CN114438111B - Construction of PP2 strict anaerobic salmonella strain and application thereof in tumor treatment - Google Patents

Construction of PP2 strict anaerobic salmonella strain and application thereof in tumor treatment Download PDF

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CN114438111B
CN114438111B CN202011221996.0A CN202011221996A CN114438111B CN 114438111 B CN114438111 B CN 114438111B CN 202011221996 A CN202011221996 A CN 202011221996A CN 114438111 B CN114438111 B CN 114438111B
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cancer
anaerobic
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CN114438111A (en
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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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Shenzhen Institute of Advanced Technology of CAS
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to an anaerobic activated promoter PP2 gene sequence, an anaerobic gene loop regulated by the PP2 promoter, a strict anaerobic salmonella typhimurium and a vector containing the anaerobic gene loop regulated by the PP2 promoter and application thereof, a method for changing facultative anaerobic bacteria into strict anaerobic bacteria, a method for treating cancers by using bacteria regulated by the anaerobic loop and application thereof in tumor treatment.

Description

Construction of PP2 strict anaerobic salmonella strain and application thereof in tumor treatment
Technical Field
The invention relates to the field of tumor targeted therapy, in particular to an anaerobic activated promoter PP2 gene sequence, an anaerobic gene loop regulated and controlled by the PP2 promoter, a strict anaerobic salmonella typhimurium and a vector containing the anaerobic gene loop regulated and controlled by the PP2 promoter and application thereof, a method for changing facultative anaerobic bacteria into strict anaerobic bacteria and a method for treating cancers by using bacteria regulated and controlled by the anaerobic loop.
Background
Cancer is a leading cause of death worldwide. Compared with normal cells, the cancer cells have the characteristics of unlimited proliferation, conversion, easy metastasis and the like. In addition to uncontrolled division (capable of multipolar division), cancer cells can also invade surrounding normal tissues locally and metastasize to other organs even via the circulatory system or lymphatic system in the body. The development history of cancer treatment shows that the traditional cancer treatment methods such as surgical treatment, chemotherapy, radiotherapy, immunological therapy, hormone therapy, bone marrow/stem cell transplantation and other treatment means have certain defects, for example, the surgical treatment has the problems of easy recurrence, difficult operation of partial tumors and the like, and the chemotherapy can generate serious side effects on patients, so that the treatment cannot be effectively performed. The cancer treatment difficulty is caused by complex and variable etiology, so that not only is the change of the gene level of the organism existed, but also the change of the external environment is one of important factors for the development of the cancer. The disadvantage of conventional therapies such as long-term radiation therapy, chemotherapy and immunotherapy is that these treatment regimens not only produce serious toxicity to normal tissues and organs, but also produce multiple drug resistance to cancer cells and do not completely eliminate cancer cells. In recent years, studies have found that gene therapy, noninvasive radiofrequency therapy for cancer, insulin potentiation therapy, diet therapy and bacterial therapy not only can prevent cancer cells from developing multiple drug resistance, but also can enhance the efficacy of conventional therapies. Among them, bacterial therapy is a promising cancer treatment approach to overcome the shortcomings of conventional therapies.
The history of cancer treatment with live bacteria can be traced back to 150 years ago. In 1868, german physician w.bus first applied bacterial treatment to sarcoma that could not be treated by surgical methods, the patient had a half-reduced tumor volume and a reduced cervical lymph node volume within one week of receiving treatment. Unfortunately, however, the patient died after 9 days from sepsis due to bacterial infection. In 1883 german surgeon Friedrich Fehleisen identified erysipelas as being caused by streptococcus pyogenes infection. Subsequently, friedrich Fehleisen and Willian B Coley from New York Hospital surgeon each independently conducted experiments demonstrated that Streptococcus pyogenes could regress the tumor in the patient. However, the results are controversial because the experimental results are difficult to repeat and do not meet the current clinical criteria. Connell 1935 observed that the filtrate from Clostridium enzymes could regress metastases. Scientists in 1947 first injected Clostridium histolyticum spores into sarcoma-transplanted mice, and observed cancer cell lysis and tumor tissue regression. However, mice survived very poorly due to the acute toxic response caused by bacteria. BCG vaccine (attenuated Mycobacterium bovis) was successfully used in the treatment of cancer by immunization in 1959. The 2002 attenuated salmonella VNP20009 (msbB-, purI-) is subjected to a phase I clinical test, and the result shows that the strain can be planted in tumor tissues, but has an insignificant effect on tumor treatment.
Although VNP20009 did not achieve good clinical results, in view of the tumor-aggregating growth and immunomodulatory function of salmonella, researchers thought that salmonella could potentially be adapted for tumor treatment by a variety of modification. The salmonella is required to be modified because wild salmonella has toxicity, can cause symptoms such as fever, vomiting, diarrhea, abdominal cramps and the like, and can seriously cause bacteremia to endanger life. With rapid development of molecular biology technology, salmonella can be modified by different strategies to be suitable for application in tumor therapy. Can knock out genes related to salmonella and virulence, regulate bacterial growth by constructing auxotroph strains, gene loops and the like, so that the attenuated strain can be used for tumor treatment early.
Bin Yu et al 2012 published a research paper titled Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain in journal SCIENTIFIC REPORTS, and the construction of strict anaerobic salmonella strain protocols described in the paper. The prior art constructs salmonella typhimurium SL7207 knockout key gene asd strain, the asd gene deletion can influence the generation of bacterial cell walls, and the addition of an intermediate metabolite DAP (diaminopimelic acid) at the downstream of the asd gene in LB culture medium can enable the bacteria to normally synthesize the cell walls. And constructing an anaerobic strain YB1 on the basis: a gene loop (gene loop inserted into the original asd gene position) with anaerobic regulation of Cm-peptide-asd-sodA was inserted into the genome of the SL7207 knockout asd strain. FNR is a transcription regulatory factor regulated by oxygen. Under anaerobic conditions, FNR is in an activated state, and can regulate and control a forward promoter Pept to enable asd genes to be transcribed, so that bacteria can generate complete cell walls. The reverse promoter PsodA blocks leakage of the asd gene product produced by the forward promoter under aerobic conditions. The design can enable YB1 strain to grow only under anaerobic condition, and DAP must be added into the culture medium under aerobic condition.
Characterization of viability of its YB1 strain by Bin Yu et al under different oxygen conditions: under aerobic conditions, YB1 can not grow in LB (DAP-) culture medium, and can grow in LB (DAP+) culture medium; under anaerobic conditions YB1 can grow in LB (DAP+), LB (DAP-) medium. Characterization of the distribution and treatment effect of YB1 strain in tumor-bearing mice: 26 days after the YB1 strain is injected into the tail of a tumor-bearing mouse intravenously, the strain is cleared in normal tissues and organs, and bacteria exist in tumor tissues (due to the low oxygen concentration of the tumor tissues and the immune suppression environment). Compared with PBS group, YB1 strain has tumor growth inhibiting ability.
However, the YB1 strain of Bin Yu et al needs up to 26 days to be completely cleared in normal tissues and organs, and has long time consumption and low safety; compared with PBS group, after the YB1 is intravenous injected into the tail of the tumor-bearing mice, the weight of the mice is remarkably reduced (more than 5%). As an important evaluation index for the health of mice, the obvious weight reduction shows that the strain has stronger toxic effect on the mice.
The field also needs to be able to be cleared by normal tissue organs easily in a shorter time, weakening the toxic and side effects on tumor-bearing mice caused by long-term bacteria remaining in the body, making the transformed strain safer and more reliable, and not affecting the tumor treatment effect of the bacteria.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide an anaerobic activating promoter PP2 gene sequence, an anaerobic gene loop regulated and controlled by the PP2 promoter, a strict anaerobic salmonella typhimurium containing the anaerobic gene loop regulated and controlled by the PP2 promoter and application of the strict anaerobic salmonella typhimurium in tumor treatment.
In one aspect of the present invention, there is provided an anaerobically activated promoter PP2 gene sequence, the nucleotide sequence of said anaerobically activated promoter PP2 being shown in SEQ ID No. 1:
SEQ ID No.1：
5’-ttgcggccgcaagaagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctc-3’。
in one aspect of the invention, there is provided a vector which is a prokaryotic cell comprising the following elements: (a) a hypoxia or strictly anaerobically activated promoter; (b) an essential gene under the control of the promoter of (a); (c) Wherein the promoter of (a) is present at a site that binds to an anaerobically activated transcription regulator.
In one aspect of the invention, in the vector, the anaerobically activated promoter is PP2, and the nucleotide sequence of the anaerobically activated promoter PP2 is as follows:
SEQ ID No.1：
5’-ttgcggccgcaagaagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctc-3’。
in one aspect of the invention, in the vector, the transcription regulatory factor is arcA or FNR.
In one aspect of the present invention, in the vector, the essential gene is selected from dapA and dapE.
In one aspect of the invention, in said carrier, culturing under in vitro aerobic conditions requires the addition of 2,6-diaminopimelic acid or analogues thereof to the medium.
In one aspect of the invention there is provided a use of a strictly anaerobic salmonella expression drug or a drug carried as a carrier for the treatment of cancer.
In one aspect of the invention, in the application, the medicament comprises: (a) Expressing a protein substance or a polypeptide substance having a therapeutic effect on cancer; (b) expressing RNA having a therapeutic effect on cancer; (c) carrying the modified RNA drug as a carrier.
In one aspect of the present invention, a method is provided for converting facultative anaerobes into strict anaerobes by hypoxia or strict anaerobism inducing loops of expression essential genes, and the strict anaerobes can inhibit tumor growth and reduce tumor volume when applied to in vivo tumor therapy.
In one aspect of the invention, in the above method, the facultative anaerobic bacteria is Salmonella species (Salmonella).
In one aspect of the present invention, in the above method, the strictly anaerobic bacterium is cultured in vitro under aerobic conditions, and 2,6-diaminopimelic acid (alias: 2, 6-diaminosyzygic acid; 2,6-Diaminopimelic acid) and the like are added to the medium.
In one aspect of the present invention, in the above method, the essential gene is selected from dapA and dapE, but is not limited to these two genes, and one or more of dapB, dapD, argD, dapF, murE, murF and lysA and the like are also included.
In one aspect of the invention, in the above method, the stringent anaerobic regulatory gene loop consists of an anaerobically activated promoter and essential genes.
In one aspect of the invention, in the above method, the gene loop of the essential gene for hypoxia or strict anaerobically induced expression regulation is present in a chromosome or other plasmid vector.
In one aspect of the invention, in the above method, the anaerobically activated promoter is PP2.
In one aspect of the present invention, in the above method, the tumor cancer includes blood cancer (chronic leukemia, acute leukemia), bone cancer, lymph cancer (non-hodgkin lymphoma, hodgkin lymphoma), intestinal cancer (colon cancer, rectal cancer), liver cancer, stomach cancer, pelvic cancer (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer, and the like.
In one aspect of the invention, methods are provided for treating cancer using anaerobic loop regulated bacteria comprising strictly hypoxia regulated essential gene expression.
In one aspect of the invention, the method of treatment further comprises: used in combination with other therapeutic cancer therapies, but are not limited to, radiotherapy, chemotherapy, immunotherapy, etc.
In one aspect of the invention, the bacterium is Salmonella typhi (Salmonella typhi) in the method of treatment.
In one aspect of the invention, the method of treatment wherein the facultative anaerobic bacteria is salmonella typhimurium (Salmonella typhimurium).
In one aspect of the invention, the method of treatment comprises the facultative anaerobic salmonella strain being of human, chicken, dog, cow, and the like.
In one aspect of the invention, the method of treatment, the facultative anaerobe genus comprises: bacteria of the Enterobacteriaceae family (Escherichia coli, klebsiella pneumoniae, proteus, enterobacter, typhoid bacillus, salmonella, shigella, etc.), staphylococcus, streptococcus, pneumococcus, bacillus anthracis, diphtheria bacillus, etc.
In one aspect of the invention, the method of treatment, the combination of the other methods of cancer treatment, comprises: (a) bacterial therapy of anaerobic strains in combination with surgical therapy; (b) bacterial therapy of anaerobic strains in combination with radiation therapy; (c) bacterial therapy of anaerobic strains in combination with a chemical: chemotherapeutic agents include alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, glimepiride, etc.), antimetabolites (doxifluridine, 6-mercaptopurine, cytarabine, fluoroguanosine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, you fodine, ancitabine, etc.), antitumor antibiotics (actinomycin, doxorubicin, epirubicin, mitomycin, pelomycin, pingyancin, picoline, etc.), botanical anticancer agents (irinotecan, cephalotaxine, hydroxycamptothecine, vinorelbine, taxol, taxotere, topotecan, vincristine, vindesine, vinblastine, etc.), immunosuppressants (almitant, anastrozole, an Lumi, letrozole, medetoram, moxifene, etc.), anticancer agents such as colpitan, carboplatin, oxazine, procarbazine, etc.; (d) bacterial therapy of anaerobic strains in combination with biological therapy; (e) Bacterial therapy of anaerobic strains is combined with traditional Chinese medicine treatment.
Drawings
FIG. 1 is a diagram showing the construction of SL7207 (. DELTA.dapA) -PP 2-BBa-B0033-dapA strain.
FIG. 2 is an electrophoretogram of the SL7207 (ΔdapA) -PP2-BBa_B0033-dapA strain and the SL7207 (ΔdapE) -PP2-BBa_B0033-dapE strain.
FIGS. 3A, 3B and 3C are in vitro experiments of SL7207 (ΔdapA) -PP2-BBa_B0033-dapA strain, FIG. 3A is a photograph of strain aerobically cultured for 24 and 48 hours, FIG. 3B is a photograph of strain anaerobically cultured for 24 hours, FIG. 3C shows that the strain of the present invention grows in DAP+ under aerobically conditions, and does not grow in DAP-.
FIGS. 4A, 4B, 4C and 4D are in vivo experiments of SL7207 (ΔdapA) -PP2-BBa_B0033-dapA strain.
FIGS. 5A, 5B and 5C are in vitro representations of SL7207 (ΔdapE) -PP 2-BBa_B0033-dapE.
FIG. 6 is an in vivo experiment of SL7207 (ΔdapE) -PP2-BBa_B0033-dapE strain.
Detailed Description
While the invention is susceptible to various modifications and alternative forms, specific examples are described and illustrated in detail below. It should be understood, however, that there is no intention to limit the invention to the specific disclosure, and the invention includes all modifications, equivalents, or alternatives thereof without departing from the spirit and technical scope of the invention.
Hereinafter, the anaerobic activated promoter PP2 gene sequence, the anaerobic gene circuit regulated by the PP2 promoter, the strictly anaerobic salmonella typhimurium and vectors comprising the anaerobic gene circuit regulated by the PP2 promoter and the use thereof, the method of converting facultative anaerobic bacteria into strictly anaerobic bacteria, the method of treating cancer using the bacteria regulated by the anaerobic circuit, and the use thereof in tumor treatment according to the specific embodiments of the present invention will be explained in more detail.
In one or more embodiments of the invention, the vector of the invention is a prokaryotic cell comprising the following elements: (a) a hypoxia or strictly anaerobically activated promoter; and (b) an essential gene under the control of the promoter in (a); wherein the promoter of (a) is present at a site that binds to an anaerobically activated transcription regulator.
In one or more embodiments of the invention, (a) the hypoxia or strictly anaerobic activated promoter may be, for example, PP2 and PP3; PP3 is preferred. In particular PP2.
In one or more embodiments of the invention, the nucleotide sequence of the anaerobic activation promoter PP2 is as follows:
SEQ ID No.1：
5’-ttgcggccgcaagaagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctc-3’。
in one or more embodiments of the present invention, the essential gene (b) under the control of the promoter in (a) may be dapA, dapB, dapD, argD, dapE, dapF, murE, murF, lysA or the like, for example; in particular dapA and dapE.
In one or more embodiments of the invention, (c) wherein the promoter in (a) is present at an anaerobically activated transcription regulator binding site may 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 converting facultative anaerobes into strict anaerobes by hypoxia or strict anaerobism inducing loops expressing essential genes.
In one or more embodiments of the invention, the stringent anaerobic regulatory gene loop consists of an anaerobically activated promoter and essential genes.
In one or more embodiments of the invention, the anaerobically activated promoters may be, for example, PP2 and PP3; PP3 is preferred. In particular PP2.
In one or more embodiments of the present invention, the essential gene may be dapA, dapB, dapD, argD, dapE, dapF, murE, murF, lysA or the like, for example; in particular dapA and dapE
When the essential genes are dapA and dapE genes, the vector of the present invention requires the addition of 2,6-diaminopimelic acid (alias: 2, 6-diaminosyzygic acid; 2,6-Diaminopimelic acid) or an analogue thereof to the medium when the vector is cultured under aerobic conditions.
When the strict anaerobe is applied to in vivo tumor treatment, the strict anaerobe can inhibit tumor growth and reduce tumor volume.
The facultative anaerobe may be any species from any of the bacterial genera escherichia coli, pneumobacterium, proteus, enterobacterium, typhoid bacillus, salmonella, shigella, etc., streptococcus, pneumococcus, bacillus anthracis, diphtheria bacillus, etc.
The source of the facultative anaerobic salmonella strain is not limited as long as it is facultative anaerobic, and for example, facultative anaerobic salmonella strains derived from humans, chickens, dogs, cows, and the like are included.
The facultative anaerobic bacteria is salmonella typhimurium (Salmonella typhimurium).
The invention also provides bacterial therapies utilizing the strains of the invention that are incapable of growing under both aerobic and anaerobic conditions for the treatment of cancer.
The cancers include blood cancer (chronic leukemia, acute leukemia), bone cancer, lymph cancer (non-hodgkin lymphoma, hodgkin lymphoma), intestinal cancer (colon cancer, rectal cancer), liver cancer, stomach cancer, pelvic cancer (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer, and the like.
In one or more embodiments of the invention, the vector of the invention as a prokaryotic cell, or the strictly anaerobic bacteria obtained by the method of the invention, may be used as bacterial therapy for anti-tumor or cancer treatment.
In one or more embodiments of the invention, the bacterial therapies of the invention may be used in combination with other methods of cancer treatment.
In one or more embodiments of the invention, the combined use of bacterial therapies with other methods of cancer treatment include, for example: (a) bacterial therapy of anaerobic strains in combination with surgical therapy; (b) bacterial therapy of anaerobic strains in combination with radiation therapy; (c) bacterial therapy of anaerobic strains in combination with a chemical: chemotherapeutic agents include alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, glimepiride, etc.), antimetabolites (doxifluridine, 6-mercaptopurine, cytarabine, fluoroguanosine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, you fodine, ancitabine, etc.), antitumor antibiotics (actinomycin, doxorubicin, epirubicin, mitomycin, pelomycin, pingyancin, picoline, etc.), botanical anticancer agents (irinotecan, cephalotaxine, hydroxycamptothecine, vinorelbine, taxol, taxotere, topotecan, vincristine, vindesine, vinblastine, etc.), immunosuppressants (almitant, anastrozole, an Lumi, letrozole, medetoram, moxifene, etc.), anticancer agents such as colpitan, carboplatin, oxazine, procarbazine, etc.; (d) bacterial therapy of anaerobic strains in combination with biological therapy; (e) Bacterial therapy of anaerobic strains is combined with traditional Chinese medicine treatment.
The vector of the present invention as a prokaryotic cell, or the strictly anaerobic bacterium obtained by the method of the present invention can also be used for inducing expression of a drug in vitro or carrying a drug as a vector for cancer treatment.
In an embodiment of the present invention, the drug that may be carried in the carrier includes: (a) Expressing a protein substance or a polypeptide substance having a therapeutic effect on cancer; (b) expressing RNA having a therapeutic effect on cancer; (c) carrying the modified RNA drug as a carrier.
The asd gene and dapA, dapE belong to DAP metabolic pathway, and the asd gene is located upstream of dapA, dapE gene. DAP, an important constituent of bacterial cell walls, is produced downstream of the asd, dapA and dapE genes. The SL7207 knockout of the key gene asd strain is based on SL7207 knockout of the asd gene. In the embodiment of the present invention, the base bacteria SL7207 (ΔdapA) and SL7207 (ΔdapE) are obtained by knocking out dapA and dapE genes, respectively, based on SL 7207.
The invention has the advantages that:
(1) The anaerobic regulation and control module of the PP2 strain is simpler, the regulation and control system is more strict, and the background leakage problem does not exist under the aerobic condition;
(2) The PP2 strain can be completely cleared in a short time in normal tissues and organs;
(3) The PP2 strain has no influence on the weight of mice in the process of treating tumor-bearing mice, has smaller toxic and side effects and improves the safety.
The present invention cloned and constructed SL7207 (ΔdapA) -PP2-BBa_B0033-dapA strain and SL7207 (ΔdapE) -PP2-BBa_B0033-dapE strain using the primers shown in the following table. FIG. 1 is a diagram showing the construction of SL7207 (. DELTA.dapA) -PP 2-BBa-B0033-dapA strain.
Strain construction: the PP2-BBa_B0033-dapA and PP2-BBa_B0033-dapE anaerobic gene loops were integrated into the SL7207 (ΔdapA) genome (original dapA gene locus) and the SL7207 (ΔdapE) genome (original dapE gene locus), respectively, on the basis of the SL7207 (ΔdapA) strain and the SL7207 (ΔdapE) strain which have been constructed in the present laboratory. The dapA and dapE genes are key genes on lysine metabolic pathways, bacteria with the dapA or dapE genes knocked out cannot form normal cell walls, the osmotic pressure inside and outside the bacteria is unbalanced, and bacterial rupture cannot survive. The PP2 promoter is an anaerobically activated promoter, and PP2 can initiate transcription of the dapA gene or dapE gene under anaerobic or hypoxia conditions, so that the downstream key protein DAP is normally produced. Bacteria can form an intact cell wall. Under aerobic conditions, the PP2-BBa_B0033-dapA and PP2-BBa_B0033-dapE gene loops are in an inactivated state, and the bacteria cannot generate complete cell walls. The strain SL7207 (DeltadapA) -PP2-BBa_B0033-dapA and the strain SL7207 (DeltadapE) -PP2-BBa_B0033-dapE are cultivated under aerobic conditions, DAP (diaminopimelic acid) needs to be added, and the defect that the bacteria cannot form complete cell walls due to the deletion of dapA and dapE genes can be overcome. The promoter sequences used in the present invention, and primers used for cloning are shown in Table 1.
TABLE 1 promoter sequences employed in the present invention, and primers for cloning
Examples:
example 1: construction and electrophoretic verification of SL7207 (DeltadapA) -PP2-BBa_B0033-dapA Strain and SL7207 (DeltadapE) -PP2-BBa_B0033-dapE Strain
1. Construction of pSC101-BBa_B0033-dapA plasmid
a. PCR was performed using pSC101-FbFp-KnaR-loxp+promoter plasmid as a template and vector forward primer 1 and vector reverse primer 2 as primers in the above table to obtain a linear vector 1 (FIG. 2 (A));
b. PCR was performed using the salmonella SL7207 genome as a template and the dapA gene forward primer and the dapA gene reverse primer in the above table as primers to obtain dapA linear fragment (FIG. 2 (B));
c. the pSC101-BBa_B0033-dapA plasmid was obtained by a one-step cloning method. Colony PCR identification results (FIG. 2 (C))
2. Construction of pSC101-PP2-BBa_B0033-dapA plasmid
The pSC101-BBa_B0033-dapA plasmid was digested with BsaI to obtain linearized vector fragment 2 (FIG. 2 (D));
b. the primer annealing method is used for obtaining a PP2 promoter fragment;
c. the plasmid pSC101-PP2-BBa_B0033-dapA was obtained by ligase ligation. Colony PCR identification results (FIG. 2 (E)).
3. Construction of pSC101-PP2-BBa_B0033-dapE plasmid
a. PCR was performed using pSC101-PP2-BBa_B0033-dapA plasmid as a template and vector forward primer 3 and vector forward primer 4 as primers in the above table to obtain linear vector fragment 3 (FIG. 2 (H));
b. PCR was performed using the Salmonella SL7207 genome as a template and the dapE gene forward primer and the dapE gene reverse primer in the above tables as primers to obtain a dapE linear fragment (FIG. 2 (I));
c. the pSC101-PP2-BBa_B0033-dapE plasmid is obtained by a one-step cloning method. Colony PCR identification results (FIG. 2 (J)).
4. Construction of a SL7207 (ΔdapA) -PP2-BBa_B0033-dapA strain and a SL7207 (ΔdapE) -PP2-BBa_B0033-dapE strain;
a. taking pSC101-PP2BBa_B0033-dapA plasmid as a template, taking dapA homologous recombination Forward primer and dapA homologous recombination Reverse primer dapA-RE-Forward-primer in the table as primers, and obtaining homologous recombination fragment 2 by PCR (FIG. 2 (F));
b. the homologous recombination fragment 2 was integrated into the original dapA gene position of SL7207 (ΔdapA) by a lambda-red homologous recombination method to obtain a target strain of SL7207 (ΔdapA) -PP2-bba_B0033-dapA (abbreviated as: PP2 strain). Extraction of genome PCR identification results are shown in (G) of FIG. 2;
c. PCR was performed using pSC101-PP2-BBa_B0033-dapE plasmid as a template, and the forward primer for dapE homologous recombination and the reverse primer for dapE homologous recombination in the above table as primers, to obtain homologous recombination fragment 4 (FIG. 2 (K));
d. the homologous recombination fragment 4 was integrated into the original dapE gene position of SL7207 (ΔdapE) by a lambda-red homologous recombination method to obtain a target strain of SL7207 (ΔdapE) -PP2-BBa_B0033-dapE (abbreviated as: PP 2-1). The PCR identification results are shown in FIG. 2 (L).
Example 2: in vitro characterization of SL7207 (ΔdapA) -PP2-BBa_B0033-dapA
Characterization under aerobic conditions: 5 individual clones were picked and resuspended in 10. Mu.l of LB medium, respectively. Mu.l of the bacterial suspension was added to LB (DAP+) medium containing kanamycin, and the remaining 5. Mu.l of the bacterial suspension was added to LB (DAP-) medium containing kanamycin. Culturing (37 ℃ C., 220 rpm) in an air shaker for a period of time.
Characterization under anaerobic conditions: 3 of the monoclonal were picked and added to LB (DAP+) medium containing kanamycin. The cells were incubated overnight in an air shaker (37 ℃,220 rpm). The overnight cultured bacterial liquid is put into an anaerobic incubator to make 1:100 ratio for transfer. Mu.l of the bacterial liquid was added to 2ml of LB (DAP+) medium containing kanamycin; mu.l of the bacterial liquid was added to 2ml of LB (DAP-) medium containing kanamycin, and the mixture was repeated 3 times. The initial OD600 value of the samples after the transfer was measured. And (3) in an anaerobic box, standing and culturing for 24 hours at 37 ℃. The OD600 of the samples after 24h of incubation was measured.
Experimental results (as in fig. 3A-3C):
(1) Under aerobic conditions: the strain is cultured in LB (DAP+) culture medium for 24 hours, and the strain can grow normally. The strain is cultured in LB (DAP-) culture medium for 48 hours, and the strain cannot grow.
(2) Under anaerobic conditions: the strain was cultured in LB (DAP+) medium and LB (DAP-) medium for 24h. The strain can grow in LB (DAP+) culture medium and LB (DAP-) culture medium.
Conclusion of experiment: tests on the strain under aerobic and anaerobic conditions show that the facultative anaerobe strain SL7207 is successfully modified into a strict anaerobe strain.
Example 3: in vivo characterization of SL7207 (ΔdapA) -PP2-BBa_B0033-dapA (abbreviated PP 2)
Subcutaneous inoculation of C57BL/6 mice 1X 10 6 Mouse bladder cancer cells (MB 49), a mouse bladder cancer subcutaneous tumor model was established. The experiments were divided into three groups, PBS group, SL7207 strain group, SL7207 (ΔdapA) -PP2-BBa_B0033-dapA group. Tail vein inoculation 1×10 7 Bacteria. Detecting the distribution of bacteria in normal tissues and organs and tumors of tumor-bearing mice, the change of tumor volume, the change of weight of the mice and the survival rate of the mice. Experimental results (as in fig. 4A, 4B, 4C, and 4D):
(1) Bacterial distribution in tumor-bearing mice (fig. 4A): the strain can be cleared from normal tissue organs of the mice within 14 days. SL7207 group, bacteria rapidly proliferated in normal tissue and tumor within 7 days, and finally mice all died within 7 days.
(2) Tumor volume change (fig. 4B): compared with PBS group, the strain group has a certain tumor growth inhibition effect on tumors within 14 days.
(3) Mouse body weight change (fig. 4C): compared with the SL7207 group, the strain group has less weight loss, is slightly lower than the PBS group, and has no statistical difference.
(4) Survival of mice (fig. 4D): all mice in the SL7207 group died within 7 days. Mice of this strain group and PBS group did not die during the experimental period.
Conclusion of experiment: within 14 days, the engineered strain can be completely cleared in tumor-bearing mice. The tumor volume of the strain group is reduced. Mice had slightly lower body weight than the PBS group during the experimental period and were not dead. The strain has improved safety and certain tumor inhibiting effect.
Example 4: in vitro characterization of SL7207 (DeltadapE) -PP2-BBa_B0033-dapE
Characterization under aerobic conditions: 3 of the monoclonal were picked and added to LB (DAP+) medium containing kanamycin. The cells were incubated overnight in an air shaker (37 ℃,220 rpm). The overnight cultured bacterial liquid was prepared in a ratio of 1:100 ratio for transfer. Mu.l of the bacterial liquid was added to 2ml of LB (DAP+) medium containing kanamycin; mu.l of the bacterial liquid was added to 2ml of LB (DAP-) medium containing kanamycin, and the mixture was repeated 3 times. Culturing in an air shaker for 72h.
Characterization under anaerobic conditions: 3 of the monoclonal were picked and added to LB (DAP+) medium containing kanamycin. The cells were incubated overnight in an air shaker (37 ℃,220 rpm). The overnight cultured bacterial liquid is put into an anaerobic incubator to make 1:100 ratio for transfer. Mu.l of the bacterial liquid was added to 2ml of LB (DAP+) medium containing kanamycin; mu.l of the bacterial liquid was added to 2ml of LB (DAP-) medium containing kanamycin, and the mixture was repeated 3 times. The initial OD600 value of the samples after the transfer was measured. And (3) in an anaerobic box, standing and culturing for 24 hours at 37 ℃. The OD600 of the samples after 24h of incubation was measured.
Experimental results (as in fig. 5A-5C):
(1) Under aerobic conditions: the strain is cultured in LB (DAP+) culture medium for 24 hours, and the strain can grow normally. The strain is cultured in LB (DAP-) culture medium for 72 hours, and the strain cannot grow.
(2) Under anaerobic conditions: the strain was cultured in LB (DAP+) medium and LB (DAP-) medium for 24h. The strain can grow in LB (DAP+) culture medium and LB (DAP-) culture medium.
Conclusion of experiment: tests on the strain under aerobic and anaerobic conditions show that the facultative anaerobe strain SL7207 is successfully modified into a strict anaerobe strain.
Example 5: in vivo characterization of SL7207 (DeltadapE) -PP2-BBa_B0033-dapE (abbreviated as PP 2-1)
Subcutaneous inoculation of C57BL/6 mice 1X 10 6 Mouse bladder cancer cells (MB 49), established subcutaneous for mouse bladder cancerTumor model. The experiments were divided into two groups, PBS group and SL7207 (ΔdapE) -PP2-BBa_B0033-dapE group. Tail vein inoculation 1×10 7 Bacteria. Detecting the distribution of bacteria in normal tissues and organs and tumors of tumor-bearing mice, the change of tumor volume, the change of weight of the mice and the survival rate of the mice. Experimental results (as in fig. 6):
(1) Tumor volume change (a in fig. 6): compared with PBS group, the strain group has a certain tumor growth inhibition effect on tumors within 14 days.
(2) Mouse body weight change (B in fig. 6): the mice of this strain group had little change in body weight compared to the PBS group.
(3) Survival of mice (C in fig. 6): mice of this strain group and PBS group did not die during the experimental period.
Conclusion of experiment: the tumor volume of the strain group is reduced. Mice had slightly lower body weight than the PBS group during the experimental period and were not dead. The strain has improved safety and certain tumor inhibiting effect.

Claims (9)

1. A vector which is salmonella comprising the following elements:
(a) A strictly anaerobically activated promoter which is PP2 with the nucleotide sequence shown below:
SEQ ID No. 1：
5'-ttgcggccgcaagaagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctc-3'; and
(b) An essential gene under the control of the promoter in (a), wherein the essential gene is dapA and dapE;
wherein the promoter of (a) is present at a site that binds to an anaerobically activated transcription regulator.
2. The vector according to claim 1, wherein 2,6-diaminopimelic acid or an analogue thereof is added to a medium for in vitro culture under aerobic conditions.
3. Use of the vector of claim 1 for the preparation of a medicament for the treatment of cancer, said vector expressing the medicament or carrying the medicament as a carrier.
4. The use of claim 3, wherein the medicament comprises: (a) Expressing a protein substance or a polypeptide substance having a therapeutic effect on cancer; (b) expressing RNA having a therapeutic effect on cancer; (c) carrying the modified RNA drug as a carrier.
5. The use according to claim 3, in combination with other therapeutic cancer therapies.
6. The use of claim 5, wherein the combination of the other cancer treatment methods comprises: (a) bacterial therapy of anaerobic strains in combination with surgical therapy; (b) bacterial therapy of anaerobic strains in combination with radiation therapy; (c) bacterial therapy of anaerobic strains in combination with a chemical: chemotherapeutic agents include alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer agents, hormones, immunosuppressants, asparaginase, carboplatin, cisplatin, dacarbazine, oxaliplatin, losartan, platanic australicide, mitoxantrone, procarbazine; (d) The bacterial therapy of the anaerobic strain is combined with biological therapy, and (e) the bacterial therapy of the anaerobic strain is combined with traditional Chinese medicine therapy.
7. The use according to claim 3, wherein the cancer is selected from the group consisting of blood cancer, bone cancer, lymphatic cancer, intestinal cancer, liver cancer, stomach cancer, pelvic cancer, lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer.
8. A method for converting facultative anaerobic salmonella into strict anaerobe by strictly anaerobically inducing the loop of the expression essential gene, and the strict anaerobic salmonella can inhibit the growth of tumor and reduce the tumor volume when being applied to in vivo tumor treatment,
the strictly anaerobic salmonella comprises the circuit consisting of the following (a) strictly anaerobic activating promoter and essential genes under the control of the promoter in (a):
(a) A strictly anaerobically activated promoter which is PP2 with the nucleotide sequence shown below:
SEQ ID No. 1：
5'-ttgcggccgcaagaagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagcctc-3'; and
(b) An essential gene under the control of the promoter in (a), wherein the essential gene is dapA and dapE;
wherein the promoter of (a) is present at a site that binds to an anaerobically activated transcription regulator.
9. The method of claim 8, wherein the facultative anaerobic salmonella strain is derived from a human, chicken, dog, cow; and/or
Wherein 2,6-diaminopimelic acid and analogues thereof are additionally added into a culture medium when the strict anaerobe is cultured under in vitro aerobic conditions; and/or
Wherein the genetic circuit for strictly anaerobically inducing expression of essential genes is present in a chromosomal or other plasmid vector.
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NZ518354A (en) * 1999-10-04 2005-02-25 Vion Pharmaceuticals Inc Compositions and methods for tumor-targeted delivery of effector molecules
CN102477440A (en) * 2010-11-29 2012-05-30 南京大学 Therapeutic gene for anaerobic tissue targeting delivery and selectivity stabilization expression method and its application
CN102604949A (en) * 2011-04-12 2012-07-25 南京大学 Anaerobic tissue selective gene expression method driven by alcohol dehydrogenase promoter and application thereof
CN104471057A (en) * 2012-05-04 2015-03-25 香港大学 Modified bacteria and uses thereof for treatment of cancer or tumor

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
NZ518354A (en) * 1999-10-04 2005-02-25 Vion Pharmaceuticals Inc Compositions and methods for tumor-targeted delivery of effector molecules
CN102477440A (en) * 2010-11-29 2012-05-30 南京大学 Therapeutic gene for anaerobic tissue targeting delivery and selectivity stabilization expression method and its application
CN102604949A (en) * 2011-04-12 2012-07-25 南京大学 Anaerobic tissue selective gene expression method driven by alcohol dehydrogenase promoter and application thereof
CN104471057A (en) * 2012-05-04 2015-03-25 香港大学 Modified bacteria and uses thereof for treatment of cancer or tumor

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