Polypeptide modified bacterial outer membrane vesicle and application thereof in preparation of medicine for promoting cell growth

Abstract

The invention provides a polypeptide-modified bacterial outer membrane vesicle and application thereof in preparation of a drug for promoting cell growth, and relates to the technical field of biological medicines. According to the invention, a growth promoting polypeptide VTPYGGGGVLLY with a brand new structure is designed on the basis of two micromolecular polypeptides VTPY and VLLY from sea cucumbers, and the engineering bacterial outer membrane vesicle W-VTPVLY OMV with a relatively strong growth promoting effect is prepared by combining the growth promoting polypeptide VTPYGGGGVLLY with bacterial outer membrane protein ClyA by utilizing a genetic engineering technology. The W-VTPVLY OMV provided by the invention has a remarkable promoting effect on HUVEC cell proliferation, migration and revascularization, can play a role in promoting cell proliferation within a short time, and can maintain the proliferation promoting effect for a relatively long time. The W-VTPVLY OMV provided by the invention is expected to become a new medicine for promoting wound regeneration and tissue healing, and provides a new thought and application reference for the fields of tissue engineering and medical materials.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a polypeptide-modified bacterial outer membrane vesicle and its application in the preparation of drugs that promote cell growth. Background Technology

[0002] Wound healing is a complex and dynamic biological repair process. Minor skin or tissue injuries can heal spontaneously within a short time, but when skin tissue suffers severe or complex trauma, healing becomes significantly more difficult, easily leading to wound inflammation or infection, and in severe cases, even amputation or death. Wound repair involves four consecutive stages: hemostasis, inflammation, proliferation, and remodeling. These stages are not independent steps but rather intertwine and simultaneously advance the healing process. Therefore, once a foreign substance invades the wound, regardless of the tissue's stage, it will cause the repair process to stall, prolonging the wound healing period and even leading to more severe trauma. This necessitates human intervention in the wound to promote healing and prevent further deterioration.

[0003] Currently, wound intervention is mainly carried out through artificial dressings. The choice is based on factors such as wound condition, complexity, economic situation, and safety. Traditional dressings include sponges and gauze. After the wound appears, they are applied directly to the wound site to quickly absorb wound blood and exudate and protect the wound surface. However, most traditional dressings only protect the wound and cannot ensure the environmental immersion of the wound site. They have poor barrier function and are prone to bacterial invasion. In addition, there is the problem of frequent replacement, which can easily cause secondary damage. An ideal wound dressing should have: (1) good mechanical properties; (2) biodegradability; (3) breathability and biocompatibility; (4) effective moisture absorption properties; and (5) the ability to promote tissue healing and regeneration. With the development of tissue engineering, various dressings with different properties and characteristics or that promote wound healing have been developed. However, in clinical applications, there is still a lack of low-cost dressings that can effectively promote wound healing.

[0004] In recent years, with the development of molecular biology techniques, researchers have discovered a variety of bioactive substances that aid in wound healing, such as Asparagus cochinchinensis polysaccharide, catfish crude mucus, and fish collagen. These substances have the characteristics of small molecular weight and easy absorption, and have high application potential in antibacterial, anti-inflammatory, collagen synthesis promotion, and wound healing. Sea cucumber is an economically important marine organism widely distributed in the coastal areas of China. Multiple studies have shown that sea cucumber has antioxidant, antitumor, antibacterial, anti-inflammatory, antithrombotic, and wound healing promoting effects. Research has found that the tetrapeptides Val-Thr-Pro-Tyr (VTPY) and Val-Leu-Leu-Tyr (VLLY) derived from sea cucumber have antioxidant activity. Through interaction with proteins related to endothelial cell proliferation, migration, and angiogenesis, they can promote angiogenesis, thereby achieving nutrient supply and accelerating tissue healing. However, due to the poor stability, easy hydrolysis, and insufficient wound regeneration capacity of the small molecules of tetrapeptides VTPY and VLLY, their further application and promotion are severely limited.

[0005] Bacterial outer membrane vesicles (OMVs) are membrane-bound vesicles with a diameter of 20-300 nm secreted by Gram-negative bacteria during their growth. OMVs are mainly spherical bilayer nanostructures composed of bacterial outer membrane and periplasmic components. The outer membrane includes components such as glycerophospholipids, phosphatidylethanolamine, and phosphatidylglycerol. The vesicle lumen contains various compounds from the periplasm or cytoplasm, such as proteins, DNA, RNA, and peptidoglycans. The advantages of OMVs as medical biomolecules are mainly: (1) OMVs have a natural loading capacity and can carry small molecule cargoes such as drugs, nucleic acids, and proteins. (2) OMVs are easily absorbed by cells and tissues, have a long half-life, good biocompatibility, and are not easily degraded in the bloodstream. (3) OMVs can penetrate the cell barrier and penetrate deep into the tissue to fully exert their specific biological activities. Advances in genetic engineering technology have promoted the development of research on the functionalization of bacterial vesicles. By displaying functionalized peptides on the surface or inside of OMVs, the biological characteristics of OMVs can be changed. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] To address the shortcomings of existing technologies, this invention first designs a novel growth-promoting polypeptide, VTPYGGGGVLLY, using VTPY and VLLY peptides. Then, it is further modified onto the surface of OMV to obtain a functionalized bacterial outer membrane vesicle that can effectively promote cell growth and tissue healing, providing a new reference for the fields of tissue repair and artificial excipients.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides a growth-promoting polypeptide, the amino acid sequence of which is VTPYGGGGVLLY, as shown in SEQ ID NO.1.

[0011] Secondly, the present invention provides a fusion protein ClyA-VTPYGGGGVLLY, the fusion protein comprising ClyA protein and growth-promoting polypeptide VTPYGGGGVLLY, the amino acid sequence of the fusion protein being shown in SEQ ID NO.2.

[0012] Thirdly, the present invention provides a recombinant plasmid expressing the fusion protein ClyA-VTPYGGGGVLLY.

[0013] Fourthly, the present invention provides a recombinant bacterium expressing the fusion protein ClyA-VTPYGGGGVLLY.

[0014] Fifthly, the present invention provides a functionalized bacterial outer membrane vesicle, wherein the surface of the bacterial outer membrane vesicle is modified with a growth-promoting polypeptide VTPYGGGGVLLY, and the bacterial outer membrane vesicle is abbreviated as W-VTPVLY OMV.

[0015] Sixthly, the present invention provides a method for preparing the above-mentioned W-VTPVLY OMV, wherein the W-VTPVLY OMV is prepared according to the following steps:

[0016] (1) Linker protein, 3×MYC tag protein sequence, Linker2 protein, and VTPYGGGGVLLY protein were sequentially inserted into the C-terminus of Escherichia coli ClyA protein to obtain the ClyA-VTPYGGGGVLLY fusion protein with the amino acid sequence as shown in SEQ ID NO.2 or the nucleotide sequence as shown in SEQ ID NO.3.

[0017] (2) The gene of the fusion protein with the nucleotide sequence shown in SEQ ID NO.3 was cloned into the multiple cloning site on the pGEX-6P-1 vector to obtain the recombinant plasmid ClyA-VTPYGGGGVLLY-pGEX-6P-1;

[0018] (3) Transform W3110 Escherichia coli competent cells with the plasmid ClyA-VTPYGGGGVLLY-pGEX-6P-1 to obtain a single clone strain that stably expresses the ClyA-VTPYGGGGVLLY fusion protein. This strain is referred to as the W-VTPYVLY strain.

[0019] (4) Add 500 mL of ampicillin-resistant LB medium to a 1 L conical flask, inoculate 10 μL of W-VTPVLY bacterial culture into the medium, and shake the culture at 37 ℃ and 220 rpm for 4 h. Measure the OD value of the bacterial culture every 20 minutes. When the OD value reaches 0.5-0.6, add 500 μL of 1 M IPTG to make the final IPTG concentration in the medium 1 mM. Then, induce the expression of exogenous protein overnight at 21 ℃ and 200 rpm to obtain W-VTPVLY bacterial culture.

[0020] (5) After centrifuging the W-VTPVLY bacterial culture at 4 ℃ and 5,000×g for 15 min, collect the supernatant and filter it with a 0.45 μm filter membrane. Concentrate the filtrate 20 times with an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD. Filter it again with a 0.22 μm filter membrane, and then centrifuge it at 150,000×g for 3 hours. Discard the supernatant and the precipitate is W-VTPVLY OMV.

[0021] In a seventh aspect, the present invention provides the application of the growth-promoting polypeptide VTPYGGGGVLLY in the preparation of drugs that promote cell growth and tissue healing.

[0022] Eighthly, the present invention provides the application of W-VTPVLY OMV in the preparation of drugs that promote cell growth and tissue healing.

[0023] Specifically, W-VTPVLY OMV promotes cell growth and tissue healing by promoting cell proliferation and migration. The cells include, but are not limited to, any one of the following: human umbilical vein endothelial cells, human dermal microvascular endothelial cells, human skin fibroblasts, human placental mesenchymal stem cells, human adipose stem cells, human bone marrow mesenchymal stem cells, human umbilical cord mesenchymal stem cells, human neural stem cells, human epidermal stem cells, human peripheral blood mesenchymal stem cells, human tendon stem cells, human hair follicle stem cells, and human hematopoietic stem cells.

[0024] (III) Beneficial Effects

[0025] This invention designs a novel growth-promoting polypeptide, VTPYGGGGVLLY, based on two small molecule polypeptides VTPY and VLLY derived from sea cucumber. Using genetic engineering technology, it is combined with the bacterial outer membrane protein ClyA to prepare engineered bacterial outer membrane vesicles W-VTPVLY OMV with strong growth-promoting effects.

[0026] Verification has shown that wild-type OMV has no significant effect on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells. Compared with wild-type OMV, the W-VTPVLY OMV provided by this invention shows a significant promoting effect on the proliferation and migration of human umbilical vein endothelial cells. Furthermore, it has been demonstrated that the novel growth-promoting polypeptide VTPYGGGGVLLY obtained by this invention possesses biological activity that promotes cell growth.

[0027] Compared with small molecule peptides VTPY and VLLY monomers, the W-VTPVLY OMV provided by this invention has a significant promoting effect on HUVEC cell proliferation, migration and angiogenesis, and can promote cell proliferation in a short time and maintain the promoting effect for a long time.

[0028] The W-VTPVLY OMV provided by this invention has the potential to become a new drug for preparing wound regeneration and tissue healing, providing new ideas and application references for the fields of tissue engineering and medical materials. Attached Figure Description

[0029] Figure 1 The prokaryotic expression plasmid ClyA-VTPYGGGGVLLY-pGEX-6P-1 was identified by agarose gel electrophoresis.

[0030] Figure 2 Sequencing identified the pGEX-6P-1-ClyA-VTPYVLLY prokaryotic expression plasmid.

[0031] Figure 3 Western blot analysis was used to identify IPTG-induced expression of VTPYGGGGVLLY protein in W-VTPVLY Escherichia coli.

[0032] Figure 4 The structure and morphology of W3110 OMV and W-VTPVLY OMV were identified by transmission electron microscopy.

[0033] Figure 5 Average particle size of W3110 OMV and W-VTPVLY OMV.

[0034] Figure 6 PDI distributions of W3110 OMV and W-VTPVLY OMV.

[0035] Figure 7 Identification of W3110 OMV and W-VTPVLY OMV marker proteins and identification of VTPYGGGGVLLY protein exovesicle surface display.

[0036] Figure 8Effects of W3110 OMV, W-VTPVLY OMV, VTPY monopeptide and VLLY monopeptide on the proliferation rate of HUVEC cells; *P<0.05, **P<0.01.

[0037] Figure 9 Effects of W3110 OMV, W-VTPVLY OMV, VTPY monopeptide, and VLLY monopeptide on the migration rate of HUVEC cells; **P < 0.01.

[0038] Figure 10 Effects of W3110 OMV, W-VTPVLY OMV, VTPY monopeptide and VLLY monopeptide on the expression of HUVEC cell proliferation, migration and angiogenesis-related proteins (AKT, ERK1, e1F4E, FGF2, VEGFA); *P<0.05, **P<0.01. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] Preparation and Identification of W-VTPVLY OMV

[0042] 1. Design of growth-promoting peptides

[0043] By sequentially adding tetrapeptide GGGG and tetrapeptide VLLY to the C-terminus of tetrapeptide VTPY, a novel growth-promoting polypeptide with a new amino acid structure was designed, the amino acid sequence of which is VTPYGGGGVLLY (SEQ ID NO. 1).

[0044] 2. Synthesis of ClyA-VTPYGGGGVLLY fusion protein

[0045] A Linker protein (amino acid sequence as shown in SEQ ID NO. 4), a 3×MYC tag protein sequence (amino acid sequence as shown in SEQ ID NO. 6), a Linker2 protein (amino acid sequence as shown in SEQ ID NO. 7), and a VTPYGGGGVLLY protein were sequentially inserted into the C-terminus of the E. coli ClyA protein (amino acid sequence as shown in SEQ ID NO. 4). The resulting ClyA-VTPYGGGGVLLY fusion protein with the amino acid sequence as shown in SEQ ID NO. 2 or the nucleotide sequence as shown in SEQ ID NO. 3 has the structure ClyA-Linker-3×Myc-Linker2-VTPYGGGGVLLY.

[0046] 3. Construction and identification of recombinant plasmids and recombinant bacteria

[0047] The gene of the fusion protein with the nucleotide sequence shown in SEQ ID NO.3 was cloned into the pGEX-6P-1 vector between the KpnI and XhoI sites to construct the recombinant plasmid ClyA-VTPYGGGGVLLY-pGEX-6P-1, labeled W-VTPVLY. The W-VTPVLY plasmid was transformed into competent cells of *E. coli* strain W311O to obtain a single-clone strain stably expressing the ClyA-VTPYGGGGVLLY fusion protein.

[0048] The above-mentioned single-clone strains were spread onto ampicillin-resistant LB solid agar plates and cultured overnight. The next day, single colonies were picked and placed in LB agar containing ampicillin resistance for 12 hours of shaking. Plasmids were extracted from the bacterial cells using a plasmid miniprep kit and the plasmid bands were detected by 1% agarose gel electrophoresis.

[0049] The results showed that the electrophoretic band was located between 5000-5500 bp, consistent with the expected molecular weight of the plasmid of 5383 bp. Figure 1 Furthermore, the plasmid was sequenced, and the sequencing results showed that the plasmid DNA sequence was consistent with the theoretical sequence. Figure 2 This indicates that the recombinant plasmid ClyA-VTPYGGGGVLLY-pGEX-6P-1 was successfully constructed.

[0050] 4. IPTG-induced expression of exogenous proteins

[0051] The single-clonal strain was inoculated into 500 ml of LB liquid medium supplemented with ampicillin resistance. A control group consisting of the original W3110 strain was also included. After incubation at 37 ℃ and 220 rpm for 4 h, the OD value of the bacterial culture was measured every 20 minutes. When the OD value reached 0.5-0.6, 500 μL of 1 M IPTG was added to bring the final IPTG concentration in the medium to 1 mM. Subsequently, exogenous protein expression was induced overnight at 21 ℃ and 200 rpm, yielding 500 mL each of W-VTPVLY and W3110 bacterial cultures.

[0052] The proteins from the bacterial culture were extracted, and then Western blot was used to identify the expression of Myc-tagged proteins in the proteins from W-VTPVLY and W3110 bacterial cultures. The results are as follows: Figure 3 As shown, the W-VTPVLY group expressed the Myc protein tag, while no protein band was detected in W3110, indicating that IPTG successfully induced the W-VTPVLY plasmid to express the fusion protein ClyA-VTPYGGGGVLLY in the W-VTPVLY strain.

[0053] 5. Extraction of bacterial outer membrane vesicles

[0054] After IPTG-induced expression, W-VTPVLY and W3110 bacterial cultures were centrifuged at 5,000×g for 15 min at 4 ℃. The supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was concentrated 20-fold using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD. It was then filtered again through a 0.22 μm filter membrane and centrifuged at 150,000×g for 3 hours. The supernatant was discarded, and the precipitate was reselected using 200 μL of PBS. The resulting solution precipitated W-VTPVLY OMV and W3110 OMV.

[0055] 7. Structural and particle size characterization of W-VTPVLY OMV and W3110 OMV: The extracted outer membrane vesicles of W-VTPVLY OMV and W3110 OMV bacteria were observed using transmission electron microscopy. The results are as follows: Figure 4 As shown, W-VTPVLY OMV and W3110OMV exhibit a distinct saucer-like bilayer membrane structure, distributed singly, consistent with the theoretical morphology of bacterial outer membrane vesicles. Furthermore, there is no significant morphological difference between W-VTPVLY OMV and W3110 OMV. Dynamic light scattering (DLS) results indicate no significant difference in particle size between W-VTPVLY OMV and W3110 OMV, with average sizes ranging from 60 to 70 nm, consistent with theoretical particle sizes. Figure 5 The PDI values ​​for W-VTPVLYOMV and W3110 OMV were 0.492 and 0.467, respectively, indicating good particle dispersion. Figure 6 ).

[0056] The above results indicate that the W-VTPVLY OMV prepared in this invention maintains the complete bacterial outer membrane vesicle structure and particle size.

[0057] 8. Identification of target protein expression on bacterial outer membrane vesicle surface

[0058] Three parallel groups were set up for the extracted W3110 OMV and W-VTPVLY OMV: a control group with no treatment, a proteinase K digestion group, and a group that underwent both SDS lipid membrane disruption and proteinase K digestion. The expression of the tag protein Myc and the membrane protein OmpA was detected by Western blot using anti-MYC antibody and anti-OmpA antibody.

[0059] The results are as follows Figure 7 As shown, OmpA marker protein expression was detected in both W-VTPVLY OMV and W3110 OMV without SDS and proteinase K treatment, confirming at the molecular level that the extracted component is a bacterial outer membrane vesicle. Furthermore, Myc tag expression was detected in W-VTPVLY OMV, while no Myc band was observed in W3110 OMV, indicating successful expression of the fusion protein in W-VTPVLY OMV. After simultaneous treatment with the membrane-disrupting agents SDS and proteinase K, both Myc and OmpA bands disappeared in W-VTPVLY OMV, and the OmpA band disappeared in W3110 OMV. With proteinase K treatment alone, an OmpA band was detected in W-VTPVLY OMV, but the Myc tag disappeared, indicating that the growth-promoting peptide VTPYGGGGVLLY is modified on the surface of the bacterial outer vesicle membrane.

[0060] Example 2

[0061] Investigation on the growth-promoting effect of W-VTPVLY OMV on cells

[0062] 1. The promoting effect of W-VTPVLY OMV on cell proliferation

[0063] 4×10 3Personal umbilical vein endothelial cells (HUVECs) were cultured in 96-well plates for 24 h, followed by the addition of W-VTPVLY OMV containing a final concentration of 0.2 mmol of VTPYGGGGVLLY peptide. Control group (with equal volume of PBS), W3110OMV group (W3110 OMV with the same volume as W-VTPVLY OMV), VTPY group (VTPY monopeptide at a final concentration of 0.2 mmol), and VLLY group (VLLY monopeptide at a final concentration of 0.2 mmol) were also established. Cells were cultured at 37°C and 5% CO2 for 24 h, 48 h, and 72 h, respectively. Cell proliferation rate was detected at the three time points using the CCK-8 assay.

[0064] The results are as follows Figure 8 As shown, compared with the blank control, W-VTPVLY OMV significantly increased the proliferation rate of HUVECs at 24h, 48h, and 72h (P < 0.05, P < 0.01); W3110 OMV and VLLY monopeptide did not show any promoting effect on HUVEC cell proliferation at different time points (P > 0.05); VTPY monopeptide group did not show any promoting effect on HUVEC cell proliferation at 24h (P > 0.05), although it significantly promoted cell proliferation rate at 48h and 72h (P < 0.05), but the HUVEC cell proliferation rate of W-VTPVLY OMV group was significantly higher than that of VTPY monopeptide (P < 0.05, P < 0.01).

[0065] The above results indicate that, compared with VTPY and VLLY peptides, W-VTPVLY OMV has a greater advantage in promoting HUVEC cell proliferation, and can exert its cell proliferation-promoting effect in a short time and maintain the proliferation-promoting effect for a longer period of time.

[0066] 2. The promoting effect of W-VTPVLY OMV on cell migration

[0067] The effect of W-VTPVLY OMV on cell migration was detected using a cell scratch assay. 1×10⁶ cells were seeded. 5 HUVEC cells were cultured in 6-well plates until adherence. The wells were then scored with a pipette tip, and W-VTPVLY OMV containing VTPYGGGGVLLY peptide at a final concentration of 4 mmol was added. Other control groups (with an equal volume of PBS), W3110 OMV group (with an equal volume of W3110 OMV to W-VTPVLY OMV), VTPY group (with a final concentration of VTPY monopeptide of 4 mmol), and VLLY group (with a final concentration of VLLY monopeptide of 4 mmol) were also established.

[0068] Images of the cell scratched areas were recorded under a microscope at 0h and 24h, and the cell migration rate was calculated based on the scratch width.

[0069] The results are as follows Figure 9 As shown, compared with the blank control group, the migration rate of HUVEC cells in the W-VTPVLY OMV group and the VTPY monopeptide group was significantly increased (P < 0.01), while the W3110 OMV group and the VLLY monopeptide group did not show a promoting effect on the migration rate of HUVEC cells (P > 0.05). The migration rate of HUVEC cells in the W-VTPVLY OMV group was significantly higher than that in the VTPY monopeptide group (P < 0.01). These results indicate that W-VTPVLY OMV has a more advantageous effect on promoting HUVEC cell migration compared with VTPY monopeptide and VLLY monopeptide.

[0070] 3. Effects of W-VTPVLY OMV on promoting cell proliferation, migration, and angiogenesis-related proteins.

[0071] AKT protein (also known as protein kinase B, PKB) is a member of the serine / threonine kinase family and plays a core role in key physiological processes such as cell survival, proliferation, and metabolism.

[0072] ERK1 (extracellular regulatory protein kinase) participates in a variety of biological reactions, including cell proliferation and differentiation, cell morphology maintenance, cytoskeleton construction, apoptosis, and cell carcinogenesis.

[0073] elfF4E (eukaryotic translation initiation factor 4E) is closely related to cell proliferation and migration. It directly affects the cell's proliferation, migration and invasion capabilities by regulating the protein translation initiation process.

[0074] FGF2 (fibroblast growth factor 2) is an important member of the FGF family. It has the functions of promoting cell proliferation, differentiation and regulating tissue homeostasis, and is widely involved in biological processes such as embryonic development and tissue repair.

[0075] VEGFA (vascular endothelial growth factor A) is a family of proteins that regulate angiogenesis and vascular permeability, playing a crucial role in embryonic development, tissue repair, and diseases such as cancer and diabetic retinopathy. It promotes endothelial cell proliferation and migration, and the formation of new blood vessels by binding to cell surface receptors and activating signaling pathways.

[0076] We used Western blot to analyze the effects of W-VTPVLY OMV on promoting cell proliferation, migration, and angiogenesis-related proteins. Specifically, 5 × 10⁶ cells were seeded. 4HUVEC cells were cultured in 12-well plates until they adhered. The cells were then co-incubated with W-VTPVLY OMV (with a final concentration of 2 mmol), a negative control group, an equal amount of W3110 OMV (with the same concentration as W-VTPVLY OMV), a 2 mmol VTPY peptide group, and a 2 mmol VLLY peptide group for 24 h. Cellular proteins were then extracted and analyzed by Western blot.

[0077] The results are as follows Figure 10 As shown in the figure. Compared with the blank control group, the expression levels of AKT, ERK1, e1F4E, FGF2, and VEGFA in the W3110 OMV group cells were not significantly different (P>0.05); the expression levels of AKT, ERK1, e1F4E, FGF2, and VEGFA in the W-VTPVLY OMV group cells were significantly increased (P<0.05, P<0.01); the expression levels of ERK1, FGF2, and VEGFA in the VTPY single peptide group cells were significantly increased (P<0.05, P<0.01), while the expression levels of AKT and e1F4E were not significantly different (P>0.05); the expression levels of ERK1 and VEGFA in the VLLY single peptide group cells were significantly increased (P<0.05), while the expression levels of AKT, e1F4E, and FGF2 were not significantly different (P>0.05). The expression levels of ERK1 and VEGFA in the W-VTPVLY OMV group were significantly higher than those in the VTPY single peptide group and the VLLY single peptide group (P < 0.05).

[0078] The above results indicate that the W-VTPVLY OMV provided by this invention can significantly upregulate the expression levels of proteins (AKT, ERK1, e1F4E, FGF2, VEGFA) that promote cell proliferation, migration, and angiogenesis, and has a significant promoting effect on cell proliferation, migration, and angiogenesis.

[0079] 4. Conclusion

[0080] This invention designs a novel growth-promoting polypeptide, VTPYGGGGVLLY, based on two small-molecule polypeptides, VTPY and VLLY, derived from sea cucumber. Using genetic engineering, this polypeptide is combined with the bacterial outer membrane protein ClyA to prepare engineered bacterial outer membrane vesicles, W-VTPVLY OMV, which exhibits strong growth-promoting effects. Wild-type OMV has no significant effect on the proliferation and migration of human umbilical vein endothelial cells. Compared to wild-type OMV, W-VTPVLY OMV shows a significant promoting effect on the proliferation and migration of human umbilical vein endothelial cells, indicating that the novel growth-promoting polypeptide VTPYGGGGVLLY obtained in this invention possesses cell growth-promoting biological activity. Compared to the smaller-molecule polypeptides VTPY and VLLY monomers, W-VTPVLY OMV has a significant promoting effect on the proliferation, migration, and angiogenesis of HUVEC cells, exerting its cell proliferation-promoting effect quickly and maintaining a prolonged proliferative effect.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A growth-promoting polypeptide, characterized in that, The amino acid sequence of the polypeptide is VTPYGGGGVLLY, as shown in SEQ ID NO.

1.

2. A fusion protein ClyA-VTPYGGGGVLLY, characterized in that, The fusion protein comprises ClyA protein and the growth-promoting polypeptide VTPYGGGGVLLY, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.

2.

3. A recombinant plasmid expressing the fusion protein ClyA-VTPYGGGGVLLY.

4. A recombinant bacterium expressing the fusion protein ClyA-VTPYGGGGVLLY.

5. A functionalized bacterial outer membrane vesicle, characterized in that, The surface of the bacterial outer membrane vesicles is modified with a growth-promoting polypeptide VTPYGGGGVLLY, and the bacterial outer membrane vesicles are referred to as W-VTPVLY OMV.

6. The functionalized bacterial external vesicle as described in claim 5, characterized in that, The W-VTPVLY OMV is prepared according to the following steps: (1) Linker protein, 3×MYC tag protein sequence, Linker2 protein, and VTPYGGGGVLLY protein were sequentially inserted into the C-terminus of Escherichia coli ClyA protein to obtain the ClyA-VTPYGGGGVLLY fusion protein with the amino acid sequence as shown in SEQ ID NO.2 or the nucleotide sequence as shown in SEQ ID NO.

3. (2) The gene of the fusion protein with the nucleotide sequence shown in SEQ ID NO.3 was cloned into the multiple cloning site on the pGEX-6P-1 vector to obtain the recombinant plasmid ClyA-VTPYGGGGVLLY-pGEX-6P-1; (3) Transform W3110 Escherichia coli competent cells with the plasmid ClyA-VTPYGGGGVLLY-pGEX-6P-1 to obtain a single clone strain that stably expresses the ClyA-VTPYGGGGVLLY fusion protein. This strain is referred to as the W-VTPYVLY strain. (4) Add 500 mL of ampicillin-resistant LB medium to a 1 L conical flask, inoculate 10 μL of W-VTPVLY bacterial culture into the medium, and shake the culture at 37 ℃ and 220 rpm for 4 h. Measure the OD value of the bacterial culture every 20 minutes. When the OD value reaches 0.5-0.6, add 500 μL of 1 M IPTG to make the final IPTG concentration in the medium 1 mM. Then, induce the expression of exogenous protein overnight at 21 ℃ and 200 rpm to obtain W-VTPVLY bacterial culture. (5) After centrifuging the W-VTPVLY bacterial culture at 4 ℃ and 5,000×g for 15 min, collect the supernatant and filter it with a 0.45 μm filter membrane. Concentrate the filtrate 20 times with an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD. Filter it again with a 0.22 μm filter membrane, and then centrifuge it at 150,000×g for 3 hours. Discard the supernatant and the precipitate is W-VTPVLY OMV.

7. The use of the growth-promoting polypeptide as described in claim 1 in the preparation of drugs that promote cell growth and tissue healing.

8. The use of W-VTPVLY OMV as described in claim 5 in the preparation of drugs that promote cell growth and tissue healing.

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