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CN-121975822-A - MRNA vaccine, preparation method and application thereof, LNP-mRNA preparation and preparation method thereof

CN121975822ACN 121975822 ACN121975822 ACN 121975822ACN-121975822-A

Abstract

The invention relates to an mRNA vaccine and a preparation method and application thereof, an LNP-mRNA preparation and a preparation method thereof, and relates to the technical field of biological preparations, wherein the mRNA vaccine comprises a sequence segment derived from a TP0435 gene, or the gene sequence of the mRNA vaccine comprises a sequence segment derived from a TP0954 gene. The invention designs a syphilis mRNA vaccine which has remarkable beneficial effects in the aspects of syphilis prevention, gene sequence design, preparation process, delivery system, application prospect and the like, provides a new technical means for preventing syphilis infection, and has wide application prospect.

Inventors

  • WANG QIANQIU
  • ZHANG RUILI
  • LU ZHIYU
  • LU YIZHOU
  • DU FANGZHI
  • ZHANG XU
  • ZHENG XIAOLI
  • LI MENGYU

Assignees

  • 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所)

Dates

Publication Date
20260505
Application Date
20260204

Claims (10)

  1. 1. An mRNA vaccine comprising a sequence segment derived from the TP0435 gene, or wherein the gene sequence of the mRNA vaccine comprises a sequence segment derived from the TP0954 gene.
  2. 2. The method of claim 1, wherein the sequences of TP0435 to which the 5'UTR and 3' UTR regions and POLYA tails have been added are shown in SEQ ID NO. 1; and/or TP0954 sequence with 5'UTR and 3' UTR regions and POLYA tail added is shown in SEQ ID NO. 2.
  3. 3. A method for preparing the mRNA vaccine of any one of claims 1 to 3, wherein the method for preparing the mRNA vaccine comprises the following steps: Synthesizing a recombinant plasmid containing a TP0435 gene sequence or a TP0954 gene sequence; Transforming, screening, amplifying and extracting the recombinant plasmid by using Stabl3 competent cells, and linearizing the recombinant plasmid by using BspQI enzyme to obtain a linearization product; and (3) carrying out in vitro transcription, capping and purification on the linearization product serving as a template of in vitro transcription under an in vitro transcription system to obtain the mRNA vaccine.
  4. 4. The method of claim 3, wherein the steps of transforming, screening, amplifying and extracting the recombinant plasmid using Stabl3 competent cells comprise the steps of: The method comprises the steps of taking Stabl3 competent cells, adding recombinant plasmids, carrying out heat shock after ice bath, adding the heat shock-treated cells into a liquid LB culture medium after ice bath, culturing at 35-40 ℃ for 8-12 h, absorbing the cultured bacterial liquid, adding a solid LB culture medium containing 0.08-0.12% of kanamycin by mass, uniformly coating a plate, inverting, culturing at 35-40 ℃ for 8-12 h, picking colonies, adding the liquid LB culture medium containing 0.08-0.12% of kanamycin by mass, culturing at 35-40 ℃ for 8-12 h by shaking, absorbing the bacterial liquid, adding the liquid LB culture medium containing 0.08-0.12% of kanamycin by shaking at 35-40 ℃ for 8-12 h, and carrying out plasmid extraction.
  5. 5. The method for preparing an mRNA vaccine according to claim 3, wherein the step of linearizing BspQI enzyme to obtain a linearized product comprises the steps of: adding enzyme-free water into the linearization system, and reacting for 2-4 hours at 35-40 ℃; The linearization system comprises 15-25 mu g of recombinant plasmid, 15-25 mu L of BspQI restriction enzyme and 35-45 mu L of Cutting Buffer.
  6. 6. A method of preparing an mRNA vaccine according to claim 3, wherein the step of in vitro transcribing, capping and purifying the linearized product as a template for in vitro transcription under an in vitro transcription system, to obtain the mRNA vaccine comprises the steps of: Adding enzyme-free water into an in-vitro transcription system, reacting for 4-5 hours at 35-40 ℃, and purifying RNA magnetic beads to obtain mRNA; Wherein the in vitro transcription system comprises 40-50 mu L of T7 RNA polymerase, 15-25 mu L, RNA enzyme inhibitor of 10X Transcription Buffer-45 mu L, YY pyrophosphatase, 15-25 mu L of 100mM UTP 30-40 mu L, 30-40 mu L of 100mM ATP, 30-40 mu L of 100mM GTP, 30-40 mu L of 100mM CTP, 40-50 mu L of cap structural analogue and 35-45 mu g of linearization DNA.
  7. 7. Use of an mRNA vaccine according to any one of claims 1 to 3 or prepared by a preparation method according to any one of claims 4 to 6 for the preparation of a syphilis vaccine.
  8. 8. An LNP-mRNA preparation, characterized in that the LNP-mRNA preparation comprises the mRNA vaccine of any one of claims 1 to 3 or prepared by the preparation method of any one of claims 4 to 6.
  9. 9. The LNP-mRNA formulation of claim 8, wherein the LNP-mRNA formulation further comprises lipid nanoparticles coating the mRNA vaccine; and/or the PDI of the LNP-mRNA preparation is 0.05-0.11; And/or, the encapsulation efficiency of the LNP-mRNA preparation is more than or equal to 94.7%.
  10. 10. A method for preparing an LNP-mRNA preparation according to any one of claims 8 to 9, characterized in that the method for preparing an LNP-mRNA preparation comprises the steps of: Preparing a lipid nanoparticle solution; Coating the mRNA vaccine by adopting the lipid nanoparticle solution, and then filtering and centrifuging to obtain the LNP-mRNA preparation; the lipid nanoparticle solution comprises 0.6-0.7 g of SM-102, 0.05-0.06 g of PEG, 0.1-0.2 g of DSPC and 0.2-0.3 g of cholesterol.

Description

MRNA vaccine, preparation method and application thereof, LNP-mRNA preparation and preparation method thereof Technical Field The invention relates to the technical field of biological preparations, in particular to an mRNA vaccine, a preparation method and application thereof, an LNP-mRNA preparation and a preparation method thereof. Background Syphilis has long posed a serious threat to human health as a global public health problem. The traditional Chinese medicine composition has wide spreading range, is popular in different areas and different crowds, not only affects the reproductive health of patients, but also can cause a series of serious complications, such as cardiovascular syphilis, nervous syphilis and the like, brings great body pain and economic burden to the patients, and also causes heavy pressure on social public health resources. Among them, vaccination is one of the most effective and economical means of preventing and controlling infectious diseases. Therefore, the research of the syphilis vaccine is extremely important in practical significance and urgency. Currently, research on treponema pallidum (Tp) vaccines covers a variety of types, where mRNA vaccines exhibit unique advantages, firstly high mRNA vaccine safety without the potential risk of infection or insertional mutation. Secondly, mRNA vaccine is very efficient, and various modifications improve the stability and translation efficiency of mRNA. In addition, the mRNA vaccine has high productivity, has the potential of being quick, low-cost and capable of mass production, and is an important prevention and treatment measure for treating sudden acute infectious diseases. Disclosure of Invention In order to solve the problems, the invention provides an mRNA vaccine, a preparation method and application thereof, an LNP-mRNA preparation and a preparation method thereof. In a first aspect, the invention provides an mRNA vaccine comprising a sequence segment derived from the TP0435 gene, or the gene sequence of the mRNA vaccine comprises a sequence segment derived from the TP0954 gene. Further, the TP0435 sequence added with the 5'UTR and 3' UTR regions and POLYA tails is shown in SEQ ID NO:1, and the TP0435 sequence (added with the 5'UTR and 3' UTR regions and POLYA tails): TAATACGACTCACTATAAGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAAAGGATCTGTCCGCGCGCTGTGCGCGTTCCTTGGTGTTGGAGCGCTCGGTAGCGCTTTGTGTGTCTCGTGCACAACCGTGTGTCCGCACGCCGGGAAGGCCAAAGCGGAAAAGGTAGAGTGCGCGTTGAAGGGAGGTATCTTTCGGGGTACGCTACCTGCGGCCGATTGCCCGGGAATCGATACGACTGTGACGTTCAACGCGGATGGCACTGCGCAAAAGGTAGAGCTTGCCCTTGAGAAGAAGTCGGCACCTTCTCCTCTTACGTATCGCGGTACGTGGATGGTACGTGAAGACGGAATTGTCGAACTCTCGCTTGTGTCCTCGGAGCAATCGAAGGCACCGCACGAGAAAGAGCTGTACGAGCTGATAGACAGTAACTCCGTTCGCTACATGGGCGCTCCCGGCGCAGGAAAGCCTTCAAAGGAGATGGCGCCGTTTTACGTGCTCAAAAAAACAAAGAAATAGTGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAGAAGAGC. And/or TP0954 sequence with added 5'UTR and 3' UTR region and POLYA tail as shown in SEQ ID NO. 2, TP0954 sequence with added 5'UTR and 3' UTR region and POLYA tail ）：TAATACGACTCACTATAAGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCGGTATGGCACTCTTTTTAAGGTGAGTGTGCTCTTAGGTGCGCTCTTTGTATCTTGTGTTAGTACTGGATCGAACAGCGCTCGGGAGAGCGAGCGGGCACAGCTTTTAAAAAGCGAAAATCCTAATATCCGTTTTGCAGCTCAGCTCAGTGGGTTGTTGGAAAAGCAGCGGTGGGAAGAGGCGTTGCAGCTGTTCGATACGTTGAGCCCTGAGCATCGTGCCGAAAAGCGCATCCAATACCTGTATCTTTCTACGCTCATTTCCGCAGGGAAATTGACGCACGCGCAGGAGCTTGCAGAAAAGCTTGGGCAGGACGGTCCTACGGCTTCTGAAACGGTGCAACTGTGGTACGCACACGCAATGATAGCGCAAGCGAAACGAGATGTGCGCAAGAAGAAACAGTATGTAGAAAAGATTCTTGCGCAGGATCCGCACGATCTGTGGGCACTGACTGAACGCGGTTACGATTTTTTAAGTGTTAATGATTATGCCCAGGCGGTGCAGGCTTTCTCGCGCGCGCTGCGCGTGGAGCCGCGTGCGCAGGACGCGCGGGTAGGTTTGGGGAAGGTGTACTATCTGCAGGGAAAGATGCAAGAGGCAGAGGCACAGTACCGACAGGTACTGCAGGATACGCCTGAACATGAGCGCGCGCTTGCAGAATGTGCGCGGGTGAAAGCGGAAACGAACCGAGTGCTCGAAGCTATTAGGGATCTGGAACGTGTGGTGCAGCTTGATCCGCATGATCCTGCATATTGGACAGATCTCGGCACGTATTTGTCCCAGGCGGGAAAAAAGGAGCGTGCGGCAGCGGCGTTCGAGCGCGCAGTGGCGCTTTCTGCTGATGCGTATTTTGCCCACATTTATCTGGGGGGAATTTACGACGAGCTCGGGCGAGCGGAGAAAGCGATTGAGCACTATCAGCGCGCGGTGCAGCTGTATCCGAAATACCATTTTTCTTTCGAAAGCTTAGGAGTGCTATTTTGGGAGCAGCAGCGGTGGGAAGAGGCGCGTGAGGCTTTTGCAACTGCGCTTACCTACGCCCCAACGAATATCTCGTATGCGCTCATGACGGCGCTGTGTTTGTGTCAGATGGGCCAGGCACACAAGGCGCAGCACTTTATGCGGACGTTTATTCGGACCGTAGATAGAACGCGACGGGAAGTGGAGTATTTTTTGTGTCGCTTGTTTGTCGATTTGAGTGGGGAACAGGATATGGCTTCCCGTATATCTAAAATTAAGTCAGTGCCCCAGCGTATAAGATACTCGTTTTACCTTGCCTTTTTTTACGAACTCGGGGGGCGGCATCTGCTTGCCGAGAAACATTACGGTGAAGTAGAATCGGCGCGTGCTCCTTCTTCCTTTGAGCACCGACTTGCGGTGAGTGCTCTTGGGCGTTTACGAGGGAGGTCATCGTCGCTGCGTACGAATCCCTAGTAACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAAC