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CN-121975919-A - Preparation method of high molecular weight polynucleotide

CN121975919ACN 121975919 ACN121975919 ACN 121975919ACN-121975919-A

Abstract

The invention belongs to the technical field of synthetic biology, and particularly relates to an industrial preparation method of high molecular weight polynucleotide (Polynucleotide, PN). The method takes a plasmid containing PN base sequences as a template, utilizes the rolling circle amplification (Rolling Circle Amplification) technology to generate long-chain DNA, and then realizes the efficient separation of PN fragments through restriction enzyme digestion. The plasmid is optimally designed, a restriction enzyme cutting site is introduced into a replication initiation site and a screening marker gene, and after enzyme cutting treatment, the plasmid skeleton is degraded into small fragments smaller than 250 bp, and the efficient separation of target PN fragments is realized by means of molecular weight difference. According to the invention, through the optimal design and large-scale preparation of the plasmid template, the low-cost and easy-to-amplify production of the PN prepared by rolling circle amplification is realized, and meanwhile, the length and sequence of the final PN product can be accurately designed and regulated. In particular, by introducing specific enzyme cutting sites into the plasmid template, the accurate removal of the subsequent plasmid skeleton sequence is facilitated, and the purity of the target product is remarkably improved. The process successfully realizes the industrialized preparation of the high molecular weight Polynucleotide (PN) of which the molecular weight is more than 1500 bp, and provides a safe, efficient, convenient and large-scale brand new path for the production of the high molecular weight PN.

Inventors

  • Zhang gun
  • XIE HAISONG
  • DING XU
  • SONG QIANQIAN
  • LU HUILI
  • HAO SIJIA

Assignees

  • 杭州中美华东制药有限公司

Dates

Publication Date
20260505
Application Date
20260115

Claims (14)

  1. 1. A preparation method of a high molecular weight polynucleotide is characterized by comprising the steps of amplifying a plasmid containing a high molecular weight polynucleotide base sequence in a host cell, performing rolling circle amplification by taking the plasmid as a template, performing restriction enzyme digestion on an amplified fragment, and separating to obtain a target high molecular weight polynucleotide, wherein restriction enzyme digestion sites are positioned on a plasmid skeleton.
  2. 2. The method of claim 1, wherein the plasmid backbone comprises a replication origin (Ori) that initiates the replication function of the plasmid, and wherein the replication origin has restriction enzyme sites that do not affect biological function.
  3. 3. The method of claim 2, wherein the replication origin is ColE1 Ori, colE2 Ori or R6K Ori.
  4. 4. The method according to claim 2, wherein the restriction enzyme cleavage site is a restriction enzyme cleavage site recognizing 4-7 bp, preferably a cleavage site of HhaI or FaeI.
  5. 5. The method of claim 2, wherein the plasmid backbone is shown as SEQ ID NO. 5 or SEQ ID NO. 6.
  6. 6. The method of claim 2, wherein the plasmid backbone further comprises a gene element having a screening function, and the screening function gene element has a restriction enzyme cleavage site that does not affect biological function.
  7. 7. The method according to claim 6, wherein the screening functional gene element is selected from the group consisting of a resistance gene, a gene essential for growth and an auxotroph gene.
  8. 8. The method according to claim 7, wherein the resistance gene is kanamycin or bleomycin, and the gene essential for growth is infA.
  9. 9. The method according to claim 6, wherein the restriction enzyme cleavage site is a restriction enzyme cleavage site recognizing 4-7 bp, preferably a cleavage site of HhaI or FaeI.
  10. 10. The method of claim 6, wherein the plasmid backbone is shown as SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4.
  11. 11. The method of claim 1, wherein the rolling circle amplification is performed using a phi 29 DNA polymerase.
  12. 12. The method according to claim 1, wherein the percentage of A, T, G, C in the high-molecular-weight polynucleotide base sequence is 30.3.+ -. 10%, 19.5.+ -. 10% or 19.9.+ -. 10%, respectively.
  13. 13. The method according to claim 12, wherein the high molecular weight polynucleotide has a base sequence length of more than 500 bp, preferably more than 1500 bp.
  14. 14. The method of claim 1, wherein the high molecular weight polynucleotide has a nucleotide sequence shown in SEQ ID No. 7 or SEQ ID No. 8.

Description

Preparation method of high molecular weight polynucleotide Technical Field The invention relates to the technical field of synthetic biology, in particular to an industrialized preparation method of high molecular weight polynucleotide (Polynucleotide, PN). Background Polydeoxyribonucleotide (Polydeoxyribonucleotide, PDRN) is a natural low molecular weight DNA derivative, the base composition of PDRN has high similarity with human DNA up to 98%, has various pharmacological activities such as tissue repair, anti-inflammation, wound healing promotion, angiogenesis stimulation, ischemia injury resistance and the like, and is widely used in new therapies and technologies, development of artificial biological substitution systems and the like. Based on the application potential of PDRN in biomedicine, the upgrade products of PDRN have also been rapidly developed. The polynucleotide (Polynucleotide, PN) is a structure which is continuously optimized on the basis of the PDRN, and compared with the PDRN, the PN is composed of higher-order polymer molecules, which is equivalent to the secondary refining of the PDRN, has a more stable long-chain molecular structure and belongs to a macromolecular structure. Document "From Polydeoxyribonucleotides (PDRNs) to Polynucleotides (PNs): Bridging the Gap Between Scientific Definitions, Molecular Insights, and Clinical Applications of Multifunctional Biomolecules."（Biomolecules. 2025 Jan 19;15(1):148） defines a clear molecular weight boundary for PDRN/PN, while the system combes the molecular mechanisms and clinical applications of both. Studies show that the high molecular weight PN fragment shows skin moisturizing performance superior to that of the PDRN fragment due to stronger hydration capability and longer skin residence time, and PN can be used for preparing high-viscosity hydrogel or filler, and the deep recovery volume of skin can be achieved by injecting the PN fragment, so that the deoxyribonucleotide monomer released during in vivo degradation can stimulate skin cells, a longer-lasting biostimulation effect is provided, and remarkable clinical benefit is brought. The polynucleotide is used as a nucleic acid medicament with wide bioactivity, and has great application potential in the fields of anti-inflammation, immunoregulation, tissue repair and the like. However, the current preparation method of PN mainly depends on extraction from fish spermary or semen, and the traditional extraction method has a plurality of inherent limitations. On the one hand, the stability of the raw materials is extremely easy to be influenced by environmental factors, so that the fluctuation of the product quality is large, and on the other hand, protein residues possibly exist in the product in the extraction process, so that the purity of the medicine is influenced, and adverse reactions can be caused. In addition, the traditional extraction method has the disadvantages of long reaction period, low production efficiency, high cost and difficult large-scale application. More importantly, the method is difficult to accurately control the target molecular weight and sequence information, and limits the further development and application of PN. Therefore, developing safer, efficient, convenient and scalable preparation methods is the focus of current research. Disclosure of Invention Aiming at the defects existing in the prior art, the invention provides a safe, efficient, convenient and large-scale preparation method of high molecular weight polynucleotide. The preparation method comprises the steps of amplifying plasmids containing high molecular weight polynucleotide base sequences in host cells, performing rolling circle amplification by taking the plasmids as templates, performing restriction enzyme digestion on amplified fragments, and separating to obtain target high molecular weight polynucleotides, wherein restriction enzyme digestion sites are positioned on plasmid frameworks. In a preferred embodiment, the plasmid backbone comprises a replication origin (Ori) that initiates the replication function of the plasmid, and the replication origin has restriction enzyme sites that do not affect the biological function (replication), which is embodied as a variation in plasmid copy number of 10% or less. Further preferably, the replication origin in the plasmid backbone is selected from ColE1 Ori, colE2 Ori or R6K Ori. The origin of replication plays an important role in plasmid construction and genetic engineering, and ensures stable replication and maintenance of plasmids in host cells. ColE1 Ori is the replication initiation site of the ColE1 plasmid (Origin of Replication), colE2 Ori is the replication initiation site of the ColE2 plasmid, R6K Ori is the replication initiation site of the R6K plasmid, and the region specifically recognized by the R6K Rep protein to initiate DNA replication. The replication initiation sites all contain specific DNA sequences that can be recognized