CN114958698A - Attenuated salmonella recombinant engineering bacterium for expressing anti-PD 1 nano antibody and preparation method and application thereof - Google Patents

Attenuated salmonella recombinant engineering bacterium for expressing anti-PD 1 nano antibody and preparation method and application thereof Download PDF

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CN114958698A
CN114958698A CN202210182870.XA CN202210182870A CN114958698A CN 114958698 A CN114958698 A CN 114958698A CN 202210182870 A CN202210182870 A CN 202210182870A CN 114958698 A CN114958698 A CN 114958698A
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华子春
吴乐阳
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Targetpharma Laboratories Jiangsu Co ltd
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Abstract

The invention discloses an attenuated salmonella recombinant engineering bacterium for expressing an anti-PD 1 nano antibody and a preparation method and application thereof, wherein the attenuated salmonella recombinant engineering bacterium for expressing an anti-PD 1 nano antibody is attenuated salmonella typhimurium VNP20009 carrying an anti-PD 1 nano antibody expression plasmid and a genetically modified strain thereof, and the tumor targeting property of the attenuated salmonella recombinant engineering bacterium is not changed; cloning the anti-PD 1 nano antibody gene on an expression plasmid containing a promoter and a signal peptide: the promoter is a J23100 constitutive promoter or a NirB promoter or an adhE promoter; the plasmid contains an AT element that prevents plasmid loss. The attenuated salmonella VNP20009 recombinant engineering bacteria expressing the anti-PD 1 nano antibody has higher tumor targeting property and better anti-tumor curative effect, and the preparation method is simple, easy to operate and capable of realizing single administration.

Description

Attenuated salmonella recombinant engineering bacterium for expressing anti-PD 1 nano antibody and preparation method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to attenuated salmonella recombinant engineering bacteria for expressing an anti-PD 1 nano antibody, and a preparation method and application thereof.
Background
Immune checkpoint blockers are the hot spot in the development of current international antitumor drugs, and as exemplified by PD-1/PD-L1 antibody class inhibitors, PD-1 or PD-L1 inhibitors can relieve the immunosuppressive state of tumors by binding to the PD-1 receptor on the surface of T cells or blocking the binding of PD-L1 protein to cancer cells, thereby effectively inhibiting and alleviating the progression of tumors (Sharma et al, Science 2015, 348(6230): 56-61). Tumor Immune checkpoint inhibitor treatment regimens (ICI) also require a high degree of tumor infiltration by T cells (Sznol et al, Cancer J2014, 20(4): 290-. Many tumors lack infiltrating T cells and have a low response to ICI, resulting in inefficient clinical use of immune checkpoint blockers, such as PD-1/PD-L1 inhibitors, by only 10-30% (Sznol et al, Cancer J2014, 20(4): 290-. Therefore, how to induce more T cells to appear at the tumor site, the initiation of the T cell response of the tumor, is a key factor for immune checkpoint blocker therapy including PD-1/PD-L1 inhibitors. However, besides the disadvantage of large molecular weight and difficulty in penetrating tissues, PD1 monoclonal antibody still has two barriers in clinical practice, i.e. most patients (70-85%) do not respond to immunotherapy and a small proportion of patients respond to immunotherapy. One of the main reasons for the former is the potent immune cells within the tumor (e.g., CD 8) + T cells) are lower. The latter is because accumulation of an immunosuppressive agent in a normal organ or tissue induces the immune system to attack normal cells, and side effects due to off-target effects are increasingly manifested in clinical applications. In addition, due to the cyclic metabolism of the body, the antibody drugs usually require multiple injections to maintain the effective therapeutic concentration of the drugs in the body, which undoubtedly increases the operation complexity and the treatment cost, and also increases the pain of patients. These factors limit the clinical use of the medicinePopularization in bed applications. Therefore, how to improve the infiltration of immune cells in the tumor region and realize the high-efficiency enrichment of the immune blocking inhibitor in the tumor region has important challenge value and exploration significance for basic research and clinical application. The nano antibody is the smallest antibody molecule at present, and has the advantages of good stability, low cost, good water solubility, capability of penetrating through a blood brain barrier and the like.
Some obligate or facultative anaerobic bacteria are capable of specifically targeting tumor tissue, growing and multiplying in their hypoxic or necrotic areas, and exhibit an inhibitory effect on tumor cell growth. Salmonella is one such tumor-targeting bacterium. VNP20009 is Salmonella typhimurium by deletionmsbBAndpurIthe gene reduces toxicity (Luo X et al, Oncol Res 2001, 12(11-12): 501-508), passes phase I Clinical test of metastatic melanoma, has insufficient curative effect (Thamm DH et al, Clinical Cancer Research 2005, 11(13): 4827-4834), but has better safety. VNP20009 can be grown and propagated preferentially in tumor tissue to achieve a ratio of bacterial titer in tumor tissue to that in normal tissue of 1000-. The VNP20009 can be used for directly treating tumors and can be used as a gene therapy vector to carry some genes for effectively treating tumors.
Disclosure of Invention
The invention aims to provide an attenuated salmonella recombinant engineering bacterium for expressing an anti-PD 1 nano antibody, and a preparation method and application thereof.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: the attenuated salmonella recombinant engineering bacteria for expressing the anti-PD 1 nano antibody is attenuated salmonella typhimurium VNP20009 carrying anti-PD 1 nano antibody expression plasmids and a genetically modified strain thereof, and the tumor targeting is not changed; including but not limited to the strains (ZL 201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X, 202010182038.0; Acta pharmaceutical Sinica B2021, 11(10): 3165-. Compared with the theoretical value of the sum of the tumor fold growth time or the tumor delay time extension amplitude caused by two groups of curative effects after the salmonella attenuated and the anti-PD 1 nano antibody are treated independently, the tumor fold growth time extension amplitude of the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody is 4.78 times of the theoretical value of the sum of the two groups of curative effects, and the tumor delay time extension amplitude of the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody is 2.98 times of the theoretical value of the sum of the two groups of curative effects.
Cloning the anti-PD 1 nano antibody gene on an expression plasmid containing a promoter and a signal peptide: the promoter is a J23100 constitutive promoter or a NirB promoter or an adhE promoter; the plasmid contains an AT element that prevents plasmid loss.
Further, the signal peptide is a conventional bacterial signal peptide, and the bacterial signal peptide is a sseJ signal peptide, a MIS signal peptide, a Flic signal peptide, a pelB signal peptide, a SOPE signal peptide, a SpA signal peptide or an OmpA signal peptide.
The attenuated salmonella recombinant engineering bacterium agent for expressing an anti-PD 1 nano antibody is prepared from attenuated salmonella recombinant engineering bacterium for expressing an anti-PD 1 nano antibody.
Further, the active ingredient is at least one of the following (a), (b) and (c):
(a) the fermentation culture of the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody;
(b) ultrasonic lysis supernatant of the attenuated salmonella recombinant engineering bacteria cell expressing the anti-PD 1 nano antibody;
(c) and (3) carrying out ultrasonic lysis precipitation on the obtained attenuated salmonella recombinant engineering bacteria cell expressing the anti-PD 1 nano antibody.
The preparation method of the attenuated salmonella recombinant engineering bacteria for expressing the anti-PD 1 nano antibody comprises the following steps:
(1) obtaining PD1 nano antibody gene;
(2) constructing a plasmid expressing the PD1 nano antibody; step1 designing homologous recombination primer; step2 PCR to obtain DNA fragment; step3 transferring the product after connection into chemically competent cells of Escherichia coli DH5 alpha through chemical transformation;
(3) constructing attenuated salmonella recombinant engineering bacteria expressing anti-PD 1 nano antibody: and (3) electrically converting the expression plasmid into attenuated salmonella typhi VNP20009 and a mutant strain thereof to prepare the attenuated salmonella recombinant engineering bacteria for expressing the anti-PD 1 nano antibody.
Further, in step (1), a J23100 constitutive promoter or a NirB promoter or an adhE promoter is cloned on a pET32a plasmid respectively, a conventional bacterial signal peptide is fused AT the N end of the anti-PD 1 nano antibody, and the plasmid contains an AT element for preventing plasmid loss.
Further, in step (2), step1 designs homologous recombination primers; step2 respectively uses pET32a (+) -PD1Nb as template PCR to obtain target gene fragment, uses pTh01 as template PCR to obtain plasmid skeleton fragment, and uses 1.2% agarose gel electrophoresis to cut out gene product, and uses gel recovery purification kit to purify to obtain DNA fragment; recovering the product by connecting gel through a double-fragment homologous recombination kit; the connection conditions are as follows: 4-8 deg.C, 1-2 hr, or 37 deg.C, 30 min; step3 transferring 10 μ L of the ligation product to chemically competent cells of E.coli DH5 α by chemical transformation; transformation conditions are as follows: placing on ice for 10 min, thermally shocking at 42 ℃ for 60s, placing on ice for 2 min, adding 900 mu L of non-resistant LB liquid culture medium, performing shake culture at 37 ℃ for 45 min, centrifuging at 5000 rpm for 3min, discarding supernatant, adding 1 mL of non-resistant LB liquid culture medium, mixing uniformly, sucking 100 mu L of non-resistant LB liquid culture medium, coating on a resistant plate with kanamycin, and standing in an incubator at 37 ℃ overnight; step4, selecting a monoclonal antibody to be added into 3 mL LB culture solution containing kanamycin, carrying out shake culture at 37 ℃ for 16 h, and extracting recombinant plasmids by using a plasmid miniextraction kit; the plasmid was sent to sequencing company for sequencing and alignment to confirm the correct sequence.
Further, in the step (3), the plasmids pTh01 and the PD1 nano antibody expression plasmids are respectively electroporated into the electrocompetent cells of VNP20009 and the mutant strains thereof, so as to prepare the recombinant strain VNP20009 and the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody:
step1 preparation of Salmonella electrotransformation competence: inoculating fresh attenuated salmonella VNP20009 and mutant strains thereof into 30 mL LB culture medium, carrying out shake culture at 37 ℃ until OD value is 0.5-0.6, centrifuging at 4 ℃ for 5000 rpm, collecting thalli for 5 min, washing the thalli for 3 times by sterilized 10% glycerol, 10 mL each time, centrifuging at 4 ℃ for 5000 rpm for 5 min, resuspending by 200 mu L of 10% glycerol, subpackaging by 50 mu L/tube for electrotransfer;
step2 the recombinant plasmid is transformed into attenuated salmonella VNP20009 and mutant strains thereof by an electroporation method: under the aseptic condition, 0.5-5 mug of constructed plasmid is added into the electrotransformation competence, and is transferred into an electrotransfer cup with 2 mm after being mixed uniformly for electric shock, wherein the electrotransfer condition is as follows: voltage 1800-;
and Step3 is coated on a kanamycin plate for screening, the grown bacterial colony is a constructed recombinant engineering bacterium, and a monoclonal bacterial strain is selected for sequencing verification.
The application of the recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody in preparing the anti-tumor drugs, disclosed by the invention, has the effect far superior to the synergistic effect of the combined use of the anti-PD 1 nano antibody and the recombinant attenuated salmonella generated by the recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody.
The invention relates to application of recombinant attenuated salmonella engineering bacteria expressing an anti-PD 1 nano antibody in preparation of anti-tumor metastasis medicaments.
The recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody is applied to the preparation of medicines for treating tumors by combining conventional chemotherapy, traditional Chinese medicines and biological medicines.
Has the advantages that: the invention combines the PD-1 immune blocking inhibitor, the nano antibody and the attenuated salmonella, and takes the attenuated salmonella as a carrier for delivering the PD-1 nano antibody, thereby having higher targeting property, low cost, single administration, convenient administration and good anti-tumor effect. The recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody has a curative effect far superior to the synergistic effect of the combined use of the anti-PD 1 nano antibody and the recombinant attenuated salmonella.
Compared with the prior art, the invention has the following advantages: (1) the invention realizes the stable expression of the PD1 nano antibody in the tumor by the selection of the promoter. Through the selection of the signal peptide, the secretory expression of the PD1 nano antibody is realized. The salmonella engineering bacteria expressing the PD1 nano antibody has better anti-tumor curative effect than the VNP20009 bacterial strain, and the tumor targeting is not changed. The salmonella engineering bacteria expressing the PD1 nano antibody has the characteristics of easy culture of VNP20009, low cost and easy large-scale production, popularization and application.
(2) The salmonella engineering bacteria expressing the PD1 nano antibody has the characteristics that VNP20009 can be orally administered, intravenously administered, intraperitoneally administered and intratumoral administered, and the treatment effect can be achieved only by single administration. The salmonella engineering bacteria expressing the PD1 nano antibody has the characteristic of the application of VNP20009 in the preparation of medicaments for treating tumors by combining conventional chemotherapy, traditional Chinese medicines and biological medicines.
(3) The attenuated salmonella recombinant engineering strain for expressing the anti-PD 1 nano antibody is characterized in that in a mouse melanoma model, compared with a control strain VNP20009, the attenuated salmonella recombinant engineering strain can more obviously inhibit the tumor growth of a tumor-bearing mouse and prolong the survival time of the mouse, and the recombinant attenuated salmonella engineering strain for expressing the anti-PD 1 nano antibody generates a curative effect which is far better than the synergistic effect of the combined use of the anti-PD 1 nano antibody and the recombinant attenuated salmonella; the recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody also has the curative effect of resisting tumor metastasis; the tumor/liver and tumor/spleen targeting of the attenuated salmonella expressing the anti-PD 1 nano antibody is obviously improved, so that the anti-tumor effect of VNP20009 expressing the PD1 nano antibody is improved, and the toxic and side effects on normal organs such as liver, spleen and the like are reduced.
Drawings
FIG. 1 is a schematic diagram of the construction of pJ23100-flag-PD1nb plasmid of the present invention;
FIG. 2 is a diagram showing in vitro detection of expression and secretion of PD1nb by VNP-PD1nb strain by immunoblotting of the present invention; the samples were divided into total strain pellet of two monoclonal strains and supernatant of medium components. 1. Precipitating the strain; 2. culture medium supernatant.
FIG. 3 is a graph showing the tumor growth after the treatment of the engineered Salmonella bacteria expressing PD1 nanobody of the present invention;
1: a PBS group; 2: PD1nb group; 3: VNP-NC group; 4: VNP-PD1nb group.
FIG. 4 is a graph of tumor fold growth time for a melanoma model treated with engineered recombinant Salmonella expressing anti-PD 1 nanobody of the present invention; i.e. tumors from 1000mm 3 Up to 2000mm in length 3 The number of days required.
1: a PBS group; 2: PD1nb group; 3: VNP-NC group; 4: VNP-PD1nb group. Tumor growth delay time
FIG. 5 is a graph of tumor delay time for a melanoma model treated with engineered recombinant Salmonella expressing anti-PD 1 nanobody of the present invention; i.e. tumors growing to 1000mm 3 The number of days required; 1: a PBS group; 2: PD1nb group; 3: VNP-NC group; 4: VNP-PD1nb group.
FIG. 6 is a model diagram of melanoma treated by recombinant engineered Salmonella expressing anti-PD 1 nanobody according to the present invention; FIG. 6a is a graph comparing the effect of lung metastasis of anti-tumor cells in different groups according to the present invention; 1: a PBS group; 2: PD1nb group; 3: VNP-NC group; 4: VNP-PD1nb group. The scale is 5 mm; FIG. 6b is a graph showing a comparison of the localization of melanoma lung metastases between different groups according to the present invention; 1: a PBS group; 2: PD1nb group; 3: VNP-NC group; 4: VNP-PD1nb group.
FIG. 7 is a graph showing the analysis of the bacterial titer of the recombinant engineered Salmonella expressing anti-PD 1 nanobody of the present invention in tumor-bearing mouse tissues (5 days after administration). Measuring the titer of bacteria in liver, spleen and tumor tissues (unit Log10(CFU /) g); 1: VNP-NC group; 2: VNP-PD1nb group.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
The attenuated salmonella recombinant engineering bacteria for expressing the anti-PD 1 nano antibody is attenuated salmonella typhimurium VNP20009 carrying anti-PD 1 nano antibody expression plasmids and a genetically modified strain thereof, and the tumor targeting is not changed; including but not limited to the strains (ZL 201410209851.7, ZL201610946268.3, ZL201610945015.4, ZL201610945021.X, 202010182038.0; Acta pharmaceutical Sinica B2021, 11(10): 3165-. Compared with the theoretical value of the sum of the tumor fold growth time or the tumor delay time extension amplitude caused by two groups of curative effects after the salmonella attenuated and the anti-PD 1 nano antibody are treated independently, the tumor fold growth time extension amplitude of the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody is 4.78 times of the theoretical value of the sum of the two groups of curative effects, and the tumor delay time extension amplitude of the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody is 2.98 times of the theoretical value of the sum of the two groups of curative effects. The recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody has a curative effect far superior to the synergistic effect of the anti-PD 1 nano antibody and the recombinant attenuated salmonella; the recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody also has the curative effect of resisting tumor metastasis.
Cloning the anti-PD 1 nano antibody gene on an expression plasmid containing a promoter and a signal peptide: the promoter is a J23100 constitutive promoter or a NirB promoter or an adhE promoter; the plasmid contains an AT element that prevents plasmid loss.
The signal peptide is a conventional bacterial signal peptide, and the bacterial signal peptide is sseJ signal peptide, MIS signal peptide, Flic signal peptide, pelB signal peptide, SOPE signal peptide, SpA signal peptide or OmpA signal peptide.
The attenuated salmonella recombinant engineering bacterium agent for expressing an anti-PD 1 nano antibody is prepared from attenuated salmonella recombinant engineering bacterium for expressing an anti-PD 1 nano antibody. The active component is at least one of the following components (a), (b) and (c):
(a) the fermentation culture of the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody;
(b) ultrasonic lysis supernatant of the attenuated salmonella recombinant engineering bacteria cell expressing the anti-PD 1 nano antibody;
(c) and (3) carrying out ultrasonic lysis precipitation on the obtained attenuated salmonella recombinant engineering bacteria cell expressing the anti-PD 1 nano antibody.
The preparation method of the attenuated salmonella recombinant engineering bacteria for expressing the anti-PD 1 nano antibody comprises the following steps:
(1) obtaining PD1 nano antibody gene; a J23100 constitutive promoter or a NirB promoter or an adhE promoter is respectively cloned on pET32a plasmid, a conventional bacterial signal peptide is fused AT the N end of an anti-PD 1 nano antibody, and the plasmid contains an AT element for preventing plasmid loss.
(2) Constructing a plasmid expressing the PD1 nano antibody; step1 designing homologous recombination primer; step2 PCR to obtain DNA fragment; step3 transferring the product after connection into chemically competent cells of Escherichia coli DH5 alpha through chemical transformation;
step1 designing homologous recombination primer; step2 respectively uses pET32a (+) -PD1Nb as template PCR to obtain target gene fragment, uses pTh01 as template PCR to obtain plasmid skeleton fragment, and uses 1.2% agarose gel electrophoresis to cut out gene product, and uses gel recovery purification kit to purify to obtain DNA fragment; recovering the product by connecting gel through a double-fragment homologous recombination kit; the connection conditions are as follows: 4-8 deg.C, 1-2 hr, or 37 deg.C, 30 min; step3 transferring 10 μ L of the ligation product to chemically competent cells of E.coli DH5 α by chemical transformation; transformation conditions are as follows: placing on ice for 10 min, thermally shocking at 42 ℃ for 60s, placing on ice for 2 min, adding 900 mu L of non-resistant LB liquid culture medium, performing shake culture at 37 ℃ for 45 min, centrifuging at 5000 rpm for 3min, discarding supernatant, adding 1 mL of non-resistant LB liquid culture medium, mixing uniformly, sucking 100 mu L of non-resistant LB liquid culture medium, coating on a resistant plate with kanamycin, and standing in an incubator at 37 ℃ overnight; step4, selecting a monoclonal antibody to be added into 3 mL LB culture solution containing kanamycin, carrying out shake culture at 37 ℃ for 16 h, and extracting recombinant plasmids by using a plasmid miniextraction kit; the plasmid was sent to sequencing company for sequencing and alignment to confirm the correct sequence.
(3) Constructing attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody: and (3) electrically converting the expression plasmid into attenuated salmonella typhi VNP20009 and a mutant strain thereof to prepare the attenuated salmonella recombinant engineering bacteria for expressing the anti-PD 1 nano antibody. Respectively transferring the plasmid pTh01 and the PD1 nano antibody expression plasmid into electrically receptive cells of VNP20009 and a mutant strain thereof to prepare the recombinant strain VNP20009 and the attenuated salmonella recombinant engineering bacteria expressing anti-PD 1 nano antibodies:
step1 preparation of Salmonella electrotransformation competence: inoculating fresh attenuated salmonella VNP20009 and mutant strains thereof into 30 mL LB culture medium, carrying out shake culture at 37 ℃ until OD value is 0.5-0.6, centrifuging at 4 ℃ for 5000 rpm, collecting thalli for 5 min, washing the thalli for 3 times by sterilized 10% glycerol, 10 mL each time, centrifuging at 4 ℃ for 5000 rpm for 5 min, resuspending by 200 mu L of 10% glycerol, subpackaging by 50 mu L/tube for electrotransfer;
step2 the recombinant plasmid is transformed into attenuated salmonella VNP20009 and mutant strains thereof by an electroporation method: under the aseptic condition, 0.5-5 mug of constructed plasmid is added into the electrotransformation competence, and is transferred into an electrotransfer cup with 2 mm after being mixed uniformly for electric shock, wherein the electrotransfer condition is as follows: voltage 1800-;
and Step3 is coated on a kanamycin plate for screening, the grown bacterial colony is a constructed recombinant engineering bacterium, and a monoclonal bacterial strain is selected for sequencing verification.
The invention relates to application of recombinant attenuated salmonella engineering bacteria expressing an anti-PD 1 nano antibody in preparing tumor drugs. The recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody has a curative effect far superior to the synergistic effect of the combined use of the anti-PD 1 nano antibody and the recombinant attenuated salmonella.
The recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody also has the curative effect of resisting tumor metastasis.
The recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody is applied to the preparation of medicines for treating tumors by combining conventional chemotherapy, traditional Chinese medicines and biological medicines.
Example 1
Analysis of expression levels of PD1 in tumor tissue microenvironment following attenuated Salmonella treatment
(1) Establishment of mouse melanoma model
B16F10 mouse melanoma is a highly invasive and metastatic malignancy. B16F10 mouse melanoma cells were digested with 0.5% trypsin after growth to exponential growth phase in DMEM cell culture medium, then centrifuged at 1000 rpm/min for 3min, the supernatant culture solution was removed, washed with PBS for 2 times and then counted, and finally the cells were resuspended in PBS to adjust the final concentration to 2X10 6 one/mL. Each C57BL/6 mouse was inoculated with 100. mu.L of the vaccine at the mouse axillary fat pad, i.e., 2X10 5 One/only. After inoculation, mice were housed in clean-grade animal houses until the tumor volume of the mice had grown to approximately 100mm 3 The subsequent experiments were performed.
C57/B6 mice bearing B16F10 mice melanoma were randomized into PBS and VNP20009 groups. The PBS group was injected intraperitoneally with 100. mu.L PBS; VNP20009 intraperitoneal injection of 5X 10 5 CFU bacteria (in exponential growth phase, dissolved in 100. mu.L PBS). Tumor bearing mice were sacrificed on day 3 post-dosing and tumors were dissected and incubated at 37 degrees in digestion medium (10U/mL collagenase I, 400U/mL collagenase IV, 30U/mL DNase I, all diluted in HBSS) with gentle inversion shaking for 30-50 minutes. The cell pellet was removed through a 40- μm cell filter to obtain a single cell suspension. Cells were stained with a specific dead cell dye (BD,564407), incubated for 10-15 minutes at room temperature protected from light, and then stained with the following anti-mouse antibodies in combination: CD45-PE-Cy7(BD, clone number 30-F11), CD11b-APC (BD, clone number 561690), F4/80-BV421(BD, clone number T45-2342), CD86-PE (BD, clone number GL1), CD3e-FITC (BD, clone number 145-2C11), CD8-APC (BD, clone number 53-6.7), PD1-APC (BD, clone number J43), PDL1-BV421(BD, clone number MIH5), and CD38-BV421(BD, clone number MIH 90/CD 38). After incubation for 20 min at room temperature in the dark, PBS was washed 1-2 times, the collected cells were carefully resuspended and analyzed by flow cytometry (BD Canto II).
Treatment with attenuated salmonella VNP20009 does not substantially alter CD8 in tumor tissue + T cell content (CD 8 in PBS group tumors) + T cells were 1.43 ± 0.1316%, CD8+ T cells were 1.285 ± 0.1203% in VNP20009 group tumors (statistical analysis, no significant difference between the two groups). But PD1 + CD38 High CD8 + The proportion of T cells was significantly reduced by about 60% (PD 1 in PBS group tumors) + CD38 High The proportion of CD8+ T cells was 25.78. + -. 1.141%, PD1 in VNP20009 group of tumors + CD38 High CD8 + The proportion of T cells was 10.73. + -. 1.562%, P<0.001）。PD1 + CD38 High CD8 + The T cell is a depletion type CD8+ T cell, and the attenuated salmonella VNP20009 can reduce depletion type PD1 for the first time + CD38 High CD8 + T cells, which are one of the important mechanisms for the antitumor efficacy of the attenuated salmonella VNP20009, are disclosed for the first time.
The invention also discovers that the expression of PDL1 on the surface of the tumor cell is obviously improved by about 2.47 times by the treatment of the attenuated salmonella VNP20009 (the percentage of the tumor cells in/highly expressing PDL1 on the inner surface of the tumor in the PBS group is 12.73 +/-0.5677%, the percentage of the tumor cells in/highly expressing PDL1 on the inner surface of the tumor in the VNP20009 group is 34.1 +/-1.587%, and P is less than 0.0001). On one hand, the improvement of PDL-1 expression on the surface of the tumor cell indicates that the immunosuppressive activity of the tumor cell is improved after the attenuated salmonella VNP20009 is treated; on the other hand, it is predicted that tumor cells may be more sensitive to treatment with PDL-1 inhibitors.
The invention also discovers that the treatment of the attenuated salmonella VNP20009 obviously improves the expression of the PDL-1 on the surface of the mononuclear cell in the tumor tissue, and the expression is increased by about 2.52 times (the percentage of the mononuclear cell with PDL1 highly expressed on the inner surface of the tumor in PBS group is 21.13 +/-1.921 percent, the percentage of the mononuclear cell with PDL1 highly expressed on the inner surface of the tumor in VNP20009 group is 53.35 +/-2.195 percent, and P is less than 0.0001). An increase in the expression of PDL-1 on the surface of monocytes would also indicate an increase in immunosuppressive activity in the tumor microenvironment following treatment with attenuated salmonella VNP 20009.
The above findings indicate that attenuated salmonella VNP20009 treatment can produce both anti-tumor beneficial changes in the tumor microenvironment and immune checkpoint changes that are not beneficial for tumor treatment. The change of the immune check points of the tumor microenvironment unfavorable for tumor treatment indicates that the construction of the recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody is expected to improve the change which is unfavorable for tumor treatment and is caused by the treatment of attenuated salmonella VNP20009, solve the problem of the treatment of the current PD1 immune check point inhibitor and generate the synergistic curative effect.
Example 2
Construction of homologous recombinant plasmid for expressing anti-PD 1 nano antibody
(1) Acquisition of PD1 nano antibody gene
Designing homologous recombination primers, respectively taking pET32a (+) -PD1Nb1 and pET32a (+) -PD1Nb2 as templates for PCR to obtain a target anti-PD 1 nano antibody gene fragment, carrying out 1.5% agarose gel electrophoresis, cutting out a gene product, and purifying by using a gel recovery and purification kit (Biochemical industries and communications Co., Ltd.) to obtain a DNA fragment.
(2) Construction of plasmid expressing PD1 nanometer antibody
The plasmid backbone fragment was obtained by PCR using pTh01 as a template, and was subjected to 1.2% agarose gel electrophoresis and purified by a gel recovery purification kit (Biochemical Co., Ltd.) to obtain a DNA fragment.
The plasmid pTh01 was a plasmid engineered on the basis of pET32a (+) and contained the following elements: j23100 constitutive promoter or NirB promoter or adhE promoter; bacterial signal peptides including sseJ signal peptide, MIS signal peptide, Flic signal peptide, pelB signal peptide, SOPE signal peptide, SpA signal peptide, OmpA signal peptide; resistance selection genes; there is also an AT element that prevents plasmid loss. The invention aims to solve the problems in the prior art, and provides an attenuated salmonella recombinant engineering strain expressing an anti-PD 1 nano antibody, and a preparation method and application thereof.
The product was recovered by double fragment homologous recombination kit (nuozin) ligation gel. The connection conditions are as follows: 4-8 deg.C, 1-2 hr, or 37 deg.C, 30 min.
10. mu.L of the ligated product was transformed into chemically competent cells of E.coli DH 5. alpha. by chemical transformation. Transformation conditions are as follows: placing on ice for 10 min, thermally shocking at 42 ℃ for 60s, placing on ice for 2 min, adding 900 mu L of non-resistant LB liquid culture medium, shaking and culturing at 37 ℃ for 45 min, centrifuging at 5000 rpm for 3min, discarding supernatant, adding 1 mL of non-resistant LB liquid culture medium, mixing well, sucking 100 mu L of non-resistant LB liquid culture medium, coating on a resistant plate with kanamycin, and standing in an incubator at 37 ℃ overnight.
The single clone was picked up into 3 mL LB medium containing kanamycin, shake-cultured at 37 ℃ for 16 h, and plasmids were extracted separately using a plasmid miniprep kit (Biotech). The plasmid was sent to a sequencer (King of King Ltd.) for sequencing and alignment to confirm the correct sequence.
Example 3
Construction of recombinant salmonella engineering bacteria for expressing PD1 nano antibody
The expression plasmid pJ23100-flag-PD1Nb comprising a J23100 constitutive promoter, pelB signal peptide, flag-tagged PD1Nb1, and AT element is exemplified in detail below (FIG. 1). The flag tag is inserted into the N-terminal of the PD1nb protein to facilitate subsequent detection.
Construction of plasmid: and respectively electrotransfering the plasmid pTh01 and the pJ23100-flag-PD1nb into electrically competent cells of the VNP20009 to obtain the recombinant salmonella VNP20009 and the recombinant salmonella VNP20009 engineering bacteria expressing the PD1 nb. J23100 constitutive promoters or NirB promoters or adhE promoters are respectively cloned on pTh01 plasmids, and PD1 nano antibodies PD1nb1 and PD1nb2 are respectively expressed; the expression effect is similar. The N end of the PD1 nano antibody is fused with conventional bacterial signal peptides, including sseJ signal peptide, MIS signal peptide, Flic signal peptide, pelB signal peptide, SOPE signal peptide, SpA signal peptide and OmpA signal peptide, and the secretion effects of the conventional bacterial signal peptides are similar.
step1 preparation of Salmonella electrotransformation competence: inoculating fresh attenuated salmonella VNP20009 to 30 mL LB culture medium, shake culturing at 37 ℃ until OD value is 0.5-0.6, centrifuging at 4 ℃ for 5000 rpm, collecting thallus for 5 min, washing thallus for 3 times with sterilized 10% glycerol, 10 mL each time, centrifuging at 4 ℃ for 5000 rpm for 5 min, resuspending with 200 muL 10% glycerol, subpackaging with 50 muL/tube, and using for electric transfer.
Step2 recombinant plasmid pJ23100-flag-PD1nb was transformed into attenuated Salmonella VNP20009 using electroporation: under the aseptic condition, 0.5-5 mug of the constructed recombinant plasmid is added into the electrotransformation competence, and is transferred into an electrotransfer cup with 2 mm after being mixed uniformly for electric shock, wherein the electrotransfer condition is as follows: the voltage is 1800-2500V, the resistance is 400-500 omega, the capacitance is 25 muF, and the discharge time is 5 ms. After electric conversion, the bacterial colony is coated on a Carna plate for screening, and the grown bacterial colony is the recombinant engineering bacterium.
Finally, the engineered attenuated salmonella engineering strain which can stably express and secrete PD1nb is obtained and is marked as VNP-PD1 nb. The strain was designated VNP-NC as a blank by introducing an empty plasmid which does not express PD1nb protein but has the same structure.
The engineering attenuated salmonella engineering strain for stably expressing and secreting PD1nb has the characteristics of easy culture of VNP20009, low cost and easy large-scale production, popularization and application.
Example 4
Detection of expression and secretion of PD1nb of VNP-PD1nb of engineered attenuated salmonella
Aiming at expression and secretion detection of PDL1nb in VNP-PDL1nb, 2 strains of VNP-PDL1nb screened at the early stage and verified by sequencing are selected to be cultured in 1 ml of kanamycin-resistant liquid LB overnight in a 37-degree shaking table, 500 ml to 40 ml of cultured bacteria liquid LB are sucked to be cultured until OD600 is between 0.6 and 1.0, the bacteria liquid is transferred to a 50 ml centrifuge tube at 5,000 rpm, centrifugation is carried out at 4 ℃ for 30 min, and supernatant and sediment are collected.
For the precipitation, after resuspension with 2 ml of PBS, the cells were heated in a metal bath at 110 ℃ for 20 min to break up the bacteria and release the protein. And centrifuging at 13,000 rpm for 10 min to obtain supernatant as total protein in the deposited thallus. For the supernatant collected at the previous stage, proteins in the supernatant were collected by TCA-acetone precipitation, and in brief, the supernatant was transferred to a 50 ml centrifuge tube (beckman centrifuge tube), centrifuged at 15,000 g and 4 degrees for 10 min using an ultracentrifuge (beckman). The supernatant was transferred to a new 50 ml centrifuge tube, 10% TCA was added, vortexed to mix well, and allowed to stand on ice for 30 min. Another 7,000 g, 4 degrees, centrifugation for 20 min, 300 ml PBS heavy suspension precipitation, and transfer to 1.5 ml sterile EP tube. 1.2 ml of pre-cooled acetone (pre-warmed-20 ℃ C.), 17,000 g, 4 ℃ C. was added and centrifuged for 20 min. The supernatant was removed, 300 ml of PBS was added again, and the above procedure was repeated. And removing the supernatant, adding 40 ml of PBS for resuspension, and obtaining the total protein secreted by the thalli in the supernatant. Protein concentrations were determined using a BSA kit (cloudy days) for both total protein in the pellet and supernatant and adjusted to the same concentration with PBS. 40 ml of total precipitated protein and total secreted protein were added to 10 ml of 5XSDS Loading (Biyun day), respectively, and heated in a 95 ℃ metal bath for 10 min. WB assay was performed by pipetting 20 ml (about 15 mg protein). Flag-tagged antibody (Sigma, F1804) was used to detect the expression and secretion of PD1nb by the engineered strain.
The results showed that the obtained monoclonal engineered strains were all able to efficiently express and secrete PD1nb, and at least more than half of the expressed PD1nb was secreted into the culture supernatant outside the bacteria (see fig. 2).
Test example 1
Anti-tumor effect of engineered attenuated salmonella engineering strain stably expressing and secreting PD1nb on mouse melanoma model
B16F10 mouse melanoma cells were digested with 0.5% trypsin after growth to exponential growth phase in DMEM cell culture medium, then centrifuged at 1000 rpm/min for 3min, the supernatant culture solution was removed, washed with PBS for 2 times and then counted, and finally the cells were resuspended in PBS to adjust the final concentration to 2X10 6 one/mL. Each C57BL/6 mouse was inoculated with 100. mu.L of the vaccine at the mouse axillary fat pad, i.e., 2X10 5 One/only. After inoculation, mice were housed in clean-grade animal houses until the tumor volume of the mice had grown to approximately 100mm 3 The subsequent experiments were performed.
The experimental animal mice are 5-7 weeks old female BALB/C mice or C57BL/6 mice (Hezhou animal center). Mice were randomly assigned to different groups. The right forelimb axilla of the C57BL/6 mice were inoculated with 100. mu.l of 2X10 5 B16F10 cells for tumorigenesis. The tumor of the mouse is grown to about 100-150 mm 3 Then the treatment is carried out. Wherein PD1nb group is prepared by intraperitoneal injection of PD1nb (5 mg kg-1) antibody obtained by the above purification, and the medicine is injected 1 time every other day and 4 times continuously; VNP-NC group is intraperitoneal injection 1X10 6 100 μ l VNP-NC strain, inoculated only once initially; VNP-PD1nGroup b is intraperitoneal injection of 1X10 6 100 μ l VNP-NC strain, inoculated only once initially; the PBS group was intraperitoneally injected with 100. mu.l sterile PBS only. Tumor volume was calculated according to the formula V = length × width 2 X 0.52. Tumor size measurements were taken at two day intervals for a specific group of mice and calculated for plotting tumor growth (fig. 3). Values are expressed as SD + -SEM.
The engineered attenuated salmonella engineering strain stably expressing and secreting PD1nb has the characteristics of VNP20009 of oral administration, intravenous administration, intraperitoneal administration and intratumoral administration, and can achieve the treatment effect only by single administration.
B16F10 mouse melanoma is a highly invasive and metastatic malignancy. B16F10 mouse melanoma cells were digested with 0.5% trypsin after growth to exponential growth phase in DMEM cell culture medium, then centrifuged at 1000 rpm/min for 3min, the supernatant culture solution was removed, washed with PBS for 2 times and then counted, and finally the cells were resuspended in PBS to adjust the final concentration to 2X10 6 one/mL. Each C57BL/6 mouse was inoculated with 100. mu.L of the vaccine at the mouse axillary fat pad, i.e., 2X10 5 One/only. After inoculation, mice were housed in clean-grade animal houses until the tumor volume of the mice had grown to approximately 100mm 3 The subsequent experiments were performed.
C57/B6 mice bearing B16F10 mice melanoma were randomized into PBS and VNP20009 groups. The PBS group was injected intraperitoneally with 100. mu.L PBS; VNP20009 intraperitoneal injection of 5X 10 5 CFU bacteria (in exponential growth phase, dissolved in 100. mu.L PBS). Tumor bearing mice were sacrificed on day 3 post-dosing and tumors were dissected and incubated at 37 degrees in digestive medium (10U/mL collagenase I, 400U/mL collagenase IV, 30U/mL DNase I, all diluted in HBSS) with gentle shaking upside down for 30-50 minutes. The cell pellet was removed through a 40- μm cell filter to obtain a single cell suspension. Cells were stained with a specific dead cell dye (BD,564407), incubated for 10-15 minutes at room temperature protected from light, and then stained with the following anti-mouse antibodies in combination: CD45-PE-Cy7(BD, clone No. 30-F11), CD11b-APC (BD, clone No. 561690), F4/80-BV421(BD, clone No. T45-2342), CD86-PE (BD, clone No. GL1), CD3e-FITC (BD, clone No. 145-2C11), CD 8-FITCAPC (BD, clone No. 53-6.7), PD1-APC (BD, clone No. J43), PDL1-BV421(BD, clone No. MIH5) and CD38-BV421(BD, clone No. 90/CD 38). After incubation for 20 min at room temperature in the dark, PBS was washed 1-2 times, the collected cells were carefully resuspended and analyzed by flow cytometry (BD Canto II).
Test example 2
Anti-tumor metastasis effect of engineered attenuated salmonella engineering strain stably expressing and secreting PD1nb on mouse melanoma lung metastasis model
For the lung metastasis model of B16F10, 100. mu.l via tail vein injection contains 1X10 6 B16F10 cell suspension (C57 BL/6), treatment started on day 6 post-injection, treatment groups were as above, and mice were sacrificed at day 18 post-injection, lung metastases were recorded in each group of mice photographed (fig. 6 a), and ImageJ analyzed lung tumor metastasis area (fig. 6B). The results indicate that VNP-PD1nb administration significantly increased the anti-tumor lung metastasis effect compared to either single treatment modality (fig. 6). 1: a PBS group; 2: PD1nb group; 3: (ii) a 4: VNP-PD1nb group.
The lung metastasis tumor area of the PBS group was 138.6 + -12.74 mm 2 The lung metastasis tumor area of PD1nb group was 77.44. + -. 6.42 mm 2 The lung metastasis tumor area of the VNP-NC group is 38.23 +/-2.156 mm 2 The lung metastasis tumor area of VNP-PD1nb group was 12.7. + -. 1.202 mm 2 . Compared with the PBS group, the lung tumor metastasis of the PD1nb group was reduced by 21.0%, the lung tumor metastasis of the VNP-NC group was reduced by 72.4%, and the lung tumor metastasis of the VNP-PD1nb was reduced by 90.8%. Lung tumor metastasis was reduced by 83.6% in VNP-PD1nb group compared to PD1nb group; compared to the VNP-NC group, the VNP-PD1nb group had 66.8% less lung tumor metastasis. The results show that the VNP-PD1nb group has a remarkably increased tumor metastasis inhibition effect compared with any single treatment mode.
The anti-tumor effect of VNP20009 expressing PD1 nano antibody PD1nb2 is obviously better than that of VNP20009, thereby showing that the PD1 nano antibody indeed endows VNP20009 with stronger anti-tumor effect.
The recombinant attenuated salmonella engineering bacteria expressing the anti-PD 1 nano antibody also has the curative effect of resisting tumor metastasis. VNP20009 expressing PD1 nano-antibody has the characteristic that VNP20009 can be applied to preparation of drugs for treating tumors by combination with conventional chemotherapy, traditional Chinese medicines and biological medicines.
Test example 3
Tissue distribution of engineered attenuated salmonella engineering strain for stably expressing and secreting PD1nb in tumor-bearing mice
Fifth day of bacterial treatment of tumor-bearing mice, the mice were sacrificed at random, and the tumor, liver and spleen of the tumor-bearing mice were taken out under sterile conditions, weighed respectively, and placed in 2 mL of PBS to be homogenized using a tissue homogenizer (frequency: 60 Hz; time: 100 s). Different tissues are diluted according to different gradients, coated in an LB plate and inverted in a bacterial incubator at 37 ℃ for 12 hours, colony counting is carried out, and distribution of VNP20009 and the recombinant salmonella engineering strain which stably expresses and secretes PD1nb in mouse tissues is comparatively analyzed (figure 7).
Liver and spleen are normal tissues with the most distributed bacteria, so the invention focuses on detecting the titer (unit is Log10(CFU /) g) of attenuated Salmonella typhimurium in liver, spleen and tumor tissues, and analyzing the tumor targeting of bacteria. After the PD1 nano antibody is expressed, the distribution of the recombinant engineering strain in tumor, liver and spleen is basically the same as that of VNP20009, and no obvious difference is found, so that the improvement of the anti-tumor effect and anti-tumor metastasis curative effect of the engineering attenuated salmonella engineering strain expressing the PD1 nano antibody is not due to the increase of the bacterial titer in tumor tissues, but due to the increase of the anti-tumor activity of the engineering attenuated salmonella engineering strain expressing the PD1 nano antibody at the unit strain level; the distribution of the recombinant engineering strain in the liver and the spleen is basically the same as that of VNP20009, which indicates that the toxic and side effects of the recombinant engineering strain on normal organs such as the liver, the spleen and the like cannot be increased.
The foregoing shows and describes the general principles, essential 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 embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.

Claims (10)

1. An attenuated salmonella recombinant engineering bacterium for expressing an anti-PD 1 nano antibody, which is characterized in that: the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody are attenuated salmonella typhimurium VNP20009 carrying anti-PD 1 nano antibody expression plasmid and genetically modified strains thereof, and the tumor targeting is not changed; cloning the anti-PD 1 nano antibody gene on an expression plasmid containing a promoter and a signal peptide: the promoter is a J23100 constitutive promoter or a NirB promoter or an adhE promoter; the plasmid contains an AT element that prevents plasmid loss.
2. The attenuated recombinant engineered Salmonella expressing an anti-PD 1 nanobody of claim 1, characterized in that: the signal peptide is a conventional bacterial signal peptide, and the bacterial signal peptide is sseJ signal peptide, MIS signal peptide, Flic signal peptide, pelB signal peptide, SOPE signal peptide, SpA signal peptide or OmpA signal peptide.
3. The attenuated salmonella recombinant engineering bacteria agent for expressing an anti-PD 1 nano antibody, which is prepared from the attenuated salmonella recombinant engineering bacteria for expressing an anti-PD 1 nano antibody, of claim 1.
4. The attenuated salmonella recombinant engineering bacteria agent expressing the anti-PD 1 nanobody of claim 3, wherein the active ingredient is at least one of the following (a), (b) and (c):
(a) the fermentation culture of the attenuated recombinant engineered Salmonella expressing an anti-PD 1 nanobody of claim 1;
(b) the ultrasonic lysis supernatant of the attenuated salmonella recombinant engineered bacteria expressing the anti-PD 1 nanobody of claim 1;
(c) the ultrasonic lysis sediment of the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody obtained in the claim 1.
5. The method for preparing the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nanobody of claim 1, comprising the steps of:
(1) obtaining PD1 nano antibody gene;
(2) constructing a plasmid expressing the PD1 nano antibody; step1 designing homologous recombination primer; step2 PCR to obtain DNA fragment; step3 transferring the product after connection into chemically competent cells of Escherichia coli DH5 alpha through chemical transformation;
(3) constructing attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nano antibody: and (3) electrically converting the expression plasmid into attenuated salmonella typhi VNP20009 and a mutant strain thereof to prepare the attenuated salmonella recombinant engineering bacteria for expressing the anti-PD 1 nano antibody.
6. The method for preparing the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nanobody of claim 5, wherein the method comprises the following steps: in step (1), a J23100 constitutive promoter or a NirB promoter or an adhE promoter is cloned on a pET32a plasmid respectively, a conventional bacterial signal peptide is fused AT the N end of an anti-PD 1 nano antibody, and the plasmid contains an AT element for preventing plasmid loss.
7. The method for preparing the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nanobody of claim 6, wherein the method comprises the following steps: in the step (2), step1 designs homologous recombination primers; step2 respectively uses pET32a (+) -PD1Nb as template PCR to obtain target gene fragment, uses pTh01 as template PCR to obtain plasmid skeleton fragment, and uses 1.2% agarose gel electrophoresis to cut out gene product, and uses gel recovery purification kit to purify to obtain DNA fragment; recovering the product by connecting gel through a double-fragment homologous recombination kit; the connection conditions are as follows: 4-8 deg.C, 1-2 hr, or 37 deg.C, 30 min; step3 transferring 10. mu.L of the ligation product to chemically competent cells of E.coli DH 5. alpha. by chemical transformation; transformation conditions are as follows: placing on ice for 10 min, thermally shocking at 42 ℃ for 60s, placing on ice for 2 min, adding 900 mu L of non-resistant LB liquid culture medium, performing shake culture at 37 ℃ for 45 min, centrifuging at 5000 rpm for 3min, discarding supernatant, adding 1 mL of non-resistant LB liquid culture medium, mixing uniformly, sucking 100 mu L of non-resistant LB liquid culture medium, coating on a resistant plate with kanamycin, and standing in an incubator at 37 ℃ overnight; step4, selecting a monoclonal antibody to be added into 3 mL LB culture solution containing kanamycin, carrying out shake culture at 37 ℃ for 16 h, and extracting recombinant plasmids by using a plasmid miniextraction kit; the plasmid was sent to sequencing company for sequencing and alignment to confirm the correct sequence.
8. The method for preparing the attenuated salmonella recombinant engineering bacteria expressing the anti-PD 1 nanobody of claim 7, wherein: in the step (3), the plasmid pTh01 and the PD1 nano antibody expression plasmid are respectively electrotransferred into electrically competent cells of VNP20009 and mutant strains thereof to prepare recombinant strains VNP20009 and attenuated salmonella recombinant engineering bacteria expressing anti-PD 1 nano antibodies:
step1 preparation of Salmonella electrotransformation competence: inoculating fresh attenuated salmonella VNP20009 and mutant strains thereof into 30 mL LB culture medium, carrying out shake culture at 37 ℃ until OD value is 0.5-0.6, centrifuging at 4 ℃ for 5000 rpm, collecting thalli for 5 min, washing the thalli for 3 times by sterilized 10% glycerol, 10 mL each time, centrifuging at 4 ℃ for 5000 rpm for 5 min, resuspending by 200 mu L of 10% glycerol, subpackaging by 50 mu L/tube for electrotransfer;
step2 the recombinant plasmid is transformed into attenuated salmonella VNP20009 and mutant strains thereof by an electroporation method: under the aseptic condition, 0.5-5 mug of constructed plasmid is added into the electrotransformation competence, and is transferred into an electrotransfer cup with 2 mm after being mixed uniformly for electric shock, wherein the electrotransfer condition is as follows: voltage 1800-;
and Step3 is coated on a kanamycin plate for screening, the grown bacterial colony is a constructed recombinant engineering bacterium, and a monoclonal bacterial strain is selected for sequencing verification.
9. The use of the recombinant attenuated salmonella engineered strain expressing an anti-PD 1 nanobody of any one of claims 1 to 8 in the preparation of an anti-tumor drug or an anti-tumor metastasis drug.
10. The use of the engineered bacterium of recombinant attenuated salmonella of any one of claims 1 to 9 expressing an anti-PD 1 nanobody for the preparation of a medicament for the treatment of tumors in combination with conventional chemotherapy, traditional Chinese medicine, or biological drugs.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190038679A1 (en) * 2016-02-09 2019-02-07 The General Hospital Corporation D/B/A Massachusetts General Hospital Methods and compositions relating to engineered microbial cells
CN110951663A (en) * 2019-12-26 2020-04-03 深圳市前海金卓生物技术有限公司 Recombinant bacterium for expressing PD-1 antibody and construction method and application thereof
US20210308195A1 (en) * 2018-07-20 2021-10-07 The Trustees Of Columbia University In The City Of New York Programmable bacteria for the treatment of cancer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190038679A1 (en) * 2016-02-09 2019-02-07 The General Hospital Corporation D/B/A Massachusetts General Hospital Methods and compositions relating to engineered microbial cells
US20210308195A1 (en) * 2018-07-20 2021-10-07 The Trustees Of Columbia University In The City Of New York Programmable bacteria for the treatment of cancer
CN110951663A (en) * 2019-12-26 2020-04-03 深圳市前海金卓生物技术有限公司 Recombinant bacterium for expressing PD-1 antibody and construction method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CANDICE R. GURBATRI等: "Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies", 《SCIENCE TRANSLATIONAL MEDICINE》, vol. 12, pages 2 *

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