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CN-122012561-A - Expression plasmid of proteus mirabilis homologous recombination system, expression system, application and gene knockout/knocking-in method

CN122012561ACN 122012561 ACN122012561 ACN 122012561ACN-122012561-A

Abstract

The invention belongs to the field of biology, and discloses a proteus mirabilis homologous recombination system expression plasmid which comprises a pBR322 replication origin, a resistance gene, an arabinose inducible promoter and a proteus mirabilis source homologous recombination operon, wherein the homologous recombination operon is used for encoding a T_PM66325 protein, an E_PM66325 protein, a single-chain binding protein SSB and a false release protein. The expression plasmid has higher recombination efficiency in Proteus mirabilis GDMCC 66325, can play a role in recombination in Proteus mirabilis standard strain ATCC35659, and verifies the effectiveness of the expression plasmid in the recombination operation aiming at Proteus mirabilis by knocking out the Proteus mirabilis GDMCC 66325 hemolysin related gene hpmA and the biomembrane formation related gene bcsB and directly knocking in the heterologous gene after the flagellum is regulated with the related gene cluster, and simultaneously, the invention also discloses a Proteus mirabilis homologous recombination system, application and a gene knocking-out/knocking-in method.

Inventors

  • MA JINGYUN
  • GAO LONG
  • SUN YUAN
  • ZHANG JIAN
  • CHEN JIANJUN
  • TANG XIAOYU
  • LI RUYI

Assignees

  • 华南农业大学

Dates

Publication Date
20260512
Application Date
20251231

Claims (10)

  1. 1. An expression plasmid of a proteus mirabilis homologous recombination system, which is characterized by comprising a pBR322 replication origin, a resistance gene, an arabinose inducible promoter and a proteus mirabilis-derived homologous recombination operon; The homologous recombination operon is used for encoding a T_PM66325 protein, an E_PM66325 protein, a single-chain binding protein SSB and a pseudorelease protein, and is 1115822-119369 bp of a genome sequence of Proteus mirabilis GDMCC No: 66325.
  2. 2. The proteus mirabilis homologous recombination system expression plasmid according to claim 1, wherein the nucleotide sequence of the expression plasmid is shown in SEQ ID No. 1.
  3. 3. A Proteus mirabilis homologous recombination system is characterized by comprising Proteus mirabilis carrying the expression plasmid as claimed in claim 1 or 2 and an exogenous target gene, wherein the exogenous target gene is provided with a homology arm matched with a gene to be knocked out or replaced, and the exogenous target gene realizes the knocking out or replacing of a specific gene of Proteus mirabilis by means of electrotransfer.
  4. 4. The proteus mirabilis homologous recombination system according to claim 3, wherein the homology arms are not less than 75bp in length.
  5. 5. A system for homologous recombination of Proteus mirabilis according to claim 3, wherein the Proteus mirabilis has accession number GDMCC No:66325 or ATCC35659.
  6. 6. The method for preparing the expression plasmid according to claim 1 or 2, comprising the steps of: Step 1, using pBR322-P BAD -GFP as an initial plasmid, and performing inverse PCR to obtain a target linear fragment pBR322-P BAD by glue, wherein the linear fragment pBR322-P BAD is a linear fragment with a pBR322 replication origin, a resistance gene and an arabinose inducible promoter; Step 2, performing in-vitro homologous recombination on the gel recovery linear fragment pBR322-P BAD obtained in the step 1 and a synthesized recombinase fragment recET _PM66325 with a carrier homology arm respectively, and then co-transforming the recombinant into DH5 alpha for overnight culture to obtain a recombinant; The recombinase fragment recET _pm66325 comprises a homologous recombination operon and a vector homology arm; and obtaining the proteus mirabilis homologous recombination system expression plasmid after sequencing and verifying the recombinant to be correct.
  7. 7. Use of a proteus mirabilis homologous recombination system according to any of claims 3 to 5 for gene knockout/knock-in at one or more sites in proteus mirabilis.
  8. 8. A method of gene knockout/knock-in against proteus mirabilis, characterized in that it is carried out using the proteus mirabilis homologous recombination system according to any one of claims 3 to 5.
  9. 9. The method according to claim 8, characterized in that it comprises in particular: Preparing competent cells using proteus mirabilis harboring the expression plasmid of claim 1 or 2; and electrotransferring the exogenous target gene into competent cells.
  10. 10. The method of claim 8, wherein the competent cells are prepared under conditions of starting OD 600 = 0.1,37 ℃ for 2h, 37 ℃ to induce 1h, and at room temperature using 10% glycerol at room temperature; the technological condition of the exogenous target gene electrotransformation into competent cells is that the exogenous target gene amount is 800-1000 ng, the electrotransformation voltage is 1350V/mm, the electrotransformation buffer solution is10 wt% glycerol solution, the electrotransformation temperature is room temperature, and the resuscitating time is1 h.

Description

Expression plasmid of proteus mirabilis homologous recombination system, expression system, application and gene knockout/knocking-in method Technical Field The invention relates to the field of biology, in particular to an expression plasmid of a proteus mirabilis homologous recombination system, an expression system, application and a gene knockout/knock-in method. Background RecET and Red alpha beta gamma system mediated homologous recombination process is called Red/ET homologous recombination engineering. Is a genetic manipulation tool derived from phage, wherein RecE and RecT homologous recombination proteins are derived from Rac prophage, red alpha, red beta and Red gamma homologous recombination proteins are derived from Lambda phage, and also similar to Red alpha beta gamma derived from luminous bacillary phage Plu2936, plu2935 and Plu2934. The system is used for accurately knocking in, knocking out or replacing target genes at any positions of the escherichia coli genome in early stage, and can realize multi-site modification of the bacterial genome by being used together with systems such as site-specific recombinase (Cre/loxP, flp/FRT) or CRISPR-Cas. The successful application of Red/ET recombination engineering promotes the development of the whole genetic engineering technology and provides an effective means for genetic modification. RecE and Red alpha, plu2936 have 5 '-3' exonuclease activity, so that the 5 '-end of dsDNA (Double STRAND DNA) is degraded, the 3' -end of a DNA molecule is changed into a single-chain overhang, recT and Red Beta, plu2935 are ssDNA (Single Strand DNA) annealed proteins, can be stably combined with ssDNA with the length of more than 35bp and can carry out gene recombination by searching homologous sequences through a chain invasion mechanism, a chain annealing mechanism and a Beta recombination mechanism, and Red gamma and Plu2934 can inhibit the exonuclease activity of RecBCD in escherichia coli and protect exogenous linear DNA molecules from degradation, thereby improving the homologous recombination efficiency. Because phage recombinases have certain host specificity, the Red/ET system has very low recombination efficiency in bacteria with far relatedness and even has no recombination function, so that it is necessary to mine efficient homologous recombination systems applicable to different strains. A variety of strains have been used to successfully construct recombinant systems using phage recombinant function operons, such as Pseudomonas Caryophylli SYRINGAE PV, syringae, lactobacillus reuteri Lactobacillusreuteri, photorhabdus, burkholderia Burkholderiales strain, mycobacterium tuberculosis Mycobacterium tuberculosis, bacillus subtilis Bacillus subtilis, agrobacterium, etc. Proteus mirabilis (Proteus mirabilis) is a conditional pathogen of Proteus (Proteus). Gutav Hauser, at the end of the 19 th century, first isolated a group of facultative anaerobic gram-negative bacteria with variable morphology and capable of migratory growth from spoilage meats, designated Proteus (Proteus). Further studies have found that one of the specific classes is capable of producing a variety of enzymes, specific metabolites and has the ability to migrate and differentiate into populations, which is designated as Proteus mirabilis (Proteus mirabilis). Under special environmental conditions, proteus mirabilis can enter a population migration state, and the cell morphology is extremely elongated and changed from a common short rod shape (1-2 mu m) to a long thread shape (10-100 mu m), and the length is increased by about 20-40 times. And the number of the flagella is increased rapidly, the density of the flagella on the surface of a unit cell is increased by more than 50 times, the total number of the flagella can reach thousands, and the flagella is densely arranged for a period of time and covers the whole cell surface. Several studies have shown that bacterial flagella are good immunological adjuvants capable of effectively activating the immune response of the organism, but current bacterial vaccines often have poor immune effects due to insufficient short-term immune activation, so that the superflagellum state of Proteus mirabilis has a strong potential as a vaccine carrier. In addition, the latest research results in the field of anti-tumor research show that the proteus mirabilis in a migration state can be uniformly distributed in a tumor microenvironment and widely form microthrombus, so that the vascular system of tumor cells is effectively destroyed, the nutrition supply of the tumor cells is blocked, and an effective tumor killing effect is exerted. However, the application and development of the recombinant oncolytic chassis bacteria as vaccine vectors are seriously hindered because of the lack of efficient, accurate and convenient genome editing technology. Therefore, it is very necessary to establish a set of efficient and simple homologous recombination system i