MRNA molecule of horse chorionic gonadotropin, recombinant expression vector, lipid nanoparticle preparation and application

Abstract

The invention discloses mRNA (messenger ribonucleic acid) molecules of horse chorionic gonadotropin, a recombinant expression vector, a lipid nanoparticle preparation and application. Relates to the technical field of therapeutic drugs, the mRNA molecule comprises sequences for coding an eCG protein alpha subunit and a beta subunit, the amino acid sequence of the alpha subunit is shown as SEQ ID NO.5, and the amino acid sequence of the beta subunit is shown as SEQ ID NO.6; the alpha subunit and the beta subunit are expressed by connecting the same mRNA molecule through an IRES sequence or a P2A sequence, or the alpha subunit and the beta subunit are connected through a Linker sequence or a Linker-Fold-Linker sequence or a Linker-GCN4-Linker. The technical scheme of the invention aims to design a novel mRNA molecule capable of improving the content of chorionic gonadotropin.

Description

Technical Field

[0001] The present invention relates to the technical field of therapeutic drugs, and particularly relates to an mRNA molecule of equine chorionic gonadotropin, a recombinant expression vector, a lipid nanoparticle preparation and applications thereof. Background Art

[0002] Chorionic gonadotropin (Equine chorionic gonadotropin, eCG) is a glycoprotein hormone secreted by the anterior pituitary gland, and its main function is to promote the growth and development of follicles in the ovary and the secretion of estrogen. Equine chorionic gonadotropin is a hormone composed of two polypeptide chains, including an α chain and a β chain. The α chain consists of approximately 92 amino acid residues. The α chain plays an important role in the structural stability of the hormone and receptor binding. The α chain forms a complete equine gonadotropin molecule by binding to the β chain, and then exerts its biological function. The β chain consists of approximately 119 amino acid residues. The β chain contains the bioactive region of gonadotropin and is crucial for activating receptors and regulating reproductive functions. The β chain is responsible for carrying the activity of gonadotropin in equine gonadotropin. The paired binding between the α chain and the β chain of equine gonadotropin determines its biological activity and function, ensuring that equine gonadotropin plays the correct role in regulating the physiological processes of female animals.

[0003] Chorionic gonadotropin plays an important role in the body. However, under normal circumstances, the body does not require additional supplementation. In certain specific situations, supplementation strategies for chorionic gonadotropin may be considered. 1. Assisted reproductive technology: In assisted reproductive technologies such as in vitro fertilization, it is used to promote the production of multiple mature follicles in the ovary. 2. Treatment of ovarian dysfunction: Such as premature ovarian failure or hypogonadotropic amenorrhea, it is used to promote follicle development and maturation. 3. Luteal insufficiency: It is used to promote the formation of the corpus luteum and improve the situation of insufficient progesterone secretion. In assisted reproductive technology, chorionic gonadotropin is often used to promote the synchronous maturation of multiple follicles to increase the pregnancy rate. In addition, chorionic gonadotropin can also be used to treat ovarian dysfunction, such as ovarian quiescence, persistent corpus luteum, follicular development arrest, etc. The research on chorionic gonadotropin is of great significance for understanding the reproductive endocrine mechanism of mammals and also provides a basis for the development of new reproductive auxiliary drugs.

[0004] Currently, the main strategy for expressing two proteins using mRNA technology is to express them separately. Since the α-chain and β-chain of chorionic gonadotropin are structurally related in space, designing an mRNA to express two proteins has great advantages. (1) Higher efficiency: Expressing two proteins through one mRNA can synthesize multiple proteins in a single transcription event, thus improving the efficiency of protein expression. This method of transcribing multiple functional proteins at once reduces waste of energy and time and increases the efficiency of protein biosynthesis. (2) Resource savings: Traditionally, each protein requires an independent mRNA for transcription and translation. Expressing two proteins through one mRNA can reduce the demand for intracellular resources, saving resources such as the cell's energy, time, and nucleotides. (3) Coordinate regulation: One mRNA encoding multiple proteins will cause these proteins to cooperate with each other during translation and synthesis, achieving better coordinate regulation. This synergistic effect helps maintain protein balance and assembly, and improves the stability and biological activity of the complex. (4) Reducing the need for transcriptional regulation: Expressing multiple proteins through one mRNA can reduce the need for transcriptional regulation because only one promoter and one transcriptional regulatory element are needed to regulate the expression of multiple proteins, simplifying the process of gene expression regulation. (5) Utilizing post-transcriptional modification: When one mRNA encodes multiple proteins, it may utilize post-transcriptional modification processes such as splicing or RNA editing to produce different protein variants. Such a strategy can provide more protein function diversity. There are currently two common methods: (1) Internal ribosome entry site (IRES) sequence: IRES is an intron sequence that can enable ribosomes on the mRNA to initiate translation at non-traditional positions (far from the 5' end), so that the same mRNA can encode multiple proteins. IRES sequences are usually present in the mRNA of some viruses and eukaryotes for achieving multiple protein expression. (2) Internal stop codon (T2A, P2A, etc.) sequence: The internal stop codon sequence is a strategy for achieving the simultaneous expression of multiple independent proteins by one mRNA through post-transcriptional cleavage. These sequences cause one protein to be released during translation, thus producing multiple proteins.

[0005] Currently, the hormones commonly used in veterinary medicine are mainly pregnant mare serum gonadotropin (PMSG), which is mainly used to stimulate the ovaries to produce additional follicles, promote the growth, development and maturation of follicles, and induce ovulation. These hormones can be used in a variety of animals, such as sows, cows, ewes, etc., to improve reproductive efficiency and conception rate. In addition, chorionic gonadotropin also has corresponding applications due to its similar function to pregnant mare serum gonadotropin. At present, the production method of PMSG is often to collect blood from pregnant mares for extraction and refinement, with a single production technology and unstable safety. At present, there is no clinical application of directly using mRNA technology for the supplementation of chorionic gonadotropin. In summary, developing a new method for supplementing chorionic gonadotropin through the mRNA pathway has multiple advantages such as high flexibility, safety and efficiency. Summary of the Invention

[0006] The technical solution of the present invention to solve the above technical problems is to provide an mRNA molecule of equine chorionic gonadotropin. The mRNA drug includes sequences encoding the α subunit and β subunit of the eCG protein. The amino acid sequence of the α subunit is as shown in SEQ ID NO.5, and the amino acid sequence of the β subunit is as shown in SEQ ID NO.6. The α subunit and β subunit are expressed by connecting with the same mRNA molecule through the IRES sequence or P2A sequence, or the α subunit and the β subunit are connected through the Linker sequence or Linker-Folden-Linker sequence or Linker-GCN4-Linker.

[0007] Further, when the α subunit and β subunit are expressed by connecting with the same mRNA molecule through the IRES sequence or P2A sequence, the mRNA sequence corresponding to the IRES connection method is as shown in SEQ ID NO.11, and the mRNA sequence corresponding to the P2A connection method is as shown in SEQ ID NO.12.

[0008] Further, the mRNA molecule of equine chorionic gonadotropin also includes the following elements:

[0009] 5' untranslated region (UTR), the nucleotide sequence is as shown in SEQ ID NO.1;

[0010] 3' untranslated region (UTR), the nucleotide sequence is as shown in SEQ ID NO.2;

[0011] Polyadenylate tail (poly A), the nucleotide sequence is as shown in SEQ ID NO.3;

[0012] The T7 promoter sequence is as shown in SEQ ID NO.4.

[0013] Further, when the α subunit and the β subunit are connected by a Linker sequence, a Linker-Folden-Linker sequence, or a Linker-GCN4-Linker, the mRNA sequence of eCG-Linker is as shown in SEQ ID NO.15, the mRNA sequence of eCG-Folden is as shown in SEQ ID NO.18, and the mRNA sequence of eCG-GCN4 is as shown in SEQ ID NO.21.

[0014] To solve the above technical problems, the present invention also provides a recombinant expression vector, which contains the DNA sequence corresponding to the above mRNA molecule. When the α subunit and the β subunit are expressed by the same mRNA molecule connected by an IRES sequence or a P2A sequence, the DNA sequence of eCG-IRES is as shown in SEQ ID NO.9; the DNA sequence of eCG-P2A is as shown in SEQ ID NO.10;

[0015] When the α subunit and the β subunit are connected by a Linker sequence, a Linker-Folden-Linker sequence, or a Linker-GCN4-Linker, the DNA sequence of eCG-Linker is as shown in SEQ ID NO.13; the DNA sequence of eCG-Folden is as shown in SEQ ID NO.16, and the DNA sequence of eCG-GCN4 is as shown in SEQ ID NO.19.

[0016] To solve the above technical problems, the present invention also provides a lipid nanoparticle preparation, which includes:

[0017] a. The above mRNA molecule;

[0018] b. An encapsulation carrier composed of cationic lipid, phospholipid, cholesterol, and PEG-modified lipid, where the mass ratio of each component is 50:10:38.5:1.5.

[0019] The application of the above mRNA molecule, the above recombinant expression vector, or the above lipid nanoparticle in the preparation of veterinary reproductive regulation drugs or superovulation drugs, where the reproductive regulation includes estrus synchronization, ovulation induction, or ovarian function recovery.

[0020] Further, the veterinary reproductive regulation drug is delivered by intramuscular injection or subcutaneous injection.

[0021] Compared with the prior art, the technical solution of the present invention has the following technical effects:

[0022] 1. The gene sequence is based on equine chorionic gonadotropin and chemically modified to enhance the stability of mRNA. To improve its translation and expression efficiency, the codons are optimized, and a T7 promoter, 5'UTR, and "Cap101" structure are added at the front end of the sequence. Elements such as 3'UTR and polyadenylate tail (poly A) are added at the back end of the mRNA sequence encoding the eCG protein. Two designed mRNAs can express two proteins through an IRES sequence and a P2A sequence respectively, or three mRNAs can achieve the fusion expression of two proteins through Linker, Linker-Folden-Linker sequence and Linker-GCN4-Linker sequence. On the one hand, it can reduce the mRNA dosage and enhance safety. On the other hand, it can promote the in vivo fusion of two subunits and enhance the drug activity.

[0023] 2. The plasmid is prepared into mRNA through in vitro transcription technology. The mRNA drug is obtained by packaging with lipid nanoparticles. The constructed candidate mRNA drug is used to immunize mice, and the results show that the estradiol hormone level in the mice significantly increases after immunization, and it can significantly promote the uterine development of mice. Brief Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0025] Figure 1 It is the electrophoresis result diagram of in vitro transcribed mRNA (Marker: RNA marker 6000; 1: eCG-IRES mRNA; 2: eCG-P2A mRNA; 3: eCG-Linker mRNA; 4: eCG-Folden mRNA; 5: eCG-GCN4 mRNA);

[0026] Figure 2It is a figure showing the verification results of in vitro transcribed mRNA protein expression (Marker: protein marker; 1: blank control; 2: intracellular detection of eCG-IRES mRNA; 3: detection of eCG-IRES mRNA in culture medium supernatant; 4: intracellular detection of eCG-P2A mRNA; 5: detection of eCG-P2A mRNA in culture medium supernatant; 6: intracellular detection of eCG-Folden mRNA; 7: intracellular detection of eCG-GCN4 mRNA; 8: blank control; 9: intracellular detection of eCG-Linker mRNA);

[0027] Figure 3 It is a figure showing the particle size analysis results after packaging of eCG LNP-mRNA (A: particle size detection results of eCG-IRES; B: particle size detection results of eCG-P2A; C: particle size detection results of eCG-Folden; D: particle size detection results of eCG-GCN4; E: particle size detection results of eCG-Linker);

[0028] Figure 4 It is a figure showing the serum estradiol content in mice immunized with eCG mRNA-LNP (*: P < 0.05 compared with the PMSG group; **: P < 0.01; ***: P < 0.001; #: P < 0.05 compared with the Mock group; ##: P < 0.01; : P < 0.001);

[0029] Figure 5 It is a statistical chart of the average weight of both ovaries in mice immunized with eCG mRNA;

[0030] Figure 6 It is a figure of ovarian pathological sections in mice immunized with eCG mRNA;

[0031] Figure 7 It is a statistical chart of the number of follicles at different developmental stages;

[0032] Figure 8 It is a figure of liver pathological sections in mice immunized with eCG mRNA;

[0033] Figure 9 It is a figure of spleen pathological sections in mice immunized with eCG mRNA;

[0034] Figure 10 It is a figure of kidney pathological sections in mice immunized with eCG mRNA. Detailed implementation manners

[0035] The present invention provides an mRNA molecule, a recombinant expression vector, a lipid nanoparticle preparation and an application thereof of equine chorionic gonadotropin, aiming to design a novel mRNA-based drug that can increase the content of chorionic gonadotropin.

[0036] The mRNA molecule, recombinant expression vector, lipid nanoparticle preparation and application of equine chorionic gonadotropin proposed by the present invention will be described in specific embodiments below:

[0037] Example 1:

[0038] An mRNA molecule of equine chorionic gonadotropin (eCG), the mRNA drug includes sequences encoding the α subunit and β subunit of the eCG protein, the α subunit and the β subunit are expressed by connection through an IRES sequence or a P2A sequence in the same mRNA molecule, the amino acid sequence of the α subunit is as shown in SEQ ID NO.5, and the amino acid sequence of the β subunit is as shown in SEQ ID NO.6.

[0039] Further, the mRNA sequence corresponding to the IRES connection method is as shown in SEQ ID NO.11, and the mRNA sequence corresponding to the P2A connection method is as shown in SEQ ID NO.12.

[0040] Further, an mRNA drug of equine chorionic gonadotropin (eCG) also includes the following elements:

[0041] 5' untranslated region (UTR), the nucleotide sequence is as shown in SEQ ID NO.1;

[0042] Open reading frame encoding eCG protein;

[0043] 3' untranslated region (UTR), the nucleotide sequence is as shown in SEQ ID NO.2;

[0044] Polyadenylate tail (poly A), the nucleotide sequence is as shown in SEQ ID NO.3;

[0045] The T7 promoter sequence is as shown in SEQ ID NO.4.

[0046] Example 2:

[0047] An mRNA molecule of equine chorionic gonadotropin (eCG), the mRNA drug includes sequences encoding the α subunit and β subunit of the eCG protein, the amino acid sequence of the α subunit is as shown in SEQ ID NO.5, the amino acid sequence of the β subunit is as shown in SEQ ID NO.6, and the α subunit and the β subunit are connected through a Linker sequence or a Linker-Folden-Linker sequence or a Linker-GCN4-Linker.

[0048] The mRNA sequence of eCG-Linker corresponding to the Linker sequence connection method is as shown in SEQ ID NO.15, the mRNA sequence of eCG-Folden corresponding to the Linker-Folden-Linker sequence connection method is as shown in SEQ ID NO.18, and the mRNA sequence of eCG-GCN4 corresponding to the Linker-GCN4-Linker sequence connection method is as shown in SEQ ID NO.21.

[0049] Example 3:

[0050] A recombinant expression vector comprising the DNA sequence corresponding to the mRNA molecule described in Example 1, wherein the DNA sequence is selected from SEQ ID NO.9 (IRES connection) or SEQ ID NO.10 (P2A connection).

[0051] Example 4:

[0052] A recombinant expression vector comprising the DNA sequence corresponding to the mRNA molecule described in Example 2. The DNA sequence of eCG-Linker is as shown in SEQ ID NO.13 (Linker connection); the DNA sequence of eCG-Folden is as shown in SEQ ID NO.16 (Linker-Folden-Linker connection), and the DNA sequence of eCG-GCN4 is as shown in SEQ ID NO.19 (Linker-GCN4-Linker connection).

[0053] Example 5:

[0054] A lipid nanoparticle (LNP) formulation comprising:

[0055] a. The mRNA drug described in Example 1 or Example 2;

[0056] b. An encapsulation vector composed of cationic lipid, phospholipid, cholesterol and PEG-modified lipid, wherein the molar ratio of each component is 50:10:38.5:1.5.

[0057] Example 6:

[0058] Use of the mRNA drug of Example 1 or Example 2, the recombinant expression vector of Example 3 or Example 4, or the lipid nanoparticle (LNP) formulation of Example 5 in the preparation of veterinary reproductive regulation drugs, wherein the reproductive regulation includes estrus synchronization, ovulation induction or ovarian function recovery;

[0059] The veterinary reproductive regulation drug is delivered by intramuscular injection or subcutaneous injection, and the single dose is 10 - 500 μg mRNA / kg body weight.

[0060] Example 7:

[0061] A veterinary reproductive regulation pharmaceutical composition, comprising:

[0062] The lipid nanoparticles of Example 3 or Example 4;

[0063] A pharmaceutically acceptable carrier;

[0064] A sucrose stabilizer with a final concentration of 5-15%.

[0065] Verification test:

[0066] 1. Construction of the eCG mRNA drug expression vector. The method for constructing the antigen expression vector plasmid is as follows:

[0067] (1) The following gene sequences were synthesized using Shanghai Sangon Biotech Co., Ltd.:

[0068] From the 5' to the 3' end are the T7 promoter (SEQ ID NO.4), 5'UTR (SEQ ID NO.1), eCG-IRES sequence (SEQ ID NO.9), 3'UTR (SEQ ID NO.2), and polyA (SEQ ID NO.3).

[0069] From the 5' to the 3' end are the T7 promoter (SEQ ID NO.4), 5'UTR (SEQ ID NO.1), eCG-P2A sequence (SEQ ID NO.10), 3'UTR (SEQ ID NO.2), and polyA (SEQ ID NO.3).

[0070] (2) The primers were synthesized using Beijing Tsingke Biotechnology Co., Ltd., as shown in Table 1.

[0071] Table 1 Primer sequence table:

[0072]

[0073] Using the above plasmid as a template, a PCR amplification reaction was performed to obtain the eCG gene sequence. The enzyme in the reaction system was PrimeSTAR Max DNA Polymerase from TAKARA Company. The reaction system is shown in Table 2.

[0074] Table 2 PCR reaction system table:

[0075]

[0076] (3) The PCR product was subjected to 1% agarose gel electrophoresis, and the PCR product was subjected to DNA recovery using the gel recovery kit from TransGen Company.

[0077] (4) Use the XhoⅠ and KpnⅠ enzymes from TransGen to perform double digestion on the DNA fragment and the vector, and use agarose gel electrophoresis to recover the DNA fragment of the target gene and the pUC57 vector fragment.

[0078] (5) For homologous recombination, use the T4 DNA ligase from Takara. Connect the pUC57 vector fragment recovered in (4) with the fragment recovered in (4), at 16 °C overnight. After the reaction, add it to the DH5α competent cells from TransGen, incubate on ice for 30 min, heat shock at 42 °C for 90 s, incubate on ice for 2 min. After the reaction, add 1 mL of LB medium without resistance, shake at 37 °C and 200 rpm for 30 min, centrifuge at 2000 rpm for 10 min. Finally, resuspend the cells with 100 μL of LB and spread them on a kanamycin-resistant solid agar culture plate, incubate at 37 °C for 16 hours, and pick colonies for overnight culture.

[0079] (6) Use the DNA miniprep kit from TransGen to perform a small-scale plasmid extraction on the bacterial solution. The extracted plasmid is identified by double digestion using the XhoⅠ and KpnⅠ restriction endonucleases, and further sequenced by Beijing Tsingke to confirm that the recombinant plasmid sequence is correct.

[0080] 2. Synthesize eCG mRNA:

[0081] Digest the plasmid obtained above with the BsaI restriction endonuclease from Yeasen Biotech to prepare a linearized plasmid. The reaction system is shown in Table 3. After reacting at 37.0 °C for 1 hour, use the DNA recovery kit from TransGen to recover the DNA.

[0082] Table 3 Plasmid linearization reaction system

[0083]

[0084] For in vitro transcription, use the T7 Transcription Kit product from Yeasen Biotech. The reaction system is shown in Table 4. Add the cap analog (Cap101) from Cangzhou Wickxen Bio to the reaction system to add a cap structure to the synthesized mRNA. Set the synthesis conditions to 37 °C for 2 h. After the reaction, add 1.5 times the reaction system volume of 7.5 M lithium chloride to the synthesis product, centrifuge at 12000 rpm for 20 min to precipitate the mRNA, resuspend the mRNA with DEPC water, measure the RNA concentration, and store the final mRNA product at -70 °C or below.

[0085] Table 4 In vitro transcription reaction system

[0086]

[0087]

[0088] Verify the quality of in vitro transcribed mRNA by 1% agarose gel electrophoresis. The results are as Figure 1 shown. The sizes of the two mRNA bands transcribed in vitro are correct, clear, and single, demonstrating that the synthesized mRNA has good quality.

[0089] The DNA sequence of the 5'-untranslated region (5'UTR) is shown in SEQ ID NO.1:

[0090] GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC GCTAGCCTCGAG.

[0091] The DNA sequence of the 3'-untranslated region (3'UTR) is shown in SEQ ID NO.2:

[0092] GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCC TTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTG GTCTTTGAATAAAGTCTG.

[0093] The polyadenylic acid (poly A) has a sequence shown in SEQ ID NO.3 and contains 104 adenine bases:

[0094] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA.

[0095] The DNA sequence of the T7 promoter is shown in SEQ ID NO.4:

[0096] TAATACGACTCACTATAGG.

[0097] The eCG-α amino acid sequence is shown in SEQ ID NO.5:

[0098] MDYYRKHAAVILATLSVFLHILHSFPDGEFTTQDCPECKLRENKYFFK LGVPIYQCKGCCFSRAYPTPARSRKTMLVPKNITSESTCCVAKAFIRVTVMG NIKLENHTQCYCSTCYHHKIDYKDDDDK.

[0099] The eCG-β amino acid sequence is shown in SEQ ID NO.6:

[0100] METLQGLLLWMLLSVGGVWASRGPLRPLCRPINATLAAEKEACPICITFTTSICAGYCPSMVRVMPAALPAIPQPVCTYRELRFASIRLPGCPPGVDPMVSFPVALSCHCGPCQIKTTDCGVFRDQPLACAPQASSSSKDPPSQPLTSTSTPTPGASRRSSHPLPIKTSDYKDDDDK。

[0101] The eCG-α DNA sequence is shown in SEQ ID NO.7:

[0102] ATGGACTACTACCGCAAGCATGCTGCTGTGATACTGGCAACACTGTCAGTGTTTCTGCACATACTGCACAGCTTTCCAGATGGTGAGTTCACCACACAAGACTGTCCAGAGTGCAAGCTGAGAGAGAACAAGTACTTCTTCAAGCTGGGTGTGCCAATCTACCAGTGCAAAGGCTGTTGCTTCTCCCGCGCTTACCCTACACCAGCTAGAAGCCGAAAGACCATGCTGGTGCCAAAGAACATCACCTCTGAGAGCACCTGTTGTGTGGCTAAGGCCTTCATCAGGGTGACTGTGATGGGCAACATCAAGCTGGAGAACCACACACAGTGCTACTGCTCAACCTGCTACCACCACAAGATCGACTACAAAGACGACGACGATAA。

[0103] The eCG-β DNA sequence is shown in SEQ ID NO.8:

[0104] ATGGAGACACTGCAAGGTCTGCTGTTGTGGATGCTGCTGTCTGTGGGTGGAGTGTGGGCTTCTCGCGGACCTTTGAGGCCTCTGTGTAGGCCTATCAACGCTACACTGGCTGCTGAGAAAGAGGCTTGCCCTATCTGCATCACCTTCACCACCAGCATCTGTGCCGGATACTGCCCTTCTATGGTGAGAGTGATGCCTGCTGCATTGCCTGCTATACCTCAACCTGTGTGCACCTACAGAGAGCTGAGATTCGCCTCTATCAGACTGCCTGGATGTCCACCTGGAGTGGACCCAATGGTGAGCTTTCCAGTGGCTTTGTCTTGCCACTGTGGACCTTGTCAGATCAAGACCACCGATTGTGGCGTGTTCAGGGATCAACCTCTGGCTTGTGCTCCACAGGCTTCTAGCAGCAGCAAAGATCCACCTTCTCAGCCTCTGACCTCTACCAGCACACCTACACCTGGAGCTTCCAGAAGGTCTAGCCATCCACTGCCTATCAAGACCTCCGACTACAAAGACGACGATGACAAGTAA。

[0105] The eCG-IRES DNA sequence is shown in SEQ ID NO.9:

[0106]

[0107] The eCG-P2A DNA sequence is shown in SEQ ID NO. 10:

[0108] ATGGACTACTACCGCAAGCATGCTGCTGTGATACTGGCAACACTGTCAGTGTTTCTGCACATACTGCACAGCTTTCCAGATGGTGAGTTCACCACACAAGACTGTCCAGAGTGCAAGCTGAGAGAGAACAAGTACTTCTTCAAGCTGGGTGTGCCAATCTACCAGTGCAAAGGCTGTTGCTTCTCCCGCGCTTACCCTACACCAGCTAGAAGCCGAAAGACCATGCTGGTGCCAAAGAACATCACCTCTGAGAGCACCTGTTGTGTGGCTAAGGCCTTCATCAGGGTGACTGTGATGGGCAACATCAAGCTGGAGAACCACACACAGTGCTACTGCTCAACCTGCTACCACCACAAGATCGACTACAAAGACGACGACGATAAGGCCACCAACTTCTCTCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAATCCGGGACCTATGGAGACACTGCAAGGTCTGCTGTTGTGGATGCTGCTGTCTGTGGGTGGAGTGTGGGCTTCTCGCGGACCTTTGAGGCCTCTGTGTAGGCCTATCAACGCTACACTGGCTGCTGAGAAAGAGGCTTGCCCTATCTGCATCACCTTCACCACCAGCATCTGTGCCGGATACTGCCCTTCTATGGTGAGAGTGATGCCTGCTGCATTGCCTGCTATACCTCAACCTGTGTGCACCTACAGAGAGCTGAGATTCGCCTCTATCAGACTGCCTGGATGTCCACCTGGAGTGGACCCAATGGTGAGCTTTCCAGTGGCTTTGTCTTGCCACTGTGGACCTTGTCAGATCAAGACCACCGATTGTGGCGTGTTCAGGGATCAACCTCTGGCTTGTGCTCCACAGGCTTCTAGCAGCAGCAAAGATCCACCTTCTCAGCCTCTGACCTCTACCAGCACACCTACACCTGGAGCTTCCAGAAGGTCTAGCCATCCACTGCCTATCAAGACCTCCGACTACAAAGACGACGATGACAAGTAA。

[0109] The eCG-IRES mRNA sequence is shown in SEQ ID NO.11:

[0110]

[0111] The eCG-P2A mRNA sequence is shown in SEQ ID NO. 12:

[0112]

[0113] The eCG-linker DNA sequence is shown in SEQ ID NO. 13:

[0114] ATGGACTACTACCGCAAGCACGCTGCCGTGATTCTGGCCACACTGA

[0115] GCGTGTTCTTGCACATTCTGCACAGCTTTCCAGACGGCGAGTTCACCAC

[0116] ACAAGACTGTCCAGAGTGCAAGCTGCGCGAGAACAAGTACTTCTTCAA

[0117] GCTGGGCGTGCCTATCTACCAGTGCAAGGGCTGCTGCTTCAGCAGAGCC

[0118] TACCCTACACCAGCCAGAAGCAGAAAGACCATGCTGGTGCCTAAGAAC

[0119] ATCACAAGCGAGAGCACCTGTTGCGTGGCCAAAGCCTTCATCAGAGTG

[0120] ACCGTGATGGGCAACATCAAGCTGGAGAACCACACACAGTGCTACTGC

[0121] AGCACCTGCTACCACCACAAGATCGGCGGAGGCGGATCTGGCGGCGGC

[0122] GGAAGCGGCGGTGGAGGCAGCAGAGGTCCACTCAGACCTCTGTGCAGA

[0123] CCTATCAACGCTACACTGGCTGCCGAGAAAGAGGCTTGTCCAATCTGCA

[0124] TCACCTTCACCACCAGCATCTGCGCCGGCTACTGTCCAAGCATGGTGAG

[0125] GGTGATGCCTGCCGCCTTGCCTGCCATTCCACAGCCTGTGTGCACCTAC

[0126] AGGGAGCTGAGGTTCGCCAGCATCAGGCTGCCTGGCTGTCCACCTGGC

[0127] GTTGATCCAATGGTGTCATTTCCAGTGGCTCTGAGCTGTCACTGCGGACC

[0128] TTGCCAGATCAAGACCACCGACTGTGGCGTGTTCAGAGATCAGCCACTG

[0129] GCTTGTGCTCCACAGGCCAGCAGCTCCAGCAAAGATCCACCTTCTCAGC

[0130] CTCTGACCAGCACCAGCACACCTACACCTGGAGCCTCTAGGCGCAGCA

[0131] GTCACCCTCTGCCTATCAAGACCTCCGACTACAAAGACGACGACGACAAGTGA。

[0132] The amino acid sequence of eCG-linker is shown in SEQ ID NO.14:

[0133] MDYYRKHAAVILATLSVFLHILHSFPDGEFTTQDCPECKLRENKYFFKLGVPIYQCKGCCFSRAYPTPARSRKTMLVPKNITSESTCCVAKAFIRVTVMGNIKLENHTQCYCSTCYHHKIGGGGSGGGGSGGGGSRGPLRPLCRPINATLAAEKEACPICITFTTSICAGYCPSMVRVMPAALPAIPQPVCTYRELRFASIRLPGCPPGVDPMVSFPVALSCHCGPCQIKTTDCGVFRDQPLACAPQASSSSKDPPSQPLTSTSTPTPGASRRSSHPLPIKTSDYKDDDDK

[0134] The mRNA sequence of eCG-linker is shown in SEQ ID NO.15:

[0135]

[0136] The eCG-Folden DNA sequence is as shown in SEQ ID NO. 16:

[0137]

[0138] The amino acid sequence of eCG-Folden is shown in SEQ ID NO. 17:

[0139] MDYYRKHAAVILATLSVFLHILHSFPDGEFTTQDCPECKLRENKYFFKLGVPIYQCKGCCFSRAYPTPARSRKTMLVPKNITSESTCCVAKAFIRVTVMGNIKLENHTQCYCSTCYHHKIGGGGSGGGGSGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSGGGGSGGGGSRGPLRPLCRPINATLAAEKEACPICITFTTSICAGYCPSMVRVMPAALPAIPQPVCTYRELRFASIRLPGCPPGVDPMVSFPVALSCHCGPCQIKTTDCGVFRDQPLACAPQASSSSKDPPSQPLTSTSTPTPGASRRSSHPLPIKTSDYKDDDDK。

[0140] The mRNA sequence of eCG-Folden is shown in SEQ ID NO. 18:

[0141]

[0142] The eCG-GCN4 DNA sequence is shown in SEQ ID NO. 19:

[0143]

[0144] The amino acid sequence of eCG-GCN4 is shown in SEQ ID NO. 20:

[0145] MDYYRKHAAVILATLSVFLHILHSFPDGEFTTQDCPECKLRENKYFFKLGVPIYQCKGCCFSRAYPTPARSRKTMLVPKNITSESTCCVAKAFIRVTVMGNIKLENHTQCYCSTCYHHKIGGGGSGGGGSGGGGSRMKQIEDKIEEILSKQYHIENEIARIKKLIGGGGGSGGGGSGGGGSRGPLRPLCRPINATLAAEKEACPICITFTTSICAGYCPSMVRVMPAALPAIPQPVCTYRELRFASIRLPGCPPGVDPMVSFPVALSCHCGPCQIKTTDCGVFRDQPLACAPQASSSSKDPPSQPLTSTSTPTPGASRRSSHPLPIKTSDYKDDDDK。

[0146] The mRNA sequence of eCG-GCN4 is shown in SEQ ID NO. 21:

[0147]

[0148] 3. Preparation of mRNA drugs:

[0149] After packaging the mRNA molecules synthesized in Example 1 and Example 2 above with lipid nanoparticles, LNP-mRNA drugs were prepared respectively.

[0150] The experimental operations are as follows:

[0151] Preparation of the alcohol phase: Dissolve the lipids in absolute ethanol according to the ratio of cationic lipid (SM102): distearoyl phosphatidylcholine (DSPC): cholesterol: DMG-PEG2000 = 50:10:38.5:1.5 (molar ratio). Preparation of the aqueous phase: Dilute the mRNA with a citric acid buffer solution (50 mM) with pH = 4.0; according to the volume ratio of the alcohol phase to the aqueous phase (1:3), use a microfluidic device to package the mRNA. After packaging, dilute the encapsulated solution with RNase-free PBS (pH = 7.4) buffer solution, concentrate it with a 30 kDa ultrafiltration tube, and finally change the solution to RNase-free PBS (pH = 7.4). Finally, add an equal volume of PBS solution with a sucrose concentration of 20%, adjust the mRNA concentration to 100 μg / ml, the sucrose concentration to 10%, and filter it with a 0.22 μm filter membrane to finally obtain the eCGLNP-mRNA drug. After aliquoting, store it at -20°C.

[0152] The liposome nanoparticles described above contain at least one of cationic lipid, distearoyl phosphatidylcholine (DSPC), cholesterol, and DMG-2000.

[0153] The detection results of the particle size distribution by dynamic light scattering showed that the two eCG mRNA-LNP drug particles after preparation were uniform, and the particle sizes were all around 100 nm ( Figure 2 ).

[0154] Expression verification of eCG protein:

[0155] Add the five prepared eCG mRNA-LNPs to HEK-293T cells for expression verification. Spread 293T cells evenly in a 24-well plate, and add 10 μl of mRNA to each well. After 48 h, collect the transfected cells and supernatant, add loading buffer to process the samples, perform SDS-PAGE electrophoresis, and incubate them successively with a mouse anti-FLAG tag antibody from TransGen and an HRP-labeled goat anti-mouse antibody. After the incubation, develop the color to obtain the Western Blot result. The result is as Figure 3As shown, after the mRNA-LNPs expressing the two subunits were added to the cells, bands consistent in size with the eCGβ subunit appeared inside the cells, and protein bands consistent in size with the eCGα subunit appeared in the supernatant, all in line with expectations, indicating that the two eCG mRNA-LNPs prepared in Example 1 and Example 2 could be expressed inside the cells; after the three mRNA-LNPs expressing the fusion protein were added to the cells, bands consistent in size with the target protein appeared inside the cells, indicating that the three eCG mRNA-LNPs could be expressed inside the cells.

[0156] The prepared eCG mRNA-LNP was used to immunize mice, and its effect in vivo was observed. 35 8-week-old SPF Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and randomly divided into 7 groups, 5 mice per group (as shown in Table 3), and immunized with 10 μg of the mRNA drug or 100 μL of PBS by intramuscular injection in the leg, and the positive control group was intraperitoneally injected with 20 U of pregnant mare serum gonadotropin (PMSG).

[0157] Table 3 Grouping table:

[0158]

[0159]

[0160] The increase in the content of PMSG and eCG in mice will promote the secretion of sex hormones by mice. Therefore, on the 3rd day after immunization, the blood of the mice was collected and the serum was separated, and the estradiol content in the serum was detected by ELISA (Shanghai Enzyme-linked). The results are as Figure 4 shown. On the 3rd day after the five groups of eCG mRNA-LNP and PMSG immunized the mice, the estradiol content in the mouse serum increased significantly. The estradiol content in the serum of the negative control group mice was about 25 pmol / L, while that of the positive control group injected with PMSG was about 34 pmol / L, higher than the blank control group (P<0.01), and the five groups of eCG mRNA-LNP immunized groups were higher than the PMSG group. Among them, the eCG-Folden group was the highest, reaching about 43 pmol / L, all significantly higher than the positive control group (P<0.001), indicating that the five groups of eCG mRNA-LNP had significant effects ( Figure 4 ). Three days after injection, the mice were sacrificed, and the ovarian tissues of the mice were separated and weighed. The results are as Figure 5As shown, compared with the blank control, the ovarian weights of the five groups of mice injected with eCG mRNA-LNP were significantly increased, indicating that the five groups of mRNA-LNP could significantly promote ovarian development in mice. Further, the ovaries of the mice were sectioned and observed, and the number of follicles at each stage was counted. The results showed that the five groups of mRNA-LNP could significantly promote the development of oocytes in the ovaries of mice, manifested as an increase in the number of growing follicles and cystic follicles, and an increase in the number of corpora lutea ( Figures 6 - 7 ). Finally, the safety of eCG mRNA-LNP was observed. The results of pathological section observations of different tissues showed that there was no obvious damage to the organs such as the liver, spleen, and kidney of the mice after immunization with the five groups of eCG mRNA-LNP, indicating that the five groups of eCG mRNA drugs had high safety ( Figures 8 - 10 ).

[0161] As described above, only the specific preferred embodiments of the present invention are provided, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. An mRNA molecule of equine chorionic gonadotropin, characterized in that, The mRNA drug includes sequences encoding the α subunit and β subunit of the eCG protein. The amino acid sequence of the α subunit is as shown in SEQ ID NO.5, and the amino acid sequence of the β subunit is as shown in SEQ ID NO.

6. The α subunit and β subunit are expressed by connection through an IRES sequence or P2A sequence on the same mRNA molecule, or are connected between the α subunit and the β subunit through a Linker sequence or Linker-Folden-Linker sequence or Linker-GCN4-Linker.

2. The mRNA molecule of equine chorionic gonadotropin according to claim 1, characterized in that, When the α subunit and β subunit are expressed by connection through an IRES sequence or P2A sequence on the same mRNA molecule, the mRNA sequence corresponding to the IRES connection mode is as shown in SEQ ID NO.11, and the mRNA sequence corresponding to the P2A connection mode is as shown in SEQ ID NO.

12.

3. The mRNA molecule of equine chorionic gonadotropin according to claim 2, characterized in that, It also includes the following elements: 5' untranslated region (UTR), with a nucleotide sequence as shown in SEQ ID NO.1; 3' untranslated region (UTR), with a nucleotide sequence as shown in SEQ ID NO.2; Polyadenylate tail (poly A), with a nucleotide sequence as shown in SEQ ID NO.3; T7 promoter sequence as shown in SEQ ID NO.

4.

4. The mRNA molecule of equine chorionic gonadotropin according to claim 1, characterized in that, When the α subunit and the β subunit are connected through a Linker sequence or Linker-Folden-Linker sequence or Linker-GCN4-Linker, the mRNA sequence of eCG-Linker is as SEQ ID NO.15, the mRNA sequence of eCG-Folden is as SEQ ID NO.18, and the mRNA sequence of eCG-GCN4 is as SEQ ID NO.

21.

5. A recombinant expression vector, characterized in that, It contains the DNA sequence corresponding to the mRNA drug described in claim 1. When the α subunit and β subunit are expressed by connection through an IRES sequence or P2A sequence on the same mRNA molecule, the DNA sequence of eCG-IRES is as shown in SEQ ID NO.9; the DNA sequence of eCG-P2A is as shown in SEQ ID NO.10; When the α subunit and the β subunit are connected through a Linker sequence or Linker-Folden-Linker sequence or Linker-GCN4-Linker, the DNA sequence of eCG-Linker is as shown in SEQ ID NO.13; the DNA sequence of eCG-Folden is as shown in SEQ ID NO.16, and the DNA sequence of eCG-GCN4 is as shown in SEQ ID NO.

19.

6. A lipid nanoparticle formulation, characterized in that, It contains: a. The mRNA molecule as described in claim 1; b. An encapsulating carrier composed of cationic lipid, phospholipid, cholesterol, and PEG-modified lipid, where the molar ratio of each component is 50:10:38.5:1.

5.

7. Use of the mRNA molecule according to claim 1, the recombinant expression vector according to claim 5, or the lipid nanoparticle according to claim 6 in the preparation of a veterinary reproductive regulation drug or a superovulation drug, wherein the reproductive regulation includes estrus synchronization, ovulation induction, or ovarian function restoration.

8. The application according to claim 7, wherein The veterinary reproductive regulation drug is delivered by intramuscular injection or subcutaneous injection.

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