WO2022094867A1 - Pp2严格厌氧沙门氏菌菌株构建及其在肿瘤治疗方面应用 - Google Patents

Pp2严格厌氧沙门氏菌菌株构建及其在肿瘤治疗方面应用 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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French (fr)
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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
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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
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    • 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

提供了厌氧激活启动子PP2基因序列，利用PP2启动子调控的厌氧基因回路，包含PP2启动子调控的厌氧基因回路的严格厌氧鼠伤寒沙门氏菌和载体及其在肿瘤治疗中的应用。还提供了使兼性厌氧细菌变成严格厌氧菌的方法，使用厌氧回路调控的细菌治疗癌症的方法，及其在肿瘤治疗中的应用。

Description

PP2严格厌氧沙门氏菌菌株构建及其在肿瘤治疗方面应用 技术领域
本发明涉及肿瘤靶向治疗领域，具体而言本发明涉及厌氧激活启动子PP2基因序列，利用PP2启动子调控的厌氧基因回路，包含PP2启动子调控的厌氧基因回路的严格厌氧鼠伤寒沙门氏菌和载体及其应用，使兼性厌氧细菌变成严格厌氧菌的方法，使用厌氧回路调控的细菌治疗癌症的方法。
背景技术
癌症是全世界范围内引起死亡的主要原因。与正常细胞比较癌细胞具有无限增殖、可转化和易转移等特点。癌细胞除了***失控外(能进行多极***)，还会局部侵入周遭正常组织甚至经由体内循环***或淋巴***转移到其他器官。癌症治疗发展史表明，传统癌症治疗方法如手术治疗、化学疗法、放射线疗法、免疫学疗法、荷尔蒙疗法、骨髓/干细胞移植等治疗手段，均具有一定的缺陷，如手术治疗存在易复发且部分肿瘤存在不易手术等问题，化疗会对患者产生严重的副反应而导致治疗不能有效进行。癌症治疗难点源于其病因复杂多变，不仅存在机体基因水平的变化，外界环境的改变也是癌症发展的重要因素之一。长期放射疗法、化学疗法和免疫治疗等传统疗法的缺点在于这些治疗方案不仅会对正常组织器官产生严重的毒性，且会使癌细胞产生多重耐药性、不能完全清除癌细胞。近年来，多项研究发现，基因治疗、无创伤射频治疗癌症方法、胰岛素增强治疗、饮食治疗和细菌治疗不仅可以阻止癌细胞产生多重耐药性，同时也增强传统疗法的疗效。其中细菌疗法是一种很有希望克服传统治疗方法缺点的癌症治疗手段。
利用活细菌治疗癌症的历史可以追溯到150多年前。1868年德国内科医生W.Bush首次应用细菌治疗无法通过手术方法治疗的肉瘤，患者在接受治疗的一周内肿瘤体积缩小一半同时颈部***体积变小。然而不幸的是该患者于9天后死于细菌感染引起的败血症。1883年德国外科医生Friedrich Fehleisen鉴定出丹毒是酿脓链球菌感染引起的。随后，Friedrich Fehleisen和来自纽约医院外科医生Willian B Coley分别独立开展实验证明酿脓链球菌可以使患者肿瘤消退。然而由于实验结果很难重复和不符合当时临床标准，因此结果备受争议。1935年Connell观测到来自梭状杆菌酶的滤液可以使转移瘤消退。1947年科学家首次注射溶组织梭菌的孢子给移植肉瘤的小鼠，观测到癌细胞溶解和肿瘤 组织消退。然而由于细菌引起的急性毒性反应，小鼠存活率很低。1959年卡介苗(减毒牛结核分枝杆菌)成功用于癌症免疫治疗。2002年减毒的沙门氏菌VNP20009(msbB-,purI-)进行了Ⅰ期临床试验，结果显示该菌株可以在肿瘤组织定植，但是对于肿瘤治疗效果不明显。
虽然VNP20009并没有取得良好的临床结果，但鉴于沙门氏菌的肿瘤聚集性生长和免疫调节功能，研究者认为可以通过多种改造方式或许能使沙门氏菌适合于肿瘤治疗。沙门氏菌需要进行改造的原因在于野生型沙门氏菌具有毒性，可以引起发热、呕吐、腹泻及腹部绞痛等症状，严重可以引起菌血症危及生命。伴随着分子生物学技术飞速发展，可以通过不同策略改造沙门氏菌使其适合应用于肿瘤治疗。可以敲除沙门氏菌与毒力相关基因、通过构建营养缺陷型菌株、基因回路调控细菌生长等，使减毒菌株早日用于肿瘤治疗。
Bin Yu等人2012年在期刊《SCIENTIFIC REPORTS》中发表一篇题为Explicit hypoxia targeting with tumor suppression by creating an“obligate”anaerobic Salmonella Typhimurium strain研究论文，论文中表述的构建严格厌氧沙门氏菌菌株方案。该现有技术构建鼠伤寒沙门氏菌SL7207敲除关键基因asd菌株，asd基因缺失会影响细菌细胞壁生成，在LB培养基中加入asd基因下游中间代谢产物DAP(二氨基庚二酸)可以使细菌正常合成细胞壁。并在此基础上构建厌氧菌株YB1：在SL7207敲除asd菌株的基因组上***Cm-pept-asd-sodA受厌氧调控的基因回路(基因回路***到原asd基因位置)。FNR是受到氧气调控的转录调控因子。在厌氧条件下，FNR处于激活状态可以调控正向启动子Pept使asd基因发生转录，使细菌可以产生完整细胞壁。反向启动子PsodA可以阻断正向启动子在有氧条件下产生asd基因产物的泄漏。该设计可以使YB1菌株只能在厌氧条件下生长，而有氧条件下必须在培养基中加入DAP才可以生长。
Bin Yu等对其YB1菌株在不同氧气条件下生存能力表征：有氧条件下，YB1在LB(DAP-)培养基中无法生长，LB(DAP+)培养基中可以生长；厌氧条件下，YB1在LB(DAP+)、LB(DAP-)培养基中均可以生长。YB1菌株在荷瘤小鼠体内分布与***效果表征：荷瘤小鼠尾静脉注射YB1菌株后26天，该菌株在正常组织器官均被清除，肿瘤组织还有细菌存在(由于肿瘤组织氧气浓度很低和处于免疫抑制环境)。与PBS组比较，YB1菌株具有抑制肿瘤生长能力。
但Bin Yu等的YB1菌株在正常组织器官内完全清除需要多达26天，耗时长、安全性低；与PBS组比较，给荷瘤小鼠尾静脉注射YB1后，小鼠体重有显著性降低(大于5％)。 作为小鼠健康与否重要评价指标，体重明显降低表明该菌对小鼠有较强的毒性作用。
本领域还需要在更短时间内能够容易被正常组织器官清除的菌株，减弱因细菌在体内长期存留而对荷瘤小鼠产生的毒副作用，使改造菌株更加安全可靠，且不影响细菌***效果。
发明内容
为了解决现有技术的问题，本发明的目的是提供厌氧激活启动子PP2基因序列，利用PP2启动子调控的厌氧基因回路，包含PP2启动子调控的厌氧基因回路的严格厌氧鼠伤寒沙门氏菌及其在肿瘤治疗中的应用。
在本发明的一个方面，提供了厌氧激活启动子PP2基因序列，所述厌氧激活的启动子PP2的核苷酸序列如SEQ ID No.1所示：
SEQ ID No.1：
5’-ttgcggccgcaagaagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctc-3’。
在本发明的一个方面，提供了一种载体，其是包含以下元件的原核细胞：(a)低氧或者严格厌氧激活启动子；(b)受到(a)中启动子调控的必需基因；(c)其中(a)中的启动子存在与厌氧激活的转录调控因子结合位点。
在本发明的一个方面，在所述载体中，所述厌氧激活启动子是PP2，所述厌氧激活的启动子PP2的核苷酸序列如下所示：
SEQ ID No.1：
5’-ttgcggccgcaagaagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctc-3’。
在本发明的一个方面，在所述载体中，所述转录调控因子为arcA或FNR。
在本发明的一个方面，在所述载体中，所述必需基因选自dapA和dapE。
在本发明的一个方面，在所述载体中，体外有氧条件下培养需要在培养基中添加2,6-二氨基庚二酸或其类似物。
在本发明的一个方面，提供了一种严格厌氧沙门氏菌表达药物或者作为载体携载药物中的应用，所述药物用于治疗癌症。
在本发明的一个方面，在所述应用中，所述药物包括：(a)表达具有癌症治疗效果的蛋白物质或者多肽物质；(b)表达具有癌症治疗效果的RNA；(c)作为载体携载经修饰的RNA药物。
在本发明的一个方面，提供了一种通过低氧或者严格厌氧诱导表达必需基因的回路 使兼性厌氧细菌变成严格厌氧菌的方法，并且所述的严格厌氧菌应用于体内肿瘤治疗时，可以抑制肿瘤生长和减小肿瘤体积。
在本发明的一个方面，在上述方法中，所述兼性厌氧细菌是沙门氏菌属物种(Salmonella)。
在本发明的一个方面，在上述方法中，所述严格厌养菌，体外有氧条件下培养时培养基中需要额外添加2，6-二氨基庚二酸(别名：2，6-二氨基蒲桃酸；2，6-Diaminopimelic acid)及其类似物。
在本发明的一个方面，在上述方法中，所述必需基因选自dapA和dapE，但不局限于这两个基因，还包括dapB、dapD、argD、dapF、murE、murF和lysA等中的一种或多种。
在本发明的一个方面，在上述方法中，所述严格厌氧调控基因回路由厌氧激活的启动子和必需基因组成。
在本发明的一个方面，在上述方法中，所述低氧或者严格厌氧诱导表达调节的必需基因的基因回路存在于染色体或者其他质粒载体。
在本发明的一个方面，在上述方法中，所述厌氧激活的启动子是PP2。
在本发明的一个方面，在上述方法中，所述肿瘤癌症包括血癌(慢性白血病、急性白血病)，骨癌，淋巴癌(非霍奇金淋巴瘤、霍奇金淋巴瘤)，肠癌(结肠癌、直肠癌)，肝癌，胃癌，盆腔癌(子***、卵巢恶性肿瘤、子宫内膜癌、卵巢癌)，肺癌，乳腺癌，胰腺癌，膀胱癌，***癌等。
在本发明的一个方面，提供了使用厌氧回路调控的细菌治疗癌症的方法，所述细菌包含严格低氧调控必需基因表达。
在本发明的一个方面，所述治疗方法还包括：与其他治疗癌症疗法联合应用，但不限于放疗、化疗、免疫疗法等。
在本发明的一个方面，所述治疗方法中，所述细菌是伤寒沙门氏菌(Salmonella typhi)。
在本发明的一个方面，所述治疗方法中，所述兼性厌氧细菌是鼠伤寒沙门氏菌(Salmonella typhimurium)。
在本发明的一个方面，所述治疗方法中，所述兼性厌氧沙门氏菌菌株包括来源于人、鸡、狗、牛等。
在本发明的一个方面，所述治疗方法中，所述兼性厌氧细菌菌属包括：肠杆菌科细菌(大肠杆菌、肺炎杆菌、变形杆菌、肠杆菌、伤寒杆菌、沙门氏菌、志贺氏菌等)，葡 萄球菌属，链球菌属，肺炎球菌，炭疽杆菌和白喉杆菌等。
在本发明的一个方面，所述治疗方法中，所述其他癌症治疗方法联合应用包括：(a)厌氧菌株的细菌疗法联合手术疗法；(b)厌氧菌株的细菌疗法联合放射治疗；(c)厌氧菌株的细菌疗法联合化学药物：化疗药物包括烷化剂(尼莫司汀、卡莫司汀、洛莫司汀、环磷酰胺、异环磷酰胺、甘磷酰芥等)，抗代谢药(去氧氟尿苷、多西氟鸟啶、6-巯基嘌呤、阿糖胞苷、氟鸟苷、替加氟、吉西他滨、卡莫氟、羟基脲、甲氨蝶呤、优福定、安西他滨等)，抗肿瘤抗生素(放线菌素、阿柔比星、表柔比星、丝裂霉素、培洛霉素、平阳霉素、吡柔比星等)，植物类抗癌药物(伊立替康、三尖杉酯碱、羟基喜树碱、长春瑞宾、紫杉醇、泰索帝、拓扑替康、长春新碱、长春地辛、长春碱等)，激素(阿他美坦、阿那曲唑、安鲁米特、来曲唑、福美坦、甲他孕酮、他莫昔芬等)免疫抑制剂及其他抗癌药物如门冬酰胺酶、卡铂、顺铂、达卡巴嗪、奥沙利铂、乐沙定、可铂澳杀、米托蒽醌、丙卡巴肼等；(d)厌氧菌株的细菌疗法联合生物治疗；(e)厌氧菌株的细菌疗法联合中医中药治疗。
附图说明
图1是SL7207(ΔdapA)-PP2-BBa_B0033-dapA菌株构建线路图。
图2是SL7207(ΔdapA)-PP2-BBa_B0033-dapA菌株和SL7207(ΔdapE)-PP2-BBa_B0033-dapE菌株构建电泳图。
图3A、图3B和图3C是SL7207(ΔdapA)-PP2-BBa_B0033-dapA菌株体外实验，图3A是菌株有氧条件培养24和48小时的照片，图3B是菌株厌氧条件培养24小时的照片，图3C表明本发明的菌株有氧条件下在DAP+中生长，在DAP-中不生长。
图4A、图4B、图4C和图4D是SL7207(ΔdapA)-PP2-BBa_B0033-dapA菌株体内实验。
图5A、图5B和图5C是SL7207(ΔdapE)-PP2-BBa_B0033-dapE的体外表征。
图6是SL7207(ΔdapE)-PP2-BBa_B0033-dapE菌株的体内实验。
具体实施方式
尽管可以对本发明进行各种修改并且本发明可以具有各种形式，但是下面将详细说明和解释具体实例。然而，应当理解的是，这些并不旨在将本发明限制于特定的公开内容，并且本发明包括其所有修改、等同物或替代物而不脱离本发明的精神和技术范围。
在下文中，将更详细地解释根据本发明具体实施方式的厌氧激活启动子PP2基因序 列，利用PP2启动子调控的厌氧基因回路，包含PP2启动子调控的厌氧基因回路的严格厌氧鼠伤寒沙门氏菌和载体及其应用，使兼性厌氧细菌变成严格厌氧菌的方法，使用厌氧回路调控的细菌治疗癌症的方法，及其在肿瘤治疗中的应用。
在本发明的一个或多个实施方式中，本发明的载体是包含以下元件的原核细胞：(a)低氧或者严格厌氧激活启动子；和(b)受到(a)中启动子调控的必需基因；其中，(a)中的启动子存在与厌氧激活的转录调控因子结合位点。
在本发明的一个或多个实施方式中，(a)低氧或者严格厌氧激活启动子例如可以为PP2和PP3；优选PP3。特别是PP2。
在本发明的一个或多个实施方式中，所述厌氧激活启动子PP2的核苷酸序列如下所示：
SEQ ID No.1：
5’-ttgcggccgcaagaagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctc-3’。
在本发明的一个或多个实施方式中，所述(b)受到(a)中启动子调控的必需基因例如可以为dapA、dapB、dapD、argD、dapE、dapF、murE、murF、lysA等；特别是dapA和dapE。
在本发明的一个或多个实施方式中，(c)其中(a)中的启动子存在与厌氧激活的转录调控因子结合位点可以为arcA(aeobic respiratory control，有氧呼吸控制转录调控因子)或FNR(fumarate nitrate reduction regulator，富马酸盐硝酸盐还原转录调控因子)。
本发明提供了一种通过低氧或者严格厌氧诱导表达必需基因的回路使兼性厌氧细菌变成严格厌氧菌的方法。
在本发明的一个或多个实施方式中，所述严格厌氧调控基因回路由厌氧激活的启动子和必需基因组成。
在本发明的一个或多个实施方式中，厌氧激活的启动子例如可以为PP2和PP3；优选PP3。特别是PP2。
在本发明的一个或多个实施方式中，所述必需基因例如可以为dapA、dapB、dapD、argD、dapE、dapF、murE、murF、lysA等；特别是dapA和dapE
当必需基因为dapA和dapE基因时，本发明的载体进行有氧条件培养时需要在培养基中额外添加2,6-二氨基庚二酸(别名：2,6-二氨基蒲桃酸；2,6-Diaminopimelic acid)或其类似物。
本发明所述的严格厌氧菌应用于体内肿瘤治疗时，可以抑制肿瘤生长和减小肿瘤体 积。
所述兼性厌氧菌可以为来自肠杆菌科细菌(大肠杆菌、肺炎杆菌、变形杆菌、肠杆菌、伤寒杆菌、沙门氏菌、志贺氏菌等)，葡萄球菌属，链球菌属，肺炎球菌，炭疽杆菌和白喉杆菌等中任一个细菌菌属中的任意菌种。
所述兼性厌氧沙门氏菌菌株来源不限，只要是兼性厌氧即可，例如包括来源于人、鸡、狗、牛等的兼性厌氧沙门氏菌菌株。
所述兼性厌氧细菌是鼠伤寒沙门氏菌(Salmonella typhimurium)。
本发明还提供了利用本发明的在有氧与无氧条件下均无法生长菌株治疗癌症的细菌疗法。
所述癌症包括血癌(慢性白血病、急性白血病)，骨癌，淋巴癌(非霍奇金淋巴瘤、霍奇金淋巴瘤)，肠癌(结肠癌、直肠癌)，肝癌，胃癌，盆腔癌(子***、卵巢恶性肿瘤、子宫内膜癌、卵巢癌)，肺癌，乳腺癌，胰腺癌，膀胱癌，***癌等。
在本发明的一个或多个实施方式中，本发明的作为原核细胞的载体，或通过本发明的方法获得的严格厌氧菌可以作为细菌疗法用于抗肿瘤或癌症治疗。
在本发明的一个或多个实施方式中，本发明的细菌疗法可以与其他癌症治疗方法联合应用。
在本发明的一个或多个实施方式中，细菌疗法与其他癌症治疗方法的联合应用包括，例如：(a)厌氧菌株的细菌疗法联合手术疗法；(b)厌氧菌株的细菌疗法联合放射治疗；(c)厌氧菌株的细菌疗法联合化学药物：化疗药物包括烷化剂(尼莫司汀、卡莫司汀、洛莫司汀、环磷酰胺、异环磷酰胺、甘磷酰芥等)，抗代谢药(去氧氟尿苷、多西氟鸟啶、6-巯基嘌呤、阿糖胞苷、氟鸟苷、替加氟、吉西他滨、卡莫氟、羟基脲、甲氨蝶呤、优福定、安西他滨等)，抗肿瘤抗生素(放线菌素、阿柔比星、表柔比星、丝裂霉素、培洛霉素、平阳霉素、吡柔比星等)，植物类抗癌药物(伊立替康、三尖杉酯碱、羟基喜树碱、长春瑞宾、紫杉醇、泰索帝、拓扑替康、长春新碱、长春地辛、长春碱等)，激素(阿他美坦、阿那曲唑、安鲁米特、来曲唑、福美坦、甲他孕酮、他莫昔芬等)免疫抑制剂及其他抗癌药物如门冬酰胺酶、卡铂、顺铂、达卡巴嗪、奥沙利铂、乐沙定、可铂澳杀、米托蒽醌、丙卡巴肼等；(d)厌氧菌株的细菌疗法联合生物治疗；(e)厌氧菌株的细菌疗法联合中医中药治疗。
本发明的作为原核细胞的载体，或通过本发明的方法获得的严格厌氧菌还可以用于在体外诱导表达药物或者作为载体携载药物，以进行癌症治疗。
在本发明的实施方式中，可以携带在所述载体中的药物包括：(a)表达具有癌症治疗效果的蛋白物质或者多肽物质；(b)表达具有癌症治疗效果的RNA；(c)作为载体携载经修饰的RNA药物。
asd基因与dapA、dapE属于DAP代谢通路，asd基因位于dapA、dapE基因的上游。asd、dapA和dapE基因的下游都会生成DAP，DAP是细菌细胞壁的重要组成成分。SL7207敲除关键基因asd菌株就是在SL7207的基础上敲除asd基因。在本发明的实施方式中，基础菌SL7207(ΔdapA)和SL7207(ΔdapE)是在SL7207的基础上分别敲除dapA和dapE基因。
本发明优点：
(1)PP2菌株的厌氧调控模块更为简单，调控***更为严谨，有氧条件下不存在本底泄漏问题；
(2)PP2菌株在正常组织器官只需要很短时间内就会被完全清除干净；
(3)PP2菌株在治疗荷瘤小鼠过程中，几乎对小鼠体重无影响，毒副作用比较小，安全性得到了提高。
本发明采用下表中的引物克隆并构建了SL7207(ΔdapA)-PP2-BBa_B0033-dapA菌株和SL7207(ΔdapE)-PP2-BBa_B0033-dapE菌株。图1是SL7207(ΔdapA)-PP2-BBa_B0033-dapA菌株构建线路图。
菌株构建：在本实验室已经构建好的SL7207(ΔdapA)菌株和SL7207(ΔdapE)菌株基础上，将PP2-BBa_B0033-dapA和PP2-BBa_B0033-dapE厌氧基因回路分别整合到SL7207(ΔdapA)基因组(原dapA基因位置)和SL7207(ΔdapE)基因组(原dapE基因位置)。dapA和dapE基因是赖氨酸代谢通路上的关键基因，敲除dapA或dapE基因细菌无法形成正常细胞壁，细菌内外渗透压不平衡，细菌破裂无法存活。PP2启动子是厌氧激活启动子，在厌氧或者低氧条件下PP2可以起始dapA基因或dapE基因转录，从而使下游关键蛋白DAP正常生成。细菌可以形成完整细胞壁。有氧条件下，PP2-BBa_B0033-dapA和PP2-BBa_B0033-dapE基因回路处于失活状态，细菌无法生成完整细胞壁。有氧条件下培养SL7207(ΔdapA)-PP2-BBa_B0033-dapA菌株和SL7207(ΔdapE)-PP2-BBa_B0033-dapE菌株，需要添加DAP(二氨基庚二酸)，可以弥补dapA和dapE基因缺失导致细菌无法形成完整细胞壁。本发明采用的启动子序列，和用于克隆的引物如表1所示。
表1本发明采用的启动子序列，和用于克隆的引物
Figure PCTCN2020126774-appb-000001
实施例：
实施例1：SL7207(ΔdapA)-PP2-BBa_B0033-dapA菌株和SL7207(ΔdapE)-PP2-BBa_B0033-dapE菌株的构建和电泳验证
1.构建pSC101-BBa_B0033-dapA质粒
a.以pSC101-FbFp-KnaR-loxp+promoter质粒为模板，以上表中的载体正向引物1、载体反向引物2为引物，PCR获得线性载体1(图2的(A))；
b.以沙门氏菌SL7207基因组为模板，以上表中的dapA基因正向引物、dapA基因反向引物为引物，PCR获得dapA线性片段(图2的(B))；
c.一步克隆方法获得pSC101-BBa_B0033-dapA质粒。菌落PCR鉴定结果(图2的(C))
2.构建pSC101-PP2-BBa_B0033-dapA质粒
a.pSC101-BBa_B0033-dapA质粒通过BsaI酶切，获得线性化载体片段2(图2的(D))；
b.引物退火方法获得PP2启动子片段；
c.连接酶连接获得pSC101-PP2-BBa_B0033-dapA质粒。菌落PCR鉴定结果(图2的(E))。
3.构建pSC101-PP2-BBa_B0033-dapE质粒
a.以pSC101-PP2-BBa_B0033-dapA质粒为模板，以上表中载体正向引物3、载体正向引物4为引物，PCR获得线性载体片段3(图2的(H))；
b.以沙门氏菌SL7207基因组为模板，以上表中dapE基因正向引物、dapE基因反向引物为引物，PCR获得dapE线性片段(图2的(I))；
c.一步克隆方法获得pSC101-PP2-BBa_B0033-dapE质粒。菌落PCR鉴定结果(图2的(J))。
4.构建SL7207(ΔdapA)-PP2-BBa_B0033-dapA菌株和SL7207(ΔdapE)-PP2-BBa_B0033-dapE菌株；
a.以pSC101-PP2BBa_B0033-dapA质粒为模板，以上表中的dapA同源重组正向引物、dapA同源重组反向引物dapA-RE-Forward-primer、dapA-RE-Reverse-primer为引物，PCR获得同源重组片段2(图2的(F))；
b.通过λ-red同源重组方法将同源重组片段2整合到SL7207(ΔdapA)的原dapA基因位置，获得SL7207(ΔdapA)-PP2-BBa_B0033-dapA目标菌株(简称：PP2菌株)。提取基因组PCR鉴定结果见图2的(G)；
c.以pSC101-PP2-BBa_B0033-dapE质粒为模板，以上表中dapE同源重组正向引物、dapE同源重组反向引物为引物，PCR获得同源重组片段4(图2的(K))；
d.通过λ-red同源重组方法将同源重组片段4整合到SL7207(ΔdapE)原dapE基因位置，获得SL7207(ΔdapE)-PP2-BBa_B0033-dapE目标菌株(简称：PP2-1)。PCR鉴定结果见图2的(L)。
实施例2：SL7207(ΔdapA)-PP2-BBa_B0033-dapA体外表征
有氧条件下表征：挑取5个单克隆，分别重悬到10μl LB培养基中。将5μl细菌重悬液加入到含有卡那霉素的LB(DAP+)培养基中，剩下5μl细菌重悬液加入到含有卡那霉 素的LB(DAP-)培养基中。空气摇床中培养(37℃，220rpm)一段时间。
厌氧条件下表征：挑取3个单克隆加入到含有卡那霉素的LB(DAP+)培养基中。空气摇床中(37℃，220rpm)过夜培养。将过夜培养的菌液放入厌氧培养箱，以1：100比例进行转接。取20μl菌液加入到2ml含有卡那霉素的LB(DAP+)培养基中；取20μl菌液加入到2ml含有卡那霉素的LB(DAP-)培养基中，3个重复。测量转接后样品的初始OD600值。厌氧箱中，37℃，静置培养24h。测量培养24h后样品的OD600值。
实验结果(如图3A-图3C)：
(1)有氧条件下：该菌株在LB(DAP+)培养基中培养24h，该菌株可以正常生长。该菌株在LB(DAP-)培养基中培养48h，该菌株无法生长。
(2)厌氧条件下：该菌株在LB(DAP+)培养基与LB(DAP-)培养基中培养24h。该菌株在LB(DAP+)培养基与LB(DAP-)培养基中均可以生长。
实验结论：通过对该菌株在有氧与厌氧条件下的测试表明，成功将兼性厌氧菌株SL7207改造成为严格厌氧菌株。
实施例3：SL7207(ΔdapA)-PP2-BBa_B0033-dapA(简写PP2)体内表征
C57BL/6小鼠皮下接种1×10 6小鼠膀胱癌细胞(MB49)，建立小鼠膀胱癌皮下瘤模型。实验分为三组，PBS组、SL7207菌株组、SL7207(ΔdapA)-PP2-BBa_B0033-dapA组。尾静脉接种1×10 7个细菌。检测细菌在荷瘤小鼠正常组织器官与肿瘤里分布、肿瘤体积变化、小鼠体重变化、小鼠生存率。实验结果(如图4A、图4B、图4C和图4D)：
(1)细菌在荷瘤小鼠体内分布(图4A)：14天内小鼠正常组织器官就可以清除该菌株。SL7207组，7天内细菌在正常组织与肿瘤内快速繁殖，最终小鼠在7天内小鼠全部死亡。
(2)肿瘤体积变化(图4B)：与PBS组比较，该菌株组在14天内对肿瘤有一定的抑制肿瘤生长作用。
(3)小鼠体重变化(图4C)：与SL7207组比较，该菌株组小鼠体重下降比较少，略低于PBS组，无统计学差异。
(4)小鼠生存率(图4D)：SL7207组小鼠7天内全部死亡。实验周期内该菌株组别与PBS组别小鼠无死亡。
实验结论：14天内，该改造菌株可以在荷瘤小鼠体内被完全清除。该菌株组别肿瘤体积减小。小鼠在实验周期内体重略微低于PBS组，并且无死亡。说明该菌株安全性得到了提高，同时具有一定抑制肿瘤作用。
实施例4：SL7207(ΔdapE)-PP2-BBa_B0033-dapE体外表征
有氧条件下表征：挑取3个单克隆加入到含有卡那霉素的LB(DAP+)培养基中。空气摇床中(37℃，220rpm)过夜培养。将过夜培养的菌液，以1：100比例进行转接。取20μl菌液加入到2ml含有卡那霉素的LB(DAP+)培养基中；取20μl菌液加入到2ml含有卡那霉素的LB(DAP-)培养基中，3个重复。空气摇床中培养72h。
厌氧条件下表征：挑取3个单克隆加入到含有卡那霉素的LB(DAP+)培养基中。空气摇床中(37℃，220rpm)过夜培养。将过夜培养的菌液放入厌氧培养箱，以1：100比例进行转接。取20μl菌液加入到2ml含有卡那霉素的LB(DAP+)培养基中；取20μl菌液加入到2ml含有卡那霉素的LB(DAP-)培养基中，3个重复。测量转接后样品的初始OD600值。厌氧箱中，37℃，静置培养24h。测量培养24h后样品的OD600值。
实验结果(如图5A-图5C)：
(1)有氧条件下：该菌株在LB(DAP+)培养基中培养24h，该菌株可以正常生长。该菌株在LB(DAP-)培养基中培养72h，该菌株无法生长。
(2)厌氧条件下：该菌株在LB(DAP+)培养基与LB(DAP-)培养基中培养24h。该菌株在LB(DAP+)培养基与LB(DAP-)培养基中均可以生长。
实验结论：通过对该菌株在有氧与厌氧条件下的测试表明，成功将兼性厌氧菌株SL7207改造成为严格厌氧菌株。
实施例5：SL7207(ΔdapE)-PP2-BBa_B0033-dapE(简写PP2-1)体内表征
C57BL/6小鼠皮下接种1×10 6小鼠膀胱癌细胞(MB49)，建立小鼠膀胱癌皮下瘤模型。实验分为两组，PBS组和SL7207(ΔdapE)-PP2-BBa_B0033-dapE组。尾静脉接种1×10 7个细菌。检测细菌在荷瘤小鼠正常组织器官与肿瘤里分布、肿瘤体积变化、小鼠体重变化、小鼠生存率。实验结果(如图6)：
(1)肿瘤体积变化(图6中A)：与PBS组比较，该菌株组别在14天内对肿瘤有一定的抑制肿瘤生长作用。
(2)小鼠体重变化(图6中B)：与PBS组比较，该菌株组别小鼠体重变化不大。
(3)小鼠生存率(图6中C)：实验周期内该菌株组别与PBS组别小鼠无死亡。
实验结论：该菌株组别肿瘤体积减小。小鼠在实验周期内体重略微低于PBS组，并且无死亡。说明该菌株安全性得到了提高，同时具有一定抑制肿瘤作用。

Claims (10)

  1. 一种载体，其是包含以下元件的原核细胞：(a)低氧或者严格厌氧激活启动子；和(b)受到(a)中启动子调控的必需基因；其中，(a)中的启动子存在与厌氧激活的转录调控因子结合位点。
  2. 根据权利要求1所述的载体，其中所述厌氧激活启动子是PP2，所述厌氧激活的启动子PP2的核苷酸序列如下所示：
    SEQ ID No.1：
    5’-ttgcggccgcaagaagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctc-3’。
  3. 根据权利要求1所述的载体，其中所述必需基因是dapA和dapE。
  4. 根据权利要求1所述的载体，体外有氧条件下培养需要在培养基中添加2,6-二氨基庚二酸或其类似物。
  5. 一种严格厌氧沙门氏菌表达药物或者作为载体携载药物中的应用，所述药物用于治疗癌症。
  6. 根据权利要求5所述的严格厌氧沙门氏菌表达药物或者作为载体携载药物中的应用，其中所述药物包括：(a)表达具有癌症治疗效果的蛋白物质或者多肽物质；(b)表达具有癌症治疗效果的RNA；(c)作为载体携载经修饰的RNA药物。
  7. 一种通过低氧或者严格厌氧诱导表达必需基因的回路使兼性厌氧细菌变成严格厌氧菌的方法，并且所述的严格厌氧菌应用于体内肿瘤治疗时，可以抑制肿瘤生长和减小肿瘤体积。
  8. 根据权利要求7所述的方法，其中，所述兼性厌氧细菌菌属包括：肠杆菌科细菌(大肠杆菌、肺炎杆菌、变形杆菌、肠杆菌、伤寒杆菌、沙门氏菌、志贺氏菌等)，葡萄球菌属，链球菌属，肺炎球菌，炭疽杆菌和白喉杆菌，优选的是所述兼性厌氧细菌是沙门氏菌属物种(Salmonella)，优选的是所述兼性厌氧沙门氏菌菌株包括来源于人、鸡、狗、牛；和/或
    其中所述严格厌养菌，体外有氧条件下培养时培养基中需要额外添加2，6-二氨基庚二酸(别名：2，6-二氨基蒲桃酸；2，6-Diaminopimelic acid)及其类似物；和/或
    其中所述必需基因选自dapA和dapE，但不局限于这两个基因，还包括dapB,dapD,argD,dapF,murE,murF,lysA等；和/或
    其中所述严格厌氧调控基因回路由厌氧激活的启动子和必需基因组成；和/或
    其中所述低氧或者严格厌氧诱导表达调节的必需基因的基因回路存在于染色体或者其他质粒载体。
  9. 根据权利要求8所述的方法，其中所述厌氧激活的启动子是PP2。
  10. 一种使用厌氧回路调控的细菌治疗癌症的方法，所述细菌包含严格低氧调控必需基因表达；和/或
    优选的是，所述方法还包括：与其他治疗癌症疗法联合应用，但不限于放疗、化疗、免疫疗法；和/或
    优选的是，其中所述其他癌症治疗方法联合应用包括：(a)厌氧菌株的细菌疗法联合手术疗法；(b)厌氧菌株的细菌疗法联合放射治疗；(c)厌氧菌株的细菌疗法联合化学药物：化疗药物包括烷化剂(尼莫司汀、卡莫司汀、洛莫司汀、环磷酰胺、异环磷酰胺、甘磷酰芥等)，抗代谢药(去氧氟尿苷、多西氟鸟啶、6-巯基嘌呤、阿糖胞苷、氟鸟苷、替加氟、吉西他滨、卡莫氟、羟基脲、甲氨蝶呤、优福定、安西他滨等)，抗肿瘤抗生素(放线菌素、阿柔比星、表柔比星、丝裂霉素、培洛霉素、平阳霉素、吡柔比星等)，植物类抗癌药物(伊立替康、三尖杉酯碱、羟基喜树碱、长春瑞宾、紫杉醇、泰索帝、拓扑替康、长春新碱、长春地辛、长春碱等)，激素(阿他美坦、阿那曲唑、安鲁米特、来曲唑、福美坦、甲他孕酮、他莫昔芬等)免疫抑制剂及其他抗癌药物如门冬酰胺酶、卡铂、顺铂、达卡巴嗪、奥沙利铂、乐沙定、可铂澳杀、米托蒽醌、丙卡巴肼等；(d)厌氧菌株的细菌疗法联合生物治疗；(e)厌氧菌株的细菌疗法联合中医中药治疗；和/或
    优选的是，其中所述肿瘤癌症包括血癌(慢性白血病、急性白血病)，骨癌，淋巴癌(非霍奇金淋巴瘤、霍奇金淋巴瘤)，肠癌(结肠癌、直肠癌)，肝癌，胃癌，盆腔癌(子***、卵巢恶性肿瘤、子宫内膜癌、卵巢癌)，肺癌，乳腺癌，胰腺癌，膀胱癌，***癌等。
PCT/CN2020/126774 2020-11-05 2020-11-05 Pp2严格厌氧沙门氏菌菌株构建及其在肿瘤治疗方面应用 WO2022094867A1 (zh)

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Publication number Priority date Publication date Assignee Title
CN104471057A (zh) * 2012-05-04 2015-03-25 香港大学 经修饰的细菌和它们用于治疗癌症或肿瘤的用途
CN108977890A (zh) * 2017-06-05 2018-12-11 北京蓝晶微生物科技有限公司 一种启动子库以及利用其在细菌中构建不同强度表达***的方法

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CN104471057A (zh) * 2012-05-04 2015-03-25 香港大学 经修饰的细菌和它们用于治疗癌症或肿瘤的用途
CN108977890A (zh) * 2017-06-05 2018-12-11 北京蓝晶微生物科技有限公司 一种启动子库以及利用其在细菌中构建不同强度表达***的方法

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BIN YU, MEI YANG, LEI SHI, YANDAN YAO, QINQIN JIANG, XUEFEI LI, LEI-HAN TANG, BO-JIAN ZHENG, KWOK-YUNG YUEN, DAVID K. SMITH, ERWEI: "Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain", SCIENTIFIC REPORTS, NATURE PUBLISHING GROUP, US, vol. 2, US , pages 436 - 10, XP055176169, ISSN: 2045-2322, DOI: 10.1038/srep00436 *

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