A recombinant attenuated Salmonella expressing tumor endostatin Tum5 and its preparation method and application

Abstract

The present invention discloses an attenuated Salmonella recombinant bacterium expressing tumor endostatin Tum5, and a preparation method and application thereof. The attenuated Salmonella recombinant bacterium expressing tumor endostatin Tum5 of the present invention is an attenuated Salmonella typhimurium VNP20009 carrying a Tum5 expression plasmid. Tumor endostatin can target tumors through attenuated Salmonella and has anti-tumor efficacy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an attenuated Salmonella recombinant bacterium expressing tumor endostatin Tum5, and a preparation method and application thereof. Background Art

[0002] Tumor growth and metastasis depend on the formation of new blood vessels to ensure a continuous supply of oxygen and nutrition. Tumor cells grow autonomously, much faster than the formation of new blood vessels. This leads to insufficient supply of nutrition and oxygen to the new blood vessels. Therefore, some tumor cells have low blood flow, forming hypoxic necrotic zones. More and more studies have found that some obligate or facultative anaerobic bacteria can specifically target tumor tissues, grow and reproduce in their hypoxic or necrotic zones, and show the effect of inhibiting tumor cell growth. Salmonella is such a class of tumor-targeting bacteria. VNP20009 is Salmonella typhimurium, and the toxicity is reduced by deleting the msbB and purI genes (Luo et al., Oncol Res, 2001, 12: 501-508). One of its main anti-tumor mechanisms is to inhibit tumor angiogenesis (Lee et al., Int J Cancer, 2008, 122:930-935). Tumor angiogenesis inhibition is an important anti-tumor strategy (Li et al., Oncol Lett, 2018, 16:687-702). Tumstatin is an endogenous angiogenesis inhibitor produced by type IV collagen, which can inhibit the proliferation of endothelial cells and promote their apoptosis (Floquet et al., J Biol Chem, 2004, 279:2091-2100; Van et al., Sci Rep, 2016, 6:26309; Maeshima et al., Science, 2002, 295:140-143). The anti-angiogenic activity center of Tumstatin is located in the amino acid sequence 54-132 fragment (referred to as Tum5 in the present invention) (Meng et al., J Biochem, 2006, 140:299-304; Maeshima et al., J Biol How to achieve the specific delivery of Tum5 in tumor tissues and how to further improve the anti-tumor efficacy of Tum5 are the bottlenecks for Tum5 to move towards anti-tumor applications. Summary of the invention

[0003] The purpose of the present invention is to solve the problems in the prior art and to propose an attenuated Salmonella recombinant bacterial strain expressing tumor endostatin Tum5 and a preparation method and application thereof.

[0004] In order to achieve the above-mentioned object, the present invention adopts the following technical scheme: the present invention provides a recombinant attenuated Salmonella expressing tumor endostatin Tum5, wherein the recombinant attenuated Salmonella expressing tumor endostatin Tum5 is an attenuated Salmonella typhimurium VNP20009 carrying a Tum5 expression plasmid.

[0005] Further, Tum5 is cloned on an expression plasmid containing a signal peptide: J23100 constitutive promoter or NirB promoter or adhE promoter; the gene sequence of 162-396 of Tumstatin is the nucleotide sequence shown in SEQ ID No.1; the amino acid sequence of 162-396 of Tumstatin amplified is the nucleotide sequence shown in SEQ ID No.2; the gene sequence of the upstream primer Tum-F for amplifying Tum5 is the nucleotide sequence shown in SEQ ID No.3; the gene sequence of the downstream primer Tum-R for amplifying Tum5 is the nucleotide sequence shown in SEQ ID No.4; the gene sequence of the upstream primer P21-F for amplifying pTh01 is the nucleotide sequence shown in SEQ ID No.5; the gene sequence of the downstream primer P21-R for amplifying pTh01 is the nucleotide sequence shown in SEQ ID No.6; the gene sequence of the universal sequencing upstream primer pTh01-F is the nucleotide sequence shown in SEQ ID No.7; the gene sequence of the universal sequencing downstream primer pTh01-R is the nucleotide sequence shown in SEQ ID The nucleotide sequence shown in No.8.

[0006] Furthermore, 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. Resistance screening gene.

[0007] Furthermore, after infecting mouse melanoma cells B16F10, the degree of apoptosis of tumor cells was significantly increased compared with the control bacteria without exogenous genes; after infecting mouse endothelial cells MUVEC, the cell migration ability was significantly decreased compared with the control strain without exogenous genes.

[0008] The attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 is prepared by the attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 of the present invention.

[0009] Furthermore, the active ingredient is at least one of the following (a), (b), and (c):

[0010] (a) fermentation culture of the attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5;

[0011] (b) the ultrasonic lysis supernatant of the obtained attenuated Salmonella recombinant bacterial cells expressing tumor endostatin Tum5;

[0012] (c) Ultrasonic lysis precipitate of the attenuated Salmonella recombinant bacterial cells expressing tumor endostatin Tum5.

[0013] The method for preparing the attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 of the present invention comprises: (1) obtaining the Tum5 gene;

[0014] (2) construct the plasmid pTh21-Tum5; step 1 design homologous recombination primers; step 2 PCR to obtain DNA fragments; step 3 chemically transform the ligated product into chemically competent cells of Escherichia coli DH5α;

[0015] (3) Construction of recombinant strain VNP-Tum5: The expression plasmid was electroporated into attenuated Salmonella typhi VNP20009.

[0016] Furthermore, in step (1), the J23100 constitutive promoter, the NirB promoter, or the adhE promoter was cloned on the pTh01 plasmid, and a conventional bacterial signal peptide was fused to the N-terminus of Tum5.

[0017] Further, in step (2), step 1 designs homologous recombination primers;

[0018] Step 2: Use pUC19-Tum5 as template to obtain the target gene fragment and pTh01 as template to obtain the plasmid backbone fragment. Cut out the gene product through 1.2% agarose gel electrophoresis and purify it with gel recovery purification kit to obtain DNA fragment. Connect the gel recovery products with double fragment homologous recombination kit. The connection conditions are: 4-8℃, 1-2h, or 37℃, 30min.

[0019] Step 3: Take 10 μL of the ligated product and transfer it to the chemically competent cells of Escherichia coli DH5α through chemical transformation; transformation conditions: ice for 10 min, heat shock at 42°C for 60 s, ice for 2 min, add 900 μL of non-resistant LB liquid culture medium, shake culture at 37°C for 45 min, centrifuge at 5000 rpm for 3 min, discard the supernatant, add 1 mL of non-resistant LB liquid culture medium and mix well, take 100 μL and spread on the resistance plate with kanamycin, and let it stand in a 37°C incubator overnight;

[0020] Step 4: Pick a single clone and transfer it to 3 mL of LB culture medium containing kanamycin, shake and culture at 37°C for 16 hours, and extract the plasmid pTh21-Tum5 using a plasmid extraction kit. Send the plasmid to a sequencing company for sequencing and comparison to confirm the correct sequence.

[0021] Further, in step (3), plasmids pTh01 and pTh21-Tum5 were electrotransformed into electrocompetent cells of VNP20009 to obtain recombinant strains VNP20009 and VNP-Tum5:

[0022] Step 1 Preparation of Salmonella electroporation competent cells: inoculate fresh attenuated Salmonella VNP20009 into 30 mL LB medium, culture at 37°C in a shaking incubator until the OD value is between 0.5-0.6, centrifuge at 5000 rpm at 4°C for 5 min to collect the cells, wash the cells 3 times with sterilized 10% glycerol, 10 mL each time, centrifuge at 5000 rpm at 4°C for 5 min, resuspend with 200 μL 10% glycerol, and dispense 50 μL / tube for electroporation;

[0023] Step 2: Use electroporation to transform the recombinant plasmid into VNP20009: Under sterile conditions, add 0.5-2μg of the constructed plasmid into the electroporation competent medium, mix well, and transfer to a 2mm electroporation cup for electroporation. The electroporation conditions are 2000kV, 25μF, 400Ω; the discharge time is 5ms;

[0024] After electroporation, add 900 μL of LB liquid medium without resistance, culture at 37°C with shaking for 45 min, centrifuge at 5000 rpm for 3 min, discard the supernatant, add 100 μL of LB liquid medium without resistance and mix well, draw 100 μL and spread on the resistance plate with kanamycin, and leave it in a 37°C incubator overnight;

[0025] Step 3: Spread on kanamycin plates for screening. The grown colonies are the constructed recombinant bacteria. Select the monoclonal strains for sequencing verification.

[0026] The invention discloses an application of the attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 in the preparation of tumor drugs.

[0027] Beneficial effects: Compared with the control strain without exogenous genes, it has better inhibition of endothelial cell migration and induction of tumor cell apoptosis, and at the same time, higher tumor targeting; it significantly inhibits tumor growth in tumor-bearing mice and prolongs the survival time of mice. The above-mentioned attenuated Salmonella recombinant strain can be administered to animals for treatment in various ways such as intraperitoneal, intravenous and oral administration.

[0028] The present invention is characterized in that:

[0029] (1) Attenuated Salmonella VNP20009 was selected as a vector to deliver the tumor endothelial inhibitor Tumstatin; VNP20009 is a facultative anaerobic strain and has the advantage of higher tumor targeting as a vector.

[0030] (2) Attenuated Salmonella VNP20009 was selected as a carrier to deliver the tumor endothelial inhibitor Tumstatin. VNP20009 is a low-toxic and easy-to-cultivate bacterium with low cost and has the prospect of large-scale promotion and application.

[0031] (3) The recombinant attenuated Salmonella strain VNP-Tum5 not only demonstrated its ability to kill tumor cells and inhibit the migration of endothelial cells in in vitro cell experiments, but also showed better anti-tumor effects in mouse models, showing potential therapeutic prospects for tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a diagram showing the therapeutic effect of the recombinant attenuated Salmonella VNP-Tum5 of the present invention on mouse melanoma; Figure 1A It is a mouse tumor growth curve diagram of the present invention; Figure 1B It is the mouse survival curve diagram of the present invention.

[0033] Figure 2 This is the tissue distribution diagram of the recombinant attenuated Salmonella VNP-Tum5 of the present invention in mouse melanoma.

[0034] Figure 3 The figure shows the killing effect of the recombinant attenuated Salmonella VNP-Tum5 of the present invention on mouse melanoma cells B16F10.

[0035] Figure 4 This is a diagram showing the killing effect of the recombinant attenuated Salmonella VNP-Tum5 of the present invention on mouse endothelial cells MUVEC. Figure 4 A is a scratch analysis diagram of the MUVEC cells of the present invention; Figure 4 B is a statistical analysis diagram of the degree of scratch healing of the MUVEC cells of the present invention. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. However, it is easy for those skilled in the art to understand that the contents described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims. If no specific conditions are specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0037] Example 1

[0038] Construction of homologous recombination plasmid pTh21-Tum5

[0039] Acquisition of Tum5 gene

[0040] First, a nucleic acid sequence of 162-396 (Tum5) of Tumstatin was synthesized and subcloned into pUC19 (GenScript), and then the Tum5 sequence was subcloned into the pTh01 plasmid by homologous recombination to obtain the Tum5 expression plasmid pTh21-Tum5. J23100 constitutive promoter, NirB promoter or adhE promoter were cloned on the pTh01 plasmid, and their expression effects were similar. 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, were fused to the N-terminus of Tum5, and their secretion effects were similar.

[0041] The specific process is as follows: design homologous recombination primers, use pUC19-Tum5 as a template for PCR to obtain the target gene fragment, use pTh01 as a template for PCR to obtain the plasmid backbone fragment, and after 1.2% agarose gel electrophoresis, cut out the gene product and purify the DNA fragment using a gel recovery purification kit (Sangon Biotechnology Co., Ltd.).

[0042] The products were recovered by gel ligation using a double fragment homologous recombination kit (Novagen) under the following ligation conditions: 4-8°C, 1-2h, or 37°C, 30min.

[0043] Take 10 μL of the ligated product and transfer it to the chemically competent cells of E. coli DH5α through chemical transformation. Transformation conditions: ice for 10 min, heat shock at 42°C for 60 s, ice for 2 min, add 900 μL of non-resistant LB liquid medium, shake culture at 37°C for 45 min, centrifuge at 5000 rpm for 3 min, discard the supernatant, add 1 mL of non-resistant LB liquid medium and mix well, take 100 μL and spread on the resistance plate with kanamycin, and let it stand overnight in a 37°C incubator.

[0044] Construction of plasmid pTh21-Tum5

[0045] A single clone was picked and placed in 3 mL of LB culture medium containing kanamycin, cultured at 37°C with shaking for 16 h, and plasmid pTh21-Tum5 was extracted using a plasmid extraction kit (Sangon Biotech). The plasmid was sent to a sequencing company (GenScript) for sequencing and comparison to confirm the correct sequence.

[0046] Example 2

[0047] Construction of recombinant strain VNP-Tum5

[0048] Plasmids pTh01 and pTh21-Tum5 were electrotransformed into electrocompetent cells of VNP20009 to obtain recombinant strains VNP20009 and VNP-Tum5; J23100 constitutive promoter and pelB signal peptide were used as examples. The specific process is as follows:

[0049] Preparation of Salmonella electroporation competent cells: inoculate fresh attenuated Salmonella VNP20009 into 30 mL LB medium, culture at 37°C in a shaking incubator until the OD value is between 0.5-0.6, centrifuge at 5000 rpm at 4°C for 5 min to collect the cells, wash the cells 3 times with sterilized 10% glycerol, 10 mL each time, centrifuge at 5000 rpm at 4°C for 5 min, resuspend with 200 μL 10% glycerol, and dispense into 50 μL / tube for electroporation.

[0050] The recombinant plasmid was transformed into VNP20009 by electroporation: under sterile conditions, 0.5-2 μg of the constructed plasmid was added to the electroporation competent medium, mixed, and transferred to a 2 mm electroporation cup for electroporation. The electroporation conditions were 2000 kV, 25 μF, and 400 Ω.

[0051] After electroporation, add 900 μL of non-resistant LB liquid culture medium, culture at 37°C with shaking for 45 min, centrifuge at 5000 rpm for 3 min, discard the supernatant, add 100 μL of non-resistant LB liquid culture medium and mix well, aspirate 100 μL and spread on a resistant plate with kanamycin, and leave in a 37°C incubator overnight.

[0052] The strain was spread on a kanamycin plate for screening, and the colonies that grew were the constructed recombinant bacteria. Monoclonal strains were selected for sequencing verification.

[0053] Example 3

[0054] Anti-tumor effect of VNP-Tum5 strain on mouse melanoma

[0055] B16F10 mouse melanoma cells were grown in DMEM cell culture medium until the exponential growth phase and then digested with 0.5% trypsin. The cells were then centrifuged at 1000 rpm / min for 3 min. The supernatant was removed and washed twice with PBS. The cells were counted and resuspended in PBS to adjust the final cell concentration to 2 × 10 6 Each C57BL / 6 mouse was inoculated with 100 μL of the culture medium into the axillary fat pad, i.e., 2×10 5 After inoculation, the mice were kept in a clean animal room until the tumor volume of the mice grew to about 100 mm 3 Follow-up experiments will be conducted.

[0056] C57 / B6 mice bearing B16F10 mouse melanoma were randomly divided into three groups: PBS, VNP20009, and VNP-Tum5. The PBS group was intraperitoneally injected with 100 μL PBS; VNP20009 and VNP-Tum5 were intraperitoneally injected with 5×105 CFU of bacteria (in the exponential growth phase, dissolved in 100 μL PBS). The tumor volume of each group of mice was measured every two days after intraperitoneal administration ( Figure 1A ) and recorded the survival of mice during the treatment process ( Figure 1B ). Draw the mouse tumor volume growth curve ( Figure 1A ) and mouse survival time curve ( Figure 1B ).

[0057] The tumor doubling time of the three groups of animals treated with PBS, VNP20009 and VNP-Tum5 were: PBS: 2.3 days; VNP20009: 4.6 days; VNP-Tum5: 7.3 days. The tumor doubling time of the VNP-Tum5 group was 630% longer than that of the PBS untreated group; the tumor doubling time of the VNP-Tum5 group was 58.70% longer than that of the VNP20009 treated group. In contrast, the tumor doubling time of the VNP20009 treated group was 100% longer than that of the PBS untreated group.

[0058] The growth delay time of animals in the PBS, VNP20009 and VNP-Tum5 groups were: PBS: 9.7d; VNP20009: 18.4d; VNP-Tum5: 23.2d. The growth delay time of the VNP-Tum5 group was 139.18% longer than that of the PBS untreated group; the growth delay time of the VNP-Tum5 group was 22.30% longer than that of the VNP20009 treated group. In contrast, the growth delay time of the VNP20009 treated group was 89.69% longer than that of the PBS untreated group.

[0059] The VNP-Tum5 group showed much better anti-tumor efficacy than the VNP20009 treatment group.

[0060] Example 4

[0061] Tissue distribution of VNP-Tum5 strain in tumor-bearing mice

[0062] On the fifth day of bacterial treatment of tumor-bearing mice, mice were randomly killed, and the tumors, livers and spleens of tumor-bearing mice were taken under sterile conditions, weighed and placed in 2 mL PBS for homogenization using a tissue homogenizer (frequency: 60 Hz; time: 100 s). Different tissues were diluted in different gradients and spread on LB plates and inverted in a 37°C bacterial incubator for 12 h. The colonies were counted and the quantitative differences between VNP20009 and VNP-Tum5 in mouse tissues were compared and analyzed ( Figure 2 ).

[0063] The bacterial targeting of tumor / liver in VNP20009 group was 1578.95 times; the bacterial targeting of tumor / liver in VNP-Tum5 group was 1740.91 times ( Figure 2 ). The bacterial targeting of tumor / spleen in the VNP20009 group was 1212.88 times; the bacterial targeting of tumor / liver in the VNP-Tum5 group was 1255.31 times ( Figure 2 ). Therefore, the tumor targeting of VNP-Tum5 bacteria is better than that of VNP20009 bacteria.

[0064] Example 5

[0065] Killing effect of VNP-Tum5 strain on melanoma cells B16F10

[0066] (1) Bacterial infection of melanoma B16F10 cells

[0067] One day before the experiment, melanoma cells B16F10 were plated in a 12-well plate, ensuring that the number of cells in each well was 10 5 , and cultured statically at 37°C and 5% CO2 for 12 hours; single clones of VNP20009 and VNP-Tum5 strains were picked and inoculated into 3 mL of fresh LB liquid medium and cultured at 37°C overnight; the next day, the single clone bacterial solution was transferred to fresh LB liquid medium and cultured for about 2 hours. When OD ≈ 0.6-0.8, centrifuged at 5000 rpm / min and 4°C for 5 minutes, the bacterial precipitate was collected, and the bacterial OD was adjusted to a consistent value of 0.5 with sterile PBS;

[0068] B16F10 cells were infected at an infection coefficient of MOI = 100:1 for 2 h and 4 h, washed 3 times with PBS, and cultured for another 1 h using DMEM medium containing 100 μg / mL gentamicin to kill bacteria outside the cells and adhering to the cell surface.

[0069] (2) Flow cytometry to detect cell apoptosis

[0070] After the cells were treated as described above, they were digested with 1× trypsin and centrifuged at 1500rpm / min at 4℃ for 5min to collect the cells. The cells were washed with pre-cooled PBS and centrifuged at 1500rpm / min at 4℃ for 5min to collect the cells. 1× Binding buffer containing AnnexinV-APC (1μL per sample) was added to resuspend the cells and reacted on ice in the dark for 30min. 1μLPI was added to each sample before loading, and cell apoptosis was detected using flow cytometry ( Figure 3 ).

[0071] The cell apoptosis rates of the three groups treated with PBS, VNP20009 and VNP-Tum5 for 2 hours were 6.6%, 16.47% and 19.09% respectively; among them, the late apoptosis rates were 4.61%, 11.7% and 14.4% respectively. Figure 3 ). The cell apoptosis rates of the three groups treated with PBS, VNP20009 and VNP-Tum5 for 4 hours were 7.76%, 30.47% and 57.70% respectively; among them, the late apoptosis rates were 5.9%, 25.8% and 29.4% respectively. The activity of VNP-Tum5 group in inducing tumor apoptosis was much stronger than that of VNP20009 group ( Figure 3 ).

[0072] Example 6

[0073] Inhibitory effect of VNP-Tum5 strain on migration of endothelial cells MUVEC

[0074] MUVEC cells were collected at 2×10 5 Cells / well were seeded into 6-well plates and cultured overnight with DMEM containing 10% FBS. After washing twice with PBS, the monolayer was scratched across the wells with a 10 μL standard pipette tip and then incubated with VNP20009 and VNP-Tum5 for 2 h in the absence of serum medium. Cell scratch healing was photographed at different time points ( Figure 4 ).

[0075] The cell scratch healing rate of the PBS group was 78.67%, the cell scratch healing rate of the VNP20009 group was 52.33%, and the cell scratch healing rate of the VNP-Tum5 group was 29.34%. Compared with the PBS group, the cell scratch healing rate of the VNP20009 group was reduced by 33.48%, and the cell scratch healing rate of the VNP-Tum5 group was reduced by 62.70% compared with the PBS group. Compared with the VNP20009 group, the cell scratch healing rate of the VNP-Tum5 group was reduced by 43.93% ( Figure 4 ). The VNP-Tum5 group had a better inhibitory activity on the migration of endothelial cells MUVEC than the VNP20009 group.

[0076] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A recombinant attenuated Salmonella expressing tumor endostatin Tum5, characterized in that: The attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 is attenuated Salmonella typhimurium VNP20009 carrying a Tum5 expression plasmid.

2. The attenuated Salmonella recombinant bacterium expressing tumor endostatin Tum5 according to claim 1, characterized in that: Tum5 is cloned on an expression plasmid containing a signal peptide: J23100 constitutive promoter or NirB promoter or adhE promoter; the nucleotide sequence of Tum5 is shown in SEQ ID No.1; the amino acid sequence of Tum5 is shown in SEQ ID No.

2.

3. The attenuated Salmonella recombinant bacterium expressing tumor endostatin Tum5 according to claim 2, characterized in that: The signal peptide is a conventional bacterial signal peptide, and the bacterial signal peptide is an 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; after infecting mouse melanoma cells B16F10, compared with a control bacteria without exogenous genes, the degree of apoptosis of tumor cells can be very significantly increased; after infecting mouse endothelial cells MUVEC, compared with a control strain without exogenous genes, the cell migration ability is significantly decreased.

4. The attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 prepared by the attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 according to claim 1.

5. The method for preparing the attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 according to claim 1, characterized in that The method comprises the following steps: (1) obtaining the Tum5 gene; (2) Construct the plasmid pTh21-Tum5; Step 1: Design homologous recombination primers; Step 2: PCR to obtain DNA fragments; Step 3: Chemically transform the ligated product into chemically competent cells of Escherichia coli DH5α; (3) Construction of recombinant strain VNP-Tum5: The expression plasmid was electroporated into attenuated Salmonella typhi VNP20009 to obtain attenuated Salmonella recombinant strain expressing tumor endostatin Tum5.

6. The method for preparing the attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 according to claim 5, characterized in that: In step (1), the J23100 constitutive promoter, the NirB promoter, or the adhE promoter was cloned on the pTh01 plasmid, and a conventional bacterial signal peptide was fused to the N-terminus of Tum5.

7. The method for preparing the attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 according to claim 6, characterized in that: In step (2), step 1 designs homologous recombination primers; Step 2: Use pUC19-Tum5 as template to obtain the target gene fragment by PCR, and use pTh01 as template to obtain the plasmid backbone fragment by PCR. Cut out the gene product after 1.2% agarose gel electrophoresis, and purify it with gel recovery purification kit to obtain DNA fragment; connect the gel recovery products with double fragment homologous recombination kit; the connection conditions are: 4-8℃, 1-2 hr, or 37℃, 30min; Step 3: Take 10 μL of the ligated product and transfer it to the chemically competent cells of Escherichia coli DH5α through chemical transformation; transformation conditions: ice for 10 min, heat shock at 42℃ for 60s, ice for 2 min, add 900 μL of non-resistant LB liquid culture medium, shake culture at 37℃ for 45 min, centrifuge at 5000 rpm for 3 min, discard the supernatant, add 1 mL of non-resistant LB liquid culture medium and mix well, take 100 μL and spread it on the resistance plate with kanamycin, and let it stand in a 37℃ incubator overnight; Step 4: Pick a single clone and transfer it to 3 mL of LB culture medium containing kanamycin, culture it at 37°C with shaking for 16 h, and extract the plasmid pTh21-Tum5 using a plasmid extraction kit; send the plasmid to a sequencing company for sequencing and comparison to confirm the correct sequence.

8. The method for preparing the attenuated Salmonella recombinant bacteria expressing tumor endostatin Tum5 according to claim 7, characterized in that: In step (3), plasmids pTh01 and pTh21-Tum5 were electroporated into electrocompetent cells of VNP20009 to obtain recombinant strains VNP20009 and VNP-Tum5: Step 1 Preparation of Salmonella electroporation competent cells: inoculate fresh attenuated Salmonella VNP20009 into 30 mL LB medium, culture at 37°C in a shaking incubator until the OD value is between 0.5-0.6, centrifuge at 5000 rpm at 4°C for 5 min to collect the cells, wash the cells three times with sterilized 10% glycerol, 10 mL each time, centrifuge at 5000 rpm at 4°C for 5 min, resuspend with 200 μL 10% glycerol, and dispense 50 μL / tube for electroporation; Step 2: Use electroporation to transform the recombinant plasmid into VNP20009: Under sterile conditions, add 0.5-2 μg of the constructed plasmid into the electroporation competent medium, mix well, and transfer to a 2 mm electroporation cup for electroporation. The electroporation conditions are 2000 kV, 25 μF, 400 Ω; the discharge time is 5 ms; After electroporation, add 900 μL of LB liquid medium without resistance, culture at 37°C with shaking for 45 min, centrifuge at 5000 rpm for 3 min, discard the supernatant, add 100 μL of LB liquid medium without resistance and mix well, draw 100 μL and spread on the resistance plate with kanamycin, and leave it in a 37°C incubator overnight; Step 3: Spread on kanamycin plates for screening. The colonies that grow are the constructed recombinant bacteria. Select monoclonal strains for sequencing verification.

9. Use of the attenuated recombinant Salmonella strain expressing tumor endostatin Tum5 according to claim 1 in the preparation of melanoma drugs.

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