A genetically engineered bacterium that efficiently secretes nano-antibody drugs and its application

Abstract

The present invention discloses a genetically engineered bacterium that efficiently secretes nano-antibody drugs and its application, and relates to the field of bioengineering. The present invention takes into account the shortcomings of the prior art and successfully develops a nano-antibody bacterial delivery route based on periplasmic expression and outer membrane perforation mechanism. PelB The signal peptide locates the target nanobody to the periplasmic space of the engineered bacteria. The reducing periplasmic environment is conducive to the formation of disulfide bonds in the nanobody and the functional stability. TollAIII The protein "punches holes" in the bacterial outer membrane, thereby increasing the release of nanoantibodies in the periplasmic space into the extracellular environment. This delivery strategy is effective in both Escherichia coli and Salmonella chassis. At the same time, this delivery strategy is easy to combine with other gene switches such as quorum response switches to achieve efficient nanoantibody release, and has good applicability and ease of use.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a genetically engineered bacterium capable of efficiently secreting a nano antibody drug and an application thereof. Background Art

[0002] In recent years, bacterial therapy has been widely studied as an emerging anti-tumor therapy. Escherichia coli Nissle 1917 and Salmonella typhimurium have the ability to target tumors and kill tumor cells, and are very potential tumor treatment carriers. At the same time, simple Escherichia coli or Salmonella cannot completely inhibit tumor growth. In order to improve the anti-tumor efficacy, synthetic biology methods can be used to transform bacteria so that they can selectively colonize in tumor tissues, produce and release anti-tumor effector molecules, and maintain high levels of anti-tumor drugs in the tumor site for a long time, inhibiting tumor growth. At the same time, anti-tumor drugs do not diffuse into other normal tissues of the body, avoiding the problem of some anti-tumor drugs having systemic toxicity to the body.

[0003] How to achieve efficient release of anti-tumor drugs at the tumor site by engineered bacteria has always been the focus of bacterial therapy research. Currently, the type III secretion system (T3SS) of Gram-negative bacteria is widely used in bacterial therapy. The complete type III secretion structure is assembled, first embedded in the outer membrane of the bacteria, and then its needle-like extension is connected to the host cell membrane and forms pores in the host cell membrane, delivering the protein directly to the cytosol of the host cell. T3SS-type secretion mechanisms have been developed for Escherichia coli and Salmonella (Lynch, Jason P., et al. "Engineered Escherichia coli for the in situ secretion of therapeutic nanobodies in the gut." Cell Host&Microbe 31.4 (2023): 634-649; Chabloz, Antoine, et al. "Salmonella-based platform for efficient delivery of functional binding proteins to the cytosol." Communications biology 3.1 (2020):342.), secreted drugs are used to treat diseases such as tumors or enteritis. However, the secretion mechanism is complex, requires a lot of bacterial modification, has a heavy chassis burden, and has low secretion efficiency. It is urgent to develop a more efficient secretion mechanism.

[0004] Nanobodies are found in the camelid family and contain only the variable region (VHH) of the heavy chain antibody HCAb. They are small in size, simple in structure, have high antigen binding affinity, and are highly stable under extreme conditions. Therefore, nanobodies have attracted great interest in various research fields, especially in the diagnosis and treatment of diseases. At the same time, nanobodies are suitable for efficient expression in prokaryotic systems and are widely used in bacterial therapy (Lynch, Jason P., et al. "EngineeredEscherichia coli for the in situ secretion of therapeutic nanobodies in thegut." Cell Host&Microbe 31.4 (2023): 634-649). Nanobodies have a small molecular weight and can be fused with other anti-tumor effector molecules to target multiple tumor targets and kill tumors more efficiently. However, the method of releasing nanoantibody drugs in bacterial therapy reported so far is to release them by engineered bacterial lysis, which has a low drug release efficiency. At the same time, in order to maximize the drug release efficiency of the group lysis circuit, it is necessary to precisely control the expression of each gene element in the circuit and coordinate the timing of bacterial lysis (Gurbatri, Candice R., et al. "Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies." Science translational medicine 12.530 (2020): eaax0876.). Therefore, when the bacterial chassis and release load are replaced, or when combined with other genetic circuits, the circuit may need to be re-optimized, which limits its wide applicability and ease of use, and other drug release methods still need to be developed.

[0005] Therefore, there is an urgent need to develop an engineered bacterium that can efficiently secrete nanoantibody drugs and apply it to bacterial therapy. Summary of the invention

[0006] The purpose of the present invention is to provide a genetically engineered bacterium that efficiently secretes nano-antibody drugs and its application. A fusion protein of a nano-antibody drug-pelB signal peptide of the present invention is co-expressed with the third topological domain (TolAIII) of the transmembrane protein TolA (having the function of punching holes in the outer membrane, named outer membrane punching protein TolAIII), to achieve a method for efficiently secreting and expressing nano-antibody drugs in Escherichia coli Nissle 1917 and Salmonella typhimurium strain SL7207. The constructed engineered bacteria that efficiently secrete nano-antibody drugs can be used as bacterial therapy and applied to tumor treatment, etc.

[0007] The specific technical solutions of the present invention are as follows:

[0008] First, the present invention provides a gene expression cassette for efficiently secreting a nano antibody drug, wherein the gene expression cassette for efficiently secreting a nano antibody drug comprises, in sequence, a gene encoding a signal peptide, a gene encoding a nano antibody drug, and a gene encoding a third topological domain of a transmembrane protein TolA; the signal peptide is a signal peptide Pel B, and the nano antibody drug is an immune checkpoint inhibitor.

[0009] Preferably, the coding gene sequence of the signal peptide Pel B is shown as SEQ ID NO.1, and the amino acid sequence of the third topological domain of the transmembrane protein TolA is shown as SEQ ID NO.5;

[0010] The coding gene sequence of the third topological domain of the transmembrane protein TolA is the 88-486 bp region shown in SEQ ID NO.2.

[0011] In some embodiments of the present invention, the immune checkpoint inhibitor selects anti-PD L1 nanoantibody or anti-CD47 nanoantibody as an example, but is not limited thereto; wherein the encoding gene sequence of the anti-PD L1 nanoantibody is shown in SEQ ID NO.3, and the encoding gene sequence of the anti-CD47 nanoantibody is shown in SEQ ID NO.4.

[0012] In some embodiments of the present invention, the 3' end of the gene encoding the Nanobody drug is also connected to a gene encoding an HA tag, and the sequence refers to the patent publication number CN116143945A.

[0013] On the other hand, the present invention also provides a recombinant expression vector encoding the gene expression cassette. A vector containing the Plac / ara-1 promoter and the Axe-Txe system is selected to construct a vector containing both the Plac / ara-1 promoter and the Axe-Txe system. pelB and TolAIII PD L1 nanobody expression cassette.

[0014] That is, the expression vector of the recombinant expression vector is Plac / ara-1-AT, and the promoter of the recombinant expression vector is Plac / ara-1 promoter.

[0015] Furthermore, the present invention also provides a genetically engineered bacterium that efficiently secretes nano-antibody drugs, which is obtained by introducing the recombinant expression vector into a host cell.

[0016] Preferably, the host cell is Escherichia coli Nissle 1917 or Salmonella typhimurium ( Salmonella typhimurium )SL7207.

[0017] Nano-antibody drug N-terminal additionpelB Signal peptide, pelB The recombinant protein can be directed to the periplasm, and the signal peptide sequence will be removed after passing through the inner membrane. pelB The signal peptide sequence is shown in SEQ ID NO.1; Nanobody Drug- pelB Signal peptide fusion protein and outer membrane puncture protein TolAIII Co-expression, TolAIII It can "punch holes" in the outer membrane to promote the release of proteins in the periplasmic space to the extracellular space. TolAIII The sequence is shown in SEQ ID NO.2; Escherichia coli Nissle 1917 and Salmonella strain SL7207 were used as expression hosts, Plac / ara-1-AT was used as expression vector, and the nano antibody drug- pelB The HA tag is fused to the C-terminus of the signal peptide fusion protein to construct an engineered bacterium that can efficiently secrete and express nano-antibody drugs.

[0018] The present invention further provides a method for developing an engineered bacterium that efficiently secretes nanoantibody drugs, comprising the following steps:

[0019] 1. Synthesis pelB , TolAIII , PD L1 or CD47 nucleic acid sequence;

[0020] 2. The above nucleic acid sequence is connected to a vector to obtain an expression vector;

[0021] 3. Transform the expression vector into the recipient cell to obtain the engineered bacteria;

[0022] 4. Cultivate the engineered bacteria, collect the supernatant and bacteria, and detect the target protein.

[0023] In the present embodiment, (1) in the construction of the expression vector in step 2, a vector containing the Plac / ara-1 promoter and the Axe-Txe system is selected to construct a vector containing both the Plac / ara-1 promoter and the Axe-Txe system. pelB and TolAIII PD L1 Nanobody Expression Cassette; only contains pelB , not including TolAIII PD L1 Nanobody Expression Cassette; Does Not Contain pelB , which only contains TolAIII PD L1 nanobody expression cassette; pelB and TolAIII None of them contain the PD L1 nanobody expression cassette;

[0024] (2) In step 2, the expression vector is constructed by selecting a vector containing the Plac / ara-1 promoter and the Axe-Txe system. pelB and TolAIIIThe PD L1 nanobody expression cassette was constructed, and IPTG-induced expression, constitutive expression, and quorum sensing-induced expression vectors were constructed.

[0025] (3) Construction of the expression vector in step 2, adding an HA tag to the C-terminus of the PD L1 nanobody;

[0026] (4) The recipient cell in step 3 is Escherichia coli Nissle 1917 or Salmonella SL7207;

[0027] (5) The transformation process in step 3 is as follows: take 50 μL of freshly prepared competent cells, add 0.01 ng DNA, mix gently, and electroporate in a 0.2 cm electroporation cup at 2.5 KV for 5 ms; after electroporation, immediately resuspend the bacteria in 950 μL of low-salt LB culture medium, culture for 45 min-1 h to revive the bacteria, and take 10 μL (1%) and 100 μL (10%) to plate (low-salt LB plate containing 50 μg / ml Zeocin antibiotics and 0.2% glucose);

[0028] (6) Step 4 specifically comprises: picking a single clone obtained in step 3, inoculating it into 3 ml of low-salt LB liquid medium containing 50 μg / ml Zeocin antibiotics and 0.2% glucose, and culturing it at 37°C, 220 rpm, overnight; inoculating the overnight bacterial liquid into 50 ml of LB liquid medium containing the corresponding antibiotics and 0.2% glucose at a ratio of 1:100, and culturing it at 37°C, 220 rpm, until OD 600 The concentration of IPTG was 0.5-0.8. IPTG was added to a final concentration of 1mM and arabinose to a final concentration of 0.5%. The mixture was induced at 37°C and 160rpm for 6h. The induced E. coli culture was centrifuged at 5000rpm for 15min. The cells were resuspended with an equal volume of PBS and the cell proteins were collected for detection. The supernatant was collected and centrifuged again at 13000rpm for 15min. The supernatant was filtered with a 0.2μm filter membrane and the secreted protein was collected for detection.

[0029] The present invention also provides the use of the coding gene expression cassette of the nano-antibody, the recombinant expression vector or the genetically engineered bacteria in the preparation of anti-tumor drugs.

[0030] The present invention also provides an anti-tumor drug composition, the active ingredients of which include the genetically engineered bacteria, which can be injected intravenously.

[0031] The present invention also provides a method for preparing a nano-antibody drug, comprising the following steps: inoculating the constructed expression strain into 3 ml of a low-salt LB liquid culture medium containing 50 μg / ml Zeocin antibiotics and 0.2% glucose, and culturing at 37°C, 220 rpm, overnight; inoculating the overnight bacterial solution into 50 ml of a LB liquid culture medium containing corresponding antibiotics and 0.2% glucose at a ratio of 1:100, and culturing at 37°C, 220 rpm, until OD 600 If induction is required, add IPTG to a final concentration of 1mM and arabinose to a final concentration of 0.5%, 37°C, 160rpm, and induce for 6h; for constitutively expressing engineered bacteria, after transferring the overnight bacterial solution, culture to an OD of 600 The samples were collected for testing when the pH value was 0.5-0.8. For quorum sensing induced expression engineered bacteria, the samples were collected for testing after 6-12 hours of culture after the overnight bacterial solution was transferred.

[0032] The induced E. coli liquid was centrifuged at 5000 rpm for 15 min. The cells were resuspended with an equal volume of PBS and the cell proteins were collected for detection. The supernatant was collected and centrifuged again at 13000 rpm for 15 min. The supernatant was filtered with a 0.2 μm filter membrane and the secretory expression protein was collected for detection.

[0033] The present invention takes into account the shortcomings of the prior art and successfully develops a nano-antibody bacterial delivery route based on periplasmic expression and outer membrane perforation mechanism. PelB The signal peptide locates the target nanobody to the periplasmic space of the engineered bacteria. The reducing periplasmic environment is conducive to the formation of disulfide bonds in the nanobody and the functional stability. TolAIII The protein "punches holes" in the bacterial outer membrane, thereby increasing the release of nanoantibodies in the periplasmic space into the extracellular environment. This delivery strategy is effective in both Escherichia coli and Salmonella chassis. At the same time, this delivery strategy is easy to combine with other gene switches such as quorum response switches to achieve efficient nanoantibody release, and has good applicability and ease of use. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the construction of an engineering bacterial expression module for efficient secretion of nanoantibody drugs (taking PD L1 nanoantibody as an example) (A) and pelB Signal peptide and TolAIII Schematic diagram of the efficient secretion mechanism coordinated by outer membrane pore-perforating proteins (B);

[0035] Figure 2 For existence pelB Signal peptide and TolAIIISchematic diagram of the construction of different combination expression modules of outer membrane pore-forming proteins (A) and Western Blot detection results of Escherichia coli Nissle 1917 engineered bacteria induced to express proteins with different expression modules (B), where M is protein marker, S is bacterial culture fluid, and C is bacterial cells;

[0036] Figure 3 This is a Western Blot test result of the protein induced by the E. coli Nissle 1917 engineered bacteria that efficiently secretes nanoantibody drugs (using CD47 nanoantibody as an example), where M is the protein marker, S is the bacterial culture solution, and C is the bacteria;

[0037] Figure 4 The Western Blot test results of the Salmonella strain SL7207 engineered bacteria that efficiently secretes nanoantibody drugs (taking PD L1 nanoantibody as an example) to induce protein expression, where M is the protein marker, S is the bacterial culture solution, and C is the bacteria;

[0038] Figure 5 Schematic diagram of the construction of the expression module of Escherichia coli Nissle 1917 engineered bacteria for efficient secretion of nano-antibody drugs (taking PD L1 nano-antibody as an example) under different induction mechanisms (A) and the Western Blot detection results of the engineered bacteria expressing proteins with different expression modules (B), where M is the protein marker, S is the bacterial culture solution, and C is the bacteria;

[0039] Figure 6 Figure 3 is the tumor immunotherapy effect of the new nanoantibody delivery route; A is the MC38 tumor model established in C57BL6 mice and the different treatments of the mice; B is the tumor growth curve of different groups (n=6); C is the summary of individual mouse tumor growth curves; D is the tumor picture of the control group and each treatment group at the experimental endpoint. DETAILED DESCRIPTION

[0040] The present invention pelB Signal peptide and TolAIII The efficient secretion mechanism of outer membrane pore-perforating proteins is shown below: Figure 1 As shown in B, using PelB The signal peptide locates the target nanobody to the periplasmic space of the engineered bacteria. The reducing periplasmic environment is conducive to the formation of disulfide bonds in the nanobody and the functional stability. TolAIII The protein "punches holes" in the bacterial outer membrane, thereby enhancing the release of nanoantibodies in the periplasmic space into the extracellular environment.

[0041] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0042] Example 1: Construction of an engineered bacterium that efficiently secretes nanoantibody drugs

[0043] Nanobody drug N-terminal addition pelB The signal peptide nucleic acid sequence is shown in SEQ ID NO.1; outer membrane pore-perforating protein TolAIII The nucleic acid sequence is shown in SEQ ID NO.2, wherein the 1-87 bp region is a signal peptide sequence, and the rest is an outer membrane pore protein TolAIII The coding gene sequence; the nucleic acid sequence of the codon-optimized PD L1 nanobody is shown in SEQ ID NO.3.

[0044] Will pelB Signal peptide sequence and outer membrane puncture protein TolAIII The sequence and the codon-optimized PD L1 nanobody sequence were constructed into the expression vector Plac / ara-1-AT (map and sequence refer to the patent publication number CN116143945A) containing the Plac / ara-1 promoter and Axe-Txe system. Figure 1 As shown in A, PD L1 nanobody expression vectors with or without pelB and TolAIII combination were constructed (including pelB , does not include TolAIII The PD L1 nanobody expression vector does not contain pelB , only contains TolAIII PD L1 nanobody expression vector, pelB and TolAIII The PDL1 nanobody expression vector that does not contain any of them, the specific schematic diagram is as follows Figure 2 coli Nissle 1917.

[0045] The constructed expression strain was inoculated into 3 ml of low-salt LB liquid medium containing 50 μg / ml Zeocin antibiotics and 0.2% glucose, and cultured overnight at 37°C, 220 rpm; the overnight bacterial solution was inoculated into 50 ml of LB liquid medium (Tryptone 10 g / L, Yeast extract 5 g / L, NaCl 10 g / L) containing the corresponding antibiotics and 0.2% glucose at a volume ratio of 1:100, and cultured at 37°C, 220 rpm until OD600 was 0.5-0.8. IPTG was added to a final concentration of 1 mM, and arabinose was added to a final concentration of 0.5%, and induced at 37°C, 160 rpm for 6 hours.

[0046] The induced E. coli liquid was centrifuged at 5000 rpm for 15 min. The cells were resuspended with an equal volume of PBS and the cell proteins were collected for detection. The supernatant was collected and centrifuged again at 13000 rpm for 15 min. The supernatant was filtered with a 0.2 μm filter membrane and the secretory expression protein was collected for detection.

[0047] After completing SDS-PAGE protein electrophoresis, Western Blot identification of the target protein was performed. Methanol activated the PVDF membrane for 2-4 minutes, and the sponge, filter paper and gel were soaked in transfer solution. The transfer conditions of the wet transfer electrophoresis instrument were: 350mA, 30min. The membrane was washed 3 times with tris buffer (TBST) containing 0.1% Tween20, 10min each time; after adding blocking solution (1×TBS+0.5% skim milk powder+0.05% tween20), the membrane was blocked at room temperature for 4 hours, and then the membrane was washed 3 times with TBST, 10min each time; 1:3000 mouse anti-HA primary antibody dilution was added, and it was kept at 4℃ overnight. The membrane was washed 3 times with TBST, 10min each time, and 1:5000 secondary antibody reaction solution (horseradish peroxidase labeled goat anti-mouse secondary antibody, invitrogen, cat#:31430) was added, and it was kept at room temperature for 1h, and the membrane was washed 3 times with TBST, 10min each time. A solution and B solution were mixed in a ratio of 1:1, dropped onto the PVDF membrane, and photographed. Figure 2 B shows, no pelB ,none TolAIII When , PDL1 nanobody can only be detected in bacteria; pelB ,have TolAIII and have pelB ,none TolAIII When , PD L1 nanobody can only be detected in the bacteria, and no PD L1 nanobody is secreted in the supernatant; pelB ,have TolAIII , PD L1 nanobody secretion can be detected in the supernatant; pelB Signal peptide and TolAIII An efficient secretion strategy for the combination of outer membrane pore-perforating proteins. pelB Signal peptide and outer membrane puncture protein TolAIII The PD L1 nanobody expression vector showed high secretory expression in Escherichia coli Nissle 1917.

[0048] Example 2: Test on the effect of secretion of different types of nanobodies by efficient secretion system

[0049] The nucleic acid sequence of the codon-optimized CD47 nanobody is shown in SEQ ID NO.4.

[0050] The PD L1 nanobody sequence in the expression vector constructed in Example 1 was replaced with the CD47 nanobody sequence, and thepelB Signal peptide, yes or no TolAIII The expression vectors of the outer membrane perforating protein were transformed into Escherichia coli Nissle 1917, and the secretion effect of CD47 nanobody was tested in the same way as in Example 1. Figure 3 As shown, there are pelB ,none TolAIII When , a trace amount of CD47 nanoantibody secretion can be detected in the supernatant; pelB ,have TolAIII When the CD47 nanobody was secreted, a large amount of CD47 nanobody secretion was detected in the supernatant; pelB Signal peptide and TolAIII The applicability of the efficient secretion strategy of outer membrane perforating protein combination to other types of nanobodies.

[0051] Example 3: Effect test of efficient secretion system in different engineered bacteria chassis

[0052] The PD L1 nanobody expression vector constructed in Example 1 was selected to construct pelB Signal peptide, yes or no TolAIII The expression vector of the outer membrane perforating protein was transformed into the Salmonella typhimurium strain SL7207 to test the secretion effect of the PD L1 nanobody. The test method was the same as in Example 1. The test results are shown in Figure 4 As shown, there are pelB ,none TolAIII When , trace amounts of PD L1 nanobody secretion could be detected in the supernatant; pelB ,have TolAIII When the PD L1 nanobody was secreted, a large amount of PD L1 nanobody could be detected in the supernatant; pelB Signal peptide and TolAIII The applicability of the efficient secretion strategy of outer membrane perforating protein combinations to other types of engineered bacterial chassis.

[0053] Example 4: Effect test of coupling efficient secretion system with different gene circuit switches (constitutive expression, inducible expression, and population response expression)

[0054] The method constructed in Example 1 includes pelB Signal peptide and TolAIII The PD L1 nanoantibody expression vector of the outer membrane pore-perforating protein is coupled with different gene circuit switches to construct constitutive expression, inducible expression, and population response expression vectors. The schematic diagram of the module construction is shown in the figure. Figure 5 The successfully constructed vectors were transformed into E. coli Nissle1917, and the secretion effect of PD L1 nanobody was tested in the same way as in Example 1. The test results are shown in Figure 5 As shown in B, pelB Signal peptide and TolAIIIThe efficient secretion strategy of combining outer membrane perforating proteins with gene switches of different induction mechanisms can achieve the effective secretion of PD L1 nanoantibodies, which proves that this delivery strategy can be easily combined with other gene switches such as quorum response switches to achieve efficient nanoantibody release, and has good applicability and ease of use.

[0055] Example 5: Anti-tumor effect test of engineered bacteria that efficiently secrete nanoantibody drugs

[0056] Select the one constructed in Example 4 and include pelB Signal peptide and TolAIII The PD L1 nanoantibody population response expression vector of the outer membrane perforating protein was transformed into Escherichia coli Nissle 1917, and the successfully constructed engineering bacteria were streaked on a plate (a low-salt LB plate containing 50 μg / ml Zeocin antibiotics and 0.2% glucose). After overnight culture, a single clone was picked and inoculated into 3 ml of low-salt LB liquid medium containing 50 μg / ml Zeocin antibiotics and 0.2% glucose, and cultured overnight at 37°C, 220 rpm; the overnight bacterial solution was inoculated into 50 ml of LB liquid medium containing the corresponding antibiotics and 0.2% glucose at a ratio of 1:100, and cultured at 37°C, 220 rpm until the early logarithmic growth stage of the strain, and the bacteria were collected by centrifugation at 4500 rpm for 5 min, and resuspended with 10 ml of pre-cooled sterile PBS, and washed three times at 4500 rpm for 5 min. Finally, the bacterial solution was resuspended with pre-cooled sterile PBS to adjust the OD 600 =0.1. For the constructed MC38 mouse subcutaneous tumor model, 100 μl of engineered bacteria was injected into the tail vein, and then the tumor size of the mouse was measured regularly to observe the trend of tumor volume changes.

[0057] in, Figure 6 A shows the MC38 tumor model established in C57BL6 mice. When the tumor grew to about 100 mm 3 Around 20 ℃, 2 × 10 7 CFU of different engineered bacteria were used as controls. 100 μg αPD-L1 was injected intraperitoneally (injected once every 3 days, for a total of 4 times), and tumor growth was recorded. In the MC38 colorectal cancer subcutaneous tumor mouse model, the tumor treatment effects were compared by intravenous injection of engineered bacteria QStolA-pelB-PDL1 nb loaded with a population response-activated delivery line or intraperitoneal administration of PD-L1 monoclonal antibody. The results showed that the periplasmic guidance signal peptide PelB and the outer membrane perforation protein TolAIII are essential for the anti-tumor effect of engineered bacteria. The lack of PelB (G3 group) or TolAIII (G4 group) and the chassis bacteria control (G2 group) lacking the PD-L1 nb load could not effectively inhibit tumor growth ( Figure 6B), while a single intravenous injection of engineered bacteria loaded with a complete delivery system (Group G6) can achieve a tumor suppression effect comparable to that of PD-L1 monoclonal antibody (Group G5), achieving complete tumor regression in some mice (2 / 6) ( Figure 6 The above studies indicate that this novel nanoantibody delivery route has potential application prospects in tumor immunotherapy.

Claims

1. A gene expression cassette for efficiently secreting nanoantibody drugs, characterized in that: The encoding gene expression cassette for efficient secretion of nano antibody drugs includes, in sequence, a gene encoding a signal peptide, a gene encoding a nano antibody drug, and a gene encoding the third topological domain of the transmembrane protein TolA; the signal peptide is a signal peptide Pel B, and the nano antibody drug is an immune checkpoint inhibitor; The coding gene sequence of the signal peptide Pel B is shown in SEQ ID NO.1, and the amino acid sequence of the third topological domain of the transmembrane protein TolA is shown in SEQ ID NO.5; The coding gene sequence of the third topological domain of the transmembrane protein TolA is the 88-486 bp region shown in SEQ ID NO.2; The immune checkpoint inhibitor is an anti-PD L1 nanobody or an anti-CD47 nanobody, The coding gene sequence of the anti-PD L1 nanobody is shown in SEQ ID NO.3, and the coding gene sequence of the anti-CD47 nanobody is shown in SEQ ID NO.4; The encoding gene expression cassette for efficient secretion of nano antibody drugs also includes RBS33, RBS31 or RBS34.

2. The gene expression cassette encoding the Nanobody according to claim 1, characterized in that The 3' end of the gene encoding the nanobody drug is also connected to a gene encoding an HA tag.

3. A recombinant expression vector comprising the encoding gene expression cassette according to claim 1 or 2.

4. The recombinant expression vector according to claim 3, characterized in that The expression vector of the recombinant expression vector is Plac / ara-1-AT, and the promoter of the recombinant expression vector is Plac / ara-1 promoter.

5. A genetically engineered bacterium that efficiently secretes nanoantibody drugs, characterized in that: The method is obtained by introducing the recombinant expression vector as claimed in claim 3 into a host cell.

6. The genetically engineered bacterium according to claim 5, characterized in that The host cell is Escherichia coli Nissle1917 or Salmonella typhimurium ( Salmonella typhimurium )SL7207.

7. Use of the encoding gene expression cassette according to claim 1 or 2, the recombinant expression vector according to claim 3 or the genetically engineered bacteria according to claim 5 in the preparation of antitumor drugs.

8. An anti-tumor drug composition, characterized in that: The active ingredient comprises the genetically engineered bacteria according to claim 5 or 6.

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