WO2023015441A1 - 一种光控裂解工程菌及其构建方法和应用 - Google Patents

一种光控裂解工程菌及其构建方法和应用 Download PDF

Info

Publication number
WO2023015441A1
WO2023015441A1 PCT/CN2021/111757 CN2021111757W WO2023015441A1 WO 2023015441 A1 WO2023015441 A1 WO 2023015441A1 CN 2021111757 W CN2021111757 W CN 2021111757W WO 2023015441 A1 WO2023015441 A1 WO 2023015441A1
Authority
WO
WIPO (PCT)
Prior art keywords
light
gene expression
protein
lysis
termination
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/111757
Other languages
English (en)
French (fr)
Inventor
付生伟
张荣荣
金帆
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute of Advanced Technology of CAS
Original Assignee
Shenzhen Institute of Advanced Technology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Institute of Advanced Technology of CAS filed Critical Shenzhen Institute of Advanced Technology of CAS
Priority to PCT/CN2021/111757 priority Critical patent/WO2023015441A1/zh
Publication of WO2023015441A1 publication Critical patent/WO2023015441A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • 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
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a light-controlled lysis engineering bacterium and its construction method and application.
  • the main purpose of the present invention is to provide a light-controlled lysis engineered bacterium, which aims to solve the problem of uncontrollable and toxic side effects of induced engineering bacterium lysis and release of drugs in the prior art.
  • the first aspect of the present invention proposes a light-controlled lysis engineering bacterium, including:
  • a light-sensitive gene expression cassette which is expressed to obtain a light-sensitive protein, and the light-sensitive protein is regulated by light to synthesize a messenger molecule;
  • An anti-termination gene expression cassette the promoter of the anti-termination gene expression cassette is regulated by the messenger molecule, and an anti-termination protein is expressed;
  • the promoter of the cleavage gene expression cassette is regulated by the anti-termination protein, and the cleavage protein is obtained by expression, which is used to lyse the engineering bacteria;
  • the expression level of the lysis protein will lyse the engineered bacteria.
  • the light-sensitive gene expression cassette and the cleavage gene expression cassette are integrated into the genome of the engineered bacteria, and the anti-termination gene expression cassette exists in an exogenous plasmid.
  • the engineering bacterium also includes:
  • the drug gene expression cassette is used to express a drug protein, and when the engineered bacteria is lysed, the drug protein is released from the engineered bacteria.
  • the drug gene expression cassette and the anti-termination gene expression cassette exist in the same exogenous plasmid.
  • the photosensitive gene includes any one of BphS, IlaC*, IlaD9, IlaM4 and IlaM5;
  • the messenger molecule comprises c-di-GMP or cAMP;
  • the promoter of the anti-termination gene expression cassette includes cdrA promoter, pel promoter or psl promoter regulated by c-di-GMP, or the lac promoter regulated by cAMP;
  • the anti-termination gene includes Q protein of lambda phage
  • the cleavage genes include LKD16.
  • the second aspect of the present invention proposes a method for constructing light-controlled lysis engineering bacteria, including plasmid construction and plasmid transformation, and the plasmid construction includes:
  • Construction of a light-sensitive gene expression vector the light-sensitive gene is expressed to obtain a light-sensitive protein, and the light-sensitive protein is regulated by light to synthesize a messenger molecule;
  • an anti-termination gene expression vector the promoter of the anti-termination gene expression vector is regulated by the messenger molecule, and an anti-termination protein is expressed;
  • the construction of the cleavage gene expression vector, the promoter of the cleavage gene expression vector is regulated by the anti-termination protein, and the cleavage protein is obtained by expression, which is used to lyse the engineering bacteria;
  • the plasmid transformation comprises:
  • the light-sensitive gene expression vector and the cleavage gene expression vector are constructed from a suicide plasmid
  • the anti-termination gene expression vector is constructed from a replication plasmid
  • the light-sensitive gene and the cleavage gene are integrated into the engineering In the genome of bacteria, the anti-termination gene exists in the form of a plasmid.
  • the plasmid construction also includes:
  • the construction of the drug gene expression vector the drug gene is expressed to obtain the drug protein
  • the plasmid transformation also includes:
  • the drug protein is released from the engineered bacteria.
  • the drug gene expression vector and the anti-termination gene expression vector are constructed on the same plasmid.
  • the photosensitive gene includes any one of BphS, IlaC*, IlaD9, IlaM4 and IlaM5;
  • the messenger molecule comprises c-di-GMP or cAMP;
  • the promoter of the anti-termination gene expression vector includes cdrA promoter, pel promoter or psl promoter regulated by c-di-GMP, or the lac promoter regulated by cAMP;
  • the anti-termination gene includes Q protein of lambda phage
  • the cleavage genes include LKD16.
  • the third aspect of the present invention provides a pharmaceutical composition, including a light-controlled lysis engineered bacterium described in any one of the above-mentioned first aspects.
  • the fourth aspect of the present invention proposes the application of a light-controlled lysis engineered bacterium described in any one of the above-mentioned first aspects in the preparation of a drug for treating tumors.
  • the fifth aspect of the present invention provides a tumor treatment system, which is characterized in that it includes a tumor treatment device and a tumor treatment drug;
  • the tumor treatment drug includes a light-controlled lysis engineered bacterium according to any one of the first aspect above;
  • the tumor treatment device includes a laser element that can emit a light source; wherein,
  • the tumor treatment system When the tumor treatment system is in use, inject the tumor treatment drug into the tumor site to be treated, and then use the light source of the tumor treatment device to irradiate the tumor site,
  • the light-controlled lysis engineered bacteria lyse and release the therapeutic substance in the bacteria.
  • the sixth aspect of the present invention proposes the use of a light-controlled lysis engineered bacterium as described in any one of the first aspect in treating diseases.
  • the technical scheme of the present invention constructs a photosensitive gene expression cassette, an anti-termination gene expression cassette, and a cleavage gene expression cassette in engineering bacteria, which can be respectively expressed to obtain a photosensitive protein, an anti-termination protein, and a cleavage protein, and the photosensitive protein is used to synthesize messenger molecules under light regulation.
  • the messenger molecule induces the expression of the anti-termination protein
  • the anti-termination protein induces the expression of the cleavage protein
  • the expression level of the cleavage protein can lyse the light-controlled lysis engineered bacteria, thereby releasing the bacteria inner substance.
  • the light-controlled lysis engineering bacteria obtained by the technical solution of the present invention can grow normally when the light intensity is lower than the lysis threshold; Controlled lysis, and no toxic side effects.
  • Fig. 1 is a schematic diagram of the gene structure of an embodiment of the light-controlled lysis engineering bacteria of the present invention
  • Fig. 2 is a schematic diagram of the gene structure of another embodiment of the light-controlled lysis engineering bacteria of the present invention.
  • Figure 3 is a schematic map of the mini-CTX2 plasmid
  • Figure 4 is a schematic map of the PUCP20 plasmid
  • Figure 5 is a schematic map of the miniTn7 plasmid
  • Figure 6 is a schematic diagram of the map of the pEX18Gm plasmid
  • Fig. 7 is the result figure of the RBS screening part of the light-controlled lysis engineering bacteria anti-termination gene expression cassette of the present invention.
  • Fig. 8a is a graph showing the tumor volume results of the light-controlled lysis engineered bacteria of the present invention to treat tumors in mice;
  • Figure 8b is a graph showing the results of mouse body weight in the experiment of treating mouse tumors with light-controlled lysis engineered bacteria of the present invention.
  • Figure 8c is a graph showing the tumor weight results of the light-controlled lysis engineering bacteria of the present invention in the treatment of tumors in mice;
  • Fig. 9a to Fig. 9d are the result graphs of the experiment of treating tumor in mice with the control strain
  • Fig. 10 is a diagram showing the staining results of tissue sections in the experiment of light-controlled lysis engineering bacteria of the present invention for treating tumors in mice;
  • Fig. 11a is the structural representation of pBAD-B0034-Q-pJN105 plasmid and pR'-tR'-mScarlet-miniTn7 plasmid;
  • Figure 11b is the microscopic observation result of testing the background expression level and induced expression level of the pR'-tR' promoter-terminator transcription system in PAO1 strain;
  • Figure 11c is the quantitative result of mScarlet fluorescence intensity
  • Figure 12a is a schematic diagram of the plasmid structure for calibrating RBS light response intensity according to the embodiment of the present invention.
  • Fig. 12b is a diagram of relative intensity calibration results of randomly synthesized RBS in the embodiment of the present invention.
  • gene expression cassette refers to a set of DNA sequences that are composed of promoters, target genes, screening genes and terminators, can be expressed in specific tissues and are easily detected, and can be present in foreign expression vectors It can also be integrated into the genome of specific tissues.
  • expression vectors refers to vectors that add expression elements (such as promoters, RBS, terminators, etc.) on the basis of the basic skeleton of the cloning vector to enable expression of the target gene.
  • the expression vector has four parts: target gene, promoter, terminator, and marker gene.
  • the present invention includes, but is not limited to, prokaryotic expression vectors, eukaryotic expression vectors, or other cellular expression vectors.
  • Plasmid is a small double-stranded circular DNA molecule that is naked, simple in structure, independent of bacterial nucleoid DNA, and capable of self-replication. It is a commonly used cloning vector for constructing expression vectors.
  • suicide plasmid refers to a derivative of the R plasmid, often characterized by a broad host range, with conjugative transfer of genes. Its replication requires a special protein that most bacteria do not produce, so when it enters the host cell, it is either unable to replicate, is eliminated, or is integrated into the chromosome and replicates with the chromosome, taking advantage of the suicide plasmid This property allows the integration of foreign genes into the bacterial genome.
  • the first aspect of the present invention proposes a light-controlled cracking engineering bacteria, please refer to Figure 1, including:
  • a light-sensitive gene expression cassette which is expressed to obtain a light-sensitive protein, and the light-sensitive protein is regulated by light to synthesize a messenger molecule;
  • An anti-termination gene expression cassette the promoter of the anti-termination gene expression cassette is regulated by the messenger molecule, and an anti-termination protein is expressed;
  • the promoter of the cleavage gene expression cassette is regulated by the anti-termination protein, and the cleavage protein is obtained by expression, which is used to lyse the engineering bacteria;
  • the expression level of the lysis protein will lyse the engineered bacteria.
  • the above-mentioned light-sensitive gene expression cassette, anti-termination gene expression cassette, and cleavage gene expression cassette can be integrated into the bacterial genome or exist in the form of exogenous plasmids in any combination.
  • the light-sensitive gene expression cassette can be integrated into the bacterial genome, and the anti-termination gene expression cassette and the cleavage gene expression cassette exist in the form of plasmids; Gene expression cassettes exist as plasmids.
  • the form of the above-mentioned gene expression cassettes is not limited to these examples, and other embodiments are no longer exemplified one by one.
  • the promoter of the light-sensitive gene expression cassette is a constitutive promoter, which can continuously express the light-sensitive protein, and the light-sensitive protein can synthesize messenger molecules under light irradiation. Terminates protein expression and promotes bacterial lysis.
  • the light intensity is positively correlated with the concentration of the synthesized messenger molecule, therefore, the light intensity is also positively correlated with the concentration of the anti-termination protein.
  • the light-sensitive gene expression cassette is constructed in a suicide plasmid and integrated into the bacterial genome to improve the genetic stability of the light-sensitive gene.
  • the promoter of the anti-termination gene expression cassette is an inducible promoter, and the expression of the anti-termination protein is regulated by the concentration of the messenger molecule.
  • the anti-termination protein can specifically prevent the function of the terminator, so that the enzyme can continue to transcribe beyond the terminator.
  • the promoter of the cleavage gene expression cassette is a promoter-terminator (PR'-tR') system, wherein, when the anti-termination protein is not expressed or the expression concentration is low, the PR'-tR' system of the cleavage gene expression cassette is in Under the action of the terminator, it does not transcribe, so the cleavage protein cannot be expressed.
  • PR'-tR' promoter-terminator
  • the anti-termination protein can prevent the function of the terminator of the PR'-tR' system, so that the normal transcription and expression of the cleavage gene can be cleaved protein, thereby lysing the bacteria.
  • the concentration of the anti-termination protein is positively correlated with the concentration of the lysed protein, and therefore, the light intensity of the light-sensitive protein is positively correlated with the expression concentration of the lysed protein. Therefore, it can be understood that the lysis threshold for regulating the light intensity of the light-sensitive protein is the light intensity value that can cause the expression of the cleavage protein to lyse the engineered bacteria. Reaching the lysis threshold may be the same as the lysis threshold, or exceeding the lysis threshold. It should also be noted that for different engineering bacteria, due to their different properties, even if they are transformed by the technical solution of the present invention, their lysis threshold may be different. Therefore, the technical solution of the present invention does not specifically limit the specific lysis threshold.
  • the technical scheme of the present invention constructs a photosensitive gene expression cassette, an anti-termination gene expression cassette, and a cleavage gene expression cassette in engineering bacteria, which can be respectively expressed to obtain a photosensitive protein, an anti-termination protein, and a cleavage protein, and the photosensitive protein is used to synthesize messenger molecules under light regulation.
  • the messenger molecule induces the expression of the anti-termination protein
  • the anti-termination protein induces the expression of the cleavage protein
  • the expression level of the cleavage protein can lyse the light-controlled lysis engineered bacteria, thereby releasing the bacteria inner substance.
  • the light-controlled lysis engineering bacteria obtained by the technical solution of the present invention can grow normally when the light intensity is lower than the lysis threshold; Controlled lysis, and no toxic side effects.
  • the light-sensitive gene expression cassette and the cleavage gene expression cassette are integrated into the genome of the engineered bacteria, and the anti-termination gene expression cassette exists in an exogenous plasmid.
  • described engineering bacterium also includes:
  • the drug gene expression cassette is used to express a drug protein, and when the engineered bacteria is lysed, the drug protein is released from the engineered bacteria.
  • the drug gene expression cassette and the anti-termination gene expression cassette exist in the same exogenous plasmid.
  • the photosensitive gene includes any one of BphS, IlaC*, IlaD9, IlaM4 and IlaM5;
  • the messenger molecule comprises c-di-GMP or cAMP;
  • the promoter of the anti-termination gene expression cassette includes cdrA promoter, pel promoter or psl promoter regulated by c-di-GMP, or the lac promoter regulated by cAMP;
  • the anti-termination gene includes Q protein of lambda phage
  • the cleavage genes include LKD16.
  • BphS can synthesize the messenger molecule c-di-GMP under the regulation of near-infrared light
  • IlaC*, IlaD9, IlaM4 and IlaM5 can synthesize the messenger molecule cAMP under the regulation of near-infrared light.
  • the photosensitive gene is BphS; the messenger molecule is c-di-GMP; the promoter of the anti-termination gene expression cassette is cdrA promoter.
  • the second aspect of the present invention proposes a method for constructing light-controlled lysis engineering bacteria, including plasmid construction and plasmid transformation, and the plasmid construction includes:
  • Construction of a light-sensitive gene expression vector the light-sensitive gene is expressed to obtain a light-sensitive protein, and the light-sensitive protein is regulated by light to synthesize a messenger molecule;
  • an anti-termination gene expression vector the promoter of the anti-termination gene expression vector is regulated by the messenger molecule, and an anti-termination protein is expressed;
  • the construction of the cleavage gene expression vector, the promoter of the cleavage gene expression vector is regulated by the anti-termination protein, and the cleavage protein is obtained by expression, which is used to lyse the engineering bacteria;
  • the plasmid transformation comprises:
  • the starting bacteria may be, for example, Pseudomonas aeruginosa, Escherichia coli or Salmonella.
  • the light-sensitive gene expression vector and the cleavage gene expression vector are constructed from a suicide plasmid
  • the anti-termination gene expression vector is constructed from a replication plasmid
  • the light-sensitive gene and the cleavage gene are integrated into the engineering In the genome of bacteria, the anti-termination gene exists in the form of a plasmid.
  • the plasmid construction also includes:
  • the construction of the drug gene expression vector the drug gene is expressed to obtain the drug protein
  • the plasmid transformation also includes:
  • the drug protein is released from the engineered bacteria.
  • the drug gene expression vector and the anti-termination gene expression vector are constructed on the same plasmid.
  • the photosensitive gene includes any one of BphS, IlaC*, IlaD9, IlaM4 and IlaM5;
  • the messenger molecule comprises c-di-GMP or cAMP;
  • the promoter of the anti-termination gene expression vector includes cdrA promoter, pel promoter or psl promoter regulated by c-di-GMP, or the lac promoter regulated by cAMP;
  • the anti-termination gene includes Q protein of lambda phage
  • the cleavage genes include LKD16.
  • BphS can synthesize the messenger molecule c-di-GMP under the regulation of near-infrared light
  • IlaC*, IlaD9, IlaM4 and IlaM5 can synthesize the messenger molecule cAMP under the regulation of near-infrared light.
  • the photosensitive gene is BphS; the messenger molecule is c-di-GMP; the promoter of the anti-termination gene expression cassette is cdrA promoter.
  • the third aspect of the present invention provides a pharmaceutical composition, including a light-controlled lysis engineered bacterium described in any one of the above-mentioned first aspects.
  • the fourth aspect of the present invention proposes the application of a light-controlled lysis engineered bacterium described in any one of the above-mentioned first aspects in the preparation of a drug for treating tumors.
  • the fifth aspect of the present invention provides a tumor treatment system, which is characterized in that it includes a tumor treatment device and a tumor treatment drug;
  • the tumor treatment drug includes a light-controlled lysis engineered bacterium according to any one of the first aspect above;
  • the tumor treatment device includes a laser element that can emit a light source; wherein,
  • the tumor treatment system When the tumor treatment system is in use, inject the tumor treatment drug into the tumor site to be treated, and then use the light source of the tumor treatment device to irradiate the tumor site,
  • the light-controlled lysis engineered bacteria lyse and release the therapeutic substance in the bacteria.
  • the sixth aspect of the present invention proposes the use of a light-controlled lysis engineered bacterium as described in any one of the first aspect in treating diseases.
  • Table 1 is the list of primer sequences used
  • pR'-LKD-ass-R ctgcaggaattcctcgagaagctttcagtctccttgattcagggcg Tn7-pR'-ass-F: ctgcaggaattcctcgagaagctttcagtctccttgattcagggcg Tn7-ass-F: aagcttctcgaggaattcctgcag Tn7-ass-R: ggtacctcgcgaaggccttg BphS-CTX-F: gataccgtcgacctcgaacccccacgcccctcga BphS-CTX-R: ggtacccaattcgccctatagtgagtcgtattacg PA1O4O3-F: ctcactatagggcgaattggg
  • PUCP-F aattcgtgagctaactcacattaattgcgttgcg PUCP-R: aagcttggcactggccgtcgttttacaacgtcgtg PUCP-PcdrA-Q-F: actagtgggttcgaggtcgacggtatcgataagctagcttt J23118-F: taaaacgacggccagtgccaagcttttgacggctagctcagtcctagta HlyE-PUCP-R: ataccgtcgacctcgaacccactagtttagacttcaggtacctcaaagagtgtctttttttttttt
  • Embodiment 1 The construction of light-controlled lysis engineering bacteria
  • Pseudomonas aeruginosa PAO1 which can effectively colonize the lungs, was used as the starting bacterium, and based on this, light-controlled lysis engineering bacteria were constructed.
  • the light-sensitive gene BphS (SEQ ID NO.1) encodes the light-sensitive protein BphS (SEQ ID NO.2).
  • the light-sensitive protein BphS can synthesize c-di-GMP under the regulation of near-infrared light.
  • the BphS expression vector is constructed in the suicide plasmid miniCTX2 (see Figure 3 for the plasmid map), so as to integrate the BphS gene and its related expression elements into the bacterial genome through the suicide plasmid, and the related expression elements include constitutive promoters PA1/O4/O3 (SEQ ID NO.3), the photoresponse component BphO (SEQ ID NO.4), enables the continuous expression of BphS protein.
  • the PA1O4O3-BphS-BphO gene fragment was synthesized from Shanghai Sangon Biological Co., Ltd., and the primer pair PA1O4O3-F and BphS-R was used for polymerase chain PCR reaction, and the miniCTX2 plasmid was used for the primer pair BphS-CTX-F and BphS-CTX-R was subjected to polymerase chain PCR reaction to obtain gene fragments capable of sustainably expressing light response components BphO and BphS with 25 base homology arms and linearized vector plasmid miniCTX2, and finally Gibson ligation was used The method (Gibson assembly) was used to connect the two gene fragments to construct the BphS expression vector PA1/O4/O3-BphS-CTX2.
  • the anti-termination gene Q comes from the lambda phage genome, and is constructed into the carrier plasmid PUCP20 (see Figure 4 for the plasmid map) by Gibson assembly.
  • the PUCP20 plasmid is an exogenous plasmid that can replicate autonomously. Construct the anti-termination gene Q expression vector, the anti-termination gene Q will not be integrated into the bacterial genome, but exists in the form of exogenous plasmids, which can facilitate the adjustment of the expression of the anti-termination gene Q.
  • the promoter used by the anti-termination gene Q expression vector is the cdrA promoter (SEQ ID NO.6) that responds to c-di-GMP, the higher the concentration of c-di-GMP, the anti-termination expression obtained by the anti-termination gene Q expression vector The more protein Q (SEQ ID NO.7).
  • the hlyE (hemolysin E) gene adopts the constitutive promoter J23118 (SEQ ID NO.9) to continuously express the hlyE protein (SEQ ID NO.10), and obtain the J23118-hlyE gene fragment by PCR (primers are J23118-F and HlyE-PUCP-R), and the template is artificially synthesized; followed by Gibson ligation in the PcdrA-RBS-Q-T0/T1-PUCP20 plasmid (linearized vector primers are PUCP-PcdrA-Q-F and PUCP Insert the J23118-hlyE gene fragment after the T0/T1 double terminator of -R), and finally obtain the PcdrA-RBS-Q-T0/T1-J23118-hlyE-PUCP20 plasmid, which can simultaneously express the anti-terminator protein Q and the drug hlyE.
  • the cleavage gene LKD16 (SEQ ID NO.11) was expressed to obtain the cleavage protein LKD16 (SEQ ID NO.12).
  • the promoter-terminator transcription system (pR'- tR') (SEQ ID NO.13) is used as the promoter of the LKD16 expression vector. This system can normally transcribe downstream genes when the anti-terminator protein Q exists. The sub-pR' cannot express the downstream LKD16 gene.
  • the cleavage gene LKD16 is designed to be placed under the control of pR'-tR', and it is hoped that it will be integrated into the genome of the engineered bacteria to further reduce its expression while improving its stability. Therefore, the cleavage gene LKD16 is constructed into the suicide plasmid miniTn7( The plasmid map is shown in Figure 5).
  • a ssrA hydrolysis tag SEQ ID NO.14
  • the PR'-tR'-LKD16 gene fragment was artificially synthesized, and the PR'-tR'-LKD16 gene fragment was obtained by PCR (primers: pR'-LKD-ass-R/Tn7-pR'-ass-F) and linearized miniTn7 plasmid (primers: Tn7-ass-F/Tn7-ass-R), thereby inserting the synthetic cleavage gene LKD16 and the pR'-tR' gene sequence into the miniTn7 plasmid, thereby obtaining pR'-tR'-B0034-LKD-T0/ T1-miniTn7 plasmid.
  • the pR'-tR'-B0034-LKD-T0/T1-miniTn7 plasmid was electrotransformed into wild-type Pseudomonas aeruginosa PAO1 with the help of the helper plasmid PTNS2, and then spread on LB+Gen30 (30 micrograms per milliliter Gentamicin) on the agar culture plate, pick the monoclonal point that grows out after 12 hours, use L B+Gen30 liquid medium 37 °C, shake the bacteria at 250 rpm for 8 hours, and electrotransfer the plasmid pflp2 into Among them, smear on the agar plate of LB+Carb300 (300 micrograms per milliliter carbenicillin).
  • Plasmid PA1/O4/O3-BphS-CTX2 is also electrotransformed into pR'-tR'-B0034-LK D-T0/T1-miniTn7-PAO1 by the same method, only need to change the resistance from Gen30 to Tc100 (100 micrograms per milliliter of tetracycline), to obtain the bacterial strain PA1/O4/O3-BphS-CTX2-pR'-tR'-B0034-LKD-T0/T1-miniTn7-PAO1, which is labeled as BphS-LKD-PAO1.
  • the wild-type PAO1 strain is highly toxic.
  • three genes were knocked out on the basis of B phS-LKD-PAO1 to control its toxicity. They are vfR (genome number PA0652) , exoS (genome number PA3841), exoT (genome number PA0044), and the finally obtained attenuated strain was named ExoST.
  • vfR gene number PA0652
  • exoS gene number PA3841
  • exoT gene number PA0044
  • the gene knockout adopts the method of seamless recombination knockout. Taking the construction of the vfR gene knockout vector as an example, it is first necessary to obtain a fragment of 1000 bases upstream and downstream of the target fragment from the genome by PCR technology, which is recorded as vfR- up and vfR-dn; then they were inserted between the restriction sites EcoRI and HindIII of the suicide vector pEX18Gm by the Gibson assembly method (see Figure 6 for the plasmid map) to construct the vector vfR-pEX18Gm.
  • the constructed knockout plasmid vfR-pEX18Gm was electrotransformed into the target bacterial species, and monoclonal plaques were screened on a plate containing gentamycin; then streaked on an LB plate containing 15% sucrose without sodium chloride Wire. After culturing at 37°C for 16 hours, single-clonal plaque PCR was selected to identify whether the target gene vfR was successfully knocked out. After sequencing verification, the second gene was knocked out in the same way until all three virulence genes were knocked out successfully. This strain was designated as ExoST.
  • the expression level of anti-termination protein Q directly affects the expression level of the cleavage gene LKD16. If the background expression level is high, the engineered bacteria may have begun to lyse without light, and if the background expression level is too low, the engineered bacteria may be lysed under high light intensity. It is also impossible to crack.
  • Both the promoter and the ribosome binding site (RBS) can affect the background expression level of Q.
  • RBS refers to a purine-rich untranslated region upstream of the gene start codon AUG. If the background expression level of Q is relatively high, that is, when there is no light, the expression level of Q is enough to turn on the expression of the lysis gene to lyse the bacteria, and the bacteria will be lysed only with weak light or even no light. In contrast, background expression levels of Q that are too low will not induce sufficient Q expression to lyse bacteria, even at high light intensities. Therefore, different RBS sequences lead to different response behaviors of the whole system to light intensity, so it is necessary to screen the RBS before the anti-termination gene Q.
  • the operation process of the microfluidic experiment the microfluidic channel is bonded by PDMS and the cover glass.
  • the channel width is 50 microns
  • the height is 200 microns
  • the shape is a single straight channel.
  • One end of the channel is the liquid inlet, and the other end is the waste water. liquid mouth.
  • the selected medium is McCoy's 5A, which contains 30 micromolar ferric chloride, 30 micrograms per milliliter of gentamicin (Gen30) and 10% fetal bovine serum (FBS). placed at room temperature, and the air bubbles were exhausted the next day, and the experimental temperature was controlled at 30°C.
  • FIG. 7 shows the microfluidic experiment results of the bacterial strain PcdrA-GFP that cannot be cleaved, the bacterial strain RBS004-Q constructed by RBS004, and the bacterial strain RBS017-Q constructed by RBS017.
  • RBS017-Q can It grows normally under the low light intensity of 16.2 ⁇ W/cm 2 , but lyses under the high light intensity of 50.8 ⁇ W/cm 2 , proving that RBS017 meets the requirements. It can also be known from Figure 7 that the lysis threshold of the RBS017-Q strain can be 50.8 ⁇ W/cm 2 , or any value between 16.2 ⁇ W/cm 2 and 50.8 ⁇ W/cm 2 , for example, it can be (20, 30 or 40) ⁇ W/cm 2 .
  • RBS017 (sequence: AACGCGGTGCAA) had a good response to light, so RBS017 was used to construct a Q protein expression plasmid and transformed, and the strains that met the requirements were: Transferred into the ExoST strain, the obtained strain was named Q017, as a control; the PcdrA-RBS017-Q-T0/T1-J23118-hlyE-PUCP20 plasmid was electrotransformed into the ExoST strain, and the obtained strain was named H017, The constructed light-controlled lysis engineering bacteria were obtained.
  • Embodiment 2 tests the background expression level and induced expression level of pR'-tR' promoter-terminator transcription system in PAO1 strain
  • the pR'-tR' promoter-terminator transcription system directly determines the background expression level of the lytic protein LKD16. If the background expression level of the LKD16 protein is too high, the bacteria will be lysed at low or no light intensity, and the induction of the LKD16 protein If the expression level is too low, the bacteria cannot be lysed under high light intensity. Therefore, it is necessary to test the background expression level and the induced expression level of the pR'-tR' promoter-terminator transcription system in PAO1 strains.
  • the upper image is the bright field image of the microscope
  • the lower image is the fluorescence image of the microscope.
  • pR'-mScarlet is listed as the bright field image and fluorescence image of pR'-mScarlet-PAO1 when there is no terminator tR', and the fluorescence intensity is very high. Strong, it proves that pR' can normally drive the expression of red fluorescent protein
  • pR'-tR'-mScarlet is listed as the bright field picture and fluorescence picture of pR'-tR'-mScarlet-PAO1 after adding the terminator tR', almost no Fluorescence, demonstrating that tR' can prevent the expression of DsRed with low background expression levels.
  • pR'-tR'-mScarlet-Q-pJN105+0.6%L-ara is the picture of PBAD-B0034-Q-pJN105-pR'-tR'-mScarlet-miniTn7-PAO1 strain induced by adding 0.6% arabinose, A certain intensity of fluorescence was generated, which proved that arabinose could induce the expression of red fluorescent protein.
  • Figure 11c is the quantitative data of the fluorescence intensity of three strains of mScarlet. The above experimental results show that the background expression level of the pR'-tR' promoter-terminator transcription system is low, and the induced expression level is high, which verifies the feasibility of the system.
  • strains to be used were streaked on the LB+Gen30 agar plate in a -80°C refrigerator the night before the injection, and the next morning, pick a single colony spot and shake it in 1mL FAB+++ until the OD600 value was about 0.6. After centrifugation, reconstitute with PBS. Wash twice by hanging, and pay attention to avoid light during the whole process.
  • mice Take 100 microliters of strains to be used and inject them into tumor-bearing (A549 cell line) Balb/c mice, and divide the mice into a high-intensity irradiation group (H017-Light) and a dark treatment group (H017-Dark), wherein The mice in the high-intensity irradiation group were irradiated with a light intensity of 10mW/cm 2 . Considering that the light penetrated through the animal tissue and reached the tumor site, the light intensity would be weakened. Therefore, the light intensity of the animal experiments was higher than that of the bacteria experiments.
  • TUNEL staining is that when the genomic DNA is broken, the exposed 3'-OH can be deoxynucleotide at the end.
  • TdT Terminal Deoxynucleotidyl Transferase
  • FITC fluorescein-labeled dUTP
  • fluorescein-dUTP fluorescein-dUTP

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种光控裂解工程菌及其构建方法和应用，该光控裂解工程菌通过构建光敏基因表达盒、抗终止基因表达盒和裂解基因表达盒，可分别表达得到光敏蛋白、抗终止蛋白和裂解蛋白，通过光敏蛋白在近红外光调控下合成信使分子，信使分子诱导抗终止蛋白表达，抗终止蛋白诱导裂解蛋白表达，当调控所述光敏蛋白的光强大于裂解阈值时，所述裂解蛋白的表达量使所述工程菌裂解，从而释放菌内物质。该光控裂解工程菌在外界近红外光光源的刺激下，当光强低于裂解阈值时，细菌可以正常生长；当光强达到裂解阈值时，细菌裂解，实现了工程菌的可控裂解，并且无毒副作用。

Description

一种光控裂解工程菌及其构建方法和应用 技术领域
本发明涉及生物医药技术领域，特别涉及一种光控裂解工程菌及其构建方法和应用。
背景技术
现有的癌症疗法存在对系统的高毒性、对肿瘤的低靶向性以及对肿瘤组织内部的低渗透性问题，高风险以及低收益的治疗手段使得发展新的肿瘤治疗技术迫在眉睫。工程化细菌由于自身特殊的性质可以完美解决这些问题，它可以靶向肿瘤组织的所有区域，在其中定植，并且可以作为携带治疗药物的载体。为了避免传统药物造成的系统性毒性，如何严格控制细菌在到达肿瘤组织时释放药物是一个需要解决的问题。
有研究报道通过对细菌加以改造后，施加2戈瑞电离辐射强度的刺激，细菌的基因组会被破坏而激活SOS响应，recA启动子开启表达合成可分泌的治疗药物，对肿瘤进行治疗。SOS系统的开启虽然可以让recA启动子表达，但是由于自身基因组遭到破坏，细菌是否继续维持其功能性值得商榷。还有工作在沙门氏菌内构建了一个由tet启动子控制的λ噬菌体裂解基因表达系统，在外源加入四环素或者四环素类似物之后，细菌可以被诱导裂解从而释放治疗药物。类似的工作还有采用阿拉伯糖诱导启动子表达来自另一噬菌体iEPS5的裂解基因使沙门氏菌裂解释放治疗药物，或者用乙酰水杨酸诱导。
现有的技术主要依赖于外界化学小分子或者肿瘤内部微环境刺激细菌产生治疗药物，进入体内后外界无法对其行为进行控制，且化学小分子的诱导是一种不可逆过程，一旦发生就无法停止。并且化学小分子诱导物可能存在毒副作用以及被代谢系统降解，例如沙门氏菌的代谢系统可以将阿拉伯糖转化为木酮糖，低浓度的阿拉伯糖无法诱导相应启动子的表达。另一种基于recA修复系统的触发方式由于会对工程菌基因组产生破坏，系统本身的鲁棒性会受到难以估量的影响，且辐射源不易获得，因此也不是理想的诱导方式。
发明内容
本发明的主要目的是提供一种光控裂解工程菌，旨在解决现有技术诱导工程菌裂解释放药物不可控且有毒副作用的问题。
为实现上述目的，本发明第一方面提出一种光控裂解工程菌，包括：
光敏基因表达盒，表达得到光敏蛋白，所述光敏蛋白受光调控合成信使分子；
抗终止基因表达盒，所述抗终止基因表达盒的启动子受所述信使分子调控，表达得到抗终止蛋白；
裂解基因表达盒，所述裂解基因表达盒的启动子受所述抗终止蛋白调控，表达得到裂解蛋白，用于裂解所述工程菌；其中，
当调控所述光敏蛋白的光强达到裂解阈值时，所述裂解蛋白的表达量使所述工程菌裂解。
可选地，所述光敏基因表达盒和所述裂解基因表达盒整合到所述工程菌的基因组中，所述抗终止基因表达盒存在于外源质粒中。
可选地，所述工程菌还包括：
药物基因表达盒，表达得到药物蛋白，当所述工程菌裂解后，所述药物蛋白从所述工程菌释放。
可选地，所述药物基因表达盒与所述抗终止基因表达盒存在于同一个外源质粒中。
可选地，所述光敏基因包括BphS、IlaC*、IlaD9、IlaM4和IlaM5中的任一种；
所述信使分子包括c-di-GMP或cAMP；
所述抗终止基因表达盒的启动子包括受c-di-GMP调控的cdrA启动子、pel启动子或psl启动子，或者受cAMP调控的lac启动子；
所述抗终止基因包括λ噬菌体的Q蛋白；
所述裂解基因包括LKD16。
本发明第二方面提出一种光控裂解工程菌的构建方法，包括质粒构建和质粒转化，所述质粒构建包括：
光敏基因表达载体的构建，所述光敏基因表达得到光敏蛋白，所述光敏蛋白受光调控合成信使分子；
抗终止基因表达载体的构建，所述抗终止基因表达载体的启动子受所述信使分子调控，表达得到抗终止蛋白；
裂解基因表达载体的构建，所述裂解基因表达载体的启动子受所述抗终止蛋白调控，表达得到裂解蛋白，用于裂解所述工程菌；
所述质粒转化包括：
将上述构建好的光敏基因表达载体、抗终止基因表达载体和裂解基因表达载体转化到出发菌中得到所述光控裂解工程菌。
可选地，所述光敏基因表达载体和所述裂解基因表达载体由自杀质粒构建得到，所述抗终止基因表达载体由复制质粒构建得到，所述光敏基因和所述裂解基因整合到所述工程菌的基因组中，所述抗终止基因以质粒形式存在。
可选地，所述质粒构建还包括：
药物基因表达载体的构建，所述药物基因表达得到药物蛋白；
所述质粒转化还包括：
将构建的药物基因表达载体转化到出发菌中得到所述光控裂解工程菌；其中，
当所述工程菌裂解后，所述药物蛋白从所述工程菌释放。
可选地，所述药物基因表达载体和所述抗终止基因表达载体构建到同一质粒上。
可选地，所述光敏基因包括BphS、IlaC*、IlaD9、IlaM4和IlaM5中的任一种；
所述信使分子包括c-di-GMP或cAMP；
所述抗终止基因表达载体的启动子包括受c-di-GMP调控的cdrA启动子、pel启动子或psl启动子，或者受cAMP调控的lac启动子；
所述抗终止基因包括λ噬菌体的Q蛋白；
所述裂解基因包括LKD16。
本发明第三方面提出一种药物组合物，包括上述第一方面任一项所述的一种光控裂解工程菌。
本发明第四方面提出上述第一方面任一项所述的一种光控裂解工程菌在制备治疗肿瘤药物中的应用。
本发明第五方面提出一种肿瘤治疗系统，其特征在于，包括肿瘤治疗装置和肿瘤治疗药物；
所述肿瘤治疗药物包括上述第一方面任一项所述的一种光控裂解工程菌；
所述肿瘤治疗装置包括激光元件，所述激光元件可发射光源；其中，
所述肿瘤治疗系统在使用的时候，将所述肿瘤治疗药物注射到待治疗肿瘤部位，然后使用所述肿瘤治疗装置的光源照射所述肿瘤部位，
当所述光强达到裂解阈值时，所述光控裂解工程菌裂解释放菌内治疗物。
本发明第六方面提出如第一方面任一项所述的一种光控裂解工程菌在治疗疾病中的用途。
本发明技术方案在工程菌中构建光敏基因表达盒、抗终止基因表达盒和裂解基因表达盒，可分别表达得到光敏蛋白、抗终止蛋白和裂解蛋白，通过光敏蛋白在光调控下合成信使分子，信使分子诱导抗终止蛋白表达，抗终止蛋白诱导裂解蛋白表达，并且当调控光敏蛋白的光强达到裂解阈值时，所述裂解蛋白的表达量能够使所述光控裂解工程菌裂解，从而释放菌内物质。从而本发明技术方案得到的光控裂解工程菌在外界光源的刺激下，当光强低于裂解阈值时，细菌可以正常生长；当光强达到裂解阈值时，细菌裂解，实现了工程菌的可控裂解，并且无毒副作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面 描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图示出的结构获得其他的附图。
图1为本发明光控裂解工程菌一实施例的基因结构示意图；
图2为本发明光控裂解工程菌另一实施例的基因结构示意图；
图3为mini-CTX2质粒的图谱示意图；
图4为PUCP20质粒的图谱示意图；
图5为miniTn7质粒的图谱示意图；
图6为pEX18Gm质粒的图谱示意图；
图7为本发明光控裂解工程菌抗终止基因表达盒的RBS筛选部分结果图；
图8a为本发明光控裂解工程菌治疗小鼠肿瘤实验的肿瘤体积结果图；
图8b为本发明光控裂解工程菌治疗小鼠肿瘤实验的小鼠体重结果图；
图8c为本发明光控裂解工程菌治疗小鼠肿瘤实验的肿瘤重量结果图；
图9a至图9d为对照菌株治疗小鼠肿瘤实验的结果图；
图10为本发明光控裂解工程菌治疗小鼠肿瘤实验的组织切片染色结果图；
图11a为PBAD-B0034-Q-pJN105质粒和pR’-tR’-mScarlet-miniTn7质粒的结构示意图；
图11b为测试pR’-tR’启动子-终止子转录系统在PAO1菌种中的背景表达水平以及诱导表达水平的显微镜观察结果；
图11c为mScarlet荧光强度的定量结果；
图12a为本发明实施例标定RBS光响应强度的质粒结构示意图；
图12b为本发明实施例随机合成的RBS的相对强度标定结果图。
本发明目的的实现、功能特点及优点将结合实施例，参照附图做进一步说明。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有 作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本文所用的术语“基因表达盒”是指由启动子，靶基因，筛选基因和终止子组成，能在特定组织中表达并易于检测的一组DNA序列，该DNA序列可以存在于外源表达载体中，也可以整合到特定组织的基因组中。
术语“表达载体(Expression vectors)”是指在克隆载体基本骨架的基础上增加表达元件(如启动子、RBS、终止子等)，使目的基因能够表达的载体。表达载体四部分：目的基因、启动子、终止子、标记基因。本发明包括但不限于原核细胞表达载体、真核细胞表达载体或其它细胞表达载体。
术语“质粒”是一种裸露的、结构简单、独立于细菌拟核DNA之外，并具有自我复制能力的很小的双链环状DNA分子，是常用的克隆载体，用于构建表达载体。
术语“自杀质粒”为R质粒的衍生质粒，常有宿主范围广的特点，具有接合转移基因。它的复制需要一种特殊的蛋白，大多数细菌不产生这种蛋白质，因此，当进入寄主细胞时，要么不能复制，被消除，要么被整合入染色体上，和染色体一起复制，利用自杀质粒的该特性可以将外源基因整合到细菌基因组中。
本发明第一方面提出一种光控裂解工程菌，请参阅图1，包括：
光敏基因表达盒，表达得到光敏蛋白，所述光敏蛋白受光调控合成信使分子；
抗终止基因表达盒，所述抗终止基因表达盒的启动子受所述信使分子调控，表达得到抗终止蛋白；
裂解基因表达盒，所述裂解基因表达盒的启动子受所述抗终止蛋白调控，表达得到裂解蛋白，用于裂解所述工程菌；其中，
当调控所述光敏蛋白的光强达到裂解阈值时，所述裂解蛋白的表达量使所述工程菌裂解。
上述光敏基因表达盒、抗终止基因表达盒、裂解基因表达盒整合到细菌基因组，或者以外源质粒形式存在可以为任意组合。详细而言，例如可以为光敏基因表达盒整合到细菌基因组，抗终止基因表达盒和裂解基因表达盒以质粒形式存在；还可以为裂解基因表达盒整合到细菌基因组，光敏基因表达盒和抗终止基因表达盒以质粒形式存在。上述基因表达盒的形式不限于所举 例这些，其他实施例不再一一例举，可以理解的是，凡是通过基因工程构建的菌株包括上述光敏基因表达盒、抗终止基因表达盒以及裂解基因表达盒都属于本发明技术方案保护的范围。
具体的，光敏基因表达盒的启动子为组成型启动子，可以持续表达光敏蛋白，光敏蛋白在光照射下可以合成信使分子，信使分子的浓度一方面促进生物被膜的形成，另一方面促进抗终止蛋白的表达，促进细菌裂解。光强大小与合成的信使分子浓度成正相关，因此，光强大小也与抗终止蛋白的产生浓度成正相关。在一优选实施例中，所述光敏基因表达盒通过构建在自杀质粒中整合到细菌基因组中，提高光敏基因遗传稳定性。
具体的，抗终止基因表达盒的启动子为诱导型启动子，受信使分子的浓度调控表达得到抗终止蛋白，抗终止蛋白可以特异的阻止终止子的作用，使酶越过终止子继续转录。
具体的，裂解基因表达盒的启动子为启动子-终止子(PR’-tR’)系统，其中，抗终止蛋白不表达或表达浓度低时，裂解基因表达盒的PR’-tR’系统在终止子的作用下不转录，因而无法表达得到裂解蛋白，当抗终止蛋白的表达浓度足够多时，抗终止蛋白可以阻止PR’-tR’系统的终止子的作用，使得裂解基因正常转录表达得到裂解蛋白，从而对细菌进行裂解。
由此可见，抗终止蛋白的浓度与裂解蛋白的浓度成正相关，因而，调控光敏蛋白的光强大小与裂解蛋白的表达浓度成正相关。因此可以理解，调控光敏蛋白的光强的裂解阈值即为可以使得裂解蛋白的表达量使工程菌裂解的光强值。达到裂解阈值可以为与裂解阈值相同，也可以为超过裂解阈值。还应当说明的是，对于不同的工程菌，由于其性质不同，即使其采用本发明技术方案进行改造，其裂解阈值也有可能不同，因此，本发明技术方案对于具体的裂解阈值不做具体限定。
本发明技术方案在工程菌中构建光敏基因表达盒、抗终止基因表达盒和裂解基因表达盒，可分别表达得到光敏蛋白、抗终止蛋白和裂解蛋白，通过光敏蛋白在光调控下合成信使分子，信使分子诱导抗终止蛋白表达，抗终止蛋白诱导裂解蛋白表达，并且当调控光敏蛋白的光强达到裂解阈值时，所述裂解蛋白的表达量能够使所述光控裂解工程菌裂解，从而释放菌内物质。从而本发明技术方案得到的光控裂解工程菌在外界光源的刺激下，当光强低于 裂解阈值时，细菌可以正常生长；当光强达到裂解阈值时，细菌裂解，实现了工程菌的可控裂解，并且无毒副作用。
可选地，所述光敏基因表达盒和所述裂解基因表达盒整合到所述工程菌的基因组中，所述抗终止基因表达盒存在于外源质粒中。
可选地，请参阅图2，所述工程菌还包括：
药物基因表达盒，表达得到药物蛋白，当所述工程菌裂解后，所述药物蛋白从所述工程菌释放。
可选地，所述药物基因表达盒与所述抗终止基因表达盒存在于同一个外源质粒中。
可选地，所述光敏基因包括BphS、IlaC*、IlaD9、IlaM4和IlaM5中的任一种；
所述信使分子包括c-di-GMP或cAMP；
所述抗终止基因表达盒的启动子包括受c-di-GMP调控的cdrA启动子、pel启动子或psl启动子，或者受cAMP调控的lac启动子；
所述抗终止基因包括λ噬菌体的Q蛋白；
所述裂解基因包括LKD16。
具体的，BphS受近红外光调控可合成信使分子c-di-GMP；IlaC*、IlaD9、IlaM4和IlaM5受近红外光调控可合成信使分子cAMP。
在一实施例中，所述光敏基因为BphS；所述信使分子为c-di-GMP；所述抗终止基因表达盒的启动子为cdrA启动子。
本发明第二方面提出一种光控裂解工程菌的构建方法，包括质粒构建和质粒转化，所述质粒构建包括：
光敏基因表达载体的构建，所述光敏基因表达得到光敏蛋白，所述光敏蛋白受光调控合成信使分子；
抗终止基因表达载体的构建，所述抗终止基因表达载体的启动子受所述信使分子调控，表达得到抗终止蛋白；
裂解基因表达载体的构建，所述裂解基因表达载体的启动子受所述抗终止蛋白调控，表达得到裂解蛋白，用于裂解所述工程菌；
所述质粒转化包括：
将上述构建好的光敏基因表达载体、抗终止基因表达载体和裂解基因表达载体转化到出发菌中得到所述光控裂解工程菌。
具体的，所述出发菌例如可以为铜绿假单胞菌、大肠杆菌或沙门氏菌等。
可选地，所述光敏基因表达载体和所述裂解基因表达载体由自杀质粒构建得到，所述抗终止基因表达载体由复制质粒构建得到，所述光敏基因和所述裂解基因整合到所述工程菌的基因组中，所述抗终止基因以质粒形式存在。
可选地，所述质粒构建还包括：
药物基因表达载体的构建，所述药物基因表达得到药物蛋白；
所述质粒转化还包括：
将构建的药物基因表达载体转化到出发菌中得到所述光控裂解工程菌；其中，
当所述工程菌裂解后，所述药物蛋白从所述工程菌释放。
可选地，所述药物基因表达载体和所述抗终止基因表达载体构建到同一质粒上。
可选地，所述光敏基因包括BphS、IlaC*、IlaD9、IlaM4和IlaM5中的任一种；
所述信使分子包括c-di-GMP或cAMP；
所述抗终止基因表达载体的启动子包括受c-di-GMP调控的cdrA启动子、pel启动子或psl启动子，或者受cAMP调控的lac启动子；
所述抗终止基因包括λ噬菌体的Q蛋白；
所述裂解基因包括LKD16。
具体的，BphS受近红外光调控可合成信使分子c-di-GMP；IlaC*、IlaD9、IlaM4和IlaM5受近红外光调控可合成信使分子cAMP。
在一实施例中，所述光敏基因为BphS；所述信使分子为c-di-GMP；所述抗终止基因表达盒的启动子为cdrA启动子。
本发明第三方面提出一种药物组合物，包括上述第一方面任一项所述的一种光控裂解工程菌。
本发明第四方面提出上述第一方面任一项所述的一种光控裂解工程菌在制备治疗肿瘤药物中的应用。
本发明第五方面提出一种肿瘤治疗系统，其特征在于，包括肿瘤治疗装置和肿瘤治疗药物；
所述肿瘤治疗药物包括上述第一方面任一项所述的一种光控裂解工程菌；
所述肿瘤治疗装置包括激光元件，所述激光元件可发射光源；其中，
所述肿瘤治疗系统在使用的时候，将所述肿瘤治疗药物注射到待治疗肿瘤部位，然后使用所述肿瘤治疗装置的光源照射所述肿瘤部位，
当所述光强达到裂解阈值时，所述光控裂解工程菌裂解释放菌内治疗物。
本发明第六方面提出如第一方面任一项所述的一种光控裂解工程菌在治疗疾病中的用途。
下面将结合具体实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下述实施例中所使用的引物序列如表1所示。
表1为所用引物序列表
pR'-LKD-ass-R： ctgcaggaattcctcgagaagctttcagtctccttgattcagggcg
Tn7-pR'-ass-F： ctgcaggaattcctcgagaagctttcagtctccttgattcagggcg
Tn7-ass-F： aagcttctcgaggaattcctgcag
Tn7-ass-R： ggtacctcgcgaaggccttg
BphS-CTX-F： gataccgtcgacctcgaaccccacgcccctcga
BphS-CTX-R: ggtacccaattcgccctatagtgagtcgtattacg
PA1O4O3-F: ctcactatagggcgaattgggtacctgccacctgacgtctaagaaaccat
BphS-R: cggccgctctagaactagttccttcatacccgccgggc
Q-F： tgagtaggacaaatccgcccccgggctaaactgatgcagcgtagttttcgtcgtttgc
PcdrA-R： attaatgtgagttagctcacgaattctggaaggttccttggcggcagcgga
PUCP-Q-R： gacgaaaactacgctgcatcagtttagcccgggggcggatttgtcctactcagga
PUCP-F： aattcgtgagctaactcacattaattgcgttgcg
PUCP-R： aagcttggcactggccgtcgttttacaacgtcgtg
PUCP-PcdrA-Q-F： actagtgggttcgaggtcgacggtatcgataagctagctt
J23118-F： taaaacgacggccagtgccaagcttttgacggctagctcagtcctaggta
HlyE-PUCP-R： ataccgtcgacctcgaacccactagtttagacttcaggtacctcaaagagtgtcttttt
实施例1光控裂解工程菌的构建
1.1质粒构建
1.1.1光敏基因BphS表达载体的构建
本方案中以能够在肺部有效定植的铜绿假单胞菌PAO1作为出发菌，以此为基础构建光控裂解工程菌。
光敏基因BphS(SEQ ID NO.1)编码得到光敏蛋白BphS(SEQ ID NO.2)，光敏蛋白BphS可以在近红外光调控下合成c-di-GMP，光强大小与合成的c-di-GMP浓度成正相关，BphS表达载体构建在自杀质粒miniCTX2中(质粒图谱见图3所示)，以通过自杀质粒将BphS基因及其相关表达元件整合到细菌基因组中，相关表达元件包括组成型启动子PA1/O4/O3(SEQ ID NO.3)，光响应组件BphO(SEQ ID NO.4)，使得BphS蛋白可以持续表达。其中，从上海生工生物有限公司合成PA1O4O3-BphS-BphO基因片段，并利用引物对PA1O4O3-F和BphS-R对其进行聚合酶链式PCR反应，miniCTX2质粒利用引物对BphS-CTX-F和BphS-CTX-R进行聚合酶链式PCR反应，以获得带有25个碱基同源臂的能持续性表达光响应组件BphO和BphS的基因片段以及线性化的载体质粒miniCTX2，最后利用吉布森连接法(Gibson assembly)将2个基因片段连接，即构建得到BphS表达载体PA1/O4/O3-BphS-CTX2。
1.1.2抗终止基因Q表达载体的构建
其中抗终止基因Q(SEQ ID NO.5)来自λ噬菌体基因组，通过Gibson assembly构建到载体质粒PUCP20(质粒图谱见图4所示)中，PUCP20质粒为可自主复制的外源质粒，通过该质粒构建抗终止基因Q表达载体，抗终止基因Q不会整合到细菌基因组中，而是以外源质粒形式存在，这样可以方便对抗终止基因Q的表达进行调整。
抗终止基因Q表达载体使用的启动子为响应c-di-GMP的cdrA启动子(SEQ ID NO.6)，c-di-GMP的浓度越高，抗终止基因Q表达载体表达得到的抗终止蛋白Q(SEQ ID NO.7)越多。
首先人工合成以PAO1为宿主进行碱基优化的PcdrA-Q基因片段，分别通过引物Q-F和PcdrA-R进行聚合酶链式PCR反应得到带有同源臂的PcdrA-Q片段，通过引物PUCP-Q-R和PUCP-F进行聚合酶链式PCR反应得到线性化的PUCP20载体片段；然后利用Gibson assembly方法将PcdrA-Q碱基片段插入到PUCP20中，从而构建载体PcdrA-Q-PUCP20。然后将抗终止基因Q前的RBS进行批量置换筛选，即得到一系列PcdrA-RBS-Q-T0/T1-PUCP20质粒。
1.1.3 hlyE基因表达载体的构建
hlyE(血溶素E)基因(SEQ ID NO.8)采用组成型启动子J23118(SEQ ID NO.9)，持续表达hlyE蛋白(SEQ ID NO.10)，通过PCR方法获得J23118-hlyE基因片段(引物为J23118-F和HlyE-PUCP-R)，模板为人工合成；随后通过吉布森连接法在PcdrA-RBS-Q-T0/T1-PUCP20质粒(线性化载体引物为PUCP-PcdrA-Q-F和PUCP-R)的T0/T1双终止子后插入J23118-hlyE基因片段，最终得到PcdrA-RBS-Q-T0/T1-J23118-hlyE-PUCP20质粒，可以同时表达抗终止蛋白Q和药物hlyE。
1.1.4裂解基因LKD16表达载体的构建
裂解基因LKD16(SEQ ID NO.11)表达得到裂解蛋白LKD16(SEQ ID NO.12)，为了降低裂解基因LKD16的背景表达水平，选用了来自λ噬菌体的启动子-终止子转录系统(pR’-tR’)(SEQ ID NO.13)作为LKD16表达载体的启动子，该系统在抗终止蛋白Q存在时可以正常转录下游基因，当抗终止蛋白Q不存在时，由于终止子的存在，导致启动子pR’无法表达下游LKD16基因。因此设计将裂解基因LKD16放置在pR’-tR’控制下，同时希望将其整合到工程菌基因组上，提升其稳定性的同时进一步降低其表达量，因此将裂解基因LKD16构建到自杀质粒miniTn7(质粒图谱见图5所示)中。为了避免抗终止蛋白Q累计造成的泄露表达，我们在抗终止基因Q后加上了ssrA水解标签(SEQ ID NO.14)。
首先人工合成PR’-tR’-LKD16基因片段，通过PCR获得PR’-tR’-LKD16基因片段(引物：pR'-LKD-ass-R/Tn7-pR'-ass-F)和线性化的miniTn7质粒(引物：Tn7-ass-F/Tn7-ass-R)，从而将合成裂解基因LKD16和pR’-tR’基因序列插入miniTn7质粒，从而得到pR’-tR’-B0034-LKD-T0/T1-miniTn7质粒。
1.2质粒转化
将上述构建出来的质粒在测序正确后通过电转的方法转入铜绿假单胞菌PAO1中对其进行工程化改造。
1.2.1 LKD16表达载体和BphS表达载体的转化
首先将pR'-tR'-B0034-LKD-T0/T1-miniTn7质粒在辅助质粒PTNS2的帮助下，电转至野生型铜绿假单胞菌PAO1中，然后涂板于LB+Gen30(30微克每毫升庆大霉素)的琼脂培养板上，挑取12小时后长出来的单克隆点，用L B+Gen30的液体培养基37℃，250转每分钟摇菌8小时，将质粒pflp2电转入其中，涂于LB+Carb300(300微克每毫升羧苄青霉素)的琼脂培养板上。
将长出来的点划线于LB+5％sucrose(蔗糖)板上，37℃培养12小时后挑单克隆点溶解于20微升无菌水中，每个点分别点于LB、LB+gen30、LB+c arb300培养板上，选取只在LB琼脂培养板上生长但在Gen、Carb两种抗性板上均未生长的点做PCR鉴定并送测序，得到将pR'-tR'-B0034-LKD-T0/T1-miniTn7质粒插入基因组的菌种pR'-tR'-B0034-LKD-T0/T1-miniTn7-PAO1。
质粒PA1/O4/O3-BphS-CTX2也采用同样的方法电转入pR'-tR'-B0034-LK D-T0/T1-miniTn7-PAO1中，只需将抗性由Gen30更改为Tc100(100微克每毫升的四环素)，得到菌种PA1/O4/O3-BphS-CTX2-pR'-tR'-B0034-LKD-T0/T1-miniTn7-PAO1，此菌种标记为BphS-LKD-PAO1。
1.2.2工程菌毒性的减弱
野生型的PAO1菌种毒性较大，为了减少毒性，采用无抗敲除的办法在B phS-LKD-PAO1的基础上敲除了三个基因以控制其毒性，分别为vfR(基因组编号为PA0652)、exoS(基因组编号为PA3841)、exoT(基因组编号为PA0044)，最终得到的减毒菌株命名为ExoST，相较于野生型PAO1菌株，Exo ST菌株的毒性大大降低。
基因敲除采用无缝重组敲除的方法，以vfR基因敲除的载体构建为例，首先需要通过PCR技术从基因组上获得目的片段的上游及下游的1000个碱基的片段，记为vfR-up和vfR-dn；随后通过Gibson assembly方法将他们插入到自杀型载体pEX18Gm的酶切位点EcoRI和HindIII之间(质粒图谱见图6所示)，构建载体vfR-pEX18Gm。
首先将构建的敲除质粒vfR-pEX18Gm电转入目的菌种，在含有庆大霉素的平板上筛选出单克隆菌斑；随后在含有15％蔗糖的不含氯化钠的LB平板上划线。37℃培养16小时后，挑选单克隆菌斑PCR鉴定目的基因vfR是否成功敲除。测序验证后，以相同的方法进行第二个基因的敲除直至三个毒力基因全部敲除成功，该菌种记为ExoST。
1.2.3 PcdrA-RBS-Q-T0/T1-J23118-hlyE-PUCP20质粒的转化
抗终止蛋白Q的表达量直接影响了裂解基因LKD16的表达量，若背景表达量较高则工程菌可能在未光照时就已经开始裂解，而若背景表达量过低，则工程菌在高光强照射时也无法裂解。
启动子与核糖体结合位点(RBS)均可以影响Q的背景表达水平，RBS是指基因起始密码子AUG上游的一段富含嘌呤的非翻译区。Q的背景表达水平如果比较高，即没有光照时，Q的表达量也足够开启裂解基因的表达使细菌裂解，细菌只需微弱的光甚至不光照就会裂解。与之相对，Q的背景表达水平如果太低，即使光强很强，也无法诱导足够的Q表达来使细菌裂解。所以说不同RBS序列导致整个系统对光强的响应行为是不一样的，因此对抗终止基因Q前的RBS进行筛选是必要的。
选用随机引物PCR的方法构建了一个可以标定RBS光响应强度的质粒库，请参阅图12a，通过公司合成了一批随机引物，其序列未知，后面连接绿色荧光蛋白，而橙色荧光蛋白则有持续性启动子和固定的已知RBS表达，我们通过绿色荧光强度与橙色荧光强度的比值来标定不同RBS的相对强度，结果如图12b所示，相对强度越高，说明RBS对光响应越强。
根据RBS的相对强度，利用两头夹的方法，将不同RBS序列放置于Q基因前，通过微流实验筛选得到可在低光生长、高光裂解的菌株。由于微流实验筛选过程比较麻烦，所以当得到一次实验结果的时候，需要知道Q蛋白表达量是高还是低，如果高就要往低调，换用更弱的RBS，如果低就要往高调，换用更强的RBS，就好比两头夹击。
微流实验前期准备：将不同RBS构建的PcdrA-RBS-Q-T0/T1-J23118-hlyE-PUCP20质粒转化ExoST菌株得到的菌株在实验前一天晚上划线于LB+Gen30琼脂板上，第二天挑菌用FAB培养基加1 微摩氯化铁、30毫摩谷氨酸盐、30微克每毫升的庆大抗生素(简记为FAB+++)的一毫升培养基于37℃，250rpm摇床培养至OD600值约为1.0，离心后用PBS重悬清洗两次，备用。注意全程避光操作。
微流实验的操作流程：微流通道由PDMS与盖玻片粘接而成，通道宽度为50微米，高度为200微米，形状为单直通道，通道的一端为进液口，另一端为废液口。选用的培养基为Mccoy’s 5A，其中含30微摩的氯化铁，30微克每毫升的庆大霉素(Gen30)以及10％的胎牛血清(FBS)，实验前一晚配置完成后吸入注射器内，放置于室温，第二天排空气泡，实验温度控制为30℃。将准备好的菌液由进液口注入，设定流速为3mL/h，在激光共聚焦显微镜下拍摄。选用的BphS激发光源为640nm激光器，由功率计测量60倍物镜处640nm激光光照强度，根据设定的拍摄程序，计算后得到约化后60倍物镜一个视野内的平均光强。部分筛选结果如图7所示，图7为不能裂解的菌株PcdrA-GFP、RBS004构建的菌株RBS004-Q和RBS017构建的菌株RBS017-Q的微流实验结果，根据图7可知，RBS017-Q可以在低光强16.2μW/cm 2下正常生长，而在高光强50.8μW/cm 2下裂解，证明RBS017符合要求。根据图7还可以知道，RBS017-Q菌株的裂解阈值可以为50.8μW/cm 2，也可以为16.2μW/cm 2～50.8μW/cm 2之间的任一值，例如可以为(20、30或40)μW/cm 2。
经过微流实验筛选后，发现RBS017(序列为：AACGCGGTGCAA)对光响应效果好，因此使用RBS017构建Q蛋白表达质粒并转化，得到符合要求的菌株分别为：将pcdrA-RBS017-Q-PUCP20质粒电转入ExoST菌种内，得到的菌株命名为Q017，作为对照；将PcdrA-RBS017-Q-T0/T1-J23118-hlyE-PUCP20质粒电转入ExoST菌种内，得到的菌种命名为H017，得到构建好的光控裂解工程菌。
实施例2测试pR’-tR’启动子-终止子转录系统在PAO1菌种中的背景表达水平以及诱导表达水平
pR’-tR’启动子-终止子转录系统直接决定裂解蛋白LKD16的背景表达水平，LKD16蛋白的背景表达水平太高，则在低光强或无光强的时候细菌都会裂解，LKD16蛋白的诱导表达水平太低，则在高光强时细菌也无法裂解，因此，测试pR’-tR’启动子-终止子转录系统在PAO1菌种中的背景表达水平以及 诱导表达水平是需要的。
如图11a所示，通过构建阿拉伯糖诱导的PBAD-B0034-Q-pJN105载体质粒，以及pR’-tR’启动子-终止子转录系统控制的红色荧光蛋白表达体系pR’-tR’-mScarlet-miniTn7质粒，在体系中添加阿拉伯糖会诱导PBAD启动子后基因的表达，PBAD-B0034-Q-pJN105载质粒通过电转的方法导入pR’-tR’-mScarlet-miniTn7-PAO1中，并用阿拉伯糖诱导Q蛋白的表达，以观察红色荧光蛋白的表达情况，并使用只有pR’启动表达的pR’-mScarlet-miniTn7质粒和pR’-tR’启动表达的pR’-tR’-mScarlet-miniTn7质粒做对照，结果如图11b和图11c所示。
图11b中上排图像为显微镜明场图片，下排图像为显微镜荧光图片，pR’-mScarlet列为没有终止子tR’时，pR’-mScarlet-PAO1的明场图片和荧光图片，荧光强度很强，证明pR’可以正常驱动红色荧光蛋白的表达，pR’-tR’-mScarlet列为加上终止子tR’后，pR’-tR’-mScarlet-PAO1的明场图片和荧光图片，几乎没有荧光，证明tR’可以阻止红色荧光蛋白的表达，背景表达水平较低。pR’-tR’-mScarlet-Q-pJN105+0.6％L-ara是PBAD-B0034-Q-pJN105-pR’-tR’-mScarlet-miniTn7-PAO1菌种在加入0.6％阿拉伯糖诱导之后的图片，有一定强度的荧光产生，证明阿拉伯糖可以诱导红色荧光蛋白的表达，图11c为三种菌种mScarlet荧光强度的定量数据。以上实验结果说明pR’-tR’启动子-终止子转录系统的背景表达水平低，诱导表达水平较高，验证了该系统的可行性。
实施例3光控裂解工程菌的小鼠体内实验
待用菌种在注菌前一夜由-80℃冰箱划线于LB+Gen30琼脂板上，第二天早上挑取单克隆点于1mL FAB+++摇菌至OD600值约为0.6，离心后用PBS重悬清洗两次，注意全程避光操作。
取100微升待用菌种注射到荷瘤(A549细胞系)Balb/c小鼠的瘤内，将小鼠分为高光强照射组(H017-Light)和黑暗处理组(H017-Dark)，其中高光强照射组小鼠采用10mW/cm 2的光强照射，考虑到光穿透动物组织到达肿瘤部位会减弱的问题，因此，动物实验照射的光强高于细菌实验照射的光强。于第1、2、7、8、12、14、15、21天再次注菌，并在第1、2、6、10、14、19、21天测量肿瘤体积、小鼠体重的变化以及肿瘤重量，结果分别如图8a、 图8b和图8c所示。说明菌株H017在光照条件下可以释放药物治疗肿瘤，而避光条件下不行。同时小鼠的体重未受到明显影响，表明释放药物不会给小鼠带来显著的生存压力。
将工程菌更换为可裂解释放红色荧光蛋白(mScarlet)或者不可裂解的菌株(ExoST)时，在光照与避光条件下均无法抑制肿瘤生长，如图9a和图9c所示，同时对小鼠体重也无显著影响，如图9b和图9d所示。
对上述初次注菌21天后的肿瘤进行组织切片染色分析，分别进行HE染色、DAPI染色、TUNEL染色，其中TUNEL染色的原理是基因组DNA断裂时，暴露的3'-OH可以在末端脱氧核苷酸转移酶(Terminal Deoxynucleotidyl Transferase,TdT)的催化下加上荧光素(FITC)标记的dUTP(fluorescein-dUTP)，从而可以通过荧光显微镜或流式细胞仪进行检测，荧光强度越强，细胞凋亡越多。结果如图10所示，发现H017光照组肿瘤组织内部大量细胞凋亡，而避光组几乎没有检测到凋亡的细胞，证实了药物的可控释放程度非常高，本发明实施例的光控裂解工程菌仅在高光强照射下才会释放治疗药物诱导细胞凋亡。
以上所述仅为本发明的可选实施例，并非因此限制本发明的专利范围，凡是在本发明的发明构思下，利用本发明说明书及附图内容所作的等效结构变换，或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。

Claims (14)

  1. 一种光控裂解工程菌，其特征在于，包括：
    光敏基因表达盒，表达得到光敏蛋白，所述光敏蛋白受光调控合成信使分子；
    抗终止基因表达盒，所述抗终止基因表达盒的启动子受所述信使分子调控，表达得到抗终止蛋白；
    裂解基因表达盒，所述裂解基因表达盒的启动子受所述抗终止蛋白调控，表达得到裂解蛋白，用于裂解所述工程菌；其中，
    当调控所述光敏蛋白的光强达到裂解阈值时，所述裂解蛋白的表达量使所述工程菌裂解。
  2. 如权利要求1所述的一种光控裂解工程菌，其特征在于，所述光敏基因表达盒和所述裂解基因表达盒整合到所述工程菌的基因组中，所述抗终止基因表达盒存在于外源质粒中。
  3. 如权利要求2所述的一种光控裂解工程菌，其特征在于，所述工程菌还包括：
    药物基因表达盒，表达得到药物蛋白，当所述工程菌裂解后，所述药物蛋白从所述工程菌释放。
  4. 如权利要求3所述的一种光控裂解工程菌，其特征在于，所述药物基因表达盒与所述抗终止基因表达盒存在于同一个外源质粒中。
  5. 如权利要求1至4中任意一项所述的一种光控裂解工程菌，其特征在于，所述光敏基因包括BphS、IlaC*、IlaD9、IlaM4和IlaM5中的任一种；
    所述信使分子包括c-di-GMP或cAMP；
    所述抗终止基因表达盒的启动子包括受c-di-GMP调控的cdrA启动子、pel启动子或psl启动子，或者受cAMP调控的lac启动子；
    所述抗终止基因包括λ噬菌体的Q蛋白；
    所述裂解基因包括LKD16。
  6. 一种光控裂解工程菌的构建方法，其特征在于，包括质粒构建和质粒转化，所述质粒构建包括：
    光敏基因表达载体的构建，所述光敏基因表达得到光敏蛋白，所述光敏蛋白受光调控合成信使分子；
    抗终止基因表达载体的构建，所述抗终止基因表达载体的启动子受所述信使分子调控，表达得到抗终止蛋白；
    裂解基因表达载体的构建，所述裂解基因表达载体的启动子受所述抗终止蛋白调控，表达得到裂解蛋白，用于裂解所述工程菌；
    所述质粒转化包括：
    将上述构建好的光敏基因表达载体、抗终止基因表达载体和裂解基因表达载体转化到出发菌中得到所述光控裂解工程菌。
  7. 如权利要求6所述的一种光控裂解工程菌的构建方法，其特征在于，所述光敏基因表达载体和所述裂解基因表达载体由自杀质粒构建得到，所述抗终止基因表达载体由复制质粒构建得到，所述光敏基因和所述裂解基因整合到所述工程菌的基因组中，所述抗终止基因以质粒形式存在。
  8. 如权利要求7所述的一种光控裂解工程菌的构建方法，其特征在于，所述质粒构建还包括：
    药物基因表达载体的构建，所述药物基因表达得到药物蛋白；
    所述质粒转化还包括：
    将构建的药物基因表达载体转化到出发菌中得到所述光控裂解工程菌；其中，
    当所述工程菌裂解后，所述药物蛋白从所述工程菌释放。
  9. 如权利要求8所述的一种光控裂解工程菌的构建方法，其特征在于，所述药物基因表达载体和所述抗终止基因表达载体构建到同一质粒上。
  10. 如权利要求6至9任一项所述的一种光控裂解工程菌的构建方法，其特征在于，所述光敏基因包括BphS、IlaC*、IlaD9、IlaM4和IlaM5中的任一种；
    所述信使分子包括c-di-GMP或cAMP；
    所述抗终止基因表达载体的启动子包括受c-di-GMP调控的cdrA启动子、pel启动子或psl启动子，或者受cAMP调控的lac启动子；
    所述抗终止基因包括λ噬菌体的Q蛋白；
    所述裂解基因包括LKD16。
  11. 一种药物组合物，其特征在于，包括权利要求1至5任一项所述的一种光控裂解工程菌。
  12. 如权利要求1至5任一项所述的一种光控裂解工程菌在制备治疗肿瘤药物中的应用。
  13. 一种肿瘤治疗系统，其特征在于，包括肿瘤治疗装置和肿瘤治疗药物；
    所述肿瘤治疗药物包括权利要求1至5任一项所述的一种光控裂解工程菌；
    所述肿瘤治疗装置包括激光元件，所述激光元件可发射光源；其中，
    所述肿瘤治疗系统在使用的时候，将所述肿瘤治疗药物注射到待治疗肿瘤部位，然后使用所述肿瘤治疗装置的光源照射所述肿瘤部位，
    当所述光强达到裂解阈值时，所述光控裂解工程菌裂解释放菌内治疗物。
  14. 如权利要求1至5任一项所述的一种光控裂解工程菌在治疗疾病中的用途。
PCT/CN2021/111757 2021-08-10 2021-08-10 一种光控裂解工程菌及其构建方法和应用 Ceased WO2023015441A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/111757 WO2023015441A1 (zh) 2021-08-10 2021-08-10 一种光控裂解工程菌及其构建方法和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/111757 WO2023015441A1 (zh) 2021-08-10 2021-08-10 一种光控裂解工程菌及其构建方法和应用

Publications (1)

Publication Number Publication Date
WO2023015441A1 true WO2023015441A1 (zh) 2023-02-16

Family

ID=85200346

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/111757 Ceased WO2023015441A1 (zh) 2021-08-10 2021-08-10 一种光控裂解工程菌及其构建方法和应用

Country Status (1)

Country Link
WO (1) WO2023015441A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4486898A4 (en) * 2022-03-01 2026-03-25 Univ Cincinnati GENETICALLY MODIFIED BACTERIA CONTAINING A LYSIS GENE

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031561A1 (en) * 1994-05-12 1995-11-23 Quest International B.V. Process for the lysis of a culture of lactic acid bacteria by means of a lysin, and uses of the resulting lysed culture
US20060040393A1 (en) * 2004-08-17 2006-02-23 Xiyu Jia Controlled lysis of bacteria
CN106244613A (zh) * 2016-08-25 2016-12-21 江南大学 一种稳定期自裂解的枯草芽孢杆菌及其应用
CN107177621A (zh) * 2016-03-10 2017-09-19 叶海峰 远红光基因环路表达控制系统进行转基因调控表达的方法
CN107174655A (zh) * 2016-03-10 2017-09-19 华东师范大学 一种远红光基因环路表达控制系统在治疗糖尿病中的应用
WO2019068006A1 (en) * 2017-09-29 2019-04-04 The Charles Stark Draper Laboratory, Inc. CELLULAR LYSE CONTROLLED BY STIMULUS
CN113136396A (zh) * 2020-01-20 2021-07-20 华东理工大学 细菌光控基因表达系统及其调控基因表达的方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031561A1 (en) * 1994-05-12 1995-11-23 Quest International B.V. Process for the lysis of a culture of lactic acid bacteria by means of a lysin, and uses of the resulting lysed culture
US20060040393A1 (en) * 2004-08-17 2006-02-23 Xiyu Jia Controlled lysis of bacteria
CN107177621A (zh) * 2016-03-10 2017-09-19 叶海峰 远红光基因环路表达控制系统进行转基因调控表达的方法
CN107174655A (zh) * 2016-03-10 2017-09-19 华东师范大学 一种远红光基因环路表达控制系统在治疗糖尿病中的应用
CN106244613A (zh) * 2016-08-25 2016-12-21 江南大学 一种稳定期自裂解的枯草芽孢杆菌及其应用
WO2019068006A1 (en) * 2017-09-29 2019-04-04 The Charles Stark Draper Laboratory, Inc. CELLULAR LYSE CONTROLLED BY STIMULUS
CN113136396A (zh) * 2020-01-20 2021-07-20 华东理工大学 细菌光控基因表达系统及其调控基因表达的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4486898A4 (en) * 2022-03-01 2026-03-25 Univ Cincinnati GENETICALLY MODIFIED BACTERIA CONTAINING A LYSIS GENE

Similar Documents

Publication Publication Date Title
Kuriyama et al. Particle-mediated gene transfer into murine livers using a newly developed gene gun
JP7189943B2 (ja) 細胞の遺伝子修飾のための非組込みｄｎａベクター
JP4750203B2 (ja) 条件複製起点をもつ環状ｄｎａ分子、それらの製造方法、及び、遺伝子治療におけるそれらの使用
JP6976249B2 (ja) 免疫を工学操作するための方法および組成物
US20190247436A1 (en) Methods and compositions for modifying genomic dna
JP4021197B2 (ja) 遺伝子発現の全身光学イメージングとその使用
Singer et al. Engineered bacteria launch and control an oncolytic virus
Zhang et al. Engineered bacteria breach tumor physical barriers to enhance radio-immunotherapy
CN112941107A (zh) 原核Argonaute蛋白的基因编辑应用
Aoyama et al. RETRACTED: Enhanced expression of plasmid DNA–cationized gelatin complex by ultrasound in murine muscle
WO2023015441A1 (zh) 一种光控裂解工程菌及其构建方法和应用
Chen et al. Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system
CN104130977B (zh) 一种抗肿瘤药物筛选细胞模型及其应用
Rana et al. Coexpression after electroporation of plasmid mixtures into muscle in vivo
Dong et al. A modular mRNA platform for programmable induction of tumour-specific immunogenic cell death
US7595386B2 (en) Methods and compositions for heat activated gene therapy using cytolethal distending toxin
Radzevičiūtė-Valčiukė et al. Effects of buffer composition and plasmid toxicity on electroporation-based non-viral gene delivery in mammalian cells using bursts of nanosecond and microsecond pulses
CN115704041A (zh) 一种光控裂解工程菌及其构建方法和应用
Kiga et al. Development of a nonreplicative phage-based DNA delivery system and its application to antimicrobial therapies
Howells et al. A drug‐selectable acoustic reporter gene system for human cell ultrasound imaging
CN115704006A (zh) 基于细菌生物被膜的肿瘤治疗工程菌及其构建方法和应用
Qudsia et al. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies
WO2022117658A1 (en) Bacterial delivery of gene silencing tools into eukaryotic cells
Qu et al. Development of ultrasound-visualized tumor-targeting engineered bacteria for precise tumor therapy
Ferguson et al. Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21953073

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21953073

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 21953073

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 31/01/2025)

122 Ep: pct application non-entry in european phase

Ref document number: 21953073

Country of ref document: EP

Kind code of ref document: A1