WO2022094867A1 - Construction d'une souche de salmonella anaérobie obligatoire pp2 et application de celle-ci dans le traitement des tumeurs - Google Patents

Construction d'une souche de salmonella anaérobie obligatoire pp2 et application de celle-ci dans le traitement des tumeurs Download PDF

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WO2022094867A1
WO2022094867A1 PCT/CN2020/126774 CN2020126774W WO2022094867A1 WO 2022094867 A1 WO2022094867 A1 WO 2022094867A1 CN 2020126774 W CN2020126774 W CN 2020126774W WO 2022094867 A1 WO2022094867 A1 WO 2022094867A1
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anaerobic
cancer
bacteria
strain
salmonella
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刘陈立
盛方芊
王作伟
曾正阳
卢伟琪
郭旋
黄雄亮
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/42—Salmonella

Definitions

  • the invention relates to the field of tumor targeted therapy, in particular to the anaerobic activation promoter PP2 gene sequence, the anaerobic gene circuit regulated by the PP2 promoter, and the strict anaerobic mouse comprising the anaerobic gene circuit regulated by the PP2 promoter Salmonella typhi and vectors and their applications, methods for turning facultative anaerobic bacteria into strict anaerobic bacteria, and methods for treating cancer using bacteria regulated by anaerobic circuits.
  • Cancer is the leading cause of death worldwide. Compared with normal cells, cancer cells have the characteristics of infinite proliferation, transformation and easy metastasis. In addition to uncontrolled division (multipolar division), cancer cells also locally invade surrounding normal tissues and even metastasize to other organs via the circulatory system or lymphatic system in the body.
  • traditional cancer treatment methods such as surgery, chemotherapy, radiotherapy, immunotherapy, hormone therapy, bone marrow/stem cell transplantation, etc., all have certain defects. For example, surgical treatment is prone to recurrence and some tumors There are problems such as difficulty in surgery, chemotherapy will cause serious side effects to patients and the treatment cannot be carried out effectively.
  • the difficulty of cancer treatment stems from the complex and changeable etiology of cancer.
  • VNP20009 did not achieve good clinical results, in view of Salmonella's tumor-aggregating growth and immunomodulatory functions, researchers believe that various modifications may make Salmonella suitable for tumor therapy.
  • Salmonella needs to be modified is that wild-type Salmonella is virulent and can cause symptoms such as fever, vomiting, diarrhea, and abdominal cramps, and in severe cases, bacteremia can be life-threatening.
  • different strategies can be used to transform Salmonella to make it suitable for tumor therapy. It can knock out Salmonella virulence-related genes, construct auxotrophic strains, regulate bacterial growth through gene circuits, etc., so that attenuated strains can be used for tumor treatment as soon as possible.
  • Bin Yu et al. published a research paper titled Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain in the journal “SCIENTIFIC REPORTS” in 2012.
  • the paper described the construction of a strict anaerobic Salmonella strain scheme.
  • the prior art constructs Salmonella typhimurium SL7207 knockout key gene asd strain, asd gene deletion will affect bacterial cell wall formation, adding asd gene downstream intermediate metabolite DAP (diaminopimelic acid) in LB medium can make bacteria to synthesize normally cell wall.
  • DAP diaminopimelic acid
  • an anaerobic strain YB1 was constructed: the gene circuit of Cm-pept-asd-sodA regulated by anaerobic was inserted into the genome of the SL7207 knockout asd strain (the gene circuit was inserted into the original asd gene position).
  • FNR is an oxygen-regulated transcriptional regulator. Under anaerobic conditions, the activation of FNR can regulate the positive promoter Pept to transcribe the asd gene, so that bacteria can produce a complete cell wall.
  • the reverse promoter PsodA can block the leakage of the asd gene product from the forward promoter under aerobic conditions. This design allows the YB1 strain to grow only under anaerobic conditions, while DAP must be added to the medium for growth under aerobic conditions.
  • Bin Yu et al. characterized the viability of YB1 strains under different oxygen conditions: under aerobic conditions, YB1 could not grow in LB (DAP-) medium, but could grow in LB (DAP+) medium; under anaerobic conditions, YB1 It can grow in both LB (DAP+) and LB (DAP-) medium.
  • the distribution of YB1 strain in tumor-bearing mice and the characterization of its therapeutic effect on tumors 26 days after the YB1 strain was injected into the tail vein of tumor-bearing mice, the strain was cleared in normal tissues and organs, and bacteria still existed in tumor tissue (due to the oxygen concentration in tumor tissue). very low and in an immunosuppressive environment). Compared with the PBS group, the YB1 strain had the ability to inhibit tumor growth.
  • the YB1 strain of Bin Yu et al. takes up to 26 days to completely clear the normal tissues and organs, which is time-consuming and has low safety.
  • the weight of the mice was significantly reduced. (greater than 5%).
  • the significant decrease in body weight indicated that the bacteria had a strong toxic effect on mice.
  • the purpose of the present invention is to provide an anaerobic activation promoter PP2 gene sequence, an anaerobic gene circuit regulated by the PP2 promoter, and a strict anaerobic typhimurium comprising an anaerobic gene circuit regulated by the PP2 promoter Salmonella and its use in tumor therapy.
  • an anaerobic activation promoter PP2 gene sequence is provided, and the nucleotide sequence of the anaerobic activation promoter PP2 is as shown in SEQ ID No.1:
  • a vector which is a prokaryotic cell comprising the following elements: (a) a hypoxia or strict anaerobic activated promoter; (b) an essential gene regulated by the promoter in (a); (c) wherein the promoter in (a) has a binding site for an anaerobic activated transcriptional regulator.
  • the anaerobic activated promoter is PP2
  • the nucleotide sequence of the anaerobic activated promoter PP2 is as follows:
  • the transcriptional regulator in the vector, is arcA or FNR.
  • the essential gene is selected from the group consisting of dapA and dapE.
  • culturing under aerobic conditions in vitro requires adding 2,6-diaminopimelic acid or an analog thereof to the medium.
  • a strictly anaerobic Salmonella to express a drug or carry a drug as a carrier, and the drug is used for the treatment of cancer.
  • the medicament comprises: (a) expressing a protein substance or polypeptide substance with a cancer treatment effect; (b) expressing an RNA with a cancer treatment effect; (c) as a carrier for carrying Loaded with modified RNA drugs.
  • a method for turning facultative anaerobic bacteria into strict anaerobic bacteria by inducing a circuit expressing essential genes through hypoxia or strict anaerobicity, and the strict anaerobic bacteria are used in vivo During tumor therapy, tumor growth can be inhibited and tumor volume reduced.
  • the facultative anaerobic bacterium is Salmonella spp.
  • the strictly anaerobic bacteria need to be additionally added with 2,6-diaminopimelic acid (alias: 2,6-diaminopimelic acid) when cultured under aerobic conditions in vitro 2,6-Diaminopimelic acid) and its analogs.
  • 2,6-diaminopimelic acid alias: 2,6-diaminopimelic acid
  • the essential genes are selected from dapA and dapE, but are not limited to these two genes, but also include one of dapB, dapD, argD, dapF, murE, murF and lysA, etc. one or more.
  • the strictly anaerobic regulated gene circuit consists of an anaerobic activated promoter and an essential gene.
  • hypoxia or strict anaerobic inducible gene circuit for expression regulation of essential genes exists in a chromosome or other plasmid vector.
  • the anaerobic activated promoter is PP2.
  • the tumor cancer includes blood cancer (chronic leukemia, acute leukemia), bone cancer, lymphoma (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal cancer (colon). cancer, rectal cancer), liver cancer, stomach cancer, pelvic cancer (cervical cancer, ovarian cancer, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer, etc.
  • blood cancer chronic leukemia, acute leukemia
  • bone cancer lymphoma (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal cancer (colon).
  • lymphoma non-Hodgkin's lymphoma, Hodgkin's lymphoma
  • intestinal cancer colon
  • cancer rectal cancer
  • liver cancer stomach cancer
  • pelvic cancer cervical cancer, ovarian cancer, endometrial cancer, ovarian cancer
  • lung cancer breast cancer
  • pancreatic cancer bladder cancer
  • methods are provided for the treatment of cancer using anaerobic loop-regulated bacteria comprising strictly hypoxic regulation of essential gene expression.
  • the treatment method further includes: combined use with other cancer treatments, but not limited to radiotherapy, chemotherapy, immunotherapy and the like.
  • the bacterium in the method of treatment, is Salmonella typhi.
  • the facultative anaerobic bacterium is Salmonella typhimurium.
  • the facultative anaerobic Salmonella strains include those derived from humans, chickens, dogs, cattle, and the like.
  • the facultative anaerobic bacteria genus include: Enterobacteriaceae (Escherichia coli, Pneumococcus, Proteus, Enterobacter, Salmonella typhi, Salmonella, Shigella etc.), Staphylococcus, Streptococcus, Pneumococcus, Bacillus anthracis and Diphtheria, etc.
  • the combination of other cancer treatment methods includes: (a) bacterial therapy of anaerobic strains combined with surgical therapy; (b) bacterial therapy of anaerobic strains combined with radiotherapy; ( c) Bacterial therapy of anaerobic strains combined with chemical drugs: chemotherapy drugs include alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, pyruvate mustard, etc.), Antimetabolites (deoxyfluridine, doxefluridine, 6-mercaptopurine, cytarabine, fluoroguanosine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, eufovir Ding, amcitabine, etc.), antitumor antibiotics (actinomycin, arubicin, epirubicin, mitomycin, pelomycin, pingyangmycin, pirarubicin,
  • Figure 1 is a schematic diagram of the construction of the SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA strain.
  • Fig. 2 is the electrophoretic diagram of the construction of SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA strain and SL7207( ⁇ dapE)-PP2-BBa_B0033-dapE strain.
  • Figure 3A, Figure 3B and Figure 3C are the in vitro experiments of the SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA strain
  • Figure 3A is a photo of the strain culturing under aerobic conditions for 24 and 48 hours
  • Figure 3B is a photo of the strain culturing under anaerobic conditions for 24 hours
  • Figure 3C shows that the strain of the present invention grows in DAP+ under aerobic conditions, but does not grow in DAP-.
  • 4A, 4B, 4C and 4D are in vivo experiments of SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA strain.
  • Figures 5A, 5B and 5C are in vitro characterization of SL7207( ⁇ dapE)-PP2-BBa_B0033-dapE.
  • Figure 6 is an in vivo experiment of the SL7207( ⁇ dapE)-PP2-BBa_B0033-dapE strain.
  • anaerobic activated promoter PP2 gene sequence according to the specific embodiment of the present invention, the anaerobic gene circuit regulated by the PP2 promoter, the strict anaerobic mouse comprising the PP2 promoter regulated anaerobic gene circuit will be explained in more detail Salmonella typhi and vectors and their applications, methods for turning facultative anaerobic bacteria into strict anaerobic bacteria, methods for treating cancer using bacteria regulated by anaerobic circuits, and their applications in tumor therapy.
  • the vector of the invention is a prokaryotic cell comprising: (a) a hypoxia or strict anaerobic activated promoter; and (b) regulated by the promoter in (a) Essential genes; wherein, the promoter in (a) has a binding site for anaerobic activated transcriptional regulators.
  • the (a) hypoxic or strictly anaerobic activated promoter may be, for example, PP2 and PP3; preferably PP3. Especially PP2.
  • nucleotide sequence of the anaerobic activated promoter PP2 is as follows:
  • the (b) essential gene regulated by the promoter in (a) can be, for example, dapA, dapB, dapD, argD, dapE, dapF, murE, murF, lysA, etc.; Specifically dapA and dapE.
  • (c) wherein the promoter in (a) exists and the anaerobic activated transcription regulator binding site can be arcA (aeobic respiratory control, aerobic respiration control transcription regulator ) or FNR (fumarate nitrate reduction regulator, fumarate nitrate reduction transcription regulator).
  • the present invention provides a method for turning facultative anaerobic bacteria into strict anaerobic bacteria by inducing a circuit expressing essential genes through hypoxia or strict anaerobicity.
  • the strictly anaerobic regulated gene circuit consists of an anaerobic activated promoter and an essential gene.
  • the anaerobic activated promoters may be, for example, PP2 and PP3; preferably PP3. Especially PP2.
  • the essential genes may be, for example, dapA, dapB, dapD, argD, dapE, dapF, murE, murF, lysA, etc.; in particular dapA and dapE
  • 2,6-diaminopimelic acid (alias: 2,6-diaminopyramic acid; 2,6-diaminopimelic acid) needs to be additionally added to the medium when the vector of the present invention is cultured under aerobic conditions. , 6-Diaminopimelic acid) or its analogs.
  • the strict anaerobic bacteria of the present invention can inhibit tumor growth and reduce tumor volume when applied to in vivo tumor treatment.
  • the facultative anaerobic bacteria can be from Enterobacteriaceae bacteria (Escherichia coli, Pneumococcus, Proteus, Enterobacter, Typhoid Bacillus, Salmonella, Shigella, etc.), Staphylococcus, Streptococcus, Pneumococcus, Any species in any bacterial genus such as Bacillus anthracis and Bacillus diphtheriae.
  • Enterobacteriaceae bacteria Esscherichia coli, Pneumococcus, Proteus, Enterobacter, Typhoid Bacillus, Salmonella, Shigella, etc.
  • Staphylococcus Streptococcus
  • Pneumococcus Any species in any bacterial genus such as Bacillus anthracis and Bacillus diphtheriae.
  • the source of the facultative anaerobic Salmonella strain is not limited, as long as it is facultative anaerobic, for example, it includes facultative anaerobic Salmonella strains derived from humans, chickens, dogs, cattle and the like.
  • the facultative anaerobic bacterium is Salmonella typhimurium.
  • the present invention also provides bacterial therapy for the treatment of cancer using the strains of the present invention that cannot grow under both aerobic and anaerobic conditions.
  • the cancers include blood cancer (chronic leukemia, acute leukemia), bone cancer, lymphoma (non-Hodgkin lymphoma, Hodgkin lymphoma), bowel cancer (colon cancer, rectal cancer), liver cancer, stomach cancer, pelvic cancer ( Cervical cancer, ovarian malignant tumor, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer, etc.
  • the vectors of the present invention as prokaryotic cells, or strict anaerobes obtained by the methods of the present invention can be used as bacterial therapy for anti-tumor or cancer treatment.
  • the bacterial therapy of the present invention may be used in combination with other cancer treatment methods.
  • the use of bacterial therapy in combination with other cancer treatment methods includes, for example: (a) bacterial therapy of anaerobic strains combined with surgical therapy; (b) bacterial therapy of anaerobic strains combined with radiation Treatment; (c) Bacterial therapy of anaerobic strains in combination with chemotherapeutic drugs: chemotherapeutic drugs include alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, mustard etc.), antimetabolites (deoxyfluridine, docefluridine, 6-mercaptopurine, cytarabine, flurguanosine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate , Eufodine, Amcitabine, etc.), antitumor antibiotics (actinomycin, arubicin, epirubicin, mitomycin, pelomycin, pingyangmycin, pira
  • the vector of the present invention as a prokaryotic cell, or the strict anaerobic bacteria obtained by the method of the present invention can also be used to induce the expression of drugs in vitro or as a vector to carry drugs for cancer treatment.
  • the drugs that can be carried in the carrier include: (a) expressing a protein substance or polypeptide substance with a cancer treatment effect; (b) expressing an RNA with a cancer treatment effect; (c) as a carrier Carrying modified RNA drugs.
  • the asd gene, dapA and dapE belong to the DAP metabolic pathway, and the asd gene is located upstream of the dapA and dapE genes. Downstream of asd, dapA and dapE genes all generate DAP, which is an important component of bacterial cell walls.
  • SL7207 knockout key gene asd strain is to knock out the asd gene on the basis of SL7207.
  • the basic bacteria SL7207 ( ⁇ dapA) and SL7207 ( ⁇ dapE) are based on SL7207 to knock out the dapA and dapE genes, respectively.
  • the anaerobic regulation module of the PP2 strain is simpler, the regulation system is more rigorous, and there is no background leakage problem under aerobic conditions;
  • the PP2 strain has almost no effect on the body weight of the mice, and the toxicity and side effects are relatively small, and the safety is improved.
  • the present invention uses the primers in the following table to clone and construct the SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA strain and the SL7207( ⁇ dapE)-PP2-BBa_B0033-dapE strain.
  • Figure 1 is a schematic diagram of the construction of the SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA strain.
  • the osmotic pressure inside and outside the bacteria is unbalanced, and the bacteria cannot survive after rupture.
  • the PP2 promoter is an anaerobic activated promoter. Under anaerobic or hypoxic conditions, PP2 can initiate the transcription of dapA gene or dapE gene, so that the downstream key protein DAP is normally generated. Bacteria can form complete cell walls. Under aerobic conditions, the PP2-BBa_B0033-dapA and PP2-BBa_B0033-dapE gene circuits are inactive, and bacteria cannot generate complete cell walls.
  • Example 1 Construction and electrophoresis verification of SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA strain and SL7207( ⁇ dapE)-PP2-BBa_B0033-dapE strain
  • the pSC101-BBa_B0033-dapA plasmid was digested with BsaI to obtain the linearized vector fragment 2 ((D) of FIG. 2 );
  • dapE gene forward primer and dapE gene reverse primer in the above table are primers, PCR obtains dapE linear fragment ((1) of Figure 2);
  • the homologous recombination fragment 2 was integrated into the original dapA gene position of SL7207( ⁇ dapA) by ⁇ -red homologous recombination method to obtain the SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA target strain (abbreviation: PP2 strain). Extraction genome PCR identification results shown in Figure 2 (G);
  • PCR obtains homologous recombination fragment 4 (Fig. 2(K)) ;
  • the homologous recombination fragment 4 was integrated into the original dapE gene position of SL7207( ⁇ dapE) by ⁇ -red homologous recombination method to obtain the SL7207( ⁇ dapE)-PP2-BBa_B0033-dapE target strain (abbreviation: PP2-1).
  • the PCR identification results are shown in (L) of 2 .
  • Example 2 In vitro characterization of SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA
  • Characterization under aerobic conditions Pick 5 single clones and resuspend in 10 ⁇ l LB medium respectively. 5 [mu]l of bacterial resuspension was added to LB (DAP+) medium containing kanamycin and the remaining 5 [mu]l of bacterial resuspension was added to LB (DAP-) medium containing kanamycin. Incubate in an air shaker (37°C, 220 rpm) for a period of time.
  • Characterization under anaerobic conditions 3 single clones were picked and added to LB (DAP+) medium containing kanamycin. Incubate overnight in an air shaker (37°C, 220 rpm). The bacterial solution cultured overnight was put into an anaerobic incubator and transferred at a ratio of 1:100. Take 20 ⁇ l of the bacterial solution and add it to 2 ml of LB (DAP+) medium containing kanamycin; take 20 ⁇ l of the bacterial solution and add it to 2 ml of LB (DAP-) medium containing kanamycin, repeating 3 times. The initial OD600 value of the transferred samples was measured. In an anaerobic box, 37 °C, static culture for 24h. Measure the OD600 value of the samples after 24h incubation.
  • the strain can grow normally when cultured in LB (DAP+) medium for 24 hours.
  • the strain was cultured in LB(DAP-) medium for 48h, and the strain could not grow.
  • the strain was cultured in LB (DAP+) medium and LB (DAP-) medium for 24 hours.
  • the strain can grow in both LB (DAP+) medium and LB (DAP-) medium.
  • Example 3 In vivo characterization of SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA (abbreviated as PP2)
  • mice were subcutaneously inoculated with 1 ⁇ 10 6 mouse bladder cancer cells (MB49) to establish a mouse bladder cancer subcutaneous tumor model.
  • the experiment was divided into three groups, PBS group, SL7207 strain group, SL7207( ⁇ dapA)-PP2-BBa_B0033-dapA group.
  • the tail vein was inoculated with 1 x 107 bacteria.
  • the distribution of bacteria in normal tissues, organs and tumors of tumor-bearing mice, the changes in tumor volume, the changes in mouse body weight, and the survival rate of mice were detected.
  • Experimental results (as shown in Figure 4A, Figure 4B, Figure 4C and Figure 4D):
  • mice survival rate (Fig. 4D): All mice in the SL7207 group died within 7 days. During the experimental period, the mice in this strain group and the PBS group did not die.
  • Characterization under aerobic conditions 3 single clones were picked and added to LB (DAP+) medium containing kanamycin. Incubate overnight in an air shaker (37°C, 220 rpm). The bacteria cultured overnight was transferred at a ratio of 1:100. Take 20 ⁇ l of the bacterial solution and add it to 2 ml of LB (DAP+) medium containing kanamycin; take 20 ⁇ l of the bacterial solution and add it to 2 ml of LB (DAP-) medium containing kanamycin, repeating 3 times. Incubate in an air shaker for 72h.
  • Characterization under anaerobic conditions 3 single clones were picked and added to LB (DAP+) medium containing kanamycin. Incubate overnight in an air shaker (37°C, 220 rpm). The bacterial solution cultured overnight was put into an anaerobic incubator and transferred at a ratio of 1:100. Take 20 ⁇ l of the bacterial solution and add it to 2 ml of LB (DAP+) medium containing kanamycin; take 20 ⁇ l of the bacterial solution and add it to 2 ml of LB (DAP-) medium containing kanamycin, repeating 3 times. The initial OD600 value of the transferred samples was measured. In an anaerobic box, 37 °C, static culture for 24h. Measure the OD600 value of the samples after 24h incubation.
  • the strain can grow normally when cultured in LB (DAP+) medium for 24 hours.
  • the strain was cultured in LB(DAP-) medium for 72h, and the strain could not grow.
  • the strain was cultured in LB (DAP+) medium and LB (DAP-) medium for 24 hours.
  • the strain can grow in both LB (DAP+) medium and LB (DAP-) medium.
  • Example 5 In vivo characterization of SL7207( ⁇ dapE)-PP2-BBa_B0033-dapE (abbreviated as PP2-1)
  • mice were subcutaneously inoculated with 1 ⁇ 10 6 mouse bladder cancer cells (MB49) to establish a mouse bladder cancer subcutaneous tumor model.
  • the experiment was divided into two groups, PBS group and SL7207( ⁇ dapE)-PP2-BBa_B0033-dapE group.
  • the tail vein was inoculated with 1 x 107 bacteria.
  • the distribution of bacteria in normal tissues, organs and tumors of tumor-bearing mice, the changes in tumor volume, the changes in mouse body weight, and the survival rate of mice were detected.
  • the experimental results (as shown in Figure 6):
  • mice survival rate (C in Figure 6): During the experimental period, the mice in this strain group and the PBS group did not die.

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Abstract

La présente invention concerne une séquence de gène PP2 d'un promoteur d'activation anaérobie, une boucle de gène anaérobie régulée par un promoteur PP2, une Salmonella typhimurium anaérobie obligatoire contenant la boucle de gène anaérobie régulée par le promoteur PP2, un vecteur, et une application de la Salmonella typhimurium anaérobie obligatoire dans le traitement des tumeurs. La présente invention concerne également un procédé permettant de transformer des bactéries anaérobies facultatives en bactéries anaérobies obligatoires, un procédé de traitement des cancers utilisant des bactéries anaérobies à boucle régulée, et une application des procédés dans le traitement des tumeurs.
PCT/CN2020/126774 2020-11-05 2020-11-05 Construction d'une souche de salmonella anaérobie obligatoire pp2 et application de celle-ci dans le traitement des tumeurs WO2022094867A1 (fr)

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