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CN-122029283-A - Antibiotic-free property particle production system and application thereof

CN122029283ACN 122029283 ACN122029283 ACN 122029283ACN-122029283-A

Abstract

A non-resistance plasmid production system constructs a plasmid containing a nucleic acid fragment 3 for encoding small RNA, a host cell expression cassette containing a toxic gene expression cassette and a resistance gene expression cassette by combining and designing a toxic transcript/resistance transcript, an operon related element and the small RNA, wherein the toxic gene expression cassette comprises a promoter and a nucleic acid fragment 1 for transcribing the toxic transcript, and the resistance gene expression cassette comprises a promoter, an operon and a nucleic acid fragment 2 for transcribing the resistance transcript. The function of inhibiting toxic transcripts is realized through the steric hindrance effect generated by the reverse complementation of the nucleic acid fragment 3 and the nucleic acid fragment 1, so that the screening production of plasmids can be realized without adding antibiotics or other inhibitors in the whole plasmid production process, and the harm of the antibiotics and the inhibitors to the plasmids in the cell gene therapy is solved.

Inventors

  • SHI JINXIU
  • LIN YINGJUN
  • LAN TIAN

Assignees

  • 云舟生物科技(广州)股份有限公司

Dates

Publication Date
20260512
Application Date
20240910
Priority Date
20230911

Claims (15)

  1. A plasmid production system comprising a host cell expression cassette and a plasmid; the host cell expression cassette comprises a toxic gene expression cassette and an antitoxic gene expression cassette; the toxic gene expression cassette comprises a promoter and a nucleic acid fragment 1 for transcribing a toxic transcript; the antitoxic gene expression cassette comprises a promoter, an operator and a nucleic acid fragment 2 for transcribing an antitoxic transcript, wherein the antitoxic transcript has a resistance effect on toxicity generated by a toxic transcript; the plasmid comprises a nucleic acid fragment 3 for transcribing small RNA and an Ori replication initiation site, wherein the nucleic acid fragment 3 and the nucleic acid fragment 1 are reversely complementary to generate a steric effect, so that the function of toxic transcripts is inhibited.
  2. The plasmid production system of claim 1, wherein the nucleic acid fragment 3 is fully reverse-complementary to the nucleic acid fragment 1 or has at least 20% reverse-complementary sequence or at least 10bp reverse-complementary sequence.
  3. The plasmid production system of claim 1 or 2, wherein the nucleic acid fragment 1 comprises a UTR sequence and a virulence gene.
  4. The plasmid production system of any one of claims 1-3, wherein the virulence gene is selected from any one of ccdB, parE, mazF, kid, hicA, relE, vapC, doc, ratA, hipA, zeta, toxN, yeeV, cptA, ghoT, hok, tisB, symE, pasA, and wherein the nucleic acid fragment 2 is an antitoxic gene selected from any one of ccdA, parD, mazE, kis, hicB, relB, vapB, phd, ratB, hipB, epsilon, toxI, yeeU, cptB, ghoS, sok, istR-1, symR, pasB/C.
  5. The plasmid production system of any one of claims 1-4, wherein the operon is any one of an arabinose operon, a Lac operon, a rhamnose catabolism operon, a tryptophan operon, a gab operon, and a Gal operon.
  6. The plasmid production system of any one of claims 3-5 wherein the small RNA transcribed from nucleic acid fragment 3 is reverse-complementary to the UTR sequence in the nucleic acid fragment 1 and creates a steric effect.
  7. The plasmid production system of any one of claims 3-6 wherein the UTR sequence and toxic gene are expressed in fusion.
  8. The plasmid production system of any one of claims 1-7, wherein the number of antitoxic gene expression cassettes and the number of toxic gene expression cassettes are each selected from integers greater than or equal to 1.
  9. The plasmid production system of any one of claims 1-8, wherein the host cell expression cassette further comprises a Rep protein.
  10. The plasmid production system of any one of claims 1-9, wherein the Ori replication initiation site is selected from ColE1, pBR322, pMB1, R6K, pUC, F1, p15A, 2 μori, or oriV.
  11. The plasmid production system of any one of claims 1-10, wherein the plasmid further comprises a gene of interest.
  12. The plasmid production system of any one of claims 1-11, wherein the plasmid comprises a promoter, a gene of interest, an Ori replication initiation site, and nucleic acid fragment 3 of a transcribed small RNA, in sequence.
  13. The plasmid production system of any one of claims 1-12, further comprising a host bacterium or host cell, wherein the host bacterium and host cell are derived from escherichia coli, agrobacterium, bacillus, or yeast.
  14. The use of the plasmid production system of any one of claims 1-13 for producing plasmids or for preparing products for cellular gene therapy.
  15. A method for producing a plasmid using the plasmid production system according to any one of claims 1 to 13, wherein the plasmid is transferred into a host cell into which the host cell expression cassette is integrated, and cultured.

Description

Antibiotic-free property particle production system and application thereof Technical Field The invention relates to the technical field of biology, in particular to a non-resistance property grain production system and application thereof. Background In recent years, gene therapy approaches have attracted attention, particularly DNA or RNA vaccines, which overcome in many ways the shortcomings of conventional vaccines or viral vector vaccines, whether DNA or RNA vaccines or viral vector vaccines, are often very high in plasmid requirements. Conventional plasmid systems have many drawbacks in the use of gene therapy. On one hand, the traditional plasmid carries antibiotic marker genes, but the overexpression of the resistance genes has certain side effects on the metabolism and growth of escherichia coli, antibiotics can be transmitted in the environment to cause general generation of antibiotic resistance for people, meanwhile, residual antibiotics in the plasmid can possibly cause allergic reaction of sensitive individuals, and on the other hand, the traditional plasmid carries prokaryotic bacterial skeletons, mostly contains unmethylated CpG sequences, and is easy to induce an innate immune system. Whether carrying antibiotic marker genes or prokaryotic bacterial skeletons, the extra-exogenous gene expression cassette can cause the extra-exogenous gene expression cassette to occupy too much space in the plasmid, increasing the volume of the plasmid, not only affecting the yield of the plasmid, but also resulting in reduced transfection efficiency, which is a negative impact in gene therapy. Supercoiled plasmids of smaller size reach the nucleus more efficiently and allow sustained, long-lasting expression of exogenous genes, so plasmids lacking resistance genes and having a shorter prokaryotic backbone at the same time have greater advantages. In order to improve the safety of gene therapy, a new generation plasmid skeleton system without antibiotic drug resistance marker is developed successively, which mainly comprises the following steps of 1, modifying a strain by introducing deletion or mutation into a certain essential gene for maintaining bacterial growth based on complementation of the strain, so that the strain can not grow in malnutrition, and then restoring the strain growth after introducing the plasmid carrying the deletion gene into the strain. 2. Toxicity-antitoxic System by inserting toxic genes into the genome of bacteria to disable the growth of bacteria, cloning antitoxic genes onto plasmids, introducing strains, and restoring strain growth. 3. Operator/repressor system by introducing a repressor protein upstream of the gene necessary for bacterial genome growth, inhibiting the expression of the necessary gene, introducing a plasmid containing one or more operator sequences into the bacterium, and competitively titrating the repressor to allow expression of the gene. 4. Overexpression of growth essential genes overexpression of certain growth essential genes (fabl or murA) in E.coli can reduce their sensitivity to antibacterial compounds, but inhibitors are present in the culture medium and must be removed from the purified plasmid DNA. Although the novel antibiotic-free plasmid systems solve the use of antibiotic marker genes, the novel antibiotic-free plasmid systems still have two main problems that 1, the ratio of sequences outside a target gene expression cassette in plasmids is overlarge, so that the volume of the plasmids is increased, and therefore, under the same unit weight, part of the yield of the plasmids is contributed by a prokaryotic sequence framework without effectiveness, the copy number ratio of the target gene expression cassette is relatively reduced, and the yield of the target expression cassette is affected. 2. Although some systems do not require the use of antibiotics, other ancillary inhibitors are required which cannot guarantee 100% removal in the final plasmid product, not only increasing the cost of plasmid preparation, but also the effect of its residues on gene therapy is not controllable. So these non-resistant vector systems are not currently available for effective widespread use. Thus, there remains a need in the art to develop plasmid production systems that do not require the use of antibiotics and other co-inhibitors. Disclosure of Invention In order to solve the defects in the prior art, the invention provides a non-resistance property grain production system and application thereof. The production system does not need antibiotics and other auxiliary inhibitors, the copy number of some plasmids can be 4-5 times of the yield of the conventional plasmids, the application safety is improved, and the production cost is greatly reduced. In order to achieve the above object, the present invention provides the following technical solutions: In one aspect, the invention provides a plasmid production system comprising a host cell expression cassette and