CN118126918A - Attenuated salmonella recombinant engineering bacteria expressing IFN beta, preparation method and application thereof - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses an attenuated salmonella recombinant engineering bacterium for expressing IFN beta, a preparation method and application thereof, belonging to the technical field of microorganism. The invention proves that the attenuated salmonella recombinant engineering bacteria expressing IFN beta can inhibit the metastasis and growth of solid tumors and inhibit the development of tumors. The invention constructs recombinant engineering bacteria carrying IFN beta expression plasmid based on attenuated salmonella typhimurium VNP 20009. The attenuated salmonella recombinant engineering bacteria expressing the IFN beta can be planted in a tumor hypoxia area and continuously secrete the IFN beta, so that the growth of the tumor is effectively inhibited, the survival period of a tumor-bearing mouse is prolonged, the generation of lung metastasis focus is inhibited, the preparation is simple, and the economic cost and the side effect of the IFN beta can be reduced by single administration.
Description
Technical Field
The invention relates to the technical field of microorganism, in particular to attenuated salmonella recombinant engineering bacteria for expressing IFN beta, a preparation method and application thereof.
Background
Tumor Microenvironment (TME) is an integral part of cancer, a complex ecosystem that impairs immune surveillance while supporting tumor growth and metastasis spread (Ann Oncol 2016,27 (8), 1482-1492.). Scientists have conducted many studies in remodeling tumor microenvironments to treat cancer, including targeting tumor infiltrating T cells through immune checkpoint blockade (Science 2015,348 (6230), 56-61.), normalizing blood vessels through anti-angiogenic therapy (NAT REV CLIN Oncol 2018,15 (5), 325-340), and reprogramming Tumor Associated Macrophages (TAMs) to restore anti-tumor properties (Nat Rev Drug Discov 2018,17 (12), 887-904). Neutrophils are the first line of defense in innate immunity, an important component of the tumor microenvironment, and are actively involved in tumor progression and metastasis (Pharmacol thier 2021,221,107753.). Tumor-associated neutrophils (TANs) have been reported to have an effect of promoting tumor growth in genetically engineered mouse cancer models (GEMMs) (Nat Commun 2017,8,14381.) but it also participates in antitumor processes by interacting with other immune cells or directly killing tumor cells (NAT REV CANCER 2020,20 (9), 485-503.).
As with TAMs, TANs can also be polarized to an anti-tumor (N1) or a pro-tumor (N2) phenotype (CANCER CELL 2009,16 (3), 183-94.). N1 neutrophils can increase immune recognition of cancer cells by progressive upregulation of neutrophil-dependent self-antigens (CANCER RES 2012,72 (13), 3153-3162.) thereby inhibiting tumor growth and lung metastasis production by H2O2 production (CANCER CELL 2011,20 (3), 300-314.). Mice with impaired type I IFN signaling (Ifnar-/-) will develop more lung metastases in the 4T1 breast Cancer and LLC lung Cancer models than control mice (Int J Cancer 2015,137 (4), 837-847.). In conclusion, IFN beta has great anti-tumor potential. As a protein drug, clinical IFN beta administration usually adopts subcutaneous injection, which can avoid first pass effect and improve bioavailability. However, single-dose drug loading is low, and it is difficult to maintain a therapeutic window for a long period of time (Biomaterials 2022,287,121619). In addition, since protein drugs are easily degraded, many strategies have been proposed to release protein drugs slowly, such as by introducing organic carriers or agarose water gels to retard Drug degradation (Adv Drug Deliv rev2017,112,106-122.Adv Funct Mater 2010,20 (2), 247-258).
Engineering bacteria are of great interest as tumor targeted drug delivery vehicles in cancer treatment because of their ability to colonize solid tumors and carry therapeutic genes, such as PD-L1 nanobody (ACTA PHARM SIN B2022,12 (10), 3952-3971) or TNF- α nanobody (Signal Transduct TARGET THER 2023,8 (1), 134). Attenuated salmonella typhimurium VNP20009 (VNP) reduces immune activation and improves targeting by deleting purI and msbB genes (Cancer Biol Ther 2005,4 (8), 840-845.) is one of the most potential strains at present. Furthermore, VNP has been widely used for drug delivery as the only strain of salmonella evaluated by clinical trials, including carrying plasmids expressing therapeutic proteins (such as anti-PD 1 nanobody or interferon-gamma) (Eur J Cancer 2017,70,48-61), cytotoxic proteins (CANCER SCI 2012,103 (2), 325-333), cytokines or immunomodulators (Mol ter 2013,21 (11), 1985-1995), tumor vaccines (J Biomed Sci 2013,20 (1), 8) or shRNA against oncogenes (CANCER RES 2007,67 (12), 5859-5864).
The present invention reports a recombinant attenuated salmonella strain expressing an interferon-beta (IFN beta) protein. The strain can target solid tumors and inhibit tumor growth, and meanwhile, certain biological safety is maintained.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide an attenuated salmonella recombinant engineering bacterium for expressing IFN beta, and a preparation method and application thereof. Can be used for tumor treatment, and can inhibit lung metastasis of solid tumor including melanoma, inhibit the progress of transplanted tumor such as subcutaneous melanin, and prolong survival time of tumor-bearing mice such as melanoma.
In order to achieve the above purpose, the present application adopts the following technical scheme: in a first aspect, the application provides an attenuated salmonella recombinant engineered strain expressing IFN beta;
in a second aspect, the application provides an attenuated salmonella recombinant engineering bacterial agent for expressing IFN beta prepared by an attenuated salmonella recombinant engineering bacterial for expressing IFN beta;
In a third aspect, the present application provides a pharmaceutical composition;
In a fourth aspect, the application provides an attenuated salmonella recombinant engineering bacterium expressing IFNbeta for use in the manufacture of a medicament for preventing or treating a tumor or/and cancer.
The attenuated salmonella recombinant engineering bacterium for expressing the IFN beta can be targeted and planted in a core area of solid tumor including melanin and continuously secrete the IFN beta; the expression plasmid of IFN beta contains J23100 constitutive promoter and PelB extracellular protein secretion signal peptide and Flag label protein, and the coding sequence of IFN beta and its expression element nucleotide sequence are shown in SEQ ID NO. 1.
The invention relates to an attenuated salmonella recombinant engineering bacterial agent for expressing IFN beta, which is prepared by the attenuated salmonella recombinant engineering bacterial for expressing IFN beta.
Further, the active ingredients are at least one of the following (a), (b), (c) and (d):
(a) The attenuated salmonella recombinant engineered bacterial cell of claim 1;
(b) The ultrasonically disrupted precipitate of the attenuated salmonella recombinant engineering bacterium of claim 1;
(c) A fermentation broth primary extract of the attenuated salmonella recombinant engineering bacteria culture of claim 1;
(d) An ultrasonic lysate of the attenuated salmonella recombinant engineering bacteria cells obtained in claim 1.
The preparation method of the attenuated salmonella recombinant engineering bacteria for expressing IFN beta comprises the following steps:
(1) PCR or synthesis of IFN beta gene sequence, the primer sequence is shown as SEQ ID NO. 2-3;
(2) Constructing IFN beta expression plasmid, firstly, designing homologous recombination primer; secondly, obtaining IFN beta gene fragments by PCR or synthesis; thirdly, the connection production after homologous recombination is transferred into chemically competent cells of escherichia coli DH5 alpha;
(3) Constructing attenuated salmonella recombinant engineering bacteria expressing IFN beta: extracting and constructing a successful IFN beta expression plasmid from escherichia coli, and converting the IFN beta expression plasmid into attenuated salmonella typhi VNP20009 or a derivative strain thereof by an electrotransformation method to prepare attenuated salmonella recombinant engineering bacteria for expressing the IFN beta; the attenuated salmonella typhi VNP20009 and its derived or genetically engineered strains include, but are not limited to, recombinant engineered strains carrying an ifnβ expression plasmid by each of the strains (ZL201410209851.7,ZL201610946268.3,ZL201610945015.4,ZL201610945021.X,202010182038.0;Acta Pharmaceutica Sinica B 2021,11(10):31653177;phoP/phoQ), of the previously described invention.
Further, the expression plasmid of IFN beta can be stably inherited.
The attenuated salmonella recombinant engineering bacteria expressing the IFN beta can inhibit the generation of lung metastasis focus of solid tumors including melanoma or inhibit the progress of subcutaneous transplantation tumors including melanin or prolong the survival time of tumor-bearing mice of the solid tumors including melanoma.
Further, the preparation of the attenuated salmonella recombinant engineering bacteria for expressing the IFN beta comprises oral preparation, injection preparation, intravenous preparation or spray preparation.
Furthermore, the attenuated salmonella recombinant engineering bacteria expressing IFN beta provided by the invention are combined with other antitumor drugs to be applied to the preparation of antitumor drugs.
The beneficial effects are that: the invention provides a high tumor targeting, safe, convenient, safe, low-cost and novel delivery mode of IFN beta, namely the attenuated salmonella targeted by tumor is used for continuously secreting the IFN beta in a tumor area, and can effectively inhibit lung metastasis of solid tumors including melanoma and growth of solid tumors including melanoma, thereby showing good tumor treatment prospect.
Compared with the prior art, the invention has the following advantages:
(1) The invention proves that the IFN beta remodelling immune microenvironment is an effective strategy for realizing tumor treatment; the invention uses the attenuated salmonella VNP20009 or the derived strain thereof to load the expression plasmid of the IFN beta for the first time, namely the attenuated salmonella recombinant engineering bacteria of the IFN beta, which has similar tumor colonization rate compared with the wild type VNP;
(2) The attenuated salmonella recombinant engineering bacteria for expressing the IFN beta can inhibit the generation of lung metastasis focus of solid tumors including melanoma and inhibit the progress of subcutaneous solid tumor transplantation tumor including melanoma, and has good treatment prospect;
(3) The attenuated salmonella recombinant engineering bacterium of the IFN beta can stably and continuously secrete the IFN beta in a tumor area, can realize that single administration has good therapeutic capability, and describes a more convenient and cheaper administration mode of the IFN beta.
(4) The invention constructs an attenuated salmonella recombinant engineering bacterial strain capable of being fixedly planted in a tumor core area to stably and continuously secrete IFN beta, and the bacterial strain shows potential of anti-tumor treatment; the invention provides a strategy for IFN beta tumor targeted delivery, which realizes the convenient and low-cost delivery of protein drugs to the tumor environment.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments or the description of the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the elements of a J23100-IFN- β plasmid of the present invention;
FIG. 2 is a diagram showing the detection of IFN beta expression by recombinant engineering bacteria of Salmonella expressing IFN beta in vitro by immunoblotting of the present invention;
FIG. 3 is a graph showing the results of lung metastasis nodules in a model mouse for lung metastasis of melanoma after treatment with recombinant engineering bacteria of Salmonella expressing IFNbeta according to the present invention;
FIG. 4 is a graph showing changes in body weight of mice in a model of lung metastasis of melanoma after treatment with recombinant engineering bacteria of Salmonella expressing IFNbeta according to the present invention;
FIG. 5 shows the results of serological analysis of a model mouse for lung metastasis of melanoma after treatment with recombinant engineering bacteria of Salmonella expressing IFNbeta of the present invention;
FIG. 6 is a graph showing the ratio of organ weight to body weight of a model mouse for lung metastasis of melanoma after treatment with recombinant engineering bacteria of Salmonella expressing IFNbeta according to the present invention;
FIG. 7 is a graph showing tumor growth in mice model for melanoma solid tumors treated with recombinant engineering bacteria expressing IFNbeta of the present invention;
FIG. 8 is a graph showing tumor doubling time of mice with melanoma solid tumor models treated with recombinant engineering bacteria expressing IFN beta;
FIG. 9 is a graph showing survival time of mice with melanoma solid tumor models treated with recombinant engineering bacteria expressing IFN beta;
FIG. 10 is a graph showing the results of tissue bacterial titer in mice model melanoma solid tumors by recombinant engineering bacteria expressing IFNbeta of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.
It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The weights of the relevant components mentioned in the description of the embodiments of the present application may refer not only to the specific contents of the components, but also to the proportional relationship between the weights of the components, so long as the contents of the relevant components in the description of the embodiments of the present application are scaled up or down within the scope of the disclosure of the embodiments of the present application. Specifically, the mass in the specification of the embodiment of the application can be a mass unit which is known in the chemical industry field such as mu g, mg, g, kg and the like.
The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
An embodiment of the present application provides an attenuated salmonella recombinant engineering bacterium expressing an ifnβ, where the attenuated salmonella recombinant engineering bacterium is an attenuated salmonella typhi VNP20009 and a derivative or genetically modified strain thereof, including but not limited to the attenuated salmonella recombinant engineering bacterium expressing an ifnβ described in the foregoing strains (ZL201410209851.7,ZL201610946268.3,ZL201610945015.4,ZL201610945021.X,202010182038.0;Acta Pharmaceutica Sinica B 2021,11(10):31653177;phoP/phoQ); of the application of which an application has been filed, which can be targeted for implantation in a melanoma core area and continuously secrete an ifnβ; the expression plasmid of IFN beta contains J23100 constitutive promoter and PelB extracellular protein secretion signal peptide and Flag tag protein, and the nucleotide sequence of the expression plasmid of IFN beta is shown as SEQ ID NO. 1.
The second aspect of the embodiment of the application provides an attenuated salmonella recombinant engineering bacterial agent for expressing IFN beta, which is prepared by the attenuated salmonella recombinant engineering bacterial for expressing IFN beta. The active ingredients are at least one of the following (a), (b), (c) and (d):
(a) The attenuated salmonella recombinant engineered bacterial cell of claim 1;
(b) The ultrasonically disrupted precipitate of the attenuated salmonella recombinant engineering bacterium of claim 1;
(c) A fermentation broth primary extract of the attenuated salmonella recombinant engineering bacteria culture of claim 1;
(d) An ultrasonic lysate of the attenuated salmonella recombinant engineering bacteria cells obtained in claim 1.
The third aspect of the embodiment of the application provides a preparation method of attenuated salmonella recombinant engineering bacteria for expressing IFN beta, comprising the following steps:
(1) PCR or synthesis of IFN beta gene sequence, the primer sequence is shown as SEQ ID NO. 2-3;
(2) Constructing IFN beta expression plasmid, firstly, designing homologous recombination primer; secondly, obtaining IFN beta gene fragments by PCR or synthesis; thirdly, the connection production after homologous recombination is transferred into chemically competent cells of escherichia coli DH5 alpha; the expression plasmid of IFN beta can be inherited stably.
(3) Constructing attenuated salmonella recombinant engineering bacteria expressing IFN beta: the IFN beta expression plasmid successfully constructed is extracted from the escherichia coli and is transformed into attenuated salmonella typhi VNP20009 by an electrotransformation method, and the attenuated salmonella recombinant engineering bacteria for expressing the IFN beta are prepared.
The attenuated salmonella recombinant engineering bacteria expressing the IFN beta can inhibit the generation of lung metastasis focus of melanoma or inhibit the progress of subcutaneous melanin transplantation tumor or prolong the survival time of melanoma tumor-bearing mice.
In some embodiments, the formulation of an attenuated salmonella recombinant engineering bacterium that expresses IFN beta comprises an oral formulation.
In some embodiments, the formulation of an attenuated salmonella recombinant engineering bacterium that expresses IFN beta comprises an injectable formulation.
In some embodiments, the preparation of an attenuated salmonella recombinant engineering bacterium that expresses IFN beta comprises a intravenous preparation.
In some embodiments, the formulation of an attenuated salmonella recombinant engineering bacterium that expresses IFN beta comprises a spray formulation.
The invention relates to an application of attenuated salmonella recombinant engineering bacteria expressing IFN beta in combination with other antitumor drugs in preparing antitumor drugs.
The present invention has been developed in the attenuated salmonella typhi VNP20009 and its various derived or genetically modified strains, including but not limited to the strains (ZL201410209851.7,ZL201610946268.3,ZL201610945015.4,ZL201610945021.X,202010182038.0;Acta Pharmaceutica Sinica B 2021,11(10):31653177;phoP/phoQ) of the previously filed invention, in tumor models including melanoma, breast cancer, lung cancer, colon cancer, lymphoma, etc., and similar results have been obtained, and embodiments of the present invention will focus on the VNP20009 strain and the melanoma model.
Example 1
Construction of attenuated salmonella recombinant engineering bacteria expressing IFN beta
Obtaining the target fragment: the expression plasmid of the IFN beta is obtained by adding other elements on the basis of pTh01 for transformation, a synthetic primer is amplified by polymerase chain reaction to obtain a plasmid skeleton, and a target fragment is recovered by 1% agarose gel electrophoresis gel; amplifying the target fragment of IFN beta through a polymerase chain reaction by using a synthetic primer, recovering the target fragment through a 1% agarose gel electrophoresis gel, and connecting the target fragment to a plasmid vector through homologous recombination; amplifying to obtain J23100 constitutive promoter, flag tag protein and PelB protein secretion signal peptide, recovering target fragment through 1% agarose gel electrophoresis gel, and connecting the target fragment to a plasmid vector through homologous recombination;
Granulating and turning: 10. Mu.L of the ligation product was chemically transformed into E.coli DH 5. Alpha. Chemically competent cells under the following conditions: placing on ice for 20 min, heat-shocking at 42 ℃ for 60 seconds, placing on ice for 2 min, adding 900 mu L of non-resistant LB liquid culture medium, shake-culturing at 37 ℃ for 45min, centrifuging at 5000rpm for 3 min to remove supernatant, adding 100 mu L of non-resistant LB liquid culture medium, mixing uniformly, coating on a resistant plate with kanamycin, and standing overnight at 37 ℃; four monoclonal colonies are picked into 1mL LB containing kanamycin, shake culture is carried out for 12 hours at 37 ℃, recombinant plasmids are obtained through a plasmid small extraction kit, verification positive strips are obtained through polymerase chain reaction amplification by using verification primers, the positive strip base sequences are detected by sequencing, and the recombinant plasmids with correct sequences are obtained after comparison, wherein a plasmid skeleton diagram is shown in figure 1;
Plasmid electrotransformation: preparation of wild type VNP20009 (abbreviated as VNP) competent cells: inoculating activated VNP into 10mL LB, shaking culture at 37 ℃ until OD600 value is 0.2-0.6, centrifuging at 5000rpm and 4 ℃ for 5min to collect thalli, washing thalli 3 times with 10mL 10% glycerol, centrifuging at 5000rpm and 4 ℃ for 5min to collect thalli, re-suspending with 100 μl 10% glycerol, and sub-packaging with 50 μl/tube for electrotransformation; the positive plasmid described above was electroporated into VNP competence by electroporation: all procedures were performed under sterile conditions, 1 μg of positive plasmid was added at electrotransformation competence and transferred to a 2mm electrotransformation cup under the following conditions: the voltage is 1800-2500V, the resistance is 400-500 omega, the capacitance is 25 mu F, and the discharge time is 5ms; the electrotransfer product was plated on plates, the incubator was allowed to stand overnight at 37℃and colonies were picked for sequencing verification.
Example 2
Characterization of secretion of attenuated salmonella recombinant engineering bacteria protein expressing IFN beta
Aiming at the protein secretion situation characterization of attenuated salmonella recombinant engineering bacteria expressing IFN beta, selecting 3 positive strains, shaking overnight in 500 mu l of LB culture medium containing the resistance of the calicheamicin, inoculating 400 mu l of bacterial liquid, shaking in 40ml of LB containing the resistance of the calicheamicin, until the OD600 reaches 0.4-0.6, centrifuging at 5000rpm and 4 ℃ for 30 minutes, and collecting bacterial precipitate;
and (3) bacterial precipitation treatment: the pellet was resuspended in 1ml PBS and the cells were sonicated to release the protein. And then 13000rpm,4 ℃, centrifuging for 10 minutes, and collecting the supernatant, wherein the supernatant is the total protein in the precipitate thalli.
The protein concentration of the extracted total protein was measured using a BSA kit and adjusted to the same concentration. The same concentration of protein was added to 5X SDS Loading Buffer and heated at 100℃for 10 minutes using a metal bath. 15mg of protein was aspirated for immunoblotting. The status of the engineering strain expressing IFNbeta was detected using Flag tag antibodies (FIG. 2). The results show that the positive strains can effectively express and secrete IFN beta.
Test example 1
Identification of lung metastasis treatment effect of attenuated salmonella recombinant engineering bacterium melanoma for expressing IFN beta
Constructing a melanoma lung metastasis model: the B16F10 mouse melanoma cells were cultured in DMEM cell culture medium to an exponential growth phase, then digested with pancreatin at 1000rpm and 4℃for 3 minutes, collected by centrifugation, washed 2 times with PBS, and then subjected to cell counting, and finally used at a final cell concentration of 1X 10 6 cells/mL. Experiments were performed 3 days after tumor cell injection by 1X 10 5/mouse were inoculated intravenously at the tail of each C57BL/6 mouse.
Tumor bearing mice were grouped and bacteria were dosed: melanoma-bearing mice from B16F10 mice were randomly divided into 3 groups and were intraperitoneally injected with 1X 10 6 CFU bacteria.
Recording the weight change of the mice after administration every two days; mice were sacrificed and dissected when PBS group mice lost weight, while mouse serum was taken for analysis, and the number of melanin metastases on the lungs of mice was recorded. The test results show that the attenuated salmonella recombinant engineering bacteria expressing the IFN beta have better effect of inhibiting the lung metastasis of melanoma compared with wild type VNP (figure 3). Compared with VNP, the number of lung tumor metastasis is reduced by about 3 times after administration of attenuated salmonella recombinant engineering bacteria expressing IFN beta.
Test example 2
Identification of treatment effect of attenuated salmonella recombinant engineering bacterium melanoma solid tumor expressing IFN beta
Constructing a subcutaneous melanoma tumor model: the B16F10 mouse melanoma cells were cultured in DMEM cell culture medium to an exponential growth phase, then digested with pancreatin at 1000rpm and 4℃for 3 minutes, collected by centrifugation, washed 2 times with PBS, and then subjected to cell counting, and finally used at a final cell concentration of 1X 10 6 cells/mL. Experiments were performed when 1X 10 5 mice/mouse were vaccinated with each C57BL/6 mouse and the tumor volume of the mice was grown to about 100mm 3.
Tumor bearing mice were grouped and bacteria were dosed: melanoma-bearing mice from B16F10 mice were randomly divided into 3 groups and were intraperitoneally injected with 1X 10 6 CFU bacteria.
To assess the safety of engineering bacteria, normal mouse body weights were recorded every two days (fig. 4), mice were sacrificed five days after dosing, organs were taken and weighed to observe changes in organs (fig. 5), and mice were serum tested for biochemical indicators (fig. 6). The experimental result proves that the attenuated salmonella recombinant engineering bacteria expressing IFN beta has better safety.
Recording the weight of the mice after administration every two days, and measuring the length and width of subcutaneous melanoma by using a vernier caliper to count the tumor growth curve and the weight change condition of the mice; mice were sacrificed when their tumors grew to 2000mm 3 and the death was recorded to count the tumor-bearing mice survival curve. The experimental results show that the attenuated salmonella recombinant engineering bacteria expressing the IFN beta have better tumor treatment effect and better capability of prolonging the survival time of tumor-bearing mice compared with wild-type VNP (figures 7-9). Compared with VNP, the tumor inhibition rate of the attenuated salmonella recombinant engineering bacteria expressing the IFN beta is improved from 65.65% to 93.11% after administration (figure 7), the tumor doubling time is prolonged from 1.85 days without administration to 4.6 days after administration of the VNP (figure 8); whereas the survival time of tumor-bearing mice was prolonged from 75% to 95% (fig. 9). The data above all demonstrate that attenuated salmonella recombinant engineering bacteria expressing IFNbeta can significantly inhibit the progression of solid tumors including melanoma
Test example 3
Investigation of in vivo distribution of attenuated salmonella recombinant engineering bacterium tumor-bearing mice expressing IFN beta
Mice were sacrificed 5 days after administration, and individual organ tissues of tumor-bearing mice, including tumor, liver, spleen, kidney and heart, were collected in a sterile environment, weighed and placed in 2mL PBS for tissue homogenization to lyse cells and release bacteria. According to the difference of bacterial titer in tissues, the proper bacterial liquid is obtained according to different dilution gradients, the bacterial liquid is coated on an LB plate of the kanamycin, then the bacterial liquid is inversely cultured for 12 hours in a bacterial incubator at 37 ℃ and the colony count is carried out, so that the distribution situation of the wild-type VNP and the attenuated salmonella recombinant engineering bacteria expressing IFN beta in the tissue of tumor-bearing mice is compared and analyzed (figure 10).
In vivo distribution, except for tumors, most bacteria are distributed in the liver and spleen, so the invention focuses on the enrichment degree of the modified strain in the tumors and the liver and spleen, and simultaneously analyzes the tumor targeting of the modified strain. Experimental results show that compared with wild-type VNP, the attenuated salmonella recombinant engineering bacteria expressing IFN beta have almost unchanged bacterial titer in tumors, livers and spleens, namely no obvious difference; however, the titer of the modified strain bacteria in the lung and the heart is obviously lower than that of the wild-type VNP, and the result shows that the attenuated salmonella recombinant engineering bacteria expressing the IFN beta has better tumor targeting.
In conclusion, the attenuated salmonella recombinant engineering bacterium for expressing the IFN beta is constructed by taking the attenuated salmonella VNP20009 as a basic strain and combining a microbial synthesis biological technology, can be planted in a tumor core area, stably and continuously express and secrete the IFN beta, and promote the large-area necrosis of tumor tissues, so that good tumor treatment effect and tumor targeting are shown. The invention also describes a high efficiency, high tumor targeting, convenient, inexpensive delivery strategy for IFN beta. Bacteria are used as gene therapy vectors, and a good foundation is laid for carrying more effective genes to treat tumors.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, the scope of which is defined in the appended claims, specification and their equivalents.
Claims (8)
1. An attenuated salmonella recombinant engineering bacterium expressing IFN beta, characterized in that: the attenuated salmonella recombinant engineering bacteria expressing the IFN beta can be targeted and planted in a solid tumor core area and continuously secrete the IFN beta; the expression plasmid of the IFN beta comprises J23100 constitutive promoter, pelB extracellular protein secretion signal peptide and Flag tag protein, and the coding sequence of the IFN beta and the nucleotide sequence of the expression element thereof are shown as SEQ ID NO. 1; the attenuated salmonella recombinant engineering bacteria are attenuated salmonella recombinant engineering bacteria which express IFN beta and are derived or genetically modified based on attenuated salmonella typhimurium VNP 20009.
2. The recombinant engineering bacteria of attenuated salmonella expressing ifnβ prepared by the recombinant engineering bacteria of attenuated salmonella expressing ifnβ of claim 1.
3. The recombinant engineering bacteria agent of attenuated salmonella expressing IFN beta according to claim 2, wherein the active ingredient is at least one of the following (a) (b) (c) (d):
(a) The attenuated salmonella recombinant engineered bacterial cell of claim 1;
(b) The ultrasonically disrupted precipitate of the attenuated salmonella recombinant engineering bacterium of claim 1;
(c) A fermentation broth primary extract of the attenuated salmonella recombinant engineering bacteria culture of claim 1;
(d) An ultrasonic lysate of the attenuated salmonella recombinant engineering bacteria cells obtained in claim 1.
4. The method for preparing the attenuated salmonella recombinant engineering bacteria expressing the IFN beta as claimed in claim 1, which is characterized by comprising the following steps:
(1) PCR or synthesis of IFN beta gene sequence, the primer sequence is shown as SEQ ID NO. 2-3;
(2) Constructing IFN beta expression plasmid, firstly, designing homologous recombination primer; secondly, obtaining IFN beta gene fragments by PCR or synthesis; thirdly, the connection production after homologous recombination is transferred into chemically competent cells of escherichia coli DH5 alpha;
(3) Constructing attenuated salmonella recombinant engineering bacteria expressing IFN beta: the IFN beta expression plasmid successfully constructed is extracted from the escherichia coli and is transformed into attenuated salmonella typhi VNP20009 by an electrotransformation method, and the attenuated salmonella recombinant engineering bacteria for expressing the IFN beta are prepared.
5. The recombinant engineering bacterium of attenuated salmonella expressing IFN beta according to claim 4, wherein: the expression plasmid of IFN beta can be inherited stably.
6. The use of the recombinant engineering bacteria of attenuated salmonella expressing IFN beta according to claim 1 in the preparation of an antitumor agent, wherein: the recombinant engineering bacteria of attenuated salmonella expressing IFN beta can inhibit the generation of lung metastasis focus of melanoma, inhibit the progress of subcutaneous melanin transplantation tumor or prolong the survival time of melanoma tumor-bearing mice.
7. The use according to claim 6, characterized in that: formulations of attenuated salmonella recombinant engineering bacteria expressing IFN beta include oral formulations, injectable formulations, intravenous formulations or spray formulations.
8. The use of the recombinant engineering bacteria of attenuated salmonella expressing IFN beta of claim 1 in combination with other antitumor drugs for the preparation of antitumor drugs.
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