CN114736840A

CN114736840A - Recombinant attenuated salmonella expressing anti-PD-L1 nano antibody and preparation method and application thereof

Info

Publication number: CN114736840A
Application number: CN202210182455.4A
Authority: CN
Inventors: 华子春; 包斐斐
Original assignee: Targetpharma Laboratories Jiangsu Co ltd
Current assignee: Targetpharma Laboratories Jiangsu Co ltd
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-07-12

Abstract

The invention discloses recombinant attenuated salmonella expressing an anti-PD-L1 nano antibody and a preparation method and application thereof, and the attenuated salmonella recombinant strain expressing the anti-PD-L1 nano antibody is attenuated salmonella typhimurium VNP20009 carrying an anti-PD-L1 nano antibody expression plasmid. The invention combines the PD-L1 immune blocking inhibitor, the nano antibody and the attenuated salmonella, and takes the attenuated salmonella as a carrier for delivering the PD-L1 nano antibody, thereby having the advantages of higher targeting property, simple method, easy operation, low cost, single administration, convenient administration, good anti-tumor effect and the like. And only a single administration is needed to achieve the treatment effect.

Description

Recombinant attenuated salmonella expressing anti-PD-L1 nano antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to recombinant attenuated salmonella expressing an anti-PD-L1 nano antibody, and a preparation method and application thereof.

Background

Programmed death factor 1 ligand 1 (PD-L1), also known as CD274, B7-H1, is a member of B7 family, is a ligand of PD-1, and belongs to type I transmembrane protein (Sznol M. et al., Cancer J2014, 20(4): 290-. Under normal conditions, PD-1 on the surface of T cells can inhibit the function of T lymphocytes, thereby inhibiting autoimmune response and preventing autoimmune diseases. However, in tumors, PD-L1 expressed by tumor cells can be combined with PD-1 on the surface of T cells to cause the down regulation of T cell proliferation and even induce T cell apoptosis and promote the immune escape of tumors through the inhibition effect on T lymphocytes (Sharma and Allison, Science 2015, 348(6230): 56-61). Blocking the negative regulation pathway of PD-1/PD-L1 can activate the function of immune system and kill tumor cells. Currently, there are several monoclonal antibodies targeting PD-L1, such as atezolizumab (Tecntriq), a monoclonal antibody of Roche PD-L1, Durvaumab (trade name: Imfinzi) developed by Aslicon, avelumab (trade name: Bavencio) of Perey/Merck, and these PD-L1 monoclonal antibodies have been successfully used in clinic (Lee HT et al. Molecules 2019, 24 (6)). However, PD-L1 monoclonal antibody has the disadvantages of large molecular weight and difficulty in penetrating tissues, and two barriers still exist clinically at present, namely, most patients (70-85%) do not respond to immunotherapy and a small part of patients respond to immunotherapy. One of the main reasons for the former is the low content of potent immune cells (e.g., CD8+ T cells) within the tumor. 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 the patients. These factors all limit their popularity in clinical 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 the effect of inhibiting tumor cell growth. Salmonella is one such tumor-targeting bacterium. VNP20009 is Salmonella typhimurium by deletionmsbBAndpurIthe toxicity is reduced by gene (Luo X et al, Oncol Res 2001, 12(11-12): 501-508), and the Clinical test of phase I of metastatic melanoma is passed, the curative effect is insufficient (Thamm DH et al, Clinical Cancer Research 2005, 11(13): 4827-4834), but the safety is better. 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 1000-10000:1 (Clairmont C et al, Journal of Infectious Diseases 2000, 181(6): 1996-2002.). 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 a preparation method and application of recombinant attenuated salmonella expressing an anti-PD-L1 nano antibody.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: the attenuated salmonella recombinant strain expressing the anti-PD-L1 nano antibody is attenuated salmonella typhimurium VNP20009 carrying anti-PD-L1 nano antibody expression plasmids. 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-: pTh30-P15, pTh31-P22 and pTh32-P28, which are modified on the basis of the plasmid pTh 01.

Further, the anti-PD-L1 nanobody gene was cloned on an expression plasmid containing a signal peptide: the plasmid pTh01 is a plasmid engineered on the basis of Pet28a (+) and comprises the following elements: j23100 constitutive promoter or NirB promoter or adhE promoter; the sequence of the anti-PD-L1 nano antibody is a nucleotide sequence shown in SEQ ID No.10, or SEQ ID No.12, or SEQ ID No. 14; the sequence of the anti-PD-L1 nano antibody is an amino acid sequence shown as SEQ ID No.9, or SEQ ID No.11, or SEQ ID No. 13.

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 bacteria agent for expressing the anti-PD-L1 nano antibody is prepared from the attenuated salmonella recombinant bacteria for expressing the anti-PD-L1 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 strain expressing the anti-PD-L1 nano antibody;

(b) ultrasonic cracking supernatant of the obtained attenuated salmonella recombinant bacteria cell expressing the anti-PD-L1 nano antibody;

(c) and (3) carrying out ultrasonic lysis precipitation on the obtained attenuated salmonella recombinant bacteria cell expressing the anti-PD-L1 nano antibody.

The preparation method of the attenuated salmonella recombinant strain expressing the anti-PD-L1 nano antibody comprises the following steps:

(1) obtaining PD-L1 nano antibody gene;

(2) constructing pTh30-P15, pTh31-P22 and pTh32-P28 plasmids expressing PD-L1 nano antibodies; 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 an attenuated salmonella recombinant strain expressing an anti-PD-L1 nano antibody: and (3) electrically converting the expression plasmid into attenuated salmonella typhi VNP20009 to prepare the attenuated salmonella recombinant strain expressing the anti-PD-L1 nano antibody.

Further, in step (1), a J23100 constitutive promoter or a NirB promoter or an adhE promoter is cloned on the pTh01 plasmid respectively, and a conventional bacterial signal peptide is fused at the N end of the anti-PD-L1 nano antibody; in the step (2), step1 designs homologous recombination primers; step2 respectively uses pET32a (+) -P15, pET32a (+) -P22 and pET32a (+) -P28 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 3 min, 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 single clone to 3 mL LB culture solution containing kanamycin, shaking-culturing for 16 h at 37 ℃, and extracting plasmids pTh30-P15, pTh31-P22 and pTh32-P28 by using a plasmid miniextract kit; the plasmid was sent to sequencing company for sequencing and alignment to confirm the correct sequence.

Further, in step (3), plasmids pTh01 and pTh30-P15, pTh31-P22 and pTh32-P28 are respectively electroporated into electrocompetent cells of VNP20009, so as to prepare a recombinant strain VNP20009 and an attenuated salmonella recombinant bacterium expressing an anti-PD-L1 nano antibody:

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 between 0.5 and 0.6, centrifuging at 4 ℃ for 5000 rpm, collecting thalli for 5 min, washing the thalli for 3 times by using sterilized 10% glycerol, centrifuging at 4 ℃ for 5000 rpm and 5 min each time by using 10% glycerol of 200 mu L for heavy suspension, and subpackaging by 50 mu L/tube for electric transfer;

step2 the recombinant plasmid was transformed into VNP20009 using electroporation: under the aseptic condition, 0.5-2 mug of constructed plasmid is added into an electrotransfer competence, and is transferred into an electrotransfer cup with the diameter of 2 mm for electric shock after being uniformly mixed, wherein the electrotransfer condition is 2000 kV, 25 mug F and 400 omega; the discharge time is 5 ms;

after electrotransformation, adding 900 mu L of non-resistant LB liquid culture medium, carrying out shake culture at 37 ℃ for 45 min, centrifuging at 5000 rpm for 3 min, discarding the supernatant, adding 100 mu L 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;

and Step3 is coated on a kanamycin plate for screening, the grown bacterial colony is the constructed recombinant bacterium, and the monoclonal strain is selected for sequencing verification.

The invention relates to application of an attenuated salmonella recombinant strain expressing a PD-L1 nano antibody in preparing a tumor medicament.

The attenuated salmonella recombinant strain expressing the PD-L1 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-L1 immune blocking inhibitor, the nano antibody and the attenuated salmonella, and takes the attenuated salmonella as a carrier for delivering the PD-L1 nano antibody, thereby having the advantages of higher targeting property, simple method, easy operation, low cost, single administration, convenient administration, good anti-tumor effect and the like. The VNP20009 for expressing the PD-L1 nano antibody has the characteristics that the VNP20009 can be orally administered, intravenously administered, intraperitoneally administered and intratumoral administered, and the treatment effect can be achieved only by single administration.

Compared with the prior art, the invention has the following advantages:

(1) the invention realizes the stable expression of the PD-L1 nano antibody in the tumor by the selection of the promoter. By selecting the signal peptide, the secretory expression of the PD-L1 nano antibody is realized. The VNP20009 expressing the PD-L1 nano antibody has higher tumor targeting property than the VNP20009 strain and lower distribution in normal organs such as liver, spleen and the like, thereby reducing toxic and side effects on the normal organs such as liver, spleen and the like and having higher safety.

(2) The VNP20009 expressing the PD-L1 nano antibody has better anti-tumor curative effect than the VNP20009 strain. VNP20009 for expressing PD-L1 nano antibody has the characteristics of easy culture of VNP20009, low cost and easy large-scale production, popularization and application.

(3) The attenuated salmonella recombinant strain for expressing the anti-PD-L1 nano antibody is characterized in that in a mouse melanoma model, compared with a control strain VNP20009, the attenuated salmonella recombinant strain can more obviously inhibit the tumor growth of a tumor-bearing mouse and prolong the survival time of the mouse; the tumor/liver and tumor/spleen targeting of the attenuated salmonella expressing the anti-PD-L1 nano antibody is obviously improved, so that the anti-tumor effect of VNP20009 expressing the PD-L1 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 graph showing the tumor-suppressing effect of recombinant Salmonella expressing an anti-PD-L1 antibody according to the present invention in a melanoma model;

FIG. 2 is a graph of the survival of mouse melanoma treated with recombinant attenuated Salmonella expressing an anti-PD-L1 antibody according to the invention;

FIG. 3 is a graph of tumor doubling time for a melanoma model treated with recombinant Salmonella expressing an anti-PD-L1 antibody according to the present invention; PBS group, 2.48 days; VNP group, 3.25 days; VHH15 group, 3.82; VHH22 group, 3.98; VHH28 group, 3.45 days.

FIG. 4 is a graph of tumor growth delay time for a melanoma model treated with recombinant Salmonella expressing an anti-PD-L1 antibody according to the present invention; PBS group, 8.12 days; VNP group, 11.28 days; VHH15 group, 13.12 days; VHH22 group, 13.98 days; VHH28 group, 12.45 days.

FIG. 5 is a tissue distribution map of recombinant attenuated Salmonella of the present invention in a mouse melanoma model; the titers of bacteria in liver, spleen and tumor tissues were measured (in Log10(CFU /) g). Bacterial titers in tumors were: VNP group, 9.375; VHH15 group, 9.2825; VHH22 group, 9.55; VHH28 group, 9.356. The bacterial titer in the spleen was VNP group, 7.05; VHH15 group, 6.725; VHH22 group, 6.875; VHH28 group, 6.75. Bacterial titers in the liver were VNP group, 6.175; VHH15 group, 5.95; VHH22 group, 6.1; VHH28 group, 5.925.

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 carried out according to conventional conditions or conditions recommended by the manufacturer.

The attenuated salmonella recombinant strain expressing the anti-PD-L1 nano antibody is attenuated salmonella typhimurium VNP20009 carrying anti-PD-L1 nano antibody expression plasmids. Cloning the anti-PD-L1 nano antibody gene on an expression plasmid containing a signal peptide: j23100 constitutive promoter or NirB promoter or adhE promoter; the sequence of the anti-PD-L1 nano antibody is a nucleotide sequence shown in SEQ ID No.10, or SEQ ID No.12, or SEQ ID No. 14; the sequence of the anti-PD-L1 nano antibody is an amino acid sequence shown as SEQ ID No.9, or SEQ ID No.11, or SEQ ID No. 13.

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 bacteria agent for expressing the anti-PD-L1 nano antibody is prepared from the attenuated salmonella recombinant bacteria for expressing the anti-PD-L1 nano antibody. The active component is at least one of the following components (a), (b) and (c):

(b) ultrasonic lysis supernatant of the obtained attenuated salmonella recombinant bacteria cell expressing the anti-PD-L1 nano antibody;

The preparation method of the attenuated salmonella recombinant strain expressing the anti-PD-L1 nano antibody comprises the following steps: (1) obtaining PD-L1 nano antibody gene; a J23100 constitutive promoter or a NirB promoter or an adhE promoter is respectively cloned on pTh01 plasmids, and a conventional bacterial signal peptide is fused at the N end of an anti-PD-L1 nano antibody; in the step (2), step1 designs homologous recombination primers; step2 respectively uses pET32a (+) -P15, pET32a (+) -P22 and pET32a (+) -P28 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 3 min, 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 single clone to 3 mL LB culture solution containing kanamycin, carrying out shake culture at 37 ℃ for 16 h, and extracting plasmids pTh30-P15, pTh31-P22 and pTh32-P28 by using a plasmid miniextract kit; the plasmid was sent to sequencing company for sequencing and alignment to confirm the correct sequence.

(3) constructing an attenuated salmonella recombinant strain expressing an anti-PD-L1 nano antibody: and electrically transforming the expression plasmid to attenuated salmonella typhi VNP20009 to obtain the attenuated salmonella recombinant strain expressing the anti-PD-L1 nano antibody. Plasmids pTh01, pTh30-P15, pTh31-P22 and pTh32-P28 are respectively electroporated into electrocompetent cells of VNP20009, and the recombinant strain VNP20009 and the attenuated salmonella recombinant strain expressing anti-PD-L1 nano antibodies are prepared:

step1 preparation of Salmonella electrotransformation competence: inoculating fresh attenuated salmonella VNP20009 to 30 mL LB culture medium, shaking culturing at 37 ℃ until OD value is between 0.5 and 0.6, centrifuging at 4 ℃ for 5000 rpm, collecting thallus for 5 min, washing the thallus for 3 times with sterilized 10% glycerol, centrifuging at 4 ℃ for 5000 rpm for 5 min for 10 mL each time, resuspending with 200 mu L10% glycerol, subpackaging with 50 mu L/tube for electric transfer;

step2 the recombinant plasmid was transformed into VNP20009 by electroporation: under the aseptic condition, 0.5-2 mug of constructed plasmid is added into the electrotransfer competence, and after being uniformly mixed, the plasmid is transferred into an electrotransfer cup with the diameter of 2 mm for electric shock, wherein the electrotransfer condition is 2000 kV, 25 muF and 400 omega; the discharge time is 5 ms;

adding 900 mu L of nonresistant LB liquid culture medium after electrotransfer, performing shake culture at 37 ℃ for 45 min, centrifuging at 5000 rpm for 3 min, discarding the supernatant, adding 100 mu L of nonresistant LB liquid culture medium, mixing uniformly, sucking 100 mu L of nonresistant LB liquid culture medium, coating on a resistance flat plate with kanamycin, and standing overnight in an incubator at 37 ℃;

Example 1

Analysis of expression levels of PD-L1 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 smallMouse melanoma cell is digested with 0.5% pancreatin after growth to exponential growth phase in DMEM cell culture medium, then centrifuged at 1000 rpm/min for 3 min, the supernatant culture fluid is removed, washed with PBS for 2 times and then cell counting is carried out, and finally cell final concentration is adjusted to 2 × 10 by using PBS to resuspend cells⁶one/mL. Each C57BL/6 mouse was inoculated with 100. mu.L of the vaccine at the mouse axillary fat pad, i.e., 2X 10⁵One/only. After inoculation, mice were housed in clean-grade animal houses until the tumor volume of the mice had grown to approximately 100 mm³The 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 10⁵CFU 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 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), CD8-APC (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).

The invention finds that the treatment of the attenuated salmonella VNP20009 obviously improves the expression of PD-L1 on the surface of the tumor cells, and the expression is increased by about 2.47 times (the percentage of the tumor cells in/highly expressing PD-L1 in the tumor inner surface of the PBS group is 12.73 +/-0.5677%, the percentage of the tumor cells in/highly expressing PD-L1 in the tumor inner surface of the VNP20009 group is 34.1 +/-1.587%, and P is less than 0.0001). On one hand, the improvement of the expression of PD-L1 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 high expression PD-L1 on the inner surface of the tumor in PBS group is 21.13 +/-1.921 percent, the percentage of the mononuclear cell with high expression PD-L1 on the inner surface of the tumor in VNP20009 group is 53.35 +/-2.195 percent, and P is less than 0.0001 percent). 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 invention also discovers that the treatment of attenuated salmonella VNP20009 does not substantially change CD8 in tumor tissues⁺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^HighCD8⁺The proportion of T cells was significantly reduced by about 60% (PD 1 in PBS group tumors)⁺CD38^HighThe proportion of CD8+ T cells was 25.78. + -. 1.141%, PD1 in VNP20009 group of tumors⁺CD38^HighCD8⁺The proportion of T cells was 10.73. + -. 1.562%, P<0.001）。PD1⁺CD38^HighCD8⁺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^HighCD8⁺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 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 recombinant attenuated salmonella engineering bacteria expressing the anti-PD-L1 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 PD-L1 immune check point inhibitor and generate the synergistic curative effect.

Example 2

Construction of homologous recombinant plasmid for expressing anti-PD-L1 nano antibody

Acquisition of PD-L1 antibody Gene

Designing homologous recombination primers, respectively taking pET32a (+) -P15, pET32a (+) -P22 and pET32a (+) -P28 as templates to obtain target anti-PD-L1 nano antibody gene fragments through PCR, cutting out gene products through 1.5% agarose gel electrophoresis, and purifying by using a gel recovery and purification kit (Biochemical industries, Ltd.) to obtain DNA fragments.

Construction of plasmid expressing PD-L1

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 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 3 min, 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 expressing PD-L1 nano antibody

Plasmids pTh01, pTh30-P15, pTh31-P22 and pTh32-P28 are respectively electroporated into electrically competent cells of VNP20009 to obtain recombinant salmonella VNP20009, recombinant salmonella VNP20009 expressing VHH15, recombinant salmonella VNP20009 expressing VHH22 and recombinant salmonella VNP20009 expressing VHH 28. A J23100 constitutive promoter or a NirB promoter or an adhE promoter is cloned on pTh01 plasmid respectively and expresses PD-L1 nano antibodies VHH15, VHH22 and VHH28 respectively; the expression effect is similar. The N end of the PD-L1 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 peptides are similar.

The PD-L1 nano antibody expression strain composed of a J23100 constitutive promoter and pelB signal peptide and the specific process of tumor treatment are as follows:

preparing the salmonella electrotransformation competence: inoculating fresh attenuated salmonella VNP20009 to 30 mL LB culture medium, shake culturing at 37 ℃ until OD value is between 0.5 and 0.6, centrifuging at 4 ℃ for 5000 rpm, collecting thallus 5 min, washing thallus 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, and subpackaging with 50 muL/tube for electric transfer.

The recombinant plasmid was transformed into VNP20009 using electroporation: under the aseptic condition, 0.5-2 mug of the constructed plasmid is added into the electrotransfer competence, and after being uniformly mixed, the plasmid is transferred into an electrotransfer cup with the diameter of 2 mm for electric shock, wherein the electrotransfer condition is 2000 kV, 25 mug F and 400 omega.

Adding 900 mu L of nonresistant LB liquid culture medium after electrotransformation, shaking for 45 min at 37 ℃, centrifuging for 3 min at 5000 rpm, discarding the supernatant, adding 100 mu L of nonresistant LB liquid culture medium, mixing well, sucking 100 mu L of nonresistant LB liquid culture medium, coating on a resistant plate with kanamycin, and standing overnight in an incubator at 37 ℃.

And coating the strain on a kanamycin plate for screening, wherein the grown bacterial colony is the constructed recombinant bacterium, and selecting a monoclonal strain for sequencing verification.

VNP20009 for expressing PD-L1 nano antibody has the characteristics of easy culture of VNP20009, low cost and easy large-scale production, popularization and application.

Test example 1

Anti-tumor effect of recombinant VNP20009 bacterium expressing anti-PD-L1 nano antibody 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 3 min, 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 2X 10⁶one/mL. Each C57BL/6 mouse was inoculated with 100. mu.L of the vaccine at the mouse axillary fat pad, i.e., 2X 10⁵One/only. After inoculation, mice were housed in clean-grade animal houses until the tumor volume of the mice had grown to approximately 100 mm³The subsequent experiments were performed.

The mice C57/B6 with B16F10 mouse melanoma are randomly divided into five groups, namely PBS, VNP20009 (VNP), recombinant VNP20009 bacterium (VHH 15) expressing an anti-PD-L1 nano antibody VHH15, recombinant VNP20009 bacterium (VHH 22) expressing an anti-PD-L1 nano antibody VHH22 and recombinant VNP20009 bacterium (VHH 28) expressing an anti-PD-L1 nano antibody VHH 28. The PBS group was injected intraperitoneally with 100. mu.L PBS; VNP20009 and recombinant salmonella strain thereof are injected into abdominal cavity by 5 x 10⁵CFU bacteria (bacterial OD)₆₀₀=0.6-0.8, in exponential growth phase, dissolved in 100 μ L PBS). Tumor volume was measured every two days from the time of intraperitoneal administration, and the survival of the mice during treatment was recorded. Mouse tumor volume growth curves (figure 1) and mouse survival time curves (figure 2) were plotted.

VNP20009 expressing PD-L1 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 tumor doubling time was 2.48 days for the PBS group, 3.25 days for the VNP group, 3.82 days for the VHH15 group, 3.98 days for the VHH22 group, and 3.45 days for the VHH28 group (fig. 3). Compared with the PBS group, the tumor doubling time of the VNP group was 31.5%, the tumor doubling time of the VHH15 group was 54.0%, the tumor doubling time of the VHH22 group was 60.5%, and the tumor doubling time of the VHH28 group was 39.1%. Compared with the VNP group, the tumor doubling time was increased by 17.5% in the VHH15 group, 21.5% in the VHH22 group, and 6.2% in the VHH28 group.

The tumor growth delay time was 8.12 days for the PBS group, 11.28 days for the VNP group, 13.12 days for the VHH15 group, 13.98 days for the VHH22 group, and 12.45 days for the VHH28 group (fig. 4). Compared with the PBS group, the tumor growth delay time of the VNP group was 38.9%, the tumor growth delay time of the VHH15 group was 61.6%, the tumor growth delay time of the VHH22 group was 72.2%, and the tumor growth delay time of the VHH28 group was 53.3%. Compared with the VNP group, the tumor growth delay time was 16.3% for the VHH15 group, 23.9% for the VHH22 group, and 10.4% for the VHH28 group.

The anti-tumor effect of VNP20009 expressing three PD-L1 nano antibodies is obviously better than that of VNP20009, so that the expression of the PD-L1 nano antibody really endows the VNP20009 with stronger anti-tumor effect.

VNP20009 expressing PD-L1 nano antibody has the characteristic that VNP20009 can be applied to the preparation of medicines for treating tumors by combining conventional chemotherapy, traditional Chinese medicines and biological medicines.

Test example 2

Recombinant VNP20009 strain expressing PD-L1 antibody in tissue distribution of 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 the difference of the distribution quantity of VNP20009 and three recombinant salmonella strains thereof in mouse tissues is comparatively analyzed (figure 5).

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. The bacterial titers in tumor tissues were: VNP group: 9.375 parts; VHH15 set: 9.2825, respectively; VHH22 group: 9.55; VHH28 group: 9.356. bacterial titers in the liver were VNP group: 6.175, respectively; VHH15 set: 5.95; VHH22 group: 6.1; VHH28 group: 5.925. the bacterial titers in the spleen were: VNP group: 7.05; VHH15 set: 6.725, respectively; VHH22 set: 6.875; VHH28 group: 6.75. tumors of bacteria: the targeting of the liver is as follows: VNP group: 1585 times of; VHH15 group: 2150 times; VHH22 set: 2818 times of the total weight; VHH28 group: 2698 times. Tumors of bacteria: the targeting of the spleen is as follows: VNP group: 211 times; VHH15 set: 361 times; VHH22 group: 473 times; VHH28 group: 403 times. It can be seen that expression of the PD-L1 nanobody produces an unexpected effect: after the PD-L1 nano antibody is expressed, the tumor/liver targeting of VNP20009 is improved by 36-78%; the targeting of the tumor/spleen of the VNP20009 is improved by 71-124%, so that the anti-tumor effect of the VNP20009 expressing the PD-L1 nano antibody can be improved, and the toxic and side effects on normal organs such as liver, spleen and the like are reduced.

The foregoing shows and describes the general 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 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.

Sequence listing

<110> Jiangsu target biomedical research institute Co., Ltd

<120> recombinant attenuated salmonella expressing anti-PD-L1 nano antibody and preparation method and application thereof

<130> 2022

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial sequence (upstream primer for amplifying PD-L1 nanometer antibody gene fragment)

<400> 1

caggtgcagc tggtggagtc tgggg 25

<210> 2

<211> 25

<212> DNA

<213> Artificial sequence (downstream primer for amplifying PD-L1 nanometer antibody gene fragment)

<400> 2

tgaggagacg gtgaccaggg tcccc 25

<210> 3

<211> 50

<212> DNA

<213> Artificial sequence (upstream primer for amplifying pTh30 vector fragment)

<400> 3

ggggaccctg gtcaccgtct cctcaggatc cactagtacc aggcatcaaa 50

<210> 4

<211> 50

<212> DNA

<213> Artificial sequence (downstream primer for amplification of pTh30/pTh31/pTh32 vector fragment)

<400> 4

ccccagactc caccagctgc acctggctgc ctccacctcc gctgccgcca 50

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence (downstream primer for amplifying PD-L122 nanometer antibody gene fragment)

<400> 5

tgaggagacg gtgacctggg tccc 24

<210> 6

<211> 47

<212> DNA

<213> Artificial sequence (upstream primer for amplification of pTh31 vector fragment)

<400> 6

ggacccaggt caccgtctcc tcaggatcca ctagtaccag gcatcaa 47

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence (downstream primer for amplifying PD-L128 nanometer antibody gene fragment)

<400> 7

tactagtgga tcctgaggag acgg 24

<210> 8

<211> 49

<212> DNA

<213> Artificial sequence (upstream primer for amplification of pTh32 vector fragment)

<400> 8

ccgtctcctc aggatccact agtaccaggc atcaaataaa acgaaaggc 49

<210> 9

<211> 127

<212> PRT

<213> Artificial sequence (PD-L115 nanometer antibody amino acid sequence)

<400> 9

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Thr Ser Asn

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Tyr Thr Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Gly Asp Leu Asp Pro Ala Glu Tyr Ser Asp Tyr Asp Pro Thr

100 105 110

Val Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 10

<211> 381

<212> DNA

<213> Artificial sequence (PD-L115 nanometer antibody nucleotide sequence)

<400> 10

caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggaggggc tctgagactc 60

tcctgtgcag cctctggata cacctccact agcaactgca tgggctggtt ccgccaggct 120

ccagggaagg agcgcgaggg ggtcgcagct atttatactg gtggtggtag cacatactat 180

gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacggtgtat 240

ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc ggcaggggat 300

ttggaccccg ccgaatatag cgactatgac cctaccgtct ttaactactg gggccagggg 360

accctggtca ccgtctcctc a 381

<210> 11

<211> 118

<212> PRT

<213> Artificial sequence (PD-L122 nanometer antibody amino acid sequence)

<400> 11

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Gly Ser

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu His Cys Thr Gly Ser Trp Ala Leu Ile Met Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser

115

<210> 12

<211> 354

<212> DNA

<213> Artificial sequence (PD-L122 nanometer antibody nucleotide sequence)

<400> 12

caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgacactc 60

tcctgtgtag cctctgggtt cacttttgat ggttctgaca tgggctggta ccgccaggct 120

ccagggactg agtgcgagtt ggtgtcaact attagtagtg atggaagcac atactatgca 180

gactccgtga agggccgatt caccatctcc caagacaacg ccaagaacac ggtgtatctg 240

caaatgaaca gcctgaaacc tgaggacacg gccgtgtatt actgtgcgcg acttcactgc 300

acgggtagct gggccttgat aatgggccag gggacccagg tcaccgtctc ctca 354

<210> 13

<211> 127

<212> PRT

<213> Artificial sequence (PD-L128 nanometer antibody amino acid sequence)

<400> 13

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Gly Ser

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu Val

35 40 45

Ser Thr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Arg Leu Pro His Ile Asp Val Val Ala Thr Ala Lys Gly Cys Lys

100 105 110

Ala Asn Ser Tyr Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 14

<211> 381

<212> DNA

<213> Artificial sequence (PD-L128 nanometer antibody nucleotide sequence)

<400> 14

caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60

tcctgtacag cctctggatt cacttttgat ggttctgaca tgggctggta ccgccaggct 120

ccagggactg agtgcgagtt ggtgtcaact attagtagtg atggtagcac atactatgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac actatatctg 240

caattgaaca gcctgaaacc tgaggacact gccgtgtatt actgtgcggc gaggctgccc 300

catattgatg tggtcgctac tgctaaggga tgtaaggcga acagctactt gggccagggg 360

acccaggtca ccgtctcctc a 381

Claims

1. An attenuated salmonella recombinant bacterium expressing an anti-PD-L1 nanobody, comprising: the attenuated salmonella recombinant strain expressing the anti-PD-L1 nano antibody is attenuated salmonella typhimurium VNP20009 carrying anti-PD-L1 nano antibody expression plasmid.

2. The attenuated recombinant strain of salmonella expressing anti-PD-L1 nanobody of claim 1, wherein: cloning the anti-PD-L1 nano antibody gene on an expression plasmid containing a signal peptide: j23100 constitutive promoter or NirB promoter or adhE promoter; the sequence of the anti-PD-L1 nano antibody is a nucleotide sequence shown in SEQ ID No.10, or SEQ ID No.12, or SEQ ID No. 14; the sequence of the anti-PD-L1 nano antibody is an amino acid sequence shown in SEQ ID No.9, or SEQ ID No.11, or SEQ ID No. 13.

3. The attenuated recombinant strain of salmonella expressing anti-PD-L1 nanobody of claim 1, wherein: 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.

4. The recombinant salmonella attenuated strain agent expressing an anti-PD-L1 nm antibody, prepared from the recombinant salmonella attenuated strain expressing an anti-PD-L1 nm antibody of claim 1.

5. The attenuated salmonella recombinant bacterial agent expressing an anti-PD-L1 nanobody according to claim 4, wherein the active ingredient is at least one of the following (a), (b) and (c):

(a) a fermentation culture of the attenuated salmonella recombinant strain of claim 1 expressing an anti-PD-L1 nanobody;

(b) the ultrasonically lysed supernatant of the recombinant salmonella cell expressing an anti-PD-L1 nanobody obtained in claim 1;

(c) the sonicated pellet of the recombinant Salmonella cell expressing anti-PD-L1 nanobody of claim 1.

6. The method for preparing the recombinant bacterium of salmonella attenuated with expression of anti-PD-L1 nanobody of claim 1, comprising the following steps:

(1) obtaining PD-L1 nano antibody gene;

(2) constructing pTh30-P15, pTh31-P22 and pTh32-P28 plasmids expressing PD-L1 nano antibodies; step1 designing homologous recombination primer; step2 PCR to obtain DNA fragment; step3, transferring the product into chemically competent cell of Escherichia coli DH5 alpha through chemical transformation;

(3) constructing an attenuated salmonella recombinant bacterium expressing an anti-PD-L1 nano antibody: and (3) electrically converting the expression plasmid into attenuated salmonella typhi VNP20009 to prepare the attenuated salmonella recombinant strain expressing the anti-PD-L1 nano antibody.

7. The method for preparing the recombinant Salmonella attenuated strain expressing an anti-PD-L1 nanobody as claimed in claim 6, characterized in that: in the step (1), a J23100 constitutive promoter or a NirB promoter or an adhE promoter is cloned on pTh01 plasmid respectively, and a conventional bacterial signal peptide is fused at the N end of an anti-PD-L1 nano antibody; in the step (2), step1 designs homologous recombination primers; step2 respectively uses pET32a (+) -P15, pET32a (+) -P22 and pET32a (+) -P28 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 3 min, 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 single clone to 3 mL LB culture solution containing kanamycin, shaking-culturing for 16 h at 37 ℃, and extracting plasmids pTh30-P15, pTh31-P22 and pTh32-P28 by using a plasmid miniextract kit; the plasmid was sent to sequencing company for sequencing and alignment to confirm the correct sequence.

8. The method for preparing the attenuated recombinant salmonella strain expressing an anti-PD-L1 nanobody of claim 7, wherein: in the step (3), plasmids pTh01 and pTh30-P15, pTh31-P22 and pTh32-P28 are respectively electroporated into electrocompetent cells of VNP20009, and the recombinant strain VNP20009 and the attenuated salmonella recombinant strain expressing anti-PD-L1 nano antibody are prepared:

step2 the recombinant plasmid was transformed into VNP20009 by electroporation: under the aseptic condition, 0.5-2 mug of constructed plasmid is added into an electrotransfer competence, and is transferred into an electrotransfer cup with the diameter of 2 mm for electric shock after being uniformly mixed, wherein the electrotransfer condition is 2000 kV, 25 mug F and 400 omega; the discharge time is 5 ms;

9. The use of the recombinant strain of Salmonella attenuated expressing the PD-L1 nanobody as claimed in any of claims 1 to 8 for the preparation of a medicament for the treatment of tumors, characterized in that the engineered recombinant attenuated Salmonella attenuated strain expressing the anti-PD-L1 nanobody produces a far superior effect to the synergistic effect of the combined use of the anti-PD-L1 nanobody and the recombinant attenuated Salmonella.

10. The use of the recombinant strain of salmonella expressing PD-L1 nanobody of any one of claims 1 to 8 for the preparation of a medicament for the treatment of tumors in combination with conventional chemotherapy, traditional Chinese medicine, or biological agents.