized, the mixture was left to stand at-20 ℃ for 30 minutes, centrifuged at 14000g and 4 ℃ for 30 minutes, the supernatant was discarded and the precipitate was washed with 70% ethanol, centrifuged at 14000g and 4 ℃ for 5 minutes, the supernatant was discarded and the precipitate was air-dried for 5 minutes and dissolved in 40. mu.L of water. After quantitation by spectrophotometer, 400ng was mixed with 10. mu.L of northern Max-Gly Sample Loading Dye, incubated at 50 ℃ for 30 minutes, and electrophoresed with northern Max-Gly Gel Prep/Running buffer (1% agarose, 70V). The electrophoretic picture is shown in FIG. 2.
6, the mRNA-IL12 and lipid encapsulation steps were as follows: the mRNA is rapidly mixed with lipids (molar composition including 1, 2-dimethylsilyl-rac-3-methoxypolyethylene glycol-2000(DMG-PEG2000), cholesterol, Distearylphosphatidylcholine (DSPC) and cationic lipids, dissolved in alcohol) by a Nanoassembler mixer, causing precipitation of lipids and entrapment of the mRNA into the LNP under charge. The mRNA-LNP complex is then reconstituted by concentration and exchange into formulation solution.
Western blot was used to examine the expression of mRNA-IL12 in cells.
BHK-21 cells purchased from Shanghai cell center were subcultured until the number of cells was sufficient, trypsinized to 6-well plates per well of the cell culture, and plated overnight in a CO2 incubator at 37 ℃. The next day, liposome-encapsulated mRNA-IL12 was transfected into plated BHK-21 cells, and after 72h of culture, the cells were lysed to collect a protein sample.
The specific method comprises the following steps:
step one, cell culture and plating: inoculating the recovered BHK-21 cells into a culture bottle of 75cm2, wherein the culture medium is DMEM high-sugar medium + 5% double antibody + 10% fetal bovine serum, digesting the cells by pancreatin when the confluence of the cells at the bottom of the bottle reaches more than 80%, and counting. Appropriate numbers of cells were plated in 6-well cell culture plates overnight in a 37 ℃ CO2 incubator.
Step two, transfection of liposome mRNA into BHK-21 cells: the medium in the well-spread 6-well plate was blotted dry, washed once with PBS buffer, liposome-encapsulated mRNA was mixed with 1mL of Opti-MEM medium, added to the well-washed 6-well plate, and incubated at 37 ℃ in a CO2 incubator for 6h followed by 1mL of DMEM high-sugar medium containing 20% fetal bovine serum. Culturing in a CO2 incubator at 37 ℃ for 72 h.
Step three, processing the protein sample: after 72 hours, 200. mu.L of cell lysate and 1% PMSF were added to BHK-21 cells, and the mixture was left on ice for 5 minutes, centrifuged at 14000g for 5 minutes, and the supernatant was transferred to a fresh centrifuge tube and then added to a metal bath of 5 XSDS 95 ℃ for 12 minutes. Placing at-20 deg.C for use.
Step four, Western blot detection: electrophoresis: the concentration of polyacrylamide gel is 6%, the loading amount of protein is 20 μ L, the electrophoresis condition of laminated gel is 150V for 10min, and the electrophoresis condition of gel is 200V for 30 min. Electric conversion: using a nitrocellulose membrane, membranes were spun at 100V for 1 hour. And (3) sealing: 5% BSA was prepared as blocking solution using 1 XPBST and blocked overnight at 4 ℃. Primary antibody incubation: IL12 antibody was raised to 1: 500 were diluted and incubated at room temperature for 2 hours. Wash 3 times with 1X PBST for 5 minutes each. And (3) secondary antibody incubation: and (3) diluting the secondary antibody by using a blocking solution at a dilution ratio of 1: 10000, incubation at room temperature for 1 hour. Wash 3 times with 1X PBST for 5 minutes each. And (3) developing: developer solution A: solution B is 1:1, developing in an imaging system. The results of the experiment are shown in FIG. 3, and FIG. 3 is a graph showing the expression of IL12 protein after transfection of mRNA-IL12 into BHK cells in vitro;
as shown in FIG. 3, the expression of IL12 protein was better after in vitro transfection of BHK cells with mRNA-IL 12.
8, mRNA-IL12 immunization of mice, evaluation of tumor treatment effect
The PDX model is constructed by using NPI (non-human immunodeficiency Virus) mice with severe immunodeficiency, and tumor blocks are divided according to 3x3x3mm and transplanted to the right rear subcutaneous position on the back of the humanized mouse. When tumor volume reached approximately 80-150 mm 3, mice were randomized into 4 groups. The dosing and grouping days were set to day 0.
The first group was inoculated: saline as a control group, the second group was inoculated with: the third group was inoculated with the inventive drug LNP-srRNA (IL 12): keytruruda: vaccination with the commercial drug keytruda group, fourth group: the commercial drug keytruda + the inventive drug LNP-srRNA (IL12) was used.
The drug was prepared according to experiment one, 100 μ l was inoculated intratumorally, and equal volume of NS was inoculated in the tail vein of the saline control group. The second booster treatment, method and dose were the same as the first immunotherapy on day 10. The second booster treatment was performed on day 17, at the same method and dose as the first. A third booster treatment was performed on day 24, at the same method and dose as the first. The survival status was observed daily. The results are shown in FIG. 4, in which mRNA-IL12 drug was administered to the model mice during the booster treatment, and it was found that the survival rate of the mRNA-IL 12-treated mice was significantly improved as compared to the normal saline control mice. The mRNA-IL12 medicine prepared by experiments has effective control and treatment effects on tumors.
From experiments, the effect comparison between the second group and the third and fourth groups shows that: the single use of the medicine of the invention has the best tumor inhibition effect, so the mRNA-IL12 obtained by the method of the invention can well inhibit the tumor growth.
The invention expresses the interleukin 12 protein by using mRNA-IL12, has good effect of mobilizing an immune system, can achieve the immune effect of killing tumors, and has good application prospect.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Sequence listing
<110> Jiachen West sea (Hangzhou) Biotechnology Ltd
<120> drug for expressing interleukin 12 and directing at tumor based on mRNA and preparation method thereof
<141> 2021-04-20
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1709
<212> DNA
<213> Artificial Sequence
<400> 1
gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa 60
gatgtgccac cagcagctgg tcatcagctg gttcagcctc gttttcctcg cctcgccgct 120
ggtcgccata tgggagctca agaaggacgt atacgtggtg gagctggact ggtaccccga 180
cgcgccgggc gagatggtcg tcctgacgtg cgacacgccg gaggaggacg gcatcacgtg 240
gacgctggac cagtccagcg aggtcctcgg ctccggcaag acgctgacga tccaggtcaa 300
ggagttcggc gacgcgggcc agtacacgtg ccacaagggc ggcgaggtcc tgagccactc 360
cctcctcctg ctacacaaga aggaggacgg gatctggagc acggacatcc tcaaggacca 420
gaaggagccg aagaacaaga ccttcctgcg ctgcgaggcg aagaattact cgggccggtt 480
cacgtgctgg tggctcacca cgatcagcac ggacctgacg ttctcggtca agtcgtcgcg 540
gggctcgtcg gacccccagg gggtgacctg cggcgcggcg acgctgtcgg cggagcgggt 600
gcggggcgac aacaaggagt acgagtactc ggtcgagtgc caggaggact cggcgtgccc 660
ggcggcggag gagtcgctgc cgatcgaggt gatggtcgac gcggtccaca agctgaagta 720
cgagaactac acgtcgtcgt tcttcatccg ggacatcatc aagccggacc cgccgaagaa 780
cctgcagctg aagccgctga agaactcgcg gcaggtcgag gtctcgtggg agtacccgga 840
cacgtggtcg acgccgcact cgtacttctc gctgacgttc tgcgtccaag tgcagggcaa 900
gtcgaagcgg gagaagaagg accgggtgtt caccgacaag acgagcgcga cggtgatctg 960
ccggaagaac gcgtcgatct cggtgcgggc gcaggaccgg tactactcgt cgtcgtggtc 1020
ggagtgggcg tcggtgccgt gcagcggcgg aagcggcggc ggcagcggcg gaggctctgg 1080
cggcggctcc cggaacctgc cggtggcgac gccggacccg gggatgttcc cgtgcctgca 1140
ccacagccag aacctgctgc gggcggtgtc gaacatgctg cagaaggcgc ggcagacgct 1200
ggagttctac ccgtgcacga gcgaggagat cgaccacgag gacatcacga aggacaagac 1260
cagcacggtg gaggcgtgcc tgccgctgga gctgacgaag aacgagtcgt gcctgaactc 1320
gagggagacg tcgttcatca cgaacgggtc gtgcctggcg tcgcggaaga cgtcgttcat 1380
gatggcgctg tgcctgtcgt cgatctacga ggacctgaag atgtaccagg tggagttcaa 1440
gacgatgaac gcgaagctgc tgatggaccc gaagcggcag atcttcctcg accagaacat 1500
gctggcggtg atcgacgagc tcatgcaggc gctcaacttc aacagcgaga cggtgccgca 1560
gaagtcgtcg ctcgaggagc cggacttcta caagacgaag atcaagctct gcatcctgct 1620
gcacgctttc cggatccggg cggtgacgat cgaccgggtg atgtcgtacc tgaacgcttc 1680
gtgataaggc gcgcccaccc agcggccgc 1709
<210> 2
<211> 9636
<212> DNA
<213> Artificial Sequence
<400> 2
atgggcggcg catgagagaa gcccagacca attacctacc caaaatggag aaagttcacg 60
ttgacatcga ggaagacagc ccattcctca gagctttgca gcggagcttc ccgcagtttg 120
aggtagaagc caagcaggtc actgataatg accatgctaa tgccagagcg ttttcgcatc 180
tggcttcaaa actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa 240
gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt ccgatgagat 300
gtgcggaaga tccggacaga ttgtataagt atgcaactaa gctgaagaaa aactgtaagg 360
aaataactga taaggaattg gacaagaaaa tgaaggagct cgccgccgtc atgagcgacc 420
ctgacctgga aactgagact atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc 480
aagtcgctgt ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag 540
ccaataaggg agttagagtc gcctactgga taggctttga caccacccct tttatgttta 600
agaacttggc tggagcatat ccatcatact ctaccaactg ggccgacgaa accgtgttaa 660
cggctcgtaa cataggccta tgcagctctg acgttatgga gcggtcacgt agagggatgt 720
ccattcttag aaagaagtat ttgaaaccat ccaacaatgt tctattctct gttggctcga 780
ccatctacca cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact 840
tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc gacgggtacg 900
tcgttaaaag aatagctatc agtccaggcc tgtatgggaa gccttcaggc tatgctgcta 960
cgatgcaccg cgagggattc ttgtgctgca aagtgacaga cacattgaac ggggagaggg 1020
tctcttttcc cgtgtgcacg tatgtgccag ctacattgtg tgaccaaatg actggcatac 1080
tggcaacaga tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta 1140
tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac cttttgcccg 1200
tagtggccca ggcatttgct aggtgggcaa aggaatataa ggaagatcaa gaagatgaaa 1260
ggccactagg actacgagat agacagttag tcatggggtg ttgttgggct tttagaaggc 1320
acaagataac atctatttat aagcgcccgg atacccaaac catcatcaaa gtgaacagcg 1380
atttccactc attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa 1440
caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt accgccgagg 1500
acgtacaaga agctaagtgc gcagccgatg aggctaagga ggtgcgtgaa gccgaggagt 1560
tgcgcgcagc tctaccacct ttggcagctg atgttgagga gcccactctg gaagccgatg 1620
tcgacttgat gttacaagag gctggggccg gctcagtgga gacacctcgt ggcttgataa 1680
aggttaccag ctacgatggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg 1740
ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa gtcatagtga 1800
taacacactc tggccgaaaa gggcgttatg ccgtggaacc ataccatggt aaagtagtgg 1860
tgccagaggg acatgcaata cccgtccagg actttcaagc tctgagtgaa agtgccacca 1920
ttgtgtacaa cgaacgtgag ttcgtaaaca ggtacctgca ccatattgcc acacatggag 1980
gagcgctgaa cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg 2040
aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc actgggctag 2100
ggctcacagg cgagctggtg gatcctccct tccatgaatt cgcctacgag agtctgagaa 2160
cacgaccagc cgctccttac caagtaccaa ccataggggt gtatggcgtg ccaggatcag 2220
gcaagtctgg catcattaaa agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga 2280
aagaaaactg tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg 2340
ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag accctgtata 2400
ttgacgaagc ttttgcttgt catgcaggta ctctcagagc gctcatagcc attataagac 2460
ctaaaaaggc agtgctctgc ggggatccca aacagtgcgg tttttttaac atgatgtgcc 2520
tgaaagtgca ttttaaccac gagatttgca cacaagtctt ccacaaaagc atctctcgcc 2580
gttgcactaa atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa 2640
cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc aaacctaagc 2700
aggacgatct cattctcact tgtttcagag ggtgggtgaa gcagttgcaa atagattaca 2760
aaggcaacga aataatgacg gcagctgcct ctcaagggct gacccgtaaa ggtgtgtatg 2820
ccgttcggta caaggtgaat gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg 2880
tcctactgac ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga 2940
taaaaacact gactgccaag taccctggga atttcactgc cacgatagag gagtggcaag 3000
cagagcatga tgccatcatg aggcacatct tggagagacc ggaccctacc gacgtcttcc 3060
agaataaggc aaacgtgtgt tgggccaagg ctttagtgcc ggtgctgaag accgctggca 3120
tagacatgac cactgaacaa tggaacactg tggattattt tgaaacggac aaagctcact 3180
cagcagagat agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg 3240
gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg gataactccc 3300
cgtcgcctaa catgtacggg ctgaataaag aagtggtccg tcagctctct cgcaggtacc 3360
cacaactgcc tcgggcagtt gccactggaa gagtctatga catgaacact ggtacactgc 3420
gcaattatga tccgcgcata aacctagtac ctgtaaacag aagactgcct catgctttag 3480
tcctccacca taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg 3540
gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg gttgactggt 3600
tgtcagaccg gcctgaggct accttcagag ctcggctgga tttaggcatc ccaggtgatg 3660
tgcccaaata tgacataata tttgttaatg tgaggacccc atataaatac catcactatc 3720
agcagtgtga agaccatgcc attaagctta gcatgttgac caagaaagct tgtctgcatc 3780
tgaatcccgg cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa 3840
gcatcattgg tgctatagcg cggctgttca agttttcccg ggtatgcaaa ccgaaatcct 3900
cacttgaaga gacggaagtt ctgtttgtat tcattgggta cgatcgcaag gcccgtacgc 3960
acaatcctta caagctttca tcaaccttga ccaacattta tacaggttcc agactccacg 4020
aagccggatg tgcaccctca tatcatgtgg tgcgagggga tattgccacg gccaccgaag 4080
gagtgattat aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc 4140
tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga aaagcgcgac 4200
tggtcaaagg tgcagctaaa catatcattc atgccgtagg accaaacttc aacaaagttt 4260
cggaggttga aggtgacaaa cagttggcag aggcttatga gtccatcgct aagattgtca 4320
acgataacaa ttacaagtca gtagcgattc cactgttgtc caccggcatc ttttccggga 4380
acaaagatcg actaacccaa tcattgaacc atttgctgac agctttagac accactgatg 4440
cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag gaagcagtgg 4500
ctaggagaga agcagtggag gagatatgca tatccgacga ctcttcagtg acagaacctg 4560
atgcagagct ggtgagggtg catccgaaga gttctttggc tggaaggaag ggctacagca 4620
caagcgatgg caaaactttc tcatatttgg aagggaccaa gtttcaccag gcggccaagg 4680
atatagcaga aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca 4740
tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc gaagagtcgg 4800
aagcctccac accacctagc acgctgcctt gcttgtgcat ccatgccatg actccagaaa 4860
gagtacagcg cctaaaagcc tcacgtccag aacaaattac tgtgtgctca tcctttccat 4920
tgccgaagta tagaatcact ggtgtgcaga agatccaatg ctcccagcct atattgttct 4980
caccgaaagt gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag 5040
acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct gaacaaccac 5100
cacttataac cgaggatgag accaggacta gaacgcctga gccgatcatc atcgaagagg 5160
aagaagagga tagcataagt ttgctgtcag atggcccgac ccaccaggtg ctgcaagtcg 5220
aggcagacat tcacgggccg ccctctgtat ctagctcatc ctggtccatt cctcatgcat 5280
ccgactttga tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca 5340
gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag tttctggcgc 5400
gaccggtgcc tgcgcctcga acagtattca ggaaccctcc acatcccgct ccgcgcacaa 5460
gaacaccgtc acttgcaccc agcagggcct gctcgagaac cagcctagtt tccaccccgc 5520
caggcgtgaa tagggtgatc actagagagg agctcgaggc gcttaccccg tcacgcactc 5580
ctagcaggtc ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga 5640
ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg 5700
catacatctt ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa 5760
cggtgctatc cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc 5820
tcgaccaaga aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta 5880
acagaagcag ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta 5940
ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc taccgaaccc 6000
tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc cttttcaagc cccaaggtcg 6060
cagtggaagc ctgtaacgcc atgttgaaag agaactttcc gactgtggct tcttactgta 6120
ttattccaga gtacgatgcc tatttggaca tggttgacgg agcttcatgc tgcttagaca 6180
ctgccagttt ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac 6240
ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac gtcctggcag 6300
ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga attgcccgta ttggattcgg 6360
cggcctttaa tgtggaatgc ttcaagaaat atgcgtgtaa taatgaatat tgggaaacgt 6420
ttaaagaaaa ccccatcagg cttactgaag aaaacgtggt aaattacatt accaaattaa 6480
aaggaccaaa agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca 6540
taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact ccaggaacaa 6600
aacatactga agaacggccc aaggtacagg tgatccaggc tgccgatccg ctagcaacag 6660
cgtatctgtg cggaatccac cgagagctgg ttaggagatt aaatgcggtc ctgcttccga 6720
acattcatac actgtttgat atgtcggctg aagactttga cgctattata gccgagcact 6780
tccagcctgg ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg 6840
acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac gcagagctgt 6900
tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat acatttgccc actaaaacta 6960
aatttaaatt cggagccatg atgaaatctg gaatgttcct cacactgttt gtgaacacag 7020
tcattaacat tgtaatcgca agcagagtgt tgagagaacg gctaaccgga tcaccatgtg 7080
cagcattcat tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag 7140
acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg gtgggcgaga 7200
aagcgcctta tttctgtgga gggtttattt tgtgtgactc cgtgaccggc acagcgtgcc 7260
gtgtggcaga ccccctaaaa aggctgttta agcttggcaa acctctggca gcagacgatg 7320
aacatgatga tgacaggaga agggcattgc atgaagagtc aacacgctgg aaccgagtgg 7380
gtattctttc agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca 7440
tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc tacctgagag 7500
gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa 7560
gtaaggcgcg cccacccagc ggccgcatac agcagcaatt ggcaagctgc ttacatagaa 7620
ctcgcggcga ttggcatgcc gccttaaaat ttttatttta tttttctttt cttttccgaa 7680
tcggattttg tttttaatat ttcaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 7740
aaaagaagag ctagggataa cagggtaatt gagcaaaagg ccagcaaaag gccaggaacc 7800
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 7860
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 7920
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 7980
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 8040
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 8100
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 8160
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 8220
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 8280
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 8340
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 8400
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 8460
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 8520
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 8580
acagttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat 8640
caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt 8700
tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac 8760
aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga 8820
cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag 8880
gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg 8940
attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa 9000
tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag 9060
gatattcttc taatacctgg aatgctgttt tcccagggat cgcagtggtg agtaaccatg 9120
catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc 9180
agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca 9240
gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc 9300
cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc 9360
gcggcctaga gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt 9420
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 9480
aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 9540
aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 9600
tagggataac agggtaatta atacgactca ctatag 9636
<210> 3
<211> 11259
<212> DNA
<213> Artificial Sequence
<400> 3
ataggcggcg catgagagaa gcccagacca attacctacc caaaatggag aaagttcacg 60
ttgacatcga ggaagacagc ccattcctca gagctttgca gcggagcttc ccgcagtttg 120
aggtagaagc caagcaggtc actgataatg accatgctaa tgccagagcg ttttcgcatc 180
tggcttcaaa actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa 240
gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt ccgatgagat 300
gtgcggaaga tccggacaga ttgtataagt atgcaactaa gctgaagaaa aactgtaagg 360
aaataactga taaggaattg gacaagaaaa tgaaggagct cgccgccgtc atgagcgacc 420
ctgacctgga aactgagact atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc 480
aagtcgctgt ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag 540
ccaataaggg agttagagtc gcctactgga taggctttga caccacccct tttatgttta 600
agaacttggc tggagcatat ccatcatact ctaccaactg ggccgacgaa accgtgttaa 660
cggctcgtaa cataggccta tgcagctctg acgttatgga gcggtcacgt agagggatgt 720
ccattcttag aaagaagtat ttgaaaccat ccaacaatgt tctattctct gttggctcga 780
ccatctacca cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact 840
tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc gacgggtacg 900
tcgttaaaag aatagctatc agtccaggcc tgtatgggaa gccttcaggc tatgctgcta 960
cgatgcaccg cgagggattc ttgtgctgca aagtgacaga cacattgaac ggggagaggg 1020
tctcttttcc cgtgtgcacg tatgtgccag ctacattgtg tgaccaaatg actggcatac 1080
tggcaacaga tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta 1140
tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac cttttgcccg 1200
tagtggccca ggcatttgct aggtgggcaa aggaatataa ggaagatcaa gaagatgaaa 1260
ggccactagg actacgagat agacagttag tcatggggtg ttgttgggct tttagaaggc 1320
acaagataac atctatttat aagcgcccgg atacccaaac catcatcaaa gtgaacagcg 1380
atttccactc attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa 1440
caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt accgccgagg 1500
acgtacaaga agctaagtgc gcagccgatg aggctaagga ggtgcgtgaa gccgaggagt 1560
tgcgcgcagc tctaccacct ttggcagctg atgttgagga gcccactctg gaagccgatg 1620
tcgacttgat gttacaagag gctggggccg gctcagtgga gacacctcgt ggcttgataa 1680
aggttaccag ctacgatggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg 1740
ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa gtcatagtga 1800
taacacactc tggccgaaaa gggcgttatg ccgtggaacc ataccatggt aaagtagtgg 1860
tgccagaggg acatgcaata cccgtccagg actttcaagc tctgagtgaa agtgccacca 1920
ttgtgtacaa cgaacgtgag ttcgtaaaca ggtacctgca ccatattgcc acacatggag 1980
gagcgctgaa cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg 2040
aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc actgggctag 2100
ggctcacagg cgagctggtg gatcctccct tccatgaatt cgcctacgag agtctgagaa 2160
cacgaccagc cgctccttac caagtaccaa ccataggggt gtatggcgtg ccaggatcag 2220
gcaagtctgg catcattaaa agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga 2280
aagaaaactg tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg 2340
ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag accctgtata 2400
ttgacgaagc ttttgcttgt catgcaggta ctctcagagc gctcatagcc attataagac 2460
ctaaaaaggc agtgctctgc ggggatccca aacagtgcgg tttttttaac atgatgtgcc 2520
tgaaagtgca ttttaaccac gagatttgca cacaagtctt ccacaaaagc atctctcgcc 2580
gttgcactaa atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa 2640
cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc aaacctaagc 2700
aggacgatct cattctcact tgtttcagag ggtgggtgaa gcagttgcaa atagattaca 2760
aaggcaacga aataatgacg gcagctgcct ctcaagggct gacccgtaaa ggtgtgtatg 2820
ccgttcggta caaggtgaat gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg 2880
tcctactgac ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga 2940
taaaaacact gactgccaag taccctggga atttcactgc cacgatagag gagtggcaag 3000
cagagcatga tgccatcatg aggcacatct tggagagacc ggaccctacc gacgtcttcc 3060
agaataaggc aaacgtgtgt tgggccaagg ctttagtgcc ggtgctgaag accgctggca 3120
tagacatgac cactgaacaa tggaacactg tggattattt tgaaacggac aaagctcact 3180
cagcagagat agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg 3240
gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg gataactccc 3300
cgtcgcctaa catgtacggg ctgaataaag aagtggtccg tcagctctct cgcaggtacc 3360
cacaactgcc tcgggcagtt gccactggaa gagtctatga catgaacact ggtacactgc 3420
gcaattatga tccgcgcata aacctagtac ctgtaaacag aagactgcct catgctttag 3480
tcctccacca taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg 3540
gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg gttgactggt 3600
tgtcagaccg gcctgaggct accttcagag ctcggctgga tttaggcatc ccaggtgatg 3660
tgcccaaata tgacataata tttgttaatg tgaggacccc atataaatac catcactatc 3720
agcagtgtga agaccatgcc attaagctta gcatgttgac caagaaagct tgtctgcatc 3780
tgaatcccgg cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa 3840
gcatcattgg tgctatagcg cggcagttca agttttcccg ggtatgcaaa ccgaaatcct 3900
cacttgaaga gacggaagtt ctgtttgtat tcattgggta cgatcgcaag gcccgtacgc 3960
acaatcctta caagctttca tcaaccttga ccaacattta tacaggttcc agactccacg 4020
aagccggatg tgcaccctca tatcatgtgg tgcgagggga tattgccacg gccaccgaag 4080
gagtgattat aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc 4140
tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga aaagcgcgac 4200
tggtcaaagg tgcagctaaa catatcattc atgccgtagg accaaacttc aacaaagttt 4260
cggaggttga aggtgacaaa cagttggcag aggcttatga gtccatcgct aagattgtca 4320
acgataacaa ttacaagtca gtagcgattc cactgttgtc caccggcatc ttttccggga 4380
acaaagatcg actaacccaa tcattgaacc atttgctgac agctttagac accactgatg 4440
cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag gaagcagtgg 4500
ctaggagaga agcagtggag gagatatgca tatccgacga ctcttcagtg acagaacctg 4560
atgcagagct ggtgagggtg catccgaaga gttctttggc tggaaggaag ggctacagca 4620
caagcgatgg caaaactttc tcatatttgg aagggaccaa gtttcaccag gcggccaagg 4680
atatagcaga aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca 4740
tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc gaagagtcgg 4800
aagcctccac accacctagc acgctgcctt gcttgtgcat ccatgccatg actccagaaa 4860
gagtacagcg cctaaaagcc tcacgtccag aacaaattac tgtgtgctca tcctttccat 4920
tgccgaagta tagaatcact ggtgtgcaga agatccaatg ctcccagcct atattgttct 4980
caccgaaagt gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag 5040
acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct gaacaaccac 5100
cacttataac cgaggatgag accaggacta gaacgcctga gccgatcatc atcgaagagg 5160
aagaagagga tagcataagt ttgctgtcag atggcccgac ccaccaggtg ctgcaagtcg 5220
aggcagacat tcacgggccg ccctctgtat ctagctcatc ctggtccatt cctcatgcat 5280
ccgactttga tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca 5340
gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag tttctggcgc 5400
gaccggtgcc tgcgcctcga acagtattca ggaaccctcc acatcccgct ccgcgcacaa 5460
gaacaccgtc acttgcaccc agcagggcct gctcgagaac cagcctagtt tccaccccgc 5520
caggcgtgaa tagggtgatc actagagagg agctcgaggc gcttaccccg tcacgcactc 5580
ctagcaggtc ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga 5640
ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg 5700
catacatctt ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa 5760
cggtgctatc cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc 5820
tcgaccaaga aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta 5880
acagaagcag ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta 5940
ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc taccgaaccc 6000
tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc cttttcaagc cccaaggtcg 6060
cagtggaagc ctgtaacgcc atgttgaaag agaactttcc gactgtggct tcttactgta 6120
ttattccaga gtacgatgcc tatttggaca tggttgacgg agcttcatgc tgcttagaca 6180
ctgccagttt ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac 6240
ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac gtcctggcag 6300
ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga attgcccgta ttggattcgg 6360
cggcctttaa tgtggaatgc ttcaagaaat atgcgtgtaa taatgaatat tgggaaacgt 6420
ttaaagaaaa ccccatcagg cttactgaag aaaacgtggt aaattacatt accaaattaa 6480
aaggaccaaa agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca 6540
taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact ccaggaacaa 6600
aacatactga agaacggccc aaggtacagg tgatccaggc tgccgatccg ctagcaacag 6660
cgtatctgtg cggaatccac cgagagctgg ttaggagatt aaatgcggtc ctgcttccga 6720
acattcatac actgtttgat atgtcggctg aagactttga cgctattata gccgagcact 6780
tccagcctgg ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg 6840
acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac gcagagctgt 6900
tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat acatttgccc actaaaacta 6960
aatttaaatt cggagccatg atgaaatctg gaatgttcct cacactgttt gtgaacacag 7020
tcattaacat tgtaatcgca agcagagtgt tgagagaacg gctaaccgga tcaccatgtg 7080
cagcattcat tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag 7140
acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg gtgggcgaga 7200
aagcgcctta tttctgtgga gggtttattt tgtgtgactc cgtgaccggc acagcgtgcc 7260
gtgtggcaga ccccctaaaa aggctgttta agcttggcaa acctctggca gcagacgatg 7320
aacatgatga tgacaggaga agggcattgc atgaagagtc aacacgctgg aaccgagtgg 7380
gtattctttc agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca 7440
tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc tacctgagag 7500
gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa 7560
gatgtgccac cagcagctgg tcatcagctg gttcagcctc gttttcctcg cctcgccgct 7620
ggtcgccata tgggagctca agaaggacgt atacgtggtg gagctggact ggtaccccga 7680
cgcgccgggc gagatggtcg tcctgacgtg cgacacgccg gaggaggacg gcatcacgtg 7740
gacgctggac cagtccagcg aggtcctcgg ctccggcaag acgctgacga tccaggtcaa 7800
ggagttcggc gacgcgggcc agtacacgtg ccacaagggc ggcgaggtcc tgagccactc 7860
cctcctcctg ctacacaaga aggaggacgg gatctggagc acggacatcc tcaaggacca 7920
gaaggagccg aagaacaaga ccttcctgcg ctgcgaggcg aagaattact cgggccggtt 7980
cacgtgctgg tggctcacca cgatcagcac ggacctgacg ttctcggtca agtcgtcgcg 8040
gggctcgtcg gacccccagg gggtgacctg cggcgcggcg acgctgtcgg cggagcgggt 8100
gcggggcgac aacaaggagt acgagtactc ggtcgagtgc caggaggact cggcgtgccc 8160
ggcggcggag gagtcgctgc cgatcgaggt gatggtcgac gcggtccaca agctgaagta 8220
cgagaactac acgtcgtcgt tcttcatccg ggacatcatc aagccggacc cgccgaagaa 8280
cctgcagctg aagccgctga agaactcgcg gcaggtcgag gtctcgtggg agtacccgga 8340
cacgtggtcg acgccgcact cgtacttctc gctgacgttc tgcgtccaag tgcagggcaa 8400
gtcgaagcgg gagaagaagg accgggtgtt caccgacaag acgagcgcga cggtgatctg 8460
ccggaagaac gcgtcgatct cggtgcgggc gcaggaccgg tactactcgt cgtcgtggtc 8520
ggagtgggcg tcggtgccgt gcagcggcgg aagcggcggc ggcagcggcg gaggctctgg 8580
cggcggctcc cggaacctgc cggtggcgac gccggacccg gggatgttcc cgtgcctgca 8640
ccacagccag aacctgctgc gggcggtgtc gaacatgctg cagaaggcgc ggcagacgct 8700
ggagttctac ccgtgcacga gcgaggagat cgaccacgag gacatcacga aggacaagac 8760
cagcacggtg gaggcgtgcc tgccgctgga gctgacgaag aacgagtcgt gcctgaactc 8820
gagggagacg tcgttcatca cgaacgggtc gtgcctggcg tcgcggaaga cgtcgttcat 8880
gatggcgctg tgcctgtcgt cgatctacga ggacctgaag atgtaccagg tggagttcaa 8940
gacgatgaac gcgaagctgc tgatggaccc gaagcggcag atcttcctcg accagaacat 9000
gctggcggtg atcgacgagc tcatgcaggc gctcaacttc aacagcgaga cggtgccgca 9060
gaagtcgtcg ctcgaggagc cggacttcta caagacgaag atcaagctct gcatcctgct 9120
gcacgctttc cggatccggg cggtgacgat cgaccgggtg atgtcgtacc tgaacgcttc 9180
gtgataaggc gcgcccaccc agcggccgca tacagcagca attggcaagc tgcttacata 9240
gaactcgcgg cgattggcat gccgccttaa aatttttatt ttatttttct tttcttttcc 9300
gaatcggatt ttgtttttaa tatttcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9360
aaaaaaagaa gagctaggga taacagggta attgagcaaa aggccagcaa aaggccagga 9420
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 9480
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 9540
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 9600
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 9660
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 9720
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 9780
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 9840
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 9900
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 9960
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 10020
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 10080
acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 10140
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 10200
ctgacagtta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat 10260
tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc 10320
agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa 10380
tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag 10440
tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa 10500
caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc 10560
gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag 10620
gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat 10680
caggatattc ttctaatacc tggaatgctg ttttcccagg gatcgcagtg gtgagtaacc 10740
atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca 10800
gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt 10860
tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt 10920
gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta 10980
atcgcggcct agagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat 11040
attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 11100
agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct 11160
aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc 11220
gtctagggat aacagggtaa ttaatacgac tcactatag 11259
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 4
tatggccatg actactctag cta 23
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 5
Claims (10)
1. A drug expressing interleukin 12 against a tumor based on self-replicating mRNA, comprising: a liposome complex of self-replicating mRNA expressing interleukin-12; the self-replicating mRNA expressing interleukin-12 is mRNA-IL 12; the liposome compound of the self-replicating mRNA for expressing interleukin-12 is mRNA-LNP; the mRNA-IL12 comprises: an mRNA replicon designed based on an alphavirus genome and containing an interleukin 12 protein gene, the interleukin 12 protein gene; the sequence of the mRNA replicon comprises a gene encoding an alphavirus self-replicating function and lacks a gene that produces a structural protein having infectious alphavirus particles.
2. The drug of claim 1, wherein the lipid comprises: DMG-PEG2000, cholesterol, distearoylphosphatidylcholine or cationic lipids.
3. The drug of claim 1, wherein the alphavirus genome comprises: venezuelan equine encephalomyelitis virus genome, sindbis virus genome, chikungunya virus genome, eastern equine encephalomyelitis virus genome, western equine encephalomyelitis virus genome, equine jaru virus genome, raw linsen virus genome, or venezuelan equine encephalomyelitis virus genome.
4. The drug for expressing interleukin 12 to tumor based on mRNA of claim 1, wherein the mRNA-IL12 is synthesized by enzyme transcription reaction in vitro using linearized plasmid DNA as a template.
5. The drug for tumor-based expression of interleukin 12 as claimed in claim 4, wherein the plasmid DNA contains kanamycin resistance gene, T7 promoter gene, nsp gene, IL12 gene, polyA sequence; the gene sequence is shown in SEQ ID NO. 3.
6. A preparation method of a medicine for expressing interleukin 12 to aim at tumors based on mRNA is characterized by comprising the following steps:
step one, synthesizing an IL12 gene fragment:
inquiring an IL12 amino acid sequence, carrying out codon optimization according to a human preference codon table to obtain an optimized sequence, adding a promoter and an ApaI enzyme digestion site GGGCCC at the upstream of the optimized sequence, adding a NotI enzyme digestion site GCGGCCGC at the downstream of the optimized sequence, and synthesizing the designed sequence to obtain the optimized sequence;
step two, preparing and obtaining mRNA-IL12, wherein the mRNA-IL12 is self-replicating mRNA for expressing interleukin-12;
step three, mixing mRNA-IL12 with lipid, wrapping mRNA-IL12 into LNP to obtain mRNA-LNP, and concentrating and changing liquid to a preparation solution; the mRNA-LNP is a liposome complex of self-replicating mRNA that expresses interleukin-12.
7. The drug for expressing interleukin 12 to tumor based on mRNA of claim 6, wherein the specific steps for preparing mRNA-IL12 in the second step comprise:
step a, constructing a TC-83 self-replicating vector:
designing a self-replicating mRNA sequence containing an interleukin 12 protein gene based on an alpha virus genome, taking a plasmid constructed by the self-replicating mRNA sequence as a template for amplification, and synthesizing the designed sequence to obtain the self-replicating mRNA sequence; the sequence of the mRNA replicon comprises a gene encoding an alphavirus self-replicating function, lacking a gene that produces a structural protein having infectious alphavirus particles;
step b, preparing a recombinant plasmid JCXH-203:
inserting an IL12 gene carrying ApaI at the upstream and NotI at the downstream into the enzyme cutting sites of ApaI and NotI of a TC-83 replication vector to obtain a recombinant plasmid JCXH-203;
step c, linearizing the JCXH-203 plasmid by using restriction enzyme digestion;
and d, firstly carrying out in-vitro transcription reaction on the linearized JCXH-203 plasmid, then degrading the template DNA, and finally adding the 7-methylated guanylic acid cap structure to the 5' end of the transcribed mRNA to obtain the in-vitro transcribed mRNA-IL 12.
8. The drug for tumor expression based on mRNA of claim 7, wherein the restriction enzyme in step c is BspQI enzyme.
9. The drug for expressing interleukin 12 on tumor based on mRNA of claim 7, wherein in the step d, the linearized JCXH-203 plasmid is subjected to in vitro transcription reaction by using T7 RNA polymerase in the first step, the template DNA is degraded by using Turbo DNase in the second step, and the 7-methylated guanylic acid cap structure is added to the 5' end of the transcribed mRNA by using capping enzyme in the third step, so as to obtain in vitro transcribed mRNA-IL 12.
10. The drug for tumor expression based on mRNA of claim 6, wherein the mRNA-IL12 is mixed with lipid in step three by using a Nanolasemblr mixer.
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