Elastin-like polypeptide V 20 K 40 L, mRNA vaccines and methods of making and using the same

Abstract

The application discloses a kind of elastin-like polypeptide V 20 K 40 L, mRNA vaccine and preparation method and application in preparation tumor prevention, mitigation or treating drug.The elastin-like polypeptide carrier V 20 K 40 L provided by the application has good biocompatibility, and has no adverse reaction in vivo.The elastin-like polypeptide V 20 K 40 L provided by the application has amphiphilic structure, can be loaded Melan-A mRNA by hydrophobic self-assembly and electrostatic adsorption, and is in the state of shrinkage when higher than Tt temperature, which can prevent the uncontrollable leakage of mRNA.Furthermore, V 20 K 40 L contains lysosome escape peptide, which can further improve the transfection efficiency of mRNA by promoting lysosome escape.The elastin-like polypeptide V 20 K 40 L in the application can deliver Melan-A mRNA efficiently and safely, and realize the prevention and treatment of melanoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mRNA vaccine development, and particularly refers to an elastin-like polypeptide V 20 K 40 L, mRNA vaccine and preparation method and application thereof. BACKGROUND

[0002] mRNA vaccine is a new vaccine technology that has attracted global attention due to its high preparation efficiency. By 2024, more than 140 mRNA vaccine projects have entered clinical trials, showing its great value in the field of life sciences. mRNA itself is unstable and easily degraded by nucleases in the body. Moreover, due to its large molecular weight and negative charge, it is difficult to deliver into cells across the cell membrane, so improving the delivery efficiency of mRNA is a key challenge in mRNA vaccine development.

[0003] To overcome these problems, researchers have developed various carrier systems to protect and deliver mRNA, including lipid nanoparticles (LNPs), polymers, proteins, etc. LNPs are the most widely used mRNA delivery carrier at present, but their delivery efficiency still needs to be improved. In addition, due to the complex composition of LNPs, it contains four types of lipids, namely ionizable lipids, phosphorylated lipids, cholesterol and polyethylene glycol lipids. These components have immunogenicity, and most of them will cause side effects, such as redness, pain or hardness at the inoculation site, and even serious allergic reactions, myocardial inflammation, pericarditis, facial nerve palsy and thrombosis. Overall, these side effects are more likely to be caused by LNPs themselves, so optimizing the carrier material and reducing related side effects is a problem that needs to be solved.

[0004] Elastin is a protein that provides elasticity and resilience to tissues, mainly existing in tissues such as skin, blood vessel walls and lungs. Elastin-like polypeptides (ELPs) are a kind of polypeptides derived from natural elastin and can be artificially synthesized, which have similar physicochemical properties to elastin. ELPs have special temperature responsiveness, which aggregate when the environmental temperature exceeds the inverse transition temperature (Tt), and dissolve again when the temperature is below Tt. After coupling with other small molecules, polypeptides, nucleic acids and other substances, this temperature-sensitive property can be fully retained, so it can be used to achieve controlled and released drug delivery.

[0005] ELPs are composed of natural amino acids, which are biocompatible, easily biodegradable, low immunogenicity, and non-toxic. More importantly, the structure and function of ELPs can be precisely regulated by genetic engineering methods, reactive amino acids or polypeptides can be added to their sequences, and large-scale production can be maintained, which makes them a cost-effective biomaterial. However, ELPs for mRNA delivery still need to be developed. SUMMARY

[0006] To overcome the shortcomings of the above-mentioned technologies, the present application provides a kind of elastin-like polypeptide V 20 K 40 L, mRNA vaccine and preparation method and application thereof, solve the delivery problem of mRNA vaccine, with the advantages of good safety, small side effect.

[0007] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows:

[0008] A kind of elastin-like polypeptide V 20 K 40 L, the amino acid sequence of the elastin-like polypeptide V 20 K 40 L is shown as SEQ ID NO:1.

[0009] The preparation method of the above-mentioned elastin-like polypeptide V 20 K 40 L, comprising the following steps: making the recombinant plasmid containing the elastin-like polypeptide V 20 K 40 L coding gene in the exogenous expression system to express, the protein purification is carried out to the system after expression, and the elastin-like polypeptide V 20 K 40 L is obtained.

[0010] Preferably, the nucleotide sequence of the elastin-like polypeptide V 20 K 40 L coding gene is shown as SEQ ID NO:2.

[0011] The present application also provides an mRNA vaccine, which is formed by self-assembly of mRNA, CpG and the above-mentioned elastin-like polypeptide V 20 K 40 L.

[0012] Preferably, the mass ratio of the mRNA, CpG and elastin-like polypeptide V 20 K 40 L is mRNA:CpG:V 20 K 40 L=1:0.5:40-60.

[0013] Preferably, the mRNA is Melan-A mRNA.

[0014] The application also provides a preparation method of the mRNA vaccine, comprising the following steps: taking a water solution of mRNA, a water solution of CpG and a water solution of elastin-like polypeptide V 20 K 40 L, mixing uniformly, then rapidly injecting into water under ultrasonic state and continuously ultrasonic, namely obtaining the mRNA vaccine.

[0015] The application also provides an application of the elastin-like polypeptide V 20 K 40 L in preparation of a drug delivery system, wherein the drug in the drug delivery system is mRNA.

[0016] The application also provides an application of the elastin-like polypeptide V 20 K 40 L or the mRNA vaccine in preparation of a drug for preventing, alleviating or treating tumors.

[0017] Preferably, the tumor is melanoma.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] The elastin-like polypeptide carrier V 20 K 40 L designed in the application has good biocompatibility, which can avoid adverse reactions of LNP carriers in vivo. 20 K 40 L has an amphiphilic structure, can efficiently load Melan-A mRNA through hydrophobic self-assembly and electrostatic adsorption, and is in a compact state at a temperature higher than Tt, which can prevent uncontrollable leakage of mRNA. 20 K 40 L contains a lysosome escape peptide, which further improves the transfection efficiency of mRNA by promoting lysosome escape. The elastin-like polypeptide carrier V 20 K 40 L of the application can efficiently and safely deliver Melan-A mRNA, and achieve prevention and treatment of melanoma. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The sequence and structure diagram of the elastin-like polypeptide V 20 K 40 L in the application are shown in the following figures;

[0021] Figure 2 The synthesis route diagram of the elastin-like polypeptide V 20 K 40 L is shown in the following figure.

[0022] Figure 3 V in Example 1 20 K 40 Structure and functional sequence diagram of L plasmid;

[0023] Figure 4 V in Example 1 20 K 40 Image of L by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE);

[0024] Figure 5 V in Example 1 20 K 40 Mass spectrum of L;

[0025] Figure 6 V in Example 1 20 K 40 Circular dichroism (CD) of L;

[0026] Figure 7 V in Example 1 20 K 40 Phase transition diagram of L;

[0027] Figure 8 This is an agarose gel electrophoresis image of the Melan-Am RNA from Example 1;

[0028] Figure 9 This is a comparison diagram of the sequence information and sequencing information of Melan-AmRNA;

[0029] Figure 10 Different proportions of V in Example 2 20 K 40 L binds to Melan-A mRNA to form V. 20 K 40 Gel electrophoresis image of L@MC nanoparticles;

[0030] Figure 11 V in Example 2 20 K 40 Dynamic light scattering plots of L@MC at different temperatures;

[0031] Figure 12 V in Example 2 20 K 40 Transmission electron microscopy (TEM) images of L@MC at different temperatures;

[0032] Figure 13 For example, V in Example 2 20 K 40 Stability diagrams of L@MC in different media;

[0033] Figure 14 For different concentrations of V 20 K 40 PCR diagram of Melan-A mRNA expression after L@MC and DC 2.4 cells were incubated for 24 hours;

[0034] Figure 15 For different concentrations of V 20 K 40 Diagram of Melan-A protein expression after L@MC and DC 2.4 cells were incubated for 24 hours;

[0035] Figure 16 For V 20 K 40 Diagram of V 20 K 40 Co-localization diagram of L@MC and lysosome;

[0036] Figure 17 For V 20 K 40 Confocal image and quantitative analysis diagram of L@MC-GFP and DC 2.4 cells after incubation for 24 hours;

[0037] Figure 18 For V 20 K 40 PCR diagram of Melan-A mRNA after L@MC and LNP@MC were respectively incubated with DC 2.4 cells for 24 hours;

[0038] Figure 19 For V 20 K 40 Diagram of Melan-A protein expression after L@MC and LNP@MC were respectively incubated with DC 2.4 cells for 24 hours;

[0039] Figure 20 For V 20 K 40 Analysis of liver function indexes ALT and AST and kidney function indexes BUN and CREA of mice after L@MC and LNP@MC were respectively subcutaneously injected into mice for 24 hours;

[0040] Figure 21 For V 20 K 40 Contents of GM-CSF, IL-6 and IL-1β in serum of mice after L and LNP were respectively subcutaneously injected into mice for 24 hours;

[0041] Figure 22 For V 20 K40 Flow cytometry of memory T cells in L@MC and LNP@MC inoculated mice;

[0042] Figure 23 V 20 K 40 Lung tissue photographs of the preventive effect of L@MC and LNP@MC on B16-F10 melanoma;

[0043] Figure 24 V 20 K 40 HE staining photographs of lung tissue sections of the preventive effect of L@MC and LNP@MC on B16-F10 melanoma. DETAILED DESCRIPTION

[0044] In order to better explain the present application, the main content of the present application is further illustrated below in combination with specific examples, but the content of the present application is not limited to the following examples only.

[0045] Example 1: Elastin-like polypeptide V 20 K 40 L and the preparation method thereof

[0046] Elastin-like polypeptide V 20 K 40 The amino acid sequence of the elastin-like polypeptide V

[0047] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGWEAKLAKALAKALAKHLAKALAKLEHHHHHH*(SEQ ID NO:1), the structure of which is shown as Figure 1 The preparation method thereof is shown as Figure 2 Specifically as follows:

[0048] (1) V 20 K40 Construction of L plasmid

[0049] Select pET-28a(+) with kana resistance as plasmid empty, use BamHI and XhoI restriction endonuclease to enzyme cut empty and V 20 K 40 L target gene, then use T4 DNA ligase to connect enzyme cut target gene and empty, successfully construct a plasmid containing V 20 K 40 L target gene, that is, V 20 K 40 L plasmid, as shown in Figure 3 After placing V 20 K 40 L plasmid on ice for 30 min, through short time heat stimulation treatment at 42℃, the plasmid is transformed into DE3 competent cells, add no resistance LB medium, shake on the shaker for 45 min, shaker 37℃, rotation speed set to 200 rpm. After activation, prepare into glycerol bacteria. Plate the glycerol bacteria, culture overnight, then pick single colonies, grow and expand in the medium containing Kana antibiotic. Collect the bacterial solution, then extract and purify the plasmid, and perform sequencing verification. At the same time, perform enzyme cut verification, confirm plasmid construction success after running gel.

[0050] (II) V 20 K 40 L plasmid transfection of E. coli

[0051] Add 10 μL of DE3 E. coli transfected with V 20 K 40 L plasmid, 10 mL of LB medium and 10 μL of 100 mg / mL kanamycin into a 50 mL centrifuge tube, shake on the shaker overnight, shaker temperature 37℃, rotation speed set to 220 rpm. Then, transfer the above liquid into a conical flask containing 500 mL of LB medium (containing 500 μL of kanamycin), continue to culture for 4-6 hours until OD 600 0.6-0.8 is reached, then add 0.5 mL of inducer IPTG (1M), shake on the shaker overnight, shaker temperature 16℃, rotation speed set to 220 rpm.

[0052] (III) V 20 K 40 L separation

[0053] The bacteria solution obtained in step (two) was centrifuged at 4°C, 6000 rpm for 10 min, and the supernatant was removed. The precipitate was resuspended with 30 mL of 100 mM phosphate buffer (pH = 7.4), and the bacteria solution was broken by ultrasonic disrupter (4 s on, 8 s off, 30% power, 30 min). Finally, the broken bacteria solution was centrifuged at 4°C, 11000 rpm for 30 min, and the supernatant of the broken bacteria solution was collected.

[0054] (four) V 20 K 40 L purification

[0055] The protein was purified by reversible phase transition cycle method. The supernatant collected in step (three) was added with sodium chloride to make the final concentration of sodium chloride 5M, and heated in 37°C water bath for 15 min, and then centrifuged at 37°C, 11000 rpm for 15 min, and the bottom precipitate was collected. The precipitate was resuspended with 50 mM sodium phosphate buffer solution (pH = 7.4) containing 300 mM sodium chloride, and placed on ice for 15 min, and then centrifuged at 4°C, 11000 rpm for 15 min, and the supernatant was collected. The above steps were repeated three times, and finally the supernatant was collected and concentrated and desalted at 4°C by using ultrafiltration tube. The desalted protein sample was purified by AKTA to obtain purified V 20 K 40 L protein sample, wherein the buffer used was 50 mM sodium phosphate buffer solution (pH = 7.4) containing 150 mM sodium chloride.

[0056] (five) V 20 K 40 L characterization

[0057] 1. SDS-PAGE electrophoresis test: the V 20 K 40 L protein sample was subjected to SDS-PAGE electrophoresis test, and the test results are shown in Figure 4 , and the results show that the size of the V 20 K 40 L protein is consistent with the expected value.

[0058] 2. Mass spectrometry test: the V 20 K 40 L protein was subjected to mass spectrometry test, and the test results are shown in Figure 5 , the calculated value is 32784, and the found value is 32652. The results show that the calculated value and the found value are consistent, indicating that the V 20 K 40 L is successfully synthesized.

[0059] 3. Circular dichroism test: the structure and phase transition temperature of the synthesized V 20 K 40 L were characterized, and the results are shown inFigure 6 As shown, V was analyzed by circular dichroism (CD) chromatography. 20 K 40 The structural changes of L at different temperatures are first evaluated. 20 K 40 The secondary structure of L at 4℃ shows that V at this temperature 20 K 40 L exists in a disordered secondary structure; V is then evaluated. 20 K 40 The secondary structure of L at 37℃ shows that V at this temperature... 20 K 40 L has an α-helix secondary structure.

[0060] 4. Ultraviolet absorption spectroscopy detection: The 10 μM V was studied using ultraviolet absorption spectroscopy. 20 K 40 The change of L at 350 nm ultraviolet absorbance at different temperatures, such as Figure 7 As shown, the results indicate that V... 20 K 40 The UV absorbance of L remains almost unchanged, but increases rapidly above 35°C, at which point V... 20 K 40 L is in a clustered state. In summary, V 20 K 40 L responds to temperature, with the response temperature around 36°C. Above the response temperature, V... 20 K 40 L has a compact structure and a secondary structure with an α-helix.

[0061] Example 2: mRNA vaccine and its preparation method

[0062] (I) Extraction and purification of Melan-Am RNA

[0063] Melan-Am RNA was extracted and purified using an RNA extraction and purification kit. This part is existing technology and will not be described in detail here.

[0064] The purified Melan-Am RNA was subjected to gel electrophoresis and sequencing. The gel electrophoresis results of Melan-Am RNA are shown below. Figure 8 As shown, the results revealed a clear band at 500 bp, consistent with the molecular weight of Melan-Am RNA. The band from gel electrophoresis was transcribed in vitro, and the reverse-transcribed DNA was sequenced. The sequencing results are shown below. Figure 9The sequence of Melan-A mRNA is consistent with the sequence of the mRNA shown (the mRNA is capped for overall stability during preparation, and the capped position is at the base AGG, which becomes GGG after capping, but is actually AGG). In summary, Melan-A mRNA is extracted and purified successfully.

[0065] (II) V 20 K 40 L@MC vaccine preparation

[0066] To explore the optimal binding ratio of mRNA and V 20 K 40 L, the amount of mRNA (1 μg) and the amount of CpG (0.5 μg) were fixed, and 10, 20, 30, 40, 50, 60 μg of V 20 K 40 L was used to self-assemble with Melan-A mRNA and CpG to form V 20 K 40 L@MC vaccine, and different ratios of V 20 K 40 L@MC vaccine were characterized by gel electrophoresis. The results of gel electrophoresis are shown in Figure 10 The results show that 10, 20, 30 μg of V 20 K 40 L is not enough to completely encapsulate mRNA, and part of the mRNA leaks out, while 40 μg of V 20 K 40 L can completely encapsulate mRNA, so 40 μg of V 20 K 40 L is the optimal carrier amount, and the optimal ratio of mRNA:CpG:V 20 K 40 L is 1:0.5:40.

[0067] Therefore, the preparation process of V 20 K 40 L@MC vaccine is as follows:

[0068] Take 1.0 mg of mRNA and dissolve it in 1 mL of DEPC water to prepare mRNA stock solution; take 0.5 mg of CpG and dissolve it in 1 mL of DEPC water to prepare CpG stock solution; weigh 4.0 mg of V 20 K 40 L and dissolve it in 1 mL of ultrapure water to prepare V 20 K 40 L.

[0069] Take 10 μL of mRNA stock solution, 10 μL of CpG stock solution, and 100 μL of V 20 K 40L stock solution was mixed well. The mixture was quickly injected into 10 mL deionized water under ultrasonication and ultrasonicated for 2 min to obtain mRNA vaccine.

[0070] (Three) V 20 K 40 L@MC vaccine characterization

[0071] 1. Dynamic light scattering instrument was used to verify the size of V 20 K 40 L@MC and its temperature response properties.

[0072] The results are shown in Figure 11 , which show that the size of V 20 K 40 L@MC (mRNA concentration of 1 μg / mL) at 4℃ is 198.9 nm, and at 37℃ is 142.6 nm, which indicates that V 20 K 40 L@MC has temperature response, when the temperature is higher than Tt, the nanoparticles are in a compact state as a whole, resulting in a decrease in particle size, not only that, V 20 K 40 L@MC has good particle size distribution at both 4℃ and 37℃.

[0073] 2. In order to further explore the size and temperature response properties of V 20 K 40 L@MC (mRNA concentration of 1 μg / mL), transmission electron microscopy was used to characterize V 20 K 40 L@MC at different temperatures.

[0074] As shown in Figure 12 , the TEM images show that V 20 K 40 L@MC has a regular spherical structure at both 4℃ and 37℃, and the size of V 20 K 40 L@MC at 37℃ is smaller than that at 4℃, which is consistent with the results of dynamic light scattering instrument.

[0075] 3. Dynamic light scattering instrument was used to explore the stability of V 20 K 40 L@MC (mRNA concentration of 1 μg / mL) in different solvents.

[0076] The results are shown in Figure 13 , which show that V 20 K 40 L@MC has almost no change in particle size in PBS and ultrapure water for a week. In summary, V 20 K40 L@MC has higher particle size distribution and stability, and V 20 K 40 L@MC can be compacted into compact nanoparticles, at this time the particle size is reduced, and the leakage of mRNA is prevented.

[0077] Example 3: Elastin-like polypeptide V 20 K 40 Lapplication in the preparation of drug delivery system

[0078] V 20 K 40 L@MC is used for cell transfection of DC2.4 cells to verify the drug delivery effect of V 20 K 40 L, and compared with LNP@MC vaccine.

[0079] (I) Cell transfection

[0080] V 20 K 40 L@MC containing different concentrations (0, 125, 250, 500, 750, 1000 ng / mL) of mRNA and DC2.4 cells were incubated for 24 hours, and the level of Melan-A DNA in the cells was detected by PCR, and the expression level of Melan-A protein in the cells was detected by WB.

[0081] The PCR detection results are shown in Figure 14 As the mRNA concentration increases, the amount of Melan-A DNA also gradually increases, and when the mRNA concentration is 1000 ng / mL, the amount of Melan-A DNA in the cells significantly increases.

[0082] The WB detection results are shown in Figure 15 As the mRNA concentration increases, the expression level of Melan-A protein gradually increases, and when the mRNA concentration is 1000 ng / mL, the expression level of Melan-A protein in the cells significantly increases, which is consistent with the results of PCR, so 1000 ng / mL mRNA is used as the best experimental concentration for subsequent cell experiments.

[0083] (II) Lysosomal escape performance analysis

[0084] V 20 K 40 L@MC@Me-DPP containing 1000 ng / mL mRNA was incubated with DC2.4 cells, and the lysosomes of DC2.4 cells were stained with commercial lysosomal dye, and the co-localization of confocal fluorescence was used to explore V 20 K 40L@MC Me-DPP lysosome escape performance.

[0085] As shown in the results Figure 16 , V 20 K 40 The co-localization coefficient of L@MC Me-DPP and lysosome gradually increased before 6 hours, and gradually decreased after 6 hours to below 0.5, which indicated that V 20 K 40 L@MC Me-DPP had the ability to escape from lysosome, thereby increasing its transfection efficiency.

[0086] (Three) V 20 K 40 Stability analysis of L@MC

[0087] V 20 K 40 L@MC containing 1000 ng / mL GFP mRNA was placed at room temperature (26°C) for different times, and V 20 K 40 L@MC placed for different times was incubated with DC2.4 cells for 24 hours, and then confocal fluorescence microscopy was used to detect the fluorescence in DC2.4 cells, thereby analyzing the stability of V 20 K 40 L@MC.

[0088] As shown in the results Figure 17 , the transfection efficiency of V 20 K 40 L@MC remained basically unchanged within 7 days, indicating that V 20 K 40 L@MC-GFP had high stability.

[0089] (Four) Comparison of cell transfection effects of V 20 K 40 L@MC vaccine and LNP@MC vaccine

[0090] V 20 K 40 L@MC and LNP@MC containing 1000 ng / mL mRNA were respectively incubated with DC2.4 cells for 24 hours, and PCR was used to detect the level of Melan-A DNA in the cells, and WB was used to detect the level of Melan-A protein in the cells.

[0091] As shown in the PCR detection results Figure 18 , the level of Melan-A DNA in the cells treated by V 20 K 40 L@MC was about 10 times that of the cells treated by LNP@MC; and as shown in the WB detection resultsFigure 19 As shown in FIG. 6, the expression level of Melan-A protein in the cells treated by V 20 K 40 The expression level of Melan-A protein in the cells treated by L@MC is much higher than that in the cells treated by LNP@MC. This indicates that the V 20 K 40 The transfection effect of L@MC is much greater than that of the commercial LNP@MC.

[0092] Example 4: Preparation of the V 20 K 40 L and mRNA vaccine for use in the preparation of a tumor treatment drug

[0093] The V 20 K 40 L, V 20 K 40 L@MC were used in mouse experiments to detect the effect of mRNA vaccine in the preparation of a tumor treatment drug, and were compared with LNP and LNP@MC vaccine.

[0094] (I) Liver and kidney toxicity experiment

[0095] The V 20 K 40 L@MC and LNP@MC containing the same dose of mRNA (5 μg) were subcutaneously injected into 8-week-old healthy B57cl / 6 mice. After 24 hours, the peripheral blood was collected and the levels of liver function indicators ALT, AST and kidney function indicators BUN, CREA were analyzed. The results are shown in FIG. 7. Figure 20 As can be seen, compared with the PBS group, neither V 20 K 40 L@MC nor LNP@MC caused obvious liver and kidney toxicity, indicating that they are both relatively safe.

[0096] (II) Inflammation reaction experiment

[0097] The V 20 K 40 L and LNP were subcutaneously injected into 8-week-old healthy B57cl / 6 mice at a dose of 200 μg. After 24 hours, the peripheral blood was collected and the contents of GM-CSF, IL-6 and IL-1β were analyzed by ELISA. The results are shown in FIG. 8. Figure 21 As can be seen, compared with the PBS group, LNP caused obvious inflammatory reaction, while the pro-inflammatory effect of V 20 K 40 L was not obvious, indicating that the side effect of V 20 K 40 L may be lower.

[0098] (III) Memory T cell detection

[0099] V 20 K 40 L@MC and LNP@MC were inoculated to 8-week-old healthy B57cl / 6 mice for 3 times, and the Melan-A mRNA was inoculated at a dose of 5 μg, and the interval between inoculations was 5 days. After the inoculation was completed, the peripheral blood of the mice was taken, and the proportion of memory T cells (CD62L- / CD44+) in the blood was analyzed. The results are shown in Figure 22 , from which it can be seen that the memory T cells after V 20 K 40 L@MC inoculation were significantly higher than those of the LNP@MC and PBS groups, indicating that V 20 K 40 L@MC inoculation can obtain better immunity.

[0100] (Four) Melanoma treatment effect

[0101] V 20 K 40 L@MC (the total inoculation amount of Melan-A mRNA was 5 μg) and LNP@MC were inoculated to healthy B57cl / 6 mice for 3 times, and then B16-F10 cells were injected through the tail vein to construct a melanoma lung metastasis model. The observation was ended on the 20th day of tumor inoculation, the mice were dissected, and the metastatic cancer in the lungs was observed. The results are shown in Figure 23 and Figure 24 , it can be seen that V 20 K 40 L@MC and LNP@MC have a preventive effect on melanoma metastasis, and the effect of V 20 K 40 L@MC is obviously better than that of LNP@MC.

Claims

1. A pro-elastin polypeptide V 20 K 40 L characterized in that: The profinflammatory cytokine is IL-1β. 20 K 40 The amino acid sequence of L is set forth in SEQ ID NO:

1.

2. The profinetic polypeptide V of claim 1 20 K 40 A method of making a profinetic polypeptide L, comprising the steps of: comprising the steps of: The application relates to a method for preparing a recombinant elastin-like polypeptide V 20 K 40 L The recombinant plasmid coding the gene is expressed in an exogenous expression system, and the expression system is subjected to protein purification to obtain the elastin-like polypeptide V 20 K 40 L.

3. The method of claim 2, wherein: The elastin-like polypeptide V 20 K 40 The nucleotide sequence of the coding gene is shown in SEQ ID NO:

2.

4. An mRNA vaccine, characterized in that: The mRNA, CpG and the elastin-like polypeptide V of claim 1 20 K 40 L self-assemble.

5. The mRNA vaccine of claim 4, characterized in that: The mRNA, CpG and elastin-like polypeptide V 20 K 40 The mass ratio of mRNA:CpG:V 20 K 40 L=1:0.5:40~60.

6. The mRNA vaccine of claim 4, characterized in that: the mRNA is a Melan-A mRNA.

7. The method of producing the mRNA vaccine according to any one of claims 4 to 6, characterized in that: comprising the steps of: An aqueous solution of mRNA, an aqueous solution of CpG and a water-soluble elastin-like polypeptide V 20 K 40 L are mixed homogeneously, then rapidly injected into water under ultrasonication and continuously ultrasonicated, i.e. the mRNA vaccine is obtained.

8. The profinflammatory polypeptide V of claim 1 20 K 40 L for use in the manufacture of a drug delivery system, characterized in that: the drug in the drug delivery system is an mRNA.

9. Use of the mRNA vaccine of claim 6 for the manufacture of a medicament for the treatment of melanoma.

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