A method for preparing a recombinant ternary fusion protein dressing with epidermal cell activation function

Abstract

The application discloses a preparation method of a recombinant ternary fusion protein dressing with epidermal cell activation function. The dressing is a fusion protein designed based on the research on the functions and properties of COL3A1 and IFN-kappa proteins by international peers and the laboratory. The specific method is that the fusion gene fragment of the covalent connection of COL3A1 and IFN-kappa with GGSGG as a linker is obtained by using overlap extension PCR technology, and a 6×His label is added. The target gene is cloned into a prokaryotic expression vector pET30 (modified in the laboratory) to obtain the fusion protein COL3A1-IFN-kappa with an MBP label. After expansion culture, the MBP-IFN-kappa-COL3A1 with a purity of more than 90% can be obtained through Ni-NTA column affinity chromatography. We verified the resistance of the fusion protein to salmonella in THP-1 cells, and verified the effect of the fusion protein on promoting wound healing and resisting salmonella infection on mice. Finally, it is proved that the MBP-IFN-kappa-COL3A1 has good uniformity, is easy to purify, has high activity, has no side effects, and is very suitable for being used as an epidermal cell activation dressing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and particularly relates to molecular cloning, protein expression and purification, polyacrylamide gel electrophoresis (SDS-PAGE) and bacterial infection techniques. BACKGROUND

[0002] Skin tissue is a barrier on the surface of human body, and has multiple functions such as protecting various organs in the skin, preventing the loss of water and electrolytes and the like in the human body, and protecting the human body from pathogenic substances such as viruses and bacteria. Therefore, the integrity of the skin is very important for the health of the human body. However, as an organ directly contacting with the external environment, the skin is continuously subjected to stimulation from the external environment and receives various signals from the body. Disorders of these factors can cause various diseases of the skin. Skin diseases are one of the major diseases that plague humans. In addition to malignant diseases such as skin cancer, skin diseases caused by various autoimmune disorders, such as eczema, psoriasis, and lupus erythematosus, are also long-term diseases that plague humans and are very difficult to cure. In addition, with increasing age, skin aging is also a problem that plagues humans, especially after the overall living standard of humans improves, the life expectancy of humans is increasing, and the attention to appearance is also increasing. Therefore, skin care and repair are also increasingly valued by people. In addition, the use of a large number of cosmetics of varying quality has also caused great damage to facial skin, causing many problems such as thinning of facial skin and easy allergy. In view of these problems, many so-called medical cosmetic products with skin repair and activation effects have appeared on the market. According to our understanding, these products are mainly divided into three categories according to the claimed active ingredients:

[0003] (1) Epidermal growth factor: including EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor) and the like. These products can promote the growth and metabolism of epidermal cells after use (under the premise of maintaining protein activity), and have a certain effect on the repair of the epidermis. However, as growth factors, they often have side effects and potential risks, such as excessive cell growth, causing scars in the local area, and even cancerization. Therefore, regular skin repair and cosmetic products will recommend cautious use. Of course, due to the difficulty of protein products in liquid (including gel) storage, it is actually impossible for many claimed growth factor products to have active growth factor proteins.

[0004] (2) Hyaluronic acid: Hyaluronic acid, also known as hyaluronic acid, is an extracellular polysaccharide with good water absorption and water retention, so it is used as a skin moisturizing cosmetic filler. Hyaluronic acid is often injected subcutaneously to achieve the effect of filling, but this effect is difficult to last. As hyaluronic acid is metabolized and broken down, the skin that was previously filled will quickly return to its original appearance. If it is only applied externally, it is difficult to be absorbed and only has a temporary effect. The promotion of hyaluronic acid in the beauty industry is often exaggerated.

[0005] (3) Collagen: Collagen is one of the main components of the extracellular matrix of human skin cells, and plays an important role in maintaining the structure of the skin. The main component of collagen is a triple helix bundle formed by the repetition of three amino acids glycine-proline-hydroxyproline, which is structurally very stable and not easily degraded. As age increases, the collagen secreted by epidermal cells will decrease, and the existing collagen will also degrade and be lost over time, so there will be a loss of collagen and a condition of loose skin. To solve this problem, many products have proposed a collagen supplement plan.

[0006] To solve these problems, we wonder if we can find a way to improve the skin by strengthening the effect of collagen in situ. On the one hand, we can supplement collagen from the outside to promote the absorption of epidermal cells; on the other hand, we can also activate the epidermal cells to promote the secretion of collagen by the epidermal cells. Through the simultaneous action of these two schemes, we hope to achieve the effect of promoting the regeneration of local epidermal cells and increasing the concentration of local collagen, thereby improving the skin.

[0007] Collagen is a large molecule, which is difficult to be absorbed by the skin directly. Moreover, purified collagen is unstable in vitro and can be degraded easily. In addition, with the growth of age, the activity of epidermal cells decreases slowly, and the synthesis and secretion of collagen also decrease slowly. How to activate these epidermal cells moderately and promote the secretion of local collagen is a difficult problem. Although the use of growth factor proteins (such as EGF, PDGF, etc.) can activate these cells, this activation is not easy to control, and a large amount and long-term use can easily induce the occurrence of cancer. We have previously tried a protein, interferon kappa (IFN-κ). IFN-κ can effectively activate the local epithelial cell antiviral immune signaling pathway and inhibit and kill a variety of viruses. Compared with interferon alpha or beta, IFN-κ is limited to the expression of epithelial cells and is activated by an autocrine pathway, so it only acts locally on epithelial cells and does not enter the blood circulation when applied externally, thus avoiding the side effects of long-term use of IFN alpha and beta; in addition, recent studies have found that IFN-κ is closely related to HPV infection, and HPV infection inhibits the expression of IFN-κ, while exogenous expression of IFN-κ can inhibit the activation of HPV, which is not possessed by IFN alpha / beta. We found previously that IFN-κ is very safe when used externally, because this activation activates the innate immune pathway that already exists in the human body and only occurs in a certain space and time, without the risk of cancerization. Since natural immune activation can activate epidermal cells and promote cell synthesis and secretion, we believe that the local use of IFN-κ can moderately activate epidermal cells and promote the expression and secretion of collagen in situ; at the same time, the moderate activation of local cells can also promote the regeneration of damaged epidermal cells and promote skin repair.

[0008] There are 28 members in the collagen family, among which collagen 3 (COL3A1) is the most abundant collagen in epidermal cells and plays an important role in maintaining the state of epidermal cells. Full-length COL3A1 is difficult to express and is also unstable in vitro. Therefore, we selected the most important segment of COL3A1, namely the VWFC domain, and the triple helix bundle region of about 40 amino acids after the domain, for expression through sequence analysis of COL3A1. The VWFC domain is the region of COL3A1 that interacts with epidermal transforming factor TGF-β, and the triple helix bundle that follows it can greatly increase the stability of VWFC. In addition, this sequence contains multiple cysteines, which can form a network-like protective structure on the cell surface together with the triple helix bundle.

[0009] MBP is a common tag protein used in protein expression, which can assist protein purification and increase the stability of fusion protein. MBP can bind to some glycosyl, so it can increase the affinity to epidermal cells.

[0010] According to these ideas, we fused and expressed three proteins, MBP, IFN-κ and COL3A1, which may have activation and repair functions for skin collagen, to observe their ability to maintain and activate epidermal cell collagen. SUMMARY

[0011] The application discloses a preparation method of a recombinant ternary fusion protein dressing with epidermal cell activation function. The method is characterized in that, according to the research on the functions of COL3A1 and IFN-κ by international counterparts and the research on the protein properties of COL3A1 and IFN-κ in an Escherichia coli expression system in the laboratory, the VWFC domain of COL3A1 and IFN-κ are covalently connected through a flexible linker, 6xHis-tag is added at one end of the fusion gene, then the fusion gene is constructed into a vector containing an MBP tag and is expressed, and the MBP-IFN-κ-COL3A1 fusion protein with good solubility and stability is obtained. Specifically, the VWFC domain of the COL3A1 protein and IFN-κ are covalently connected through overlap extension polymerase chain reaction (overlap extension PCR) technology by means of GGSGG. The gene fragment of the fusion protein is obtained by using overlap extension PCR technology, the target gene is cloned into a prokaryotic expression vector pET30 (modified in the laboratory), and the plasmid is extracted and then transferred into BL21 (DE3) codon Plus Escherichia coli. The Escherichia coli into which the target gene is transferred is cultured in LB medium, and when the OD 600 = 0.6-0.8, 0.1 mM IPTG is used to induce the expression of the protein, and the target protein can be obtained through Ni-NTA column affinity chromatography. The purity and uniformity of the target protein are very good, and the expression amount is large. The anti-bacterial experiment of the fusion protein is carried out in monocyte macrophage THP-1, and it is found that the protein can effectively inhibit the invasion of Salmonella 14028 strain and D23580 strain on the cells. Then, the protein is used for promoting wound healing in mice. A 6mm-diameter wound is made on the back skin of the mice, different concentrations of MBP-IFN-κ-COL3A1 protein solution are added dropwise, and the recovery of the wound is monitored, and it is found that the protein can effectively promote the healing of the wound and resist the invasion of Salmonella.

[0012] The present application fuses three proteins MBP, IFN-κ and COL3A1 which may have the function of activating and repairing skin collagen, and can help the healing of micro-wounds of epithelial cells while resisting the invasion of Salmonella to the cells. Compared with interferon alpha or beta, IFN-κ is limited to express in epithelial cells, is activated through an autocrine pathway, and can inhibit the activation of HPV and does not enter the blood circulation when applied externally, thereby avoiding the side effects of long-term use of IFN alpha and beta. Therefore, the fusion protein MBP-IFN-κ-COL3A1 is very suitable as an epithelial cell activating dressing. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 : Design diagram and mechanism diagram of fusion protein MBP-IFNκ-COL3A1.

[0014] Figure 2 : SDS-PAGE results of expression and purification of fusion protein MBP-IFNκ-COL3A1. 1 is the precipitate after bacterial disruption and centrifugation; 2 is the supernatant after bacterial disruption and centrifugation; 3 is the sample flowed down from the His affinity column; 4 is the sample washed with Binding buffer; 5 is the sample washed with Washing buffer; 6 is the protein sample eluted with eluent.

[0015] Figure 3 : Detection of Salmonella content in THP-1 cells. The experimental group uses culture medium containing fusion protein MBP-IFNκ-COL3A1 to culture cells, and the culture medium without fusion protein is used as a control. Salmonella 14028 and D23580 are used to infect cells, and after cell disruption, they are plated on solid LB medium plates and cultured for about 12 h for counting.

[0016] Figure 4 : Statistical results of mouse skin wound healing experiment. A 6mm punch is used to make a skin wound on the back of a mouse, and different concentrations of MBP-IFNκ-COL3A1 protein solution are added to the wound. PBS solution is used as a control. The left graph shows the healing of mouse wounds after Salmonella 14028 strain infection, and the right graph shows the healing of mouse wounds after Salmonella D23580 strain infection.

[0017] Figure 5 : Detection of Salmonella content in mouse skin wounds. The mouse wounds are treated with fusion protein MBP-IFNκ-COL3A1, and the PBS solution treatment is used as a control. Then the wounds are infected with Salmonella, and the Salmonella content near the mouse wound tissue is detected three days later. DETAILED DESCRIPTION

[0018] Example 1: Construction of prokaryotic expression vector of fusion protein MBP-IFNκ-COL3A1

[0019] The DNA fragment of fusion protein COL3A1-IFN-κ with GGSGG as linker was obtained by overlap extension PCR using COL3A1 and IFN-κ as templates. The target gene fragment was cloned into the pET30 (containing MBP tag) vector modified by our laboratory by homologous recombination. The modified plasmid enables the fusion protein to carry MBP tag and 6×His tag. The MBP tag can largely solve the problems of poor stability and poor solubility of IFN-κ, while the 6×His tag is helpful for subsequent protein extraction and purification. After double digestion and sequencing, the correct plasmid was retained for subsequent experiments.

[0020] The primers used in the vector construction process are as follows:

[0021] Primer 1: IFNκ-F: 5'-CTGTACTTCCAATCCAATCTGGACTGTAACTTACTGAACGTTCAC-3' Primer 2: IFNκ-R: 5'-CATCCTCCGCCACCGCTTCCGCCTTTCCTCCTGAATAGAGC-3'

[0022] Primer 3: COL3A1-F: 5'-GAGGAAAGGCGGAAGCGGTGGCGGAGGATGTTCCCATCTTG-3' Primer 4: COL3A1-R: 5'-CCGTTATCCACTTCCAATTTAAACACCACCACAGCAAG-3'

[0023] Example 2: Expression and purification of fusion protein MBP-IFNκ-COL3A1

[0024] The constructed plasmid was transformed into BL21 (DE3) codon Plus E. coli. 1 μL plasmid was added to BL21 (DE3) codon Plus competent cells, mixed by blowing and sucking several times. After ice bath for 30 min, heat shock at 42°C for 90 s, and ice bath for 2 min. In the clean bench, 200 μL fresh LB (without antibiotics) liquid medium was added to the treated BL21 (DE3) codon Plus competent cells and mixed, and cultured at 37°C, 220 rpm constant temperature shaking for 30-60 min. The cultured BL21 (DE3) codon Plus competent cells were centrifuged at 2000-3000 rpm for 3 min, and the supernatant was removed in the clean bench. After resuspending the bacterial cells, they were plated on LB solid medium plates with Kan resistance, and cultured in a 37°C constant temperature incubator for 12-16 h. A single colony of BL21 (DE3) codon Plus was picked and inoculated in 100 mL LB (Kan resistance) liquid medium, and cultured at 37°C, 220 rpm constant temperature shaking for 6-8 h until the OD 600 = 0.6-0.8. In the clean bench, 40-50 mL of each was transferred to 1000 mL LB (Kan resistance) liquid medium, and cultured at 37°C, 220 rpm constant temperature shaking for 3-5 h until the OD 600 = 0.6-0.8. 200 μL of 1 M IPTG was added to each bottle to induce protein expression, and cultured at 16°C, 220 rpm constant temperature shaking for 12-16 h. The E. coli after induction was centrifuged at 4000 rpm for 20 min, and the supernatant was discarded. The bacterial cells were resuspended in 25 mL Binding Buffer (50 mM Tris, 0.3 M Nacl, PH = 8.0). The resuspended bacterial cells were poured into a beaker, and the beaker was placed in an ice box containing an ice-water mixture. The ice box was placed in a soundproof box for ultrasonic disruption, 5 min (break for two seconds and stop for four seconds), AMPL = 40%. The disrupted bacterial solution was placed in a centrifuge tube, and after balancing, it was centrifuged at 18000 rpm for 40 min at 4°C. The supernatant was collected, and His column affinity chromatography purification was performed. First, the supernatant was washed with Binding Buffer (50 mM Tris, 0.3 M Nacl, PH = 8.0) for three column lengths. Then the supernatant was passed through the affinity chromatography column three times, and a column volume of Washing Buffer (50 mM Tris, 0.3 M Nacl, 20 mM imidazole, PH = 8.0) was used to wash away the non-specifically adsorbed impurities. Finally, His Elute Buffer (50 mM Tris, 0.3 M Nacl, 0.5 M imidazole, PH = 9.0) was used for elution, and the eluted components were collected. After SDS-PAGE, the results were analyzed by Coomassie brilliant blue staining. Then the protein was aliquoted and stored at -80°C after freeze-drying.

[0025] Example 3: Anti-bacterial experiment of fusion protein MBP-IFNκ-COL3A1 at cell level

[0026] THP-1 cells were first plated in six-well plates. The cells were induced to adhere with PMA-containing medium for 48 h. Then the cells were cultured with medium containing 500 ng / mL MBP-IFNκ-COL3A1 fusion protein for 4 h, and the cells were cultured with normal medium as control. The cells were infected with Salmonella 14028 and D23580 for 40 min, and the ratio of cells to bacteria was 1:40. The cells were cultured with medium containing 100 ng / mL gentamicin for 2 h to kill the bacteria adhering to the cell surface. Then the medium was replaced with fresh medium containing 10 ng / mL gentamicin for 10 h to complete the whole infection experiment. The cells were lysed with RIPA lysis buffer to release the intracellular bacteria, and the cell lysate was spread on LB solid medium without resistance for 12 h at 37 °C for colony counting.

[0027] Example 4: Detection of Salmonella content in mouse skin wound

[0028] Four healthy male mice with consistent growth were selected, and the mice were anesthetized with 4% chloral hydrate, and then the hair on their backs was removed with a razor and depilatory cream. Two circular wounds with consistent size were punched on the exposed skin with a punch with a diameter of 6 mm. 10 μL of MBP-IFNκ-COL3A1 fusion protein solution with different concentrations was added to the wounds, and the same volume of PBS solution was used as control. After 18 h, 10 μL of different types of Salmonella liquid was added to the wounds to infect the mice. The above treatment was repeated for three days. On the third day, the mice were sacrificed by cervical dislocation, and the skin tissue around the wound of the mice was cut off to 2 mm and placed in 1 mL of LB liquid medium. After shaking several times, 10 μL was taken and spread on LB solid medium without resistance, and incubated at 37 °C for 12 h or so for colony counting.

Claims

1. Use of the fusion protein MBP-IFN-κ-COL3A1 in the preparation of a dressing against Salmonella infection, characterized in that, IFN-κ is in front of COL3A1 in the fusion protein; the linker connecting IFN-κ and COL3A1 is GGSGG.

2. Use according to claim 1, characterized in that, Two active proteins, COL3A1 and IFN-κ, are used.

3. Use according to claim 1, characterized in that, The fusion protein is tagged with MBP by modification of the prokaryotic expression vector pET30, so that the solubility of the fusion protein is better, the properties are more stable, and the fusion protein is more suitable as a recombinant ternary fusion protein dressing with epidermal cell activation function.

4. Use according to claim 1, characterized in that, The gene fragment of the fusion protein is obtained by overlap extension PCR technology, a 6×His tag is added to one end of the gene fragment, the target gene is cloned into the prokaryotic expression pET30, transformed into BL21(DE3) codon Plus E. coli, and then cultured at low temperature. High-purity MBP-IFN-κ-COL3A1 fusion protein can be obtained by Ni-NTA affinity column affinity chromatography.

Images