WO2022094864A1 - 构建严格厌氧沙门氏菌的方法，所构建的严格厌氧沙门氏菌及其应用 - Google Patents

构建严格厌氧沙门氏菌的方法，所构建的严格厌氧沙门氏菌及其应用 Download PDF

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WO2022094864A1
WO2022094864A1 PCT/CN2020/126769 CN2020126769W WO2022094864A1 WO 2022094864 A1 WO2022094864 A1 WO 2022094864A1 CN 2020126769 W CN2020126769 W CN 2020126769W WO 2022094864 A1 WO2022094864 A1 WO 2022094864A1
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cancer
anaerobic
salmonella
bacteria
strains
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PCT/CN2020/126769
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French (fr)
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刘陈立
王作伟
盛方芊
曾正阳
卢伟琪
郭旋
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中国科学院深圳先进技术研究院
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Priority to PCT/CN2020/126769 priority Critical patent/WO2022094864A1/zh
Priority to KR1020237019056A priority patent/KR20230120123A/ko
Priority to US18/265,400 priority patent/US20240041945A1/en
Priority to EP20960325.7A priority patent/EP4242296A1/en
Publication of WO2022094864A1 publication Critical patent/WO2022094864A1/zh

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Definitions

  • the invention relates to the field of tumor targeted therapy, in particular to a method for constructing strict anaerobic Salmonella, the strict anaerobic Salmonella constructed by the method and its application in tumor therapy.
  • 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 to knock out the key gene asd strain: the deletion of the asd gene will affect the formation of bacterial cell walls, and the addition of the downstream intermediate metabolite DAP (diaminopimelic acid) of the asd gene in the LB medium can make the bacteria 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 a method for constructing strict anaerobic Salmonella, the strict anaerobic Salmonella constructed by the method and its application in tumor treatment.
  • a method for turning facultative anaerobic bacteria into strict anaerobic bacteria by inducing a circuit expressing essential genes by hypoxia or strict anaerobicity, and said strict anaerobic bacteria are used in tumor therapy can inhibit tumor growth and reduce tumor volume.
  • the facultative anaerobic bacteria genus include: Enterobacteriaceae bacteria (Escherichia coli, Pneumococcus, Proteus, Enterobacter, Salmonella typhi, Salmonella, Shigella, etc. ), Staphylococcus, Streptococcus, Pneumococcus, Bacillus anthracis and Diphtheria.
  • the facultative anaerobic bacterium is Salmonella spp.
  • the facultative anaerobic Salmonella strains include those derived from humans, chickens, dogs, cattle, and the like.
  • 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 gene is dapA or dapE but is not limited to this gene, and also includes one or more selected from dapB, dapD, argD, dapF, murE, murF or lysA .
  • 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 selected from Pept, Fnr-SP, Hipl, I141018, Ptet-arcA, Ptet-Fnr, R1074, Ssbp1 and YsgAP.
  • the transcriptional regulator that regulates the promoter is Fnr or arcA.
  • the Fnr-SP promoter is regulated by the transcriptional regulator Fnr
  • the Ptet-arcA promoter is regulated by the transcriptional regulator arcA
  • 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 method of treatment further comprises: combined use with other therapies for the treatment of cancer.
  • 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,
  • a vector which is a prokaryotic cell comprising: (a) a hypoxia or strictly anaerobic activated promoter; and (b) an essential gene regulated by the promoter in (a) ; wherein, the promoter in (a) has a binding site for anaerobic activated transcriptional regulators.
  • the anaerobic activated promoter is selected from Pept, Fnr-SP, Hipl, I141018, Ptet-arcA, Ptet-Fnr, R1074, Ssbp1 and YsgAP.
  • the essential gene is dapA or dapE; and/or, wherein the transcriptional regulator is Fnr, arcA.
  • the Fnr-SP promoter is regulated by the transcriptional regulator Fnr;
  • the Ptet-arcA promoter is regulated by the transcriptional regulator arcA
  • culturing under aerobic conditions in vitro requires the addition of 2,6-diaminopimelic acid 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.
  • Figure 1 is a schematic diagram of the construction of the R1074 strain.
  • Figures 2A-2G are electropherograms of construction of 9 strains (SL7207( ⁇ dapA)-promoter-BBa_B0033-dapA).
  • Fig. 3A, Fig. 3B and Fig. 3C are the in vitro experiments of 9 strains
  • Fig. 3A is the photos of 9 strains (as shown by the abbreviation of the strain on the left side of the photo) cultivated under aerobic conditions for 24-144h
  • Fig. 3B is the 9 strains respectively (As shown by the abbreviations of strains on the left side of the photo) photos of 24h cultured under anaerobic conditions
  • Figure 3C shows 9 strains (as shown by the abbreviations of strains on the ordinate) cultivated under anaerobic conditions for 24h, and the value of OD600 was detected.
  • Fig. 4A-Fig. 4E are the results of in vivo experiments of the constructed 9 strains.
  • FIG. 5 are the electrophoretograms constructed by 5 strains (SL7207( ⁇ dapE)-promoter (R1074, YsgAP, Fnr-SP, Pept, Hipl)-BBa_B0033-dapE).
  • Fig. 6A, Fig. 6B and Fig. 6C are the in vitro experiments of 5 strains
  • Fig. 6A is the photos of 5 strains (as shown by the abbreviation of the strain on the left side of the photo) cultivated under aerobic conditions for 24-72h
  • Fig. 6B is the 5 strains respectively (as shown by the abbreviation of the strain on the left side of the photo) after 24h of anaerobic cultivation
  • Figure 6C shows the 5 strains (as shown by the abbreviation of the strain on the left side of the photo) cultivated under anaerobic conditions for 24h
  • the value of OD600 was detected.
  • Figure 7 shows the in vivo experiment of the SL7207( ⁇ dapE)-R1074-BBa_B0033-dapE (abbreviation: R1074-1) strain.
  • 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 in which the promoter in (a) presents a binding site to an anaerobic activated transcriptional regulator.
  • the (a) hypoxia or strict anaerobic activated promoter may be, for example, Fnr-SP, Hipl, I14018, Ptet-arcA, Ptet-Fnr, R1074, Ssbp1, YsgAP.
  • the transcriptional regulator is Fnr or arcA.
  • the (b) essential gene regulated by the promoter in (a) can be, for example, dapA, dapB, dapD, argD, dapE, dapF, murE, murF, lysA, etc.; Especially dapA or dapE.
  • 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 promoter can be, for example, Pept, Fnr-SP, Hipl, I14018, Ptet-arcA, Ptet-Fnr, R1074, Ssbp1, YsgAP.
  • the essential gene may for example be dapA, dapB, dapD, argD, dapE, dapF, murE, murF, lysA, etc.; in particular dapA or dapE.
  • 2,6-diaminopimelic acid (alias: 2,6-diaminopimelic 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, Salmonella typhi, 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, Salmonella typhi, 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 anaerobic regulation module of the transformed strain is simpler, the regulation system is more rigorous, and there is no background leakage problem under aerobic conditions;
  • the modified strain has almost no effect on the body weight of the mice, the toxic and side effects are relatively small, and the safety is improved.
  • strain construction scheme is shown in Figure 1 and Figure 5: On the basis of SL7207 ( ⁇ dapA) and SL7207 ( ⁇ dapE) that have been constructed in our laboratory, Fnr-SP, Hip1, I14018, Pept, Ptet-arcA, Ptet-Fnr , R1074, Ssbp1, YsgAP-BBa_B0033-dapA anaerobic gene circuit integrated into SL7207 ( ⁇ dapA) genome (formerly dapA position); Fnr-SP, Hip1, Pept, R1074, YsgAP-BBa_B0033-dapE anaerobic gene circuit integrated into SL7207 ( ⁇ dapE) Genome (formerly dapA position).
  • the dapA and dapE genes are key genes in the lysine metabolism pathway. If either of these two genes is knocked out, the bacteria cannot form a normal cell wall, the internal and external osmotic pressures of the bacteria are unbalanced, and the bacteria cannot survive after rupture.
  • the above-mentioned promoter is an anaerobic-activated promoter, and the above-mentioned promoter can initiate the transcription of dapA or dapE gene under anaerobic or hypoxic conditions, so that the downstream DAP protein can generate DAP. Bacteria can form complete cell walls.
  • Example 1 Construction of 9 strictly anaerobic strains (SL7207( ⁇ dapA)-Promoters-BBa_B0033-dapA) (the primers used in the following experiments are shown in Table 2)
  • the linearized vector fragment 1 was obtained by PCR;
  • 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)-R1074-BBa_B0033-dapA target strain.
  • the construction method of the other 8 target strains is basically the same as that of the SL7207( ⁇ dapA)-R1074-BBa_B0033-dapA strain. It is necessary to replace the annealed fragments of primer b in step 2 with Hip1, I14018, Ptet-Fnr, PepT, and Ptet-arcA, respectively. , Ssbp1, Fnr-SP, YsgAP promoter fragments.
  • the 9 strains are abbreviated as (Fnr-SP; Hip1; I14018; Pept; Ptet-arcA; Ptet-Fnr; R1074; Ssbp1; YsgAP), and the electrophoresis results are shown in Figures 2A-2G.
  • Characterization under aerobic conditions pick 1 single clone and add it to LB (DAP+) medium containing kanamycin; pick 3 clones and add them to LB (DAP-) medium containing kanamycin respectively . Incubate in an air shaker (37°C, 220rpm) 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. 20 ⁇ l of the bacterial solution was added to 2 ml of LB (DAP+) medium containing kanamycin; 20 ⁇ l of bacterial solution was added to 2 ml of LB (DAP-) medium containing kanamycin, and repeated three 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.
  • mice were subcutaneously inoculated with 1 ⁇ 10 6 mouse bladder cancer cells (MB49)/mouse to establish a mouse bladder cancer subcutaneous tumor model.
  • the experiment was divided into PBS group, SL7207 strain group, Fnr-SP group, Hip1 group, I14018 group, Pept group, Ptet-arcA group, Ptet-Fnr group, R1074 group, Ssbp1 group, YsgAP group.
  • the tail vein was inoculated with 1 ⁇ 10 7 various bacteria of the present invention per mouse. 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 within 6 days.
  • Experimental results (as shown in Figure 4A-4E):
  • mice survival rate all mice in SL7207 group died within 6 days. During the experimental period, the mice in the 9 strain groups and the PBS group did not die.
  • Example 4 Construction of five strictly anaerobic strains (SL7207( ⁇ dapE)-Promoters-BBa_B0033-dapE)
  • strains were abbreviated as (R1074-1, YsgAP-1, Fnr-SP-1, Pept-1, Hip1-1).
  • Example 5 In vitro characterization of 5 strict anaerobic strains (SL7207( ⁇ dapE)-Promoters(R1074, YsgAP, Fnr-SP, Pept, Hipl)-BBa_B0033-dapE)
  • Characterization under aerobic conditions 3 clones were picked and added to LB (DAP+) medium containing spectinomycin, and cultured overnight on an air shaker (37° C., 220 rpm). The overnight culture broth was transferred at a ratio of 1:100. 20 ⁇ l of the bacterial solution was added to 2 ml of LB (DAP+) medium containing spectinomycin; 20 ⁇ l of bacterial solution was added to 2 ml of LB (DAP-) medium containing spectinomycin, and repeated three times. Culture in an air shaker (37°C, 220rpm) for 72h, and observe the growth of the strain.
  • Characterization under anaerobic conditions 3 single clones were picked and added to LB (DAP+) medium containing spectinomycin, and cultured 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. 20 ⁇ l of the bacterial solution was added to 2 ml of LB (DAP+) medium containing spectinomycin; 20 ⁇ l of bacterial solution was added to 2 ml of LB (DAP-) medium containing spectinomycin, and repeated three times. In an anaerobic box (37°C), the cells were cultured for 24h. Measure the OD600 value of the samples after 24h incubation.
  • mice were subcutaneously inoculated with 1 ⁇ 10 6 mouse bladder cancer cells (MB49)/mouse to establish a mouse bladder cancer subcutaneous tumor model.
  • the experiment was divided into PBS group and R1074-1 group.
  • the tail vein was inoculated with 1 ⁇ 10 7 bacteria of the present invention per mouse. Changes in tumor volume, mouse body weight, and mouse survival rate were detected within 14 days.
  • the experimental results (as shown in Figure 7):
  • mice in the PBS group and the R1074-1 strain group survived.
  • the R1074-1 strain can inhibit the tumor growth of mice; at the same time, compared with the PBS group, there is no significant difference in the body weight of the mice and the mice survived, indicating that the strain is relatively safe.

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Abstract

提供一种构建严格厌氧沙门氏菌的方法，利用该方法构建的严格厌氧沙门氏菌及其在肿瘤治疗中的应用。

Description

构建严格厌氧沙门氏菌的方法，所构建的严格厌氧沙门氏菌及其应用 技术领域
本发明涉及肿瘤靶向治疗领域，具体而言涉及构建严格厌氧沙门氏菌的方法，利用该方法构建的严格厌氧沙门氏菌及其在肿瘤治疗中的应用。
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。癌细胞除了分裂失控外(能进行多极分裂)，还会局部侵入周遭正常组织甚至经由体内循环系统或淋巴系统转移到其他器官。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淋巴结体积变小。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菌株的基因组上插入Cm-pept-asd-sodA受厌氧调控的基因回路(基因回路插入到原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治疗肿瘤效果表征：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。
本领域还需要一种构建方法，该方法能够产生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，且不影响细菌治疗肿瘤效果。
发明内容
为了解决现有技术的问题，本发明的目的是提供构建严格厌氧沙门氏菌的方法，利用该方法构建的严格厌氧沙门氏菌及其在肿瘤治疗中的应用。
在本发明的一个方面，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。
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，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，所述必需基因是dapA或dapE但不局限于该基因，还包括选自dapB、dapD、argD、dapF、murE、murF或lysA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选自Pept、Fnr-SP、Hip1、I141018、Ptet-arcA、Ptet-Fnr、R1074、Ssbp1和YsgAP。
在本发明的一个方面，在上述方法中，调控启动子的转录调控因子是Fnr或arcA。其中Fnr-SP启动子受转录调控因子Fnr调控，Ptet-arcA启动子受转录调控因子arcA 调控
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(子宫颈癌、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，前列腺癌等。
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。
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。
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；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。
在本发明的一个方面，在载体中，所述厌氧激活启动子选自Pept、Fnr-SP、Hip1、I141018、Ptet-arcA、Ptet-Fnr、R1074、Ssbp1和YsgAP。
在本发明的一个方面，在载体中，所述必需基因是dapA或dapE；和/或，其中所述转录调控因子是Fnr、arcA。其中，Fnr-SP启动子受转录调控因子Fnr调控；Ptet-arcA启动子受转录调控因子arcA调控
在本发明的一个方面，在载体中，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。
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。
附图说明
图1是R1074菌株构建线路图。
图2A-图2G是9株菌株(SL7207(ΔdapA)-启动子-BBa_B0033-dapA)的构建电泳图。
图3A、图3B和图3C是9株菌株体外实验，图3A分别是9种菌株(如照片左侧菌株缩写所示)在有氧条件培养24-144h的照片，图3B分别是9种菌株(如照片左侧菌株缩写所示)在厌氧条件培养24h的照片，图3C分别是9种菌株(如纵坐标菌株缩写所示)在厌氧条件下培养24h，检测OD600的数值。
图4A-图4E是所构建的9株菌株的体内实验结果。
图5中(A)-(E)是5株菌株(SL7207(ΔdapE)-启动子(R1074、YsgAP、Fnr-SP、Pept、Hip1)-BBa_B0033-dapE)构建电泳图。
图6A、图6B和图6C是5株菌株体外实验，图6A分别是5种菌株(如照片左侧菌株缩写所示)在有氧条件培养24-72h的照片，图6B分别是5种菌株(如照片左侧菌株缩写所示)在厌氧培养24h的图片，图6C分别是5种菌株(如照片左侧菌株缩写所示)在厌氧条件下培养24h，检测OD600的数值。
图7是SL7207(ΔdapE)-R1074-BBa_B0033-dapE(简称：R1074-1)菌株体内实验。
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构建严格厌氧沙门氏菌方法，利用该方法构建的严格厌氧沙门氏菌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Fnr-SP、Hip1，I14018，Ptet-arcA、Ptet-Fnr、R1074、Ssbp1、YsgAP。
在本发明的一个或多个实施方式中，所述转录调控因子是Fnr或arcA。
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或dapE。
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Pept、Fnr-SP、Hip1、I14018、Ptet-arcA、Ptet-Fnr、R1074、Ssbp1、YsgAP。
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或dapE。
当必需基因为dapA或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(子宫颈癌、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，前列腺癌等。
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。
本发明优点：
(1)改造菌株的厌氧调控模块更为简单，调控系统更为严谨，有氧条件下不存在本底泄漏问题；
(2)改造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)改造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。
菌株构建方案如图1和图5所示：在本实验室已经构建好的SL7207(ΔdapA)和SL7207(ΔdapE)基础上，将Fnr-SP、Hip1、I14018、Pept、Ptet-arcA、Ptet-Fnr、R1074、Ssbp1、YsgAP-BBa_B0033-dapA厌氧基因回路整合到SL7207(ΔdapA)基因组(原dapA位置)；将Fnr-SP、Hip1、Pept、R1074、YsgAP-BBa_B0033-dapE厌氧基因回路整合到SL7207(ΔdapE)基因组(原dapA位置)。dapA和dapE基因是赖氨酸代谢通路上关键基因，敲除这两个基因中的其中任一个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。上述启动子是厌氧激活启动子，在厌氧或者低氧条件下上述启动子可以起始dapA或dapE基因转录，从而使下游DAP蛋白生成DAP。细菌可以形成完整细胞壁。有氧条件下，9个启动子-BBa_B0033-dapA或5个启动子-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)-Promoters(9个启动子，如表1所示)-BBa_B0033-dapA菌株或SL7207(ΔdapE)-Promoters(5个启动子)-BBa_B0033-dapE菌株，需要添加DAP(二氨基庚二酸)，可以弥补dapA或dapE的基因缺失导致细菌无法形成完整细胞壁。
表1本发明采用的启动子
启动子名称 序列
Fnr‐SP 5’GATCCGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCTTGCAGGTGAATGCAACGTCAAGCGAT 3’
Hip1 5’GATCGGATAAAAGTGACCTGACGCAATATTTGTCTTTTCTTGCTTAATAATGTTGTCA 3’
I14018 5’GATCTGTAAGTTTATACATAGGCGAGTACTCTGTTATGG 3’
Pept 5’GATCGCAGGGGTAAAAGTGACCTGACGCAATATTTGTCTTTTCTTGCTTCTTAATAATGTTGTCACAAAAAGTGAGGGTGACTACATGG 3’
Ptet‐arcA 5’GATCGTTAATAAAATGTTATTGACAGTTAATAAAATGTTATACTGAGC 3’
Ptet‐Fnr 5’GATCAAAATTGATCTGAATCAATATTTTGACAAAAATTGATCTGAATCAATATTTACTGAGC 3’
R1074 5’GATCTTAAATTTCCTCTCGTCAGGCCGGAATAACTCCCTATAATGCGCCACCACACTGATAGTGCTAGTGTAGATCAC 3’
Ssbp1 5’GATCAACCGAGGTCACAACATAGTAAAAGCGCTATTGGTAATGGTACAATCGCGCGTTTACACTTATTCAGAACGATTTTTTTCAGGAG 3’
YsgAP 5’GATCTCAGAAGAAGCAAAAAGACACTTTACCGAAGGGTTTAACATTTTTTCGTGATACTCATCACCATGACGCAAATGCGTTGCATAAA 3’
表2本发明克隆所用的引物
Figure PCTCN2020126769-appb-000001
实施例：
实施例1：构建9株严格厌氧的菌株(SL7207(ΔdapA)-Promoters-BBa_B0033-dapA)(以下实验中所用引物如表2所示)
1.构建pSC101-BBa_B0033-dapA质粒
a.以pSC101-FbFp-KnaR-loxp+启动子质粒为模板，以载体正向引物、载体反向引物为引物，PCR获得线性化载体片段1；
b.以沙门氏菌SL7207基因组为模板，dapA基因正向引物、dapA基因反向引物为引物，PCR获得目的片段1；
c.一步克隆方法获得pSC101-BBa_B0033-dapA质粒。
2.构建pSC101-R1074-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；
b.引物退火方法获得R1074启动子片段；
c.连接酶连接获得pSC101-R1074-BBa_B0033-dapA质粒。
3.构建SL7207(ΔdapA)-R1074-BBa_B0033-dapA菌株
a.以pSC101-R1074-BBa_B0033-dapA质粒为模板，以dapA同源重组正向引物、dapA同源重组反向引物为引物，PCR获得同源重组片段2；
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)-R1074-BBa_B0033-dapA目标菌株。
其他8株目标菌株(如下)构建方法与SL7207(ΔdapA)-R1074-BBa_B0033-dapA菌株基本一致，需要将步骤2中b引物退火片段分别置换为Hip1、I14018、Ptet-Fnr、PepT、Ptet-arcA、Ssbp1、Fnr-SP、YsgAP启动子片段。
1.SL7207(ΔdapA)-Fnr-SP-BBa_B0033-dapA；
2.SL7207(ΔdapA)-Hip1-BBa_B0033-dapA；
3.SL7207(ΔdapA)-I14018-BBa_B0033-dapA；
4.SL7207(ΔdapA)-Pept-BBa_B0033-dapA；
5.SL7207(ΔdapA)-Ptet-arcA-BBa_B0033-dapA；
6.SL7207(ΔdapA)-Ptet-Fnr-BBa_B0033-dapA；
7.SL7207(ΔdapA)-Ssbp1-BBa_B0033-dapA；
8.SL7207(ΔdapA)-YsgAP-BBa_B0033-dapA。
9株菌株分别简写成(Fnr-SP；Hip1；I14018；Pept；Ptet-arcA；Ptet-Fnr；R1074；Ssbp1；YsgAP)，电泳结果如图2A-图2G所示。
实施例2：9株菌株体外表征
有氧条件下表征：挑取1个单克隆加入到含有卡那霉素的LB(DAP+)培养基中；挑取3个克隆分别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：9株菌株在LB(DAP+)培养基中培养144h，这些菌株可以正常生长。9株菌株在LB(DAP-)培养基中培养144h，这些菌株无法生长。
(2)厌氧条件下：9株菌株在LB(DAP+)培养基与LB(DAP-)培养基中培养24h。这些菌株在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3：9株菌株体内表征
C57BL/6小鼠皮下接种1×10 6小鼠膀胱癌细胞(MB49)/只小鼠，建立小鼠膀胱癌皮下瘤模型。实验分为PBS组、SL7207菌株组、Fnr-SP组、Hip1组、I14018组、Pept组、Ptet-arcA组、Ptet-Fnr组、R1074组、Ssbp1组、YsgAP组。尾静脉接种1×10 7个本发明的各种细菌/只小鼠。检测6天内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4E)：
(1)细菌在荷瘤小鼠体内分布(图4A-图4E，左侧列)：Fnr-SP组该菌株在体内清除速度比较慢，正常组织与肿瘤组织具有大量细菌；Hip1组、I14018组、R1074组、Ssbp1组菌株在体内清除比较快。在心脏、肝脏、肺脏、肾脏、血液里细菌量比较少，脾脏和肿瘤组织存在细菌有10 4(CFU/g)。Pept组和YsgAP组细菌只在肝脏与脾脏有少量残留，其他组织器官与肿瘤均无细菌。Ptet-arcA组细菌只在心脏、肝脏、肺脏存在少量细菌，在脾脏、肾脏、血液和肿瘤组织均无细菌。Ptet-Fnr组细菌只存在肝脏与肿瘤组织，其他脏器均无细菌。
(2)肿瘤体积变化(图4A-图4E，中间列)：与PBS组比较，9株菌株组别在6天内均具有抑制肿瘤生长作用。
(3)小鼠体重变化(图4A-图4E，右侧列)：与SL7207组比较，9株菌株组别小鼠体重下降比较少，略低于PBS组。
(4)小鼠生存率：SL7207组小鼠6天内全部死亡。实验周期内9株菌株组别与PBS组别小鼠无死亡。
实验结论：6天内，9株改造菌株在体内正常组织器官与肿瘤内分布不完全相同。与 SL7207组比较，本发明的9种细菌在体内被大量清除。本发明的9株菌株组别肿瘤体积均减小。9株菌株的小鼠在实验周期内体重略微低于PBS组，并且无死亡。说明与现有技术相比，本发明的9株菌株安全性得到了提高，同时具有抑制肿瘤作用。
实施例4：构建5株严格厌氧的菌株(SL7207(ΔdapE)-Promoters-BBa_B0033-dapE)
1.构建pSC101-Promoters-BBa_B0033-dapE质粒
a.以pSC101-Promoters(R1074、YsgAP、Fnr-SP、Pept、Hip1)-BBa_B0033-dapA质粒为模板，以载体正向引物3、载体反向引物4为引物，PCR获得5个线性化载体片段(结果如图5中(A))；
b.以沙门氏菌SL7207基因组为模板，dapE基因正向引物、dapE基因反向引物为引物，PCR获得目的片段2(结果如图5中(A))；
c.一步克隆方法获得pSC101-Promoters(R1074、YsgAP、Fnr-SP、Pept、Hip1)-BBa_B0033-dapE质粒(结果如图5中(B))。
3.构建SL7207(ΔdapE)-Promoters(R1074、YsgAP、Fnr-SP、Pept、Hip1)-BBa_B0033-dapE菌株
a.以pSC101-Promoters(R1074、YsgAP、Fnr-SP、Pept、Hip1)-BBa_B0033-dapE质粒为模板，以dapE同源重组正向引物、dapE同源重组反向引物为引物，PCR获得5个同源重组片段(结果如图5中(C))；
b.通过λ-red同源重组方法将5个同源重组片段整合到SL7207(ΔdapE)的原dapE基因位置，获得SL7207(ΔdapE)-Promoters(R1074、YsgAP、Fnr-SP、Pept、Hip1)-BBa_B0033-dapE目标菌株(结果如5中(D)、(E))。
5株菌株分别简写成(R1074-1、YsgAP-1、Fnr-SP-1、Pept-1、Hip1-1)。
实施例5：5株严格厌氧菌株(SL7207(ΔdapE)-Promoters(R1074、YsgAP、Fnr-SP、Pept、Hip1)-BBa_B0033-dapE)体外表征
有氧条件下表征：挑取3个克隆加入到含有壮观霉素的LB(DAP+)培养基中，空气摇床(37℃，220rpm)过夜培养。将过夜培养菌液以1：100比例进行转接。取20μl菌液加入到2ml含有壮观霉素的LB(DAP+)培养基中；取20μl菌液加入到2ml含有壮观霉素的LB(DAP-)培养基中，3个重复。在空气摇床(37℃，220rpm)中培养72h，观察菌株生长情况。
厌氧条件下表征：挑取3个单克隆加入到含有壮观霉素的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含有壮观霉素的LB(DAP+)培养基中；取20μl菌液加入到2ml含有壮观霉素的LB(DAP-)培养基中，3个重复。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6A、图6B、图6C)：
(1)有氧条件下：5株菌株在LB(DAP+)培养基中培养72h，这些菌株可以正常生长。5株菌株在LB(DAP-)培养基中培养72h，这些菌株无法生长。
(2)厌氧条件下：5株菌株在LB(DAP+)培养基与LB(DAP-)培养基中培养24h。这些菌株在LB(DAP+)培养基与LB(DAP-)培养基中均可以生长。
实验结论：通过对5株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6：R1074-1菌株体内表征
C57BL/6小鼠皮下接种1×10 6小鼠膀胱癌细胞(MB49)/只小鼠，建立小鼠膀胱癌皮下瘤模型。实验分为PBS组、R1074-1组。尾静脉接种1×10 7个本发明的细菌/只小鼠。检测14天内肿瘤体积变化、小鼠体重变化、小鼠生存率。实验结果(如图7)：
(1)肿瘤体积变化(图7中A)：与PBS组比较，R1074-1菌株组别在实验周期内具有抑制肿瘤生长作用。
(2)小鼠体重变化(图7中B)：与PBS组比较，R1074-1菌株组别在试验周期内小鼠体重无显著性差异
(3)小鼠生存率:实验周期内，PBS组与R1074-1菌株组别小鼠均存活。
实验结论：在实验周期内，R1074-1菌株可以抑制小鼠肿瘤生长；同时与PBS组比较，小鼠体重无显著性差异和小鼠均存活，说明该菌株比较安全。

Claims (10)

  1. 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肿瘤治疗时，可以抑制肿瘤生长和减小肿瘤体积。
  2. 根据权利要求1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)或其类似物；和/或
    其中所述必需基因包括dapA或dapE但不局限于该基因，还包括选自dapB、dapD、argD、dapF、murE、murF或lysA的一种或多种；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。
  3. 根据权利要求1所述的方法，其中所述厌氧激活的启动子选自Pept、Fnr-SP、Hip1、I141018、Ptet-arcA、Ptet-Fnr、R1074、Ssbp1和YsgAP。
  4. 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；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(子宫颈癌、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，前列腺癌等。
  5. 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。
  6. 根据权利要求5所述的载体，其中所述厌氧激活启动子选自Pept、Fnr-SP、Hip1、I141018、Ptet-arcA、Ptet-Fnr、R1074、Ssbp1和YsgAP。
  7. 根据权利要求5所述的载体，其中所述必需基因所述必需基因包括dapA或dapE但不局限于该基因，还包括选自dapB、dapD、argD、dapF、murE、murF或lysA的一种或多种。
  8. 根据权利要求5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。
  9. 一种严格厌氧沙门氏菌在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。
  10. 根据权利要求9所述的严格厌氧沙门氏菌在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。
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