Construction method of Hcp, the core component of bacterial T6SS, as a drug delivery carrier

Abstract

This invention discloses a method for constructing Hcp, a core component of bacterial T6SS, as a drug delivery carrier, belonging to the field of biotechnology. This invention experimentally demonstrates that fusion proteins of peptide or protein drugs and Hcp can be expressed and synthesized within bacteria and secreted extracellularly by carrying the aforementioned T6SS core component. This drug secretion system has significant application potential in delivering macromolecular drugs to eukaryotic cells and other organisms.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for constructing a drug delivery carrier using Hcp, a core component of the bacterial type VI secretion system (T6SS); more particularly, it relates to a method for constructing a system of peptide and protein drugs secreted by Hcp, a core component of T6SS. Background Technology

[0002] In recent years, the pharmaceutical industry has trended towards complex macromolecular therapies. Macromolecular drugs include protein-based therapies such as antibodies, hormones, growth factors, and cytokines, as well as nucleic acid-based therapies such as short interfering RNA, DNA / RNA vaccines, and gene therapy. The molecular size and complexity of macromolecular drugs give them high specificity, resulting in greater potency and fewer side effects compared to small molecule drugs. Nevertheless, macromolecular drugs still face several major challenges that small molecule drugs do not, one of the key issues that needs to be addressed is efficient drug delivery strategies.

[0003] Large molecule drugs are often easily degraded in the stomach and intestines, which limits the dosage form of these drugs. They can usually only be used as injections, which are far less convenient than oral administration. In addition, their large molecular weight and hydrophilicity greatly limit their penetration into biological membranes, resulting in low bioavailability.

[0004] Bacteria, as single-celled, structurally simple prokaryotes, exhibit a wide variety of species and metabolic types. Their rapid growth facilitates large-scale culture, and their gene expression regulation mechanisms are well-understood, making genetic manipulation easy to obtain various mutant strains. Using bacteria as drug delivery vectors can overcome the difficulties of purification and drug stability in the industrial production of macromolecular drugs. Furthermore, it allows for the modification and alteration of target proteins as needed, thereby improving drug efficacy. Secondly, with a deeper understanding of the mechanisms by which bacteria antagonize their natural hosts, researchers have discovered unique secretion systems in various bacteria that can directly deliver target proteins into the cytoplasm of target cells. The type VI secretion system (T6SS) is one such system. The T6SS is a large complex assembled from 13 components, primarily located within the bacterial cytoplasm. Its core secretory apparatus includes an inner secretory tube composed of Hcp hexamers, a contractile sheath composed of TssB and TssC proteins (also known as VipA and VipB), and VgrG and PAAR forming a "spike" at the tip of the Hcp tube. During sheath contraction, the Hcp tube and tip protein pierce the cell membrane and are injected into the target cell. Currently, studies have successfully fused lactamases with T6SS for delivery assays in eukaryotic cells. Furthermore, research has shown that T6SS from Vibrio cholerae has successfully delivered Cre recombinase to recipient bacteria for gene editing without introducing exogenous DNA. This system can also inject the exogenous antimicrobial toxin TseC into adjacent Pseudomonas aeruginosa bacteria, killing it. However, there are currently no reports on using T6SS to deliver marketed or investigational peptide and protein drugs. Summary of the Invention

[0005] Based on the above technical problems, the purpose of this invention is to provide a method for constructing Hcp, a core component of bacterial T6SS, as a drug delivery carrier.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for constructing Hcp, a core component of the type VI secretion system T6SS, as a carrier for polypeptide and protein drugs. The method includes: integrating the target drug sequence into the bacterial genome and linking it with the Hcp protein sequence in Vibrio cholerae V52 strain to obtain a system for secreting polypeptide and protein drugs.

[0008] As one embodiment of the present invention, the Vibrio cholerae V52 strain is a mutant strain of Vibrio cholerae V52 with several T6SS toxic proteins inactivated and mutated, or a mutant Vibrio cholerae V52 strain with the Hcp2 coding sequence knocked out. The several T6SS toxic proteins include TseL, VasX, TseH, and VgrG1. In one specific implementation example, the Vibrio cholerae V52 strain is derived from Vibrio cholerae V52 "RHH" (ΔhlyA(VCA0219), ΔhapA(VCA0865), ΔrtxA(VC1451)) as the original strain. The aspartic acid at position 425 of the effector protein TseL(VC1418) is mutated to alanine, and the histidine at position 64 of the effector protein TseH(VCA0285) is mutated to alanine. Furthermore, 16 amino acids from positions 852 to 867 of the effector protein VasX(VCA0020) and 434 amino acids from positions 716 to 1149 of the C-terminal actin crosslinking domain (ACD) of VgrG1(VC1416) are knocked out, resulting in a background strain. Alternatively, a mutant Vibrio cholerae V52 strain with the Hcp2 coding sequence further knocked out can be obtained from this mutant strain.

[0009] As one embodiment of the present invention, the target drug fragment containing the homologous arm sequence is first cloned into the suicide plasmid pDS132 using the Goldengate cloning method; then the verified plasmid is transformed into the donor Escherichia coli WM6026, and the plasmid is transferred into V. cholerae V52 through conjugation transfer; the target drug protein sequence is inserted after the hcp1 gene in the V. cholerae V52 genome using the homologous recombination double exchange method to achieve fusion expression.

[0010] As one embodiment of the present invention, the construction method includes the following steps:

[0011] S1. Using Vibrio cholerae V52 RHH strain as a template, the upstream homologous left arm and the downstream homologous right arm of the mutant hcp1 were amplified respectively; the amplified left and right arms were ligated, and the ligated homologous arm fragments were cloned into the suicide plasmid pDS132; thus obtaining the pDS132-VChcp1-BsaI mutant plasmid.

[0012] S2. The target drug fragment is cloned into the pDS132-VChcp1-BsaI mutant plasmid to obtain the pDS132-VChcp1-X plasmid, where X refers to the target drug sequence.

[0013] S3. Transform Escherichia coli with pDS132-VChcp1-X plasmid to form a donor strain for conjugation transfer; co-culture the donor strain with Vibrio cholerae V52 strain to obtain an Hcp-X fusion protein mutant strain.

[0014] In one embodiment of the present invention, the target drug is a drug with a DNA length of less than 100 bp, including Etelcalcetide, Bradykinin, Tigapotide, and Dirucotide.

[0015] Secondly, the present invention provides a secretion system for Hcp carrying drugs constructed by the construction method described above.

[0016] As one embodiment of the present invention, the fusion protein of the target drug and Hcp can be expressed and synthesized in bacteria.

[0017] As one embodiment of the present invention, the target drug is secreted into the extracellular space of bacteria by being carried by Hcp.

[0018] Thirdly, the present invention provides a plasmid for constructing an Hcp-carrying drug secretion system, said plasmid being constructed by a method comprising the following steps:

[0019] A1. Using Vibrio cholerae V52 RHH strain as a template, the upstream homologous left arm and the downstream homologous right arm of the mutant hcp1 were amplified respectively.

[0020] A2. The left and right arms were amplified and ligated. The homologous arm fragments after ligation were cloned into the suicide plasmid pDS132 using the Gibson assembly method.

[0021] A3. Phosphorylate the two single-stranded primers containing the target drug sequence and pair them to form double-stranded DNA. Then, clone the double-stranded DNA into the vector plasmid obtained in step A2 using the Golden Gate cloning method.

[0022] A4. If the verification is correct, the plasmid is obtained.

[0023] As one embodiment of the present invention, in step A1, the primers for amplifying the upstream homologous left arm of the mutant hcp1 are VChcp1-KI-1 and BsaI-VChcp1 KI-2; the primers for amplifying the downstream homologous right arm of the mutant hcp1 are VChcp1-BsaIKI-3 and VChcp1-KI-4.

[0024] In one embodiment of the present invention, in step A2, the primers used for the amplification ligation of the left and right arms are VChcp1-KI-1 and VChcp1-KI-4.

[0025] In one embodiment of the present invention, in step A3, the single-stranded primers are BsaI-XF and X-BsaI-R, respectively, where X refers to the target drug sequence.

[0026] The present invention provides a pharmaceutical composition which is added to the secretion system in a pharmaceutically acceptable formulation.

[0027] As one embodiment of the present invention, the composition is a (oral) liquid dosage form.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) T6SS is able to secrete substrate proteins in their native conformation, an ability that is particularly suitable for proteins that are not easy to unfold and refold.

[0030] (2) T6SS can secrete multiple proteins simultaneously using its multiple protein transport pathways;

[0031] (3) T6SS can directly inject effector proteins into the prokaryotic and eukaryotic cells it comes into contact with, without the need for corresponding receptors on the cell surface;

[0032] (4) Based on the above secretion mode and characteristics, T6SS has extremely wide potential application value in synthetic biology, anti-drug resistant pathogens and whole health;

[0033] (5) When necessary, the drug delivery system can be added to a pharmaceutically acceptable formulation to prepare an orally administered liquid dosage form. Attached Figure Description

[0034] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0035] Figure 1 A. Schematic diagram of the construction of the pDS132-VChcp1-X mutant plasmid; A. Schematic diagram of the pDS132-VChcp1-BsaI mutant plasmid, which contains wild-type VChcp1, linker sequence and BsaI restriction site sequence; B. Schematic diagram of the double-stranded DNA formed by primers BsaI-XF and X-BsaI-R, which contains 5' AGGA and 3' ATTA gaps, "NNNNNNNNNNNN" represents the gene sequence of the target drug;

[0036] Figure 2The image shows the Western blot results of the secretion of the fusion protein of the target drug and Hcp; A, secretion of the fusion protein in an expression system containing wild-type Hcp; RNA polymerase subunit RpoB is used to indicate equal protein loading and bacterial lysis; WT: V. cholerae V52 rhh vipA-mCherry,tseL D425A vasX Δ16 ,tseH H64A ,vgrG1 ΔACD ΔtssM: V. cholerae V52 rhhΔtssM; B, secretion of the fusion protein in a mutant expression system with one Hcp coding sequence knocked out; RNA polymerase subunit RpoB is used to indicate equal protein loading and bacterial lysis; WT: V. cholerae V52 rhh vipA-mCherry,tseL D425A vasX Δ16 ,tseH H64A ,vgrG1 ΔACD , Δhcp2, ΔtssM: V. cholerae V52 rhhΔtssM. Detailed Implementation

[0037] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0038] This invention provides a method for constructing a system of Hcp secretory peptides and protein drugs, which are core components of the type VI secretion system (T6SS).

[0039] First, V. cholerae V52 was selected as the vector bacteria. Since relatively large proteins may not fit into the lumen of the Hcp tube, the inner tube of the Hcp tube was selected as the loading site for target proteins with a DNA length of less than 100 bp.

[0040] The genome of *V. cholerae* V52 (NCBI database ID: NZ_KQ410497) contains two homologous genes encoding the Hcp protein, hcp1 (VC1415) and hcp2 (VCA0017). Therefore, this invention constructs two expression systems for the Hcp fusion protein. One system integrates the target protein into a V52 strain containing both hcp genes, resulting in simultaneous expression of wild-type and fusion Hcp. The other system integrates the target protein into the genome of a mutant V52 strain with the Hcp2 coding sequence knocked out, resulting in expression of only the fusion Hcp. In this invention, the target drug fragment containing the homologous arm sequence is first cloned into the suicide plasmid pDS132 using Golden Gate cloning. The verified plasmid was then transformed into donor E. coli WM6026, and the plasmid was transferred into V. cholerae V52 via conjugation transfer. The target drug protein sequence was then inserted after the hcp1 gene in the V. cholerae V52 genome using homologous recombination double exchange to achieve fusion expression.

[0041] The bacterial strain expressing the target drug and Hcp fusion protein was induced to secrete the drug in a 30°C water bath for 1 hour. The bacterial cells and supernatant were then separated by centrifugation. After separate treatment, Western blot analysis confirmed that T6SS could carry and secrete peptide / protein drugs extracellularly.

[0042] The specific culture medium formulation and culture conditions involved in the embodiments of this invention are as follows:

[0043] All strains used in this invention were cultured in LB medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride) at 37°C. NaCl-free LB medium (10 g / L tryptone, 5 g / L yeast extract) was used to screen strains that had lost plasmid resistance; sucrose was added to a final concentration of 6% before use, and the medium was incubated at 22°C. The antibiotic concentrations used were as follows: 100 μg / mL streptomycin and 50 μg / mL kanamycin.

[0044] Information on the drugs that can be secreted in this invention is shown in Table 1:

[0045] Table 1. Drugs that can be secreted in this invention.

[0046] name size Applicable symptoms Etelcalcetide 1.05kD Secondary hyperthyroidism Bradykinin 1.06kD Treatment research on hypertension and type 2 diabetes Tigapotide 2.04kD Treatment of advanced hormone-refractory prostate cancer for which there is currently no effective treatment Dirucotide 2.25kD Treatment of multiple sclerosis

[0047] The bacterial strains used in this invention are shown in Table 2:

[0048] Table 2 Strains used in this invention

[0049]

[0050]

[0051] * X represents the drug name.

[0052] 1.Pukatzki,S.,et al.Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host modelsystem.Proc Natl Acad Sci US A.103,1528-33(2006).

[0053] 2. Hersch, SJ, et al. Envelope stress responses defend against type six secretion system attacks independently of immunity proteins. Nat Microbiol. 5, 706-14 (2020).

[0054] 3. Blodgett, JA, et al. Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide. Nat Chem Biol. 3, 480-5 (2007).

[0055] The primers used in this invention are shown in Table 3:

[0056] Table 3 Primers used in this invention

[0057]

[0058]

[0059] The plasmids used in this invention are shown in Table 4:

[0060] Table 4. Plasmids used in this invention

[0061] plasmid describe source pDS132 Suicide plasmids used for chromosome gene knockout 4 pDS132-VChcp1-BsaI Vector plasmids used to construct pDS132-VChcp1-X plasmid This study <![CDATA[pDS132-VChcp1-X * ]]> Suicide plasmids used to construct chromosomal hcp1-target drug fusion proteins This study

[0062] * X represents the drug name.

[0063] 4. Philippe, N., et al. Improvement of pCVD442, a suicide plasmid for geneallele exchange in bacteria. Plasmid. 51, 246-55 (2004).

[0064] The fusion protein sequences constructed in this invention are shown in Table 5:

[0065] Table 5. Fusion protein sequence of the present invention

[0066]

[0067]

[0068] Example 1: Construction of Hcp-X fusion protein expression strain (X represents drug name)

[0069] Obtaining the background strain:

[0070] The background strains are from studies already published in our laboratory. 2This strain was constructed based on the original Vibrio cholerae “RHH” (ΔhlyA(VCA0219), ΔhapA(VCA0865), ΔrtxA(VC1451)) V52 strain. The aspartic acid at position 425 of the effector protein TseL(VC1418) was mutated to alanine, and the histidine at position 64 of the effector protein TseH(VCA0285) was mutated to alanine. Additionally, 16 amino acids from positions 852 to 867 of the effector protein VasX(VCA0020) were knocked out. Furthermore, the fluorescent protein mCherry was fused to the C-terminus of the T6SS sheath protein VipA(VCA0107), resulting in a strain with mutated active sites of three toxic effector proteins and fluorescently labeled T6SS sheaths. Based on this strain, using strain V52RHH as a template, this invention amplifies the upstream homologous arm of the vgrG1 (VC1416) ACD sequence in the genome using primers VC1416 dACD-KO-1 and VC1416 dACD-KO-2 (Table 3), and amplifies the downstream homologous arm of the vgrG1 ACD sequence in the genome using primers VC1416 dACD-KO-3 and VC1416 dACD-KO-4. Then, using these upstream and downstream homologous arms as templates, primers VC1416 dACD-KO-1 and VC1416 dACD-KO-4 are used to ligate the amplified upstream and downstream homologous arms together. The ligated homologous arm fragment is cloned into the suicide plasmid pDS132 using the Gibson assembly method. The plasmid's correctness is verified by PCR (primers pDS132-F and pDS132-R (Table 3)) and Sanger sequencing. The validated plasmid was transformed into *E. coli* WM6026 to form a conjugation transfer donor strain. The overnight-cultured donor strain and the V52 strain from the previously published study were each suspended in 100 μl LB medium, mixed 1:1, and spotted onto solid LB medium containing 100 μg / mL DAP. After co-culturing at 37°C for 3 h, the bacterial cells were scraped off and resuspended in 500 μl LB medium. After recovery culture at 37°C and 950 rpm for 1 h, the cells were spread onto solid LB agar plates containing 100 μg / mL streptomycin and 50 μg / mL kanamycin. The plates were incubated overnight at 37°C. Conjugation transferons were picked and cultured in 500 μl antibiotic-free LB medium for 4 h, then spread onto LB agar plates containing 6% sucrose and free of NaCl. After incubation at 22℃ for 2 days, the mutant strain was verified to be correct using PCR (primers VC1416 dACD-KO-5 and VC1416 dACD-KO-6 from Table 3) and Sanger sequencing. The background strain V52 rhh,vipA-mCherry,tseL was finally obtained. D425A vasX Δ16 ,tseH H64A ,vgrG1 ΔACD .

[0071] 1. Construction of Hcp2(VCA0017) knockout mutant strain

[0072] Using Vibrio cholerae V52 RHH strain as a template, the upstream homologous arm of the hcp2 sequence in the genome was amplified using primers VCA0017 KO-1 and VCA0017 KO-2 (Table 3), and the downstream homologous arm of the hcp2 sequence was amplified using primers VCA0017 KO-3 and VCA0017 KO-4. Simultaneously, using these upstream and downstream homologous arms as templates, the amplified and ligated upstream and downstream homologous arms were joined together using primers VCA0017 KO-1 and VCA0017 KO-4. The ligated homologous arm fragment was cloned into the suicide plasmid pDS132 using the Gibson assembly method. The plasmid's correctness was verified by PCR (primers pDS132-F and pDS132-R (Table 3)) and Sanger sequencing. The verified plasmid was transformed into Escherichia coli WM6026 to form a conjugation transfer donor bacterium. The donor strain and the recipient background V52 strain, cultured overnight, were each suspended in 100 μl LB medium, mixed 1:1, and spotted onto solid LB medium containing 100 μg / mL DAP. After co-culturing at 37°C for 3 h, the bacterial cells were scraped off and resuspended in 500 μl LB medium. After recovery culture at 37°C and 950 rpm for 1 h, the cells were plated onto solid LB plates containing 100 μg / mL streptomycin and 50 μg / mL kanamycin. After overnight culture at 37°C, conjugation transferons were picked and cultured in 500 μl antibiotic-free LB medium for 4 h. Then, the cells were plated onto LB plates containing 6% sucrose and free of NaCl. After culturing at 22°C for 2 days, the correctness of the mutant strain was verified by PCR (primers VCA0017 KO-5 and VCA0017 KO-6 in Table 3) and Sanger sequencing.

[0073] 2. Construction of pDS132-VChcp1-BsaI mutant plasmid

[0074] Using Vibrio cholerae V52 RHH strain as a template, the upstream homologous left arm of the hcp1 sequence in the genome was amplified using primers VChcp1-KI-1 and BsaI-VChcp1-KI-2 (Table 3), and the downstream homologous right arm of the hcp1 sequence in the genome was amplified using primers VChcp1-BsaI-KI-3 and VChcp1-KI-4. Then, using these upstream and downstream homologous arms as templates, the amplified arms were ligated together using primers VChcp1-KI-1 and VChcp1-KI-4. The ligated homologous arm fragments were cloned into the suicide plasmid pDS132 using the Gibson assembly method. The plasmid was verified for correctness by PCR (primers pDS132-F and pDS132-R (Table 3)) and Sanger sequencing, finally yielding the pDS132-VChcp1-BsaI plasmid. This plasmid inserts the sequence gccgcaggaggaggaAGAGACCATTAGGTCTCT before the stop codon TAA in the wild-type hcp1 (VC1415) gene sequence. gccgcaggaggagga encodes the amino acid AAGGG, which can act as a linker, while AGAGACCATTAGGTCTCT contains two BsaI enzyme recognition sites, allowing for complete cleavage by BsaI (e.g., ...). Figure 1 A).

[0075] 3. Construction of pDS132-VChcp1-X plasmid

[0076] First, the primers BsaI-XF and X-BsaI-R from Table 3 were reacted with T4 PNK enzyme at 37℃ for 1 h to fully phosphorylate both ends of the single-stranded DNA. Then, 1M NaCl was added to the system to bring the final concentration to 200mM. The sample was first denatured at 95℃ for 3 min, then annealed in a gradient at a cooling rate of -1℃ / 10s, and finally cooled to 50℃ to allow the two single-stranded primers to pair and form double-stranded DNA with AGGA at the 5' end and ATTA at the 3' end (e.g., ...). Figure 1 B), this is the target fragment to be cloned. The target fragment is cloned into the vector pDS132-VChcp1-BsaI using the Golden Gate cloning method. The correctness of the plasmid is verified by PCR (primers are pDS132-F and pDS132-R in Table 3) and Sanger sequencing.

[0077] 3. Construction of Hcp-X fusion protein-related mutant strains

[0078] The verified plasmid was transformed into *E. coli* WM6026 to form donor bacteria for conjugation transfer. The overnight cultured donor strain and the recipient V52 background strain or the hcp2 knockout mutant strain were each suspended in 100 μl LB medium, mixed 1:1, and spotted onto solid LB medium containing 100 μg / mL DAP. After co-culturing at 37°C for 3 h, the bacterial cells were scraped off and resuspended in 500 μl LB medium. After recovery culture at 37°C and 950 rpm for 1 h, the cells were plated onto solid LB agar plates containing 100 μg / mL streptomycin and 50 μg / mL kanamycin. The plates were incubated overnight at 37°C. Conjugation transferons were picked and cultured in 500 μl antibiotic-free LB medium for 4 h, then plated onto LB agar plates containing 6% sucrose and free of NaCl. After culturing at 22°C for 2 days, the correctness of the mutant strain was verified using PCR (primers VChcp1-confirm-F, -R from Table 3) and Sanger sequencing.

[0079] Example 2: Detection of Hcp-X fusion protein secretion (X represents drug name)

[0080] 1. Induction of fusion protein secretion experiment

[0081] Vibrio cholerae expressing the fusion protein was cultured overnight at 37°C on LB agar plates containing 100 μg / mL streptomycin. The next day, an appropriate amount of bacteria was transferred to 500 μL of LB medium containing 100 μg / mL streptomycin and activated at 37°C and 950 rpm for 1 h. 300 μL of the bacterial culture was then transferred to 30 mL of LB medium containing 100 μg / mL streptomycin and cultured at 30°C and 200 rpm until OD500 was reached. 600 =0.8-1.0. At room temperature, centrifuge at 2,500×g for 5 min to collect 5 mL of bacterial culture as a cell pellet. Resuspend the pellet in 2 mL of fresh LB medium and incubate at 30°C for 1 h. After incubation, centrifuge the bacterial culture at 10,000×g for 2 min at room temperature. Resuspend the cell pellet in 600 L of fresh LB medium as a whole cell sample. Centrifuge the supernatant again at 10,000×g for 2 min at room temperature, and collect the supernatant as a secretion sample.

[0082] 2. Western blot detection of protein secretion

[0083] Protein loading buffer was added to each sample, and the mixture was incubated at 98°C for 10 min. Protein samples were separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with 5% [w / v] skim milk for 1 h, followed by incubation with primary antibody containing 1% [w / v] skim milk at room temperature for 1 h. After incubation, the membrane was washed three times with TBST (50 mM Tris, 150 mM NaCl, 0.1% [v / v] Tween-20, pH 7.6), and incubated with secondary antibody at room temperature for 1 h. After three TBST washes, the membrane was finally developed using ECL chemiluminescence buffer.

[0084] See results Figure 2 As shown in the figure, Hcp-Dirucotide and Hcp-Tigapotide can be secreted in the presence of wild-type Hcp. Figure 2 A); In the absence of wild-type Hcp, Hcp-Etelcalcetide, Hcp-Bradykinin, and Hcp-Tigapotide can be secreted ( Figure 2 B).

[0085] In summary, this invention experimentally demonstrates that fusion proteins of peptide or protein drugs and Hcp can be expressed and synthesized within bacteria and secreted extracellularly via the aforementioned T6SS core component. This drug secretion system holds significant potential for delivering macromolecular drugs to eukaryotic cells and other organisms.

[0086] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for constructing a secretion system using Hcp, a core component of the type VI secretion system T6SS, as a carrier for peptide and protein drugs, characterized in that, In Vibrio cholerae V52 strain, the target drug sequence was integrated into the bacterial genome and linked to the Hcp1 protein sequence to obtain a system of secreted peptide and protein drugs. The target drug is a drug with a DNA length of less than 100 bp, and the target drug is Bradykinin or Tigapotide; The Vibrio cholerae V52 strain is a mutant strain that uses Vibrio cholerae V52 RHH as the original strain, and has inactivated and mutated the T6SS toxicity proteins TseL, VasX, TseH and VgrG1, and further knocked out the Hcp2 coding sequence. First, the target drug fragment containing the homologous arm sequence was cloned into the suicide plasmid pDS132 using Golden Gate cloning. Then, the verified plasmid was transformed into donor *E. coli* WM6026, and the plasmid was transferred into *Vibrio cholerae* strain V52 via conjugation transfer. Homologous recombination double exchange was then performed on the *Vibrio cholerae* strain V52 genome. hcp1 The target drug protein sequence was inserted post-geneally to achieve fusion expression.

2. The construction method according to claim 1, characterized in that, The inactivation mutations TseL, VasX, TseH, and VgrG1 involve mutating the aspartic acid at position 425 of effector protein TseL to alanine, mutating the histidine at position 64 of effector protein TseH to alanine, knocking out 16 amino acids from positions 852 to 867 of effector protein VasX, and knocking out 434 amino acids from positions 716 to 1149 of the C-terminal actin crosslinking domain of VgrG1.

3. A secretion system using Hcp, the core component of the type VI secretion system T6SS, as a drug carrier for peptides and proteins, obtained by the construction method described in claim 1 or 2.

4. The secretion system according to claim 3, characterized in that, The fusion protein of the target drug and Hcp1 can be expressed and synthesized in bacteria.

5. The secretion system according to claim 3, characterized in that, The target drug is secreted into the bacterial extracellular space by carrying Hcp1.

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