CN113186145A - Tumor-targeted bacteria with visualization function, visualization method and application thereof - Google Patents
Tumor-targeted bacteria with visualization function, visualization method and application thereof Download PDFInfo
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Abstract
The invention provides a tumor-targeted bacterium with a visualization function, a visualization method and application thereof, and belongs to the technical field of genetic engineering and tumor detection. The invention constructs a series of modified strains of escherichia coli Nissle1917, the strains have a bioluminescence functional module and excellent bioluminescence activity, and can be used for tracing and imaging the bacterial targeting tumor process on the aspect of a small animal living body. The Escherichia coli Nissle1917 with the visualization function can be used as an imaging tracing tool for self tracing of tumor-targeted bacteria and detection of tumors, and has wide application value. The escherichia coli Nissle1917 with bioluminescence capability can be used as a tumor targeting vector with a visualization function, and is applied to the relevant preclinical research of tumor targeting therapy, so that the invention has good practical application value.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and tumor detection, and particularly relates to tumor-targeted bacteria with a visualization function, a visualization method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Coli Nissle1917 (EcN) is a probiotic and also a tumor targeting bacterium that has a preference for and can proliferate in large numbers within solid tumors. The Escherichia coli Nissle1917 with good tumor targeting colonization and safety has great potential to become a carrier for tumor targeting therapy. The visualization function module can endow the tumor targeting carrier with a tracing function, is a real-time monitoring tool for reflecting the conditions of bacteria migration, propagation, distribution and decay in vivo, and is a visualization tool for generating anticancer activity by bacteria targeted delivery of drugs.
The bioluminescence functional module and the bioluminescence imaging technology are important technical means for tumor monitoring and anticancer effect evaluation in preclinical research of tumors. With the development of tumor-targeted bacteria-related tumor therapy, a suitable visual imaging means is needed to assist in tracing the bacteria, and effective information is provided for the controlled release of targeted delivery of anticancer drugs. However, the inventors found that the visualization research aiming at the Escherichia coli Nissle1917 is only rarely reported at present.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a tumor targeted bacterium with a visualization function, a visualization method and application thereof, the invention takes escherichia coli Nissle1917 as an original strain, and the gene engineering strain is constructed by inserting a bioluminescence related functional gene into a wild type or a mutant strain genome of which a restriction endonuclease gene is knocked out in a recombination mode, so that the tumor targeted bacterium has good bioluminescence activity and a remarkable bioluminescence imaging effect, and has good practical application value.
In a first aspect of the invention, a genetically engineered bacterium is provided, which is constructed by inserting a bioluminescence related functional gene into a wild type escherichia coli or an escherichia coli mutant strain genome from which a restriction endonuclease gene is knocked out.
More specifically, the insertion site is downstream of the E.coli glucosamine synthetase gene (glm S).
Wherein the bioluminescence-related functional gene comprises a luciferase gene;
such luciferases include, but are not limited to, Firefly luciferase (FLuc), secreted membrane-anchored luciferase (Gluc), and Renilla luciferase (Rluc), and mutants thereof.
In a second aspect of the present invention, there is provided a method for constructing the above genetically engineered bacterium, the method comprising:
constructing a bioluminescent element plasmid, and recombining a target gene element with a bioluminescent function into an escherichia coli genome for expression to obtain the recombinant escherichia coli.
Wherein the target genetic element with bioluminescence function comprises a luciferase genetic element.
Based on the technical scheme, the genetically engineered bacterium prepared by the invention can be used as a tumor-targeted bacterium with a visualization function, so that the third aspect of the invention provides an application of the genetically engineered bacterium in any one or more of the following aspects:
a) imaging tracing and/or preparing an imaging tracing product;
b) detecting tumors and/or preparing tumor detection products;
c) tumor-targeted therapy and/or tumor-targeted therapy products;
d) tumor targeted therapy preclinical studies.
In a fourth aspect of the present invention, a tumor visual detection method is provided, which comprises administering the above genetically engineered bacterium to a subject.
The subject of the present invention refers to an animal that has been the subject of treatment, observation or experiment.
The beneficial technical effects of one or more technical schemes are as follows:
according to the technical scheme, a series of modified strains of escherichia coli Nissle1917 are constructed, the strains have a bioluminescence functional module and excellent bioluminescence activity, and can be used for tracing and imaging of bacterial target tumor processes on the aspect of a small animal living body. The Escherichia coli Nissle1917 with the visualization function can be used as an imaging tracing tool for self tracing of tumor-targeted bacteria and detection of tumors.
The escherichia coli Nissle1917 with bioluminescence capability in the technical scheme can be used as a tumor targeting vector with a visualization function, and is applied to the relevant preclinical research of tumor targeting therapy, so that the method has a good practical application prospect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of a firefly based bioluminescent element according to an embodiment of the present invention.
FIG. 2 is an agarose gel electrophoresis image of a bioluminescent element plasmid of the present invention after enzyme digestion and identification; wherein A is the result of identifying the Fluc-EGFP plasmid digested by NcoI. And B is the identification result of the NotI enzyme digestion Fluc-EGFP-LRE-mcherry plasmid.
FIG. 3 shows Fluc-LRE or Fluc-mediated bioluminescence according to an embodiment of the present invention; wherein A is a change curve of bioluminescence intensity along with time in a live bacterium state; and B is the change curve of the bioluminescence intensity of the bacterial lysate with time.
FIG. 4 is the agarose gel electrophoresis result of the colony PCR verification of the target gene of the engineering strain with the bioluminescence module according to the embodiment of the present invention; wherein A is a colony PCR result inserted with a Fluc module; b is the result of colony PCR with inserted Fluc-LRE module.
FIG. 5 shows the results of bioluminescence imaging of E.coli Nissle1917 with a knock-in bioluminescent element in the genome according to the example of the present invention; wherein A is a comparison graph of bioluminescence intensity of the engineering strains with the Fluc-LRE element knocked in and the Fluc element only knocked in. B is a comparison graph of bioluminescence persistence of the engineered strains with the knockin Fluc-LRE element and the knockin Fluc element only. And C is a bioluminescence imaging result obtained after the engineering strains with the Fluc-LRE element knocked in and the engineering strains with the Fluc element knocked in only pass through different filters. D is the quantization diagram of the diagram C. The substrate is a mixed solution of D-type fluorescein and D-type cysteine.
FIG. 6 shows the bioluminescence imaging results of the engineered E.coli Nissle1917 strain with knock-in visualization elements of the genome according to the embodiment of the present invention; wherein A is bioluminescence imaging after intratumoral injection of a strain with a knock-in visualization element. B is the quantization diagram of diagram A. And C is bioluminescence imaging after tail vein injection of the strain with the knock-in visualization element. D is the quantization diagram of the diagram C. And E is a bioluminescence continuous imaging result after the tail vein is injected with the strain with the knock-in visualization element. F is the quantization map of the map E. The substrate is a mixed solution of D-type fluorescein and D-type cysteine.
FIG. 7 is a schematic diagram of a restriction endonuclease gene knockout process in accordance with an embodiment of the present invention.
FIG. 8 shows the tumor colonization effect of the E.coli Nissle1917 mutant strain with the restriction enzyme knockout of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As mentioned above, the bioluminescent functional module and the bioluminescent imaging technology are important technical means for tumor monitoring and anticancer effect evaluation in preclinical research of tumors. With the development of tumor-targeted bacteria-related tumor therapy, a suitable visual imaging means is needed to assist in tracing the bacteria, and effective information is provided for the controlled release of targeted delivery of anticancer drugs.
In view of the above, the invention designs a series of bioluminescent elements, knocks the bioluminescent elements into escherichia coli Nissle1917 wild type or escherichia coli Nissle1917 mutant strains with restriction enzyme knocked out in a homologous recombination mode, constructs a series of tumor-targeted bacteria with bioluminescent visualization elements, can be used as tumor-targeted vectors with visualization functions, and has great potential to be applied to tumor-targeted drug delivery or targeted therapy related research.
Specifically, in an exemplary embodiment of the present invention, a genetically engineered bacterium is provided, wherein the genetically engineered bacterium is constructed by inserting a bioluminescence-related functional gene into a wild-type escherichia coli or an escherichia coli mutant strain genome in which a restriction endonuclease gene is knocked out.
In yet another embodiment of the present invention, the insertion site is downstream of the E.coli glucosamine synthetase gene (glm S).
In another embodiment of the present invention, the starting strain of Escherichia coli is specifically Escherichia coli Nissle 1917. It is a probiotic, and also a tumor-targeting bacterium, which has a preference for and can proliferate in large quantities within solid tumors. The Escherichia coli Nissle1917 with good tumor targeting colonization and safety has great potential to become a carrier for tumor targeting therapy.
In still another embodiment of the present invention, the bioluminescence-related functional gene comprises a luciferase gene;
in yet another embodiment of the invention, the luciferases include, but are not limited to, firefly luciferase (FLuc), secreted membrane-anchored luciferase (Gluc), and Renilla luciferase (Rluc), and mutants thereof.
In a further embodiment of the invention, the luciferase is selected from any one or more of the group consisting of:
in yet another embodiment of the present invention, the restriction endonuclease Gene includes, but is not limited to Gene1-Gene7, see in particular below:
in still another embodiment of the present invention, the functional genes related to bioluminescence include the above firefly luciferase and mutant genes thereof and a substrate regeneration enzyme (LRE) gene; thereby further improving the bioluminescence intensity and the luminescence time of the genetically engineered bacteria.
In another embodiment of the present invention, there is provided a method for constructing the above genetically engineered bacterium, the method comprising:
constructing a bioluminescent element plasmid, and recombining a target gene element with a bioluminescent function into an escherichia coli genome for expression to obtain the recombinant escherichia coli.
Wherein the target genetic element with bioluminescence function comprises a luciferase genetic element;
in yet another embodiment of the invention, the luciferase genetic element comprises firefly luciferase and its mutant-substrate regenerant enzyme tandem genetic element, secreted membrane-anchored luciferase and its mutant genetic element, and Renilla luciferase and its mutant genetic element.
In another embodiment of the present invention, the firefly luciferase and the mutant-substrate regeneration enzyme tandem gene element plasmid thereof are constructed by a method comprising:
the firefly luciferase (Fluc) and mutant genes are fused with EGFP label genes, substrate regeneration enzyme (LRE) genes are fused with mCherry genes, and the firefly luciferase (Fluc) and mutant genes are assembled with pMB1 replicons and constitutive lac promoters to construct firefly expression plasmids (Fluc-LRE series).
In another embodiment of the present invention, the Escherichia coli is Escherichia coli Nissle 1917;
the Escherichia coli Nissle1917 is wild type Escherichia coli Nissle1917 or Escherichia coli Nissle1917 with the restriction endonuclease gene knocked out.
In yet another embodiment of the present invention, "recombining said genetic element of interest into the genome of E.coli" is specifically recombining said genetic element of interest into the genome of E.coli downstream of the glm S gene.
Based on the technical scheme, the genetically engineered bacterium prepared by the invention can be used as a tumor-targeted bacterium with a visualization function, so that in another specific embodiment of the invention, the application of the genetically engineered bacterium in any one or more of the following is provided:
a) imaging tracing and/or preparing an imaging tracing product;
b) detecting tumors and/or preparing tumor detection products;
c) tumor-targeted therapy and/or tumor-targeted therapy products;
d) tumor targeted therapy preclinical studies.
In another embodiment of the present invention, a tumor visual detection method is provided, which comprises administering the above genetically engineered bacterium to a subject.
The subject of the present invention refers to an animal, such as a mouse, a rabbit, a monkey, etc., which has been the subject of treatment, observation or experiment.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
In the present example, the mutant strain EcN-Fluc-LRE of Escherichia coli Nissle1917 was obtained by knocking in the firefly luciferase gene and the substrate-regenerating enzyme gene into the genome. The construction process and the application method are as follows:
1. construction of firefly bioluminescent element plasmid
The C ends of firefly luciferase (Fluc) and mutant (table 1) genes are fused with the N end of an EGFP label gene, the C end of a substrate regeneration enzyme (LRE) gene is fused with the N end of an mCherry gene, and the fusion products, a pMB1 replicon and a constitutive lac promoter are assembled to construct plasmids (Fluc-LRE series) for tandem expression of Fluc and LRE. Accordingly, an expression plasmid containing only the fusion fragment of the wild-type luciferase gene and the EGFP-tagged gene served as a control plasmid. The primers required are shown in Table 2, and the relevant genes of interest are shown in Table 3.
TABLE 1 kinds and characteristics of luciferases involved in the bioluminescent elements of the present invention
TABLE 2 primers for construction of bioluminescent elements and engineering strains
TABLE 3 wild-type gene sequences of interest for construction of bioluminescent elements
(1) The target gene was PCR amplified using primers containing homology arms (shown in Table 2) and the PCR product was purified.
(2) And (3) transforming the PCR product into 10% Ala-induced engineering bacteria E.coli GB05-dir by an electrotransformation method, and obtaining a corresponding recombinant by resistance screening.
(3) Selecting monoclonals from corresponding recombinant plates, extracting plasmids, carrying out enzyme digestion identification on plasmid DNA, placing the enzyme digestion system under the optimal temperature condition of restriction enzyme, reacting for 2h, and carrying out agarose gel electrophoresis detection.
| Plasmid | 4μl |
| 10X NEB buffer | 1μl |
| Enzyme | 0.2μl |
| ddH2O | 4.8μl |
| Total | 10μl |
(4) The constructed bioluminescent element plasmid was electrically transformed into E.coli Nissle 1917.
(5) Preliminary bioluminescence Activity test
and a, inoculating a single colony of escherichia coli Nissle1917 containing the bioluminescence element plasmid into a culture medium, and placing the single colony on a constant-temperature mixer at 37 ℃ for overnight culture at 950rpm for 16 h. Transferring the bacterial liquid the next day, growing to logarithmic phase, centrifugally collecting thallus, washing with sterile water, re-suspending the bacterial liquid with fresh LB culture medium and diluting to OD600Is 1.0; the bacterial suspension was added to a black 96-well plate at 100. mu.L per well.
b, preparing a 2.5mM D-cysteine solution by using a 50mM Tris-HCl buffer solution with the pH value of 7.4, and preparing a 100 mu M D-luciferin solution by using the solution or the Tris-HCl buffer solution;
and c, adding the substrate solution into a 96-well plate, quickly mixing with the bacteria solution uniformly, and immediately carrying out bioluminescence test or imaging by using a bioluminescence enzyme-labeling instrument or a small animal living body imaging instrument.
And d, performing statistical calculation on the obtained data by using GraphPad software.
Bioluminescence test As shown in FIG. 3, the group expressing Fluc-LRE dual enzyme showed higher bioluminescence intensity and longer luminescence time than the group expressing Fluc alone.
2. Construction of Escherichia coli Nissle1917 engineering strain with bioluminescence function
(1) Plasmid pSC101-BAD-ccdA-Rha-gbaA-tet was first transformed into e.coli Nissle1917 to give strain EcN-gbaA.
(2) Primers (shown in Table 2) with homology arms (HA,50bp) on both sides of the target gene (Fluc-LRE) are used, and a PCR product with the homology arms required for recombination is obtained by PCR amplification by using a bioluminescent element plasmid as a template.
(3) Homologous recombination
The PCR product was transformed into EcN-gbaA induced by 10% Rha by electrotransformation, recombination was mediated by recombinase gbaA, and the bioluminescent element gene was inserted downstream of the glm S gene in the EcN genome. And (4) screening the recombinants by using the resistance gene carried by the inserted gene fragment. EcN-Fluc-LRE strain was obtained.
(4) Colony PCR
The insertion of the foreign gene into the EcN genome was verified by colony PCR.
and a, picking a single colony, inoculating the single colony into an LB liquid culture medium containing a proper antibiotic, and culturing at 30 ℃ and 950rpm for 3 hours until obvious turbidity appears. The cells were collected by centrifugation, washed with sterile water, and then resuspended in 1ml of sterile water as a template.
b, carrying out PCR by using corresponding primers; the PCR system was as follows
And c, carrying out gel electrophoresis detection on the PCR product.
Other methods of inserting the gene of the bioluminescent element are similar. According to the method, on the basis of the wild type of Escherichia coli Nissle1917, mutant strains EcN-Fluc, EcN-Fluc-LRE, EcN-FlucMutant1-LRE, EcN-FlucMutant2-LRE, EcN-FlucMutant3-LRE, EcN-FlucMutant4-LRE, EcN-FlucMutant5-LRE, EcN-FlucMutant6-LRE, EcN-FlucMutant7-LRE, EcN-FlucMutant8-LRE, EcN-FlucMutant9-LRE, EcN-FlucMutant10-LRE, EcN-FlucMutant11-LRE, N-Gluc EcN-Fluc EcN-sbGluc EcN-LRE, EcN-FlucMutant10-LRE, EcN-FlucMutant 3948-LRE, Rc 598-Rflucmutant 599.5-LRE, EcN-FlucMutant7-LRE, EcN-FlucMutant8-LRE, EcN-Fluc-EcN-LRE, EcN-flucmut-EcN-EcN-LRE, Rc-EcN-LRE, Rc 598-LRE, Rc 599, Rc 598-LRE, Rc 599, Rc 598-LRE, EcN-LRE, rC 3-LRE, rC 3-LRE, rC-
3. Bioluminescence activity and test method of escherichia coli Nissle1917 engineering strain with bioluminescence visualization element
a,1.5ml centrifuge tubes are punched and 1.4ml LB liquid medium containing the appropriate antibiotics is added. Inoculating a single colony of Escherichia coli Nissle1917 with a visualization function into a culture medium, and placing the single colony on a constant-temperature mixer at 37 ℃ for overnight culture at 950 rpm. Transferring the bacterial liquid the next day, growing to logarithmic phase, centrifugally collecting thallus, washing with sterile water, re-suspending the bacterial liquid with fresh LB culture medium and diluting to OD600Is 1.0; the bacterial suspension was added to a black 96-well plate at 100. mu.L per well.
b, preparing a 2.5mM D-cysteine solution with 50mM Tris-HCl buffer solution with pH7.4, and preparing a 100 mu M D-luciferin solution with the solution or the Tris-HCl buffer solution.
And c, adding the substrate solution into a 96-well plate, quickly mixing with the bacteria solution uniformly, and immediately carrying out bioluminescence test or imaging by using a bioluminescence enzyme-labeling instrument or a small animal living body imaging instrument.
And d, performing statistical calculation on the obtained data by using GraphPad software.
As shown in FIG. 5, the Escherichia coli Nissle1917 with the genome into which the bioluminescent element Fluc-LRE was knocked in had a stronger bioluminescence intensity and a better bioluminescence persistence than those of Escherichia coli Nissle1917 with the genome into which the Fluc gene was knocked in alone (FIGS. 5A and 5B); significant bioluminescence imaging effect and relative wavelength red shift (fig. 5C, 5D).
4. Small animal living body imaging effect and testing method of escherichia coli Nissle1917 engineering strain with bioluminescence visualization element
a. Culturing in RPMI 1640 medium containing 10% serum, 100U/mL penicillin and 100. mu.g/mL streptomycin at 37 deg.C in 5% carbon dioxide incubatorMurine mammary carcinoma 4T1 cells reached logarithmic growth phase. Pancreatin cells, washing cells 2 times with PBS, resuspending cells with PBS and diluting to 6X 106cells/mL。
b. Balb/c female mice, 8 weeks old, were harvested, and the hair, each axillary, was injected subcutaneously with 100. mu.L of cell suspension. After the tumor formation, the subsequent experiment is carried out.
c. Determination of CFU and OD of engineered Strain600And fitting a linear equation.
c. A single clone of the E.coli Nissle1917 engineered strain with inserted visual function was picked up and cultured overnight at 37 degrees in a liquid medium containing the appropriate antibiotic.
d. Transferring bacteria liquid the next day, centrifuging at 10000rpm for 1min to collect thalli after the bacteria liquid grows to logarithmic phase, and re-suspending the thalli with 1ml of sterile water; this step is repeated.
e.10000rpm centrifugation for 1min to collect thallus, abandoning supernatant, resuspending with sterile PBS, diluting to 2X 108CFU/mL of bacterial liquid.
f. Tumor-bearing mice were randomly grouped, and 100. mu.L of the bacterial suspension was injected into the tail vein of each tumor-bearing mouse. Or 25 mul of intratumoral injection bacterial liquid.
g. A2.5 mM D-cysteine solution was prepared in sterile PBS buffer, and a 46mM D-luciferin potassium salt solution was prepared from this solution.
h. After mice carrying tumor and injected with engineering strain via tail vein are anesthetized by isoflurane, 25 mu L of substrate solution is injected into tumor, and imaging is carried out immediately by a small animal living body imaging instrument.
The in vivo imaging result of the small animal (figure 6) shows that the escherichia coli Nissle1917 inserted with the bioluminescence element (Fluc-LRE) gene shows excellent bioluminescence imaging intensity and bioluminescence persistence in a tumor-bearing model of the small animal. The luminous intensity of EcN-Fluc-LRE was significantly better than that of EcN-Fluc, regardless of intratumoral injection of bacterial solutions (FIGS. 6A and 6B) or caudal vein injection of bacterial solutions (FIGS. 6C and 6D). EcN-Fluc-LRE also has a significant advantage over EcN-Fluc in bioluminescence imaging persistence when tail vein injection of bacteria was used (FIGS. 6E, 6F).
In addition to constructing the tumor-targeted bacteria with the visualization function based on the wild type of the escherichia coli Nissle1917, the tumor-targeted bacteria with the visualization function is constructed by using a similar method based on the escherichia coli Nissle1917 mutant strain with the restriction endonuclease gene knocked out. The implementation process is as follows.
5. Knockout of restriction endonuclease gene in Escherichia coli Nissle1917 and screening thereof
Restriction enzyme related genes were knocked out in E.coli Nissle1917 wild strain, and the information of the knocked-out genes is shown in Table 2.
TABLE 2 major restriction endonuclease genes in E.coli Nissle1917
The knock-out pattern is schematically shown in FIG. 7.
a. Plasmid pSC101-BAD-ccdA-Rha-Gbaa-tet was first transformed into E.coli Nissle1917, yielding strain EcN-Gbaa.
b. The antibiotic resistance Gene (CmR) is amplified by PCR using primers with homology arms (HA,50bp) and lox sites (lox66, lox71) at both sides of the target Gene (Gene1), and a PCR product of the resistance Gene targeting vector with the homology arms and lox sites required for recombination is obtained.
c. The PCR product is transformed into EcN-Gbcaa induced by 10% Rha by an electrotransformation method, recombinase Gbcaa mediates recombination, and a resistance Gene replaces a target restriction enzyme Gene to obtain a strain EcN-Gbcaa-CmR:: Gene 1.
d. Plasmid RK2-BAD-Cre-SacB is transformed into strain EcN-Gbaa-CmR, wherein 10% Ara is added into a culture medium to induce Cre enzyme expression, the Cre enzyme recognizes two lox sites to recombine, and a resistance screening marker between the two lox sites is deleted to obtain the strain EcN-Gbaa delta Gene 1.
e. The obtained strain RK2-BAD-Cre-SacB EcN-Gbcaa delta Gene1 is subcultured for 3 times in a culture medium containing cane sugar, and a single colony is obtained by partition streaking, so that the plasmid RK2-BAD-Cre-SacB can be eliminated.
f. A new lox site (lox6671) is left on the genome, which is neither different from the original one nor recognized by Cre, so that the strategy can be reused in this bacterium. The knockout process of the remaining restriction endonuclease genes was referred to the above procedure.
According to the knockout process, the restriction enzyme-knocked-out E.coli Nissle1917 mutant strains shown in Table 3 were obtained altogether.
Table 3: restriction enzyme-knockout Escherichia coli Nissle1917 mutant strain information
6. Evaluation of tumor-targeting colonization ability of Escherichia coli Nissle1917 knockout restriction endonuclease gene mutant
a. Mouse breast cancer 4T1 cells were cultured to logarithmic growth phase using RPMI 1640 medium containing 10% serum, 100U/mL penicillin and 100. mu.g/mL streptomycin in an incubator at 37 ℃ containing 5% carbon dioxide. Cells were trypsinized, washed 2 times with PBS, and resuspended in PBS. Balb/c female mice, 8 weeks old, were harvested, and the hair, each axillary, was injected subcutaneously with 100. mu.L of cell suspension.
And b, after the tumors are formed, randomly grouping the tumor-bearing mice, and each group comprises 5-6 mice.
c, selecting single clones of EcN mutant strain EcN-DR004, EcN-DR005, EcN-DR006 and wild type EcN strain which knock out restriction enzyme, and culturing in liquid LB culture medium for 37 ℃ overnight. The next day, the bacterial solution was transferred and grown to logarithmic phase. Centrifuging to collect thallus, washing thallus with sterile water, centrifuging, discarding supernatant, resuspending the thallus with sterile PBS, and diluting to 2 × 108CFU/mL of bacterial liquid.
d, injecting 100 mu L of bacteria solution into the tail vein of each mouse according to groups.
e, after 12h,24h,48h,72h of caudal iv injection of the bacteria, respectively, tumor size was measured, mice were sacrificed, tumors were detached and weighed. Washing tumor tissue with normal saline, shearing, grinding, and homogenizing to obtain tumor tissue homogenate.
f, diluting the tumor tissue fluid with sterile PBS according to a gradient of 10 times, and taking 10-5,10-6,10-7Gradient concentrations were plated on LB plates and counted after overnight incubation. The bacterial concentration (CFU/g) in the tumor tissue was calculated.
The tumor colonization ability of E.coli Nissle1917 mutant strain knocked out for restriction enzyme is shown in FIG. 8 and Table 4. The mutant strain with the knocked-out restriction enzyme still has tumor-targeted colonization capacity, but the tumor-targeted colonization quantity is slightly reduced compared with the wild type.
Table 4: tumor colonization effect of escherichia coli Nissle1917 mutant strain knocked out of restriction enzyme in tumor-bearing mouse model
7. Construction of restriction enzyme gene-knocked-out Escherichia coli Nissle1917 mutant strain with visualization function
On the basis of EcN-DR004, EcN-DR005 and EcN-DR006, respectively, the bioluminescent element gene is inserted into the restriction enzyme-deleted mutant strain according to the method described in 2, and the tumor-targeted bacteria with the visualization function is obtained.
8. Visual evaluation and evaluation method of Escherichia coli Nissle1917 mutant strain with restriction endonuclease gene knock-out function
Evaluation of bioluminescence activity and bioluminescence imaging test in a small animal in vivo model can be performed on the E.coli Nissle1917 mutant strain with a restriction enzyme gene knock-out function with visualization according to the methods described in 3 and 4.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
SEQUENCE LISTING
<110> Shandong university
<120> tumor-targeted bacteria with visualization function, and visualization method and application thereof
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1653
<212> DNA
<213> Fluc Gene sequence
<400> 1
atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatccgct agaggatgga 60
accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120
gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 180
gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240
tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300
gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 360
tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420
aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 480
tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540
tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa ctcctctgga 600
tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 660
catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720
gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780
cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 840
aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg 900
attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc acctctttcg 960
aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat 1020
gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080
gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140
acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1200
tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260
ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg cttgaagtct 1320
ttaattaaat acaaaggata ccaggtggcc cccgctgaat tggagtcgat attgttacaa 1380
caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt 1440
cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500
tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560
gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620
aaggccaaga agggcggaaa gtccaaattg taa 1653
<210> 2
<211> 927
<212> DNA
<213> LRE Gene sequence
<400> 2
atggggccag ttgttgaaaa aattgcagaa cttggcaagt atacggttgg agaaggtcct 60
cactgggatc atgaaactca gaccttatat ttcgtcgaca ccgtagagaa aacttttcat 120
aaatatgtac cttctcagaa aaaatacacg ttttgtaaag tagataaact ggtttcattc 180
attattcccc ttgctggatc ccctggccgt tttgtagtca gcttggaacg tgaaatagcc 240
attcttacgt gggatggcgt tagtgctgca cctacgagca tagaagctat tgttaatgtc 300
gaaccccaca ttaaaaataa cagactcaat gatggcaaag cagatcctct tggcaatcta 360
tggacaggta caatggctat tgacgctggt ctccccatag gaccggtcac tggcagttta 420
tatcatttag gggctgataa aaaggtaaaa atgcacgaga gcaacatagc tatagcaaat 480
gggctcgcgt ggagtaatga tttgaagaaa atgtattata ttgattcggg aaaaagaaga 540
gtagacgagt acgattatga tgcttctaca ttatccatca gcaatcaacg gccattattt 600
acttttgaaa agcatgaagt gcctggatat ccagatggtc aaacaattga tgaggagggt 660
aatttatggg ttgccgtttt ccaaggacag cgaattatta aaatcagtac ccaacaaccg 720
gaagtgttac tggataccgt aaaaatacca gatcctcagg tcacctctgt tgcatttggc 780
gggccgaatt tggatgaact gtatgtaaca tctgctggtc ttcagcttga cgacagttct 840
tttgacaaaa gtttagttaa tgggcacgtc tacagagtaa cgggtttagg cgtcaaaggt 900
ttcgcgggag ttaaagtgaa gctataa 927
<210> 3
<211> 720
<212> DNA
<213> EGFP Gene sequence
<400> 3
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 4
<211> 708
<212> DNA
<213> mCherry Gene sequence
<400> 4
gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg 60
cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc 120
ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc 180
gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc 240
gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg 300
atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc 360
gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg 420
cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc 480
ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag 540
gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac 600
atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc 660
gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaa 708
Claims (10)
1. A gene engineering bacterium is characterized in that the gene engineering bacterium is constructed by inserting a bioluminescence related functional gene into a wild type escherichia coli or an escherichia coli mutant strain genome with a restriction endonuclease gene knocked out;
wherein the bioluminescence-related functional gene comprises a luciferase gene.
2. The genetically engineered bacterium of claim 1, wherein the insertion site is downstream of the E.coli glucosamine synthetase gene.
3. The genetically engineered bacterium of claim 1, wherein the starting strain of Escherichia coli is Escherichia coli Nissle 1917.
4. The genetically engineered bacterium of claim 1, wherein the luciferases comprise firefly luciferase, secreted membrane-anchored luciferase and renilla luciferase, and mutants thereof;
preferably, the luciferase is selected from any one or more of the group consisting of:
5. the genetically engineered bacterium of claim 1, wherein the restriction endonuclease Gene comprises Gene1-Gene 7.
6. The genetically engineered bacterium of claim 1, wherein the bioluminescence-related functional genes comprise the firefly luciferase and mutant genes thereof and substrate regenerator genes.
7. The method for constructing a genetically engineered bacterium according to any one of claims 1 to 6, wherein the method comprises:
constructing a bioluminescent element plasmid, and recombining a target gene element with a bioluminescent function into an escherichia coli genome for expression to obtain the recombinant escherichia coli.
8. The method of claim 7, wherein the target gene element having bioluminescence function comprises a luciferase gene element;
the luciferase gene element comprises firefly luciferase and a mutant-substrate regeneration enzyme tandem gene element thereof, a secretory membrane anchored luciferase and a mutant gene element thereof, and a renilla luciferase and a mutant gene element thereof;
preferably, the firefly luciferase and the mutant-substrate regeneration enzyme tandem gene element plasmid thereof are constructed by a method comprising:
fusing the firefly luciferase and mutant genes with an EGFP label gene, fusing a substrate regeneration enzyme (LRE) gene with an mCherry gene, and assembling the firefly luciferase and mutant genes with a pMB1 replicon and a constitutive lac promoter to construct a firefly expression plasmid;
preferably, the escherichia coli is escherichia coli Nissle 1917;
preferably, the Escherichia coli Nissle1917 is wild type Escherichia coli Nissle1917 or Escherichia coli Nissle1917 with the restriction endonuclease gene knocked out;
preferably, the "recombining the target gene element into the genome of escherichia coli" is to recombine the element gene into the downstream of glm S gene in the genome of escherichia coli.
9. The use of the genetically engineered bacteria of any one of claims 1 to 6 in any one or more of:
a) imaging tracing and/or preparing an imaging tracing product;
b) detecting tumors and/or preparing tumor detection products;
c) tumor-targeted therapy and/or tumor-targeted therapy products;
d) tumor targeted therapy preclinical studies.
10. A method for visually detecting a tumor, comprising administering the genetically engineered bacterium of any one of claims 1 to 6 to a subject.
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