A recombinant protein of the C-terminal of Apis mellifera MRJP3, its preparation method and its application

Abstract

The present invention provides a C-terminal recombinant protein of Major Royal Jelly Protein 3 (MRJP3) of Apis mellifera, a preparation method thereof, and its application, belonging to the technical field of genetic engineering. The amino acid sequence of the C-terminal recombinant protein of Apis mellifera MRJP3 is shown in SEQ ID NO.1, and the sequence of the polynucleotide encoding the C-terminal recombinant protein of Apis mellifera MRJP3 is shown in SEQ ID NO.3. The present invention also constructs a plasmid for highly expressing the C-terminal recombinant protein of Apis mellifera MRJP3. The C-terminal recombinant protein of Apis mellifera MRJP3 produced by using 293F cells can promote the healing of an in vitro wound model constructed by human immortalized keratinocytes, indicating that the C-terminal recombinant protein of Apis mellifera MRJP3 provided by the present invention has good application prospects as a wound healing promoter.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant protein of the C-terminal of Apis mellifera MRJP3, a preparation method thereof, and an application thereof. Background Art

[0002] Wound healing is a complex process involving the combined action of multiple cell and matrix components, among which keratinocytes play a crucial role in skin repair. After skin injury, keratinocytes migrate to the wound site under the action of cytokines released by macrophages, and transfer from the wound edge to the wound bed through proliferation and migration, participate in re-epithelialization and form new epithelial cells, rebuild the epithelial barrier, and restore its protective function. At the same time, keratinocytes can secrete various factors to resist the invasion of pathogens. Due to the important functions of keratinocytes, their proliferation and migration are important influencing factors for wound healing. Exploring new substances that promote the proliferation and migration of keratinocytes and clarifying their action mechanisms have important clinical significance.

[0003] Royal jelly is a substance secreted by the hypopharyngeal gland and mandibular gland of nurse bees, and is used for larvae within three days old and the queen bee in the bee colony. Many studies have found that royal jelly has various biological activities such as antibacterial, anti-inflammatory, anti-tumor, and liver protection. In folk records, royal jelly has been used for wound treatment for a long time. In previous studies in this laboratory, it was found that a mixture containing major royal jelly proteins MRJP2, MRJP3, MRJP7, etc. could significantly promote the healing of in vitro wound models, but the specific active ingredients therein have not been determined. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a recombinant protein of the C-terminal of Apis mellifera MRJP3 and an efficient preparation method thereof.

[0005] Another purpose of the present invention is to provide the application of the recombinant protein of the C-terminal of Apis mellifera MRJP3 as a functional component in promoting wound healing.

[0006] To achieve the above purposes, the present invention adopts the following technical solutions:

[0007] A recombinant protein of the C-terminal of Apis mellifera MRJP3, which is composed of 113 amino acids, has a molecular weight of 13.0 KDa, an isoelectric point of 10.65, and the amino acid sequence is as shown in SEQ ID NO.1.

[0008] Furthermore, the above-mentioned recombinant protein of the C-terminal of Apis mellifera MRJP3 is encoded by a polynucleotide with a nucleotide sequence as shown in SEQ ID NO.3.

[0009] A polynucleotide encoding the C-terminal recombinant protein of Apis mellifera MRJP3, whose sequence is shown as SEQ ID NO.3.

[0010] A vector containing the polynucleotide shown as SEQ ID NO.3 above.

[0011] A host cell containing the above vector.

[0012] Furthermore, the above host cell is 293F cell.

[0013] A method for preparing the C-terminal recombinant protein of Apis mellifera MRJP3 above, the method is: constructing an expression vector containing the polynucleotide encoding the C-terminal recombinant protein of Apis mellifera MRJP3, transfecting the obtained expression vector into a host cell, culturing the recombinant host cell, collecting the culture solution and purifying it using a Protein A gel equilibration buffer chromatography column, thus obtaining the C-terminal recombinant protein of Apis mellifera MRJP3.

[0014] Furthermore, the above method for preparing the C-terminal recombinant protein of Apis mellifera MRJP3 specifically includes the following steps:

[0015] (1) Obtaining the MRJP3 protein sequence from the NCBI database, through bioinformatics analysis, selecting 113 amino acids at its C-terminal as the target protein, optimizing the nucleotide sequence corresponding to the target protein and adding a human immunoglobulin Fc fragment at the C-terminal to obtain the nucleotide sequence of the C-terminal recombinant protein of Apis mellifera MRJP3, as shown in SEQ ID NO.3, and then ligating it to the vector pcDNA3.1 and transforming Escherichia coli;

[0016] (2) Culturing the Escherichia coli obtained in step (1) in an LB agar medium containing ampicillin, performing plasmid extraction after a large amount of amplification, and transfecting the extracted plasmid into 293F cells;

[0017] (3) Culturing the 293F cells obtained in step (2), collecting the supernatant of the culture solution, and purifying it through a Protein A gel equilibration buffer chromatography column to obtain the C-terminal recombinant protein of Apis mellifera MRJP3.

[0018] The remarkable advantages of the present invention are:

[0019] The present invention successfully constructs a 293F cell expression system for the C-terminal recombinant protein of Apis mellifera MRJP3, and the C-terminal recombinant protein of Apis mellifera MRJP3 expressed by this system can be used as a wound healing agent and can significantly promote wound healing in an in vitro model. Description of the Drawings

[0020] Figure 1Electrophoresis pattern of the C-terminal recombinant protein of Apis mellifera MRJP3 provided by the embodiment of the present invention. Lane M is the prestained protein molecular weight Marker, and lane 1 is the purified C-terminal recombinant protein of Apis mellifera MRJP3.

[0021] Figure 2 Proliferation activity of the C-terminal recombinant protein of Apis mellifera MRJP3 provided by the embodiment of the present invention on HaCaT. Different concentrations of the C-terminal recombinant protein of Apis mellifera MRJP3 were added to equal amounts of HaCaT cells, and the cell proliferation map at 48 h; *P<0.05, **P<0.01, ***P<0.001; the serum-free medium was used as the control group.

[0022] Figure 3 Promoting healing activity of the C-terminal recombinant protein of Apis mellifera MRJP3 provided by the embodiment of the present invention on the in vitro scratch model. Among them, A is the microscopic image of HaCaT scratch healing at different time points after adding different concentrations of the C-terminal recombinant protein of Apis mellifera MRJP3 to the HaCaT scratch model; B is the analysis chart of the scratch healing rate of HaCaT after adding the drug for 18 h; *P<0.05; the serum-free medium was used as the control group.

[0023] Figure 4 Promoting migration activity of the C-terminal recombinant protein of Apis mellifera MRJP3 on HaCaT cells provided by the embodiment of the present invention. Among them, A is the microscopic image of HaCaT cell migration after 18 h when different concentrations of the C-terminal recombinant protein of Apis mellifera MRJP3 were added to the lower chamber of Transwell; B is the analysis chart of the cell migration rate of HaCaT after adding the drug for 18 h; *P<0.05, **P<0.01, ***P<0.001; the serum-free medium was used as the control group. Detailed implementation manners

[0024] In order to make the content described in the present invention more understandable, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. The embodiments are intended to describe the present invention by way of example, rather than to limit the present invention in any form.

[0025] Example 1 Construction of a system for highly expressing the C-terminal recombinant protein of Apis mellifera MRJP3

[0026] The recombinant protein of the C-terminal of Apis mellifera MRJP3 according to the present invention is composed of 113 amino acids at the C-terminal of the Apis mellifera MRJP3 protein, and its sequence is shown in SEQ ID NO. 1. The sequence of Apis mellifera MRJP3 has been published (GenBank: AY663104.1). Using the selected C-terminal protein of Apis mellifera MRJP3 as a template, human optimization was carried out to obtain the optimized nucleotide sequence (shown in SEQ ID NO.2). A signal peptide and an Fc tag were added before and after the optimized nucleotide sequence respectively, and finally the nucleotide sequence of the recombinant protein of the C-terminal of Apis mellifera MRJP3 was obtained (shown in SEQ ID NO.3). The nucleotide sequence shown in SEQ ID NO. 3 was ligated to the vector pcDNA3.1 using the pEASY-UniSeamless Cloning and Assembly Kit (purchased from Transgen, CU101-01) to obtain the recombinant plasmid pcDNA3.1-C113-Fc. The recombinant plasmid pcDNA3.1-C113-Fc was transformed into Escherichia coli TOP10 competent cells, and the transformed bacterial solution was spread on an LB agar medium containing 1 μg / mL ampicillin and cultured statically at 37°C for 12 h. Single colonies were picked and transferred to 1 mL of LB liquid medium, cultured on a shaker at 37°C for 6 h, and then the culture solution was added to 300 mL of LB liquid medium and cultured at 37°C for 14 h. After the culture was completed, the plasmid was extracted using the FastPure® EndoFree Plasmid Maxi Kit (purchased from Vazyme, product number DC202-01), and the plasmid concentration was adjusted to 1 mg / mL.

[0027] Among them, the amino acid sequence of the signal peptide is MTKWLLLVVCLGIACQDVTS;

[0028] The amino acid sequence of the Fc tag is EPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

[0029] PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0030] The formula of the LB agar medium is: 1% peptone, 0.5% yeast extract, 1% sodium chloride and 1.5% agar powder;

[0031] The formula of the LB medium is: 1% peptone, 0.5% yeast extract, 1% sodium chloride.

[0032] Example 2 Expression and purification of the C-terminal recombinant protein of Apis mellifera MRJP3

[0033] (1)Expression analysis of the C-terminal recombinant protein of Apis mellifera MRJP3

[0034] Absorb the SMM 293-TII cell culture medium containing 8×10 7 293F cells, centrifuge at 1000 rpm for 5 min, collect the cell pellet and supernatant respectively. Resuspend the cell pellet with 10 mL of SMM 293-TII medium, inoculate it into a cell shake flask, then add 10 mL of the supernatant after centrifugation of 293F cells and 80 mL of SMM 293-TII medium, and place it in a CO2 incubator at 37℃, and shake and suspend the culture at 160 rpm. After 12 h, take 6 mL of serum-free medium OptiPRO TM SFM, 0.5 mL of PEI and 150 μg of the plasmid extracted in Example 1 are mixed evenly, and after standing for 15 min, add it to the 293F cell shake flask for transfection. Thereafter, add 3.5 mL of SMS M293-SUPI (containing 2.5% L-glutamine) to the shake flask every 48 h, and collect the samples on the 7th day after transfection.

[0035] (2)Purification of the C-terminal recombinant protein of Apis mellifera MRJP3

[0036] 1) Collect the culture solution of 293F cells after the above transfection, and centrifuge at 1000 rpm for 5 min. Collect the supernatant and centrifuge at 12000 rpm at 4℃ for 30 min. Collect the supernatant again, and filter it with a 0.45 μm filter membrane for subsequent protein purification.

[0037] 2) The Protein A gel equilibration buffer chromatography column (purchased from Sangon Biotech, catalog number: C600951) was washed sequentially with 5 column volumes of sterile deionized water and 5 column volumes of sterile Banding / Washing Buffer. After the sterile Banding / Washing Buffer had drained completely, the filtered supernatant of the 293F cell culture medium in 1) was added to the Protein A gel equilibration buffer chromatography column, and the effluent was collected. The process of passing through the column was repeated 3 times. Then, it was washed with 15 column volumes of sterile Banding / Washing Buffer, and the target protein was eluted in batches with 35 mL of sterile Elution Buffer. The pH of the eluted protein solution was adjusted to 7.0 using Neutralizing buffer, and then it was concentrated using a 0.22 μm regenerated cellulose ultrafiltration membrane to obtain the purified recombinant MRJP3 C-terminal protein ( Figure 1 );

[0038] The formula of the Banding / Washing Buffer is: 0.15 M NaCl, 20 mM Na2HPO4, pH 7.4;

[0039] The formula of the Elution Buffer is: 0.1 M citric acid, pH 2.5 - 3.0;

[0040] The formula of the Neutralizing buffer is: 1 M Tris-HCl (pH 9.0).

[0041] Example 3 Biological Activity of the Recombinant MRJP3 C-Terminal Protein of the Western Honey Bee

[0042] (1) Proliferation Activity on HaCaT Cells

[0043] At 4.0×10 4For the cell density of cells per well, HaCaT cells were evenly seeded into a 96-well plate, 100 μL per well. After about 24 h (after the cells were completely adherent), the culture medium was aspirated, and serum-free medium was added for starvation treatment. When the cell growth status was consistent, 100 μL of recombinant MRJP3 C-terminal protein at different concentrations (0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, 200 nM) was added to each well, and the cells were cultured for another 48 h for subsequent detection. Before detection, 10 μL of 5% MTT solution was added to each well and incubated in the dark for 4 h. The culture medium was aspirated, and then 100 μL of DMSO was added to each well, and the plate was incubated on a shaker for 10 min. Finally, the absorbance was measured at 550 nm. Using the group with 0 nM recombinant protein added as the control, the results showed that the recombinant MRJP3 C-terminal protein of Apis mellifera had a significant promoting effect on the proliferation of HaCaT cells at concentrations of 12.5 nM, 6.25 nM, and 3.13 nM ( Figure 2 )

[0044] (2)Promoting healing activity on the HaCaT cell scratch model

[0045] A clean silicone insert was placed into a 24-well cell culture plate. 3.5×10 4 HaCaT cells were seeded into each insert compartment. After culturing for 12 h, the insert was removed, and the cells were washed twice with PBS (0.01 M, pH 7.4) to form a regular scratch area of 8.4 mm×0.5 mm. Subsequently, 1 mL of recombinant MRJP3 C-terminal protein at different concentrations (0, 3.12, 6.25, 12.5, 25 nM) was added. The scratch area was recorded under a microscope at 0, 12, and 18 h after adding the recombinant MRJP3 C-terminal protein. Using the group with 0 nM recombinant protein added as the control, the results showed that the recombinant MRJP3 C-terminal protein of Apis mellifera had a significant promoting effect on the healing of the cell scratch model at concentrations of 6.25 nM and 12.5 nM ( Figure 3 )

[0046] (3)Promoting migration activity on HaCaT cells

[0047] 8×10 4HaCaT cells were added, and 600 μL of recombinant proteins of the C-terminus of MRJP3 at different concentrations (0, 3.12, 6.25, 12.5, 25 nM) were added to the lower chamber. After culturing in an incubator for 18 h, the cells were taken out. The cells were fixed with polyformaldehyde and stained with crystal violet solution. The crystal violet in the chamber was aspirated, the non-migrated cells in the upper chamber were wiped off, and the bottom of the chamber was observed under a microscope and photographed. Taking the group with 0 nM recombinant protein as the control, the results showed that the recombinant protein of the C-terminus of MRJP3 of Apis mellifera had a significant promoting effect on cell migration at the concentrations of 6.25 nM and 12.5 nM ( Figure 4 ).

Claims

1. A method for preparing the C-terminal recombinant protein of Apis mellifera MRJP3, characterized in that: Construct an expression vector containing the polynucleotide encoding the C-terminal recombinant protein of Apis mellifera MRJP3, transfect the obtained expression vector into a host cell, culture the recombinant host cell, collect the culture medium and purify it using a Protein A pre-packed chromatography column to obtain the Apis mellifera MRJP3 C-terminal recombinant protein; The expression vector is the pcDNA3.1 vector; The host cell is the 293F cell; Using the selected Apis mellifera MRJP3 C-terminal protein as a template, perform human optimization to obtain the optimized nucleotide sequence, as shown in SEQ ID NO.2; Add a signal peptide and an Fc tag before and after the optimized nucleotide sequence respectively to finally obtain the nucleotide sequence of the Apis mellifera MRJP3 C-terminal recombinant protein, as shown in SEQ ID NO.3; The Apis mellifera MRJP3 C-terminal protein consists of 113 amino acids, with a molecular weight of 13.0 kDa and an isoelectric point of 10.65, and the amino acid sequence is as shown in SEQ ID NO.

1.

2. Use of the C-terminal recombinant protein of Apis mellifera MRJP3 prepared by the preparation method according to claim 1 in the preparation of a preparation for promoting wound healing.

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