CN115991760A - Western bee MRJP 3C-terminal recombinant protein and preparation method and application thereof - Google Patents

Abstract

The invention provides a C-terminal recombinant protein of a western bee royal jelly main protein 3 (Major Royal Jelly Protein 3, MRJP3 for short) and a preparation method and application thereof, belonging to the technical field of genetic engineering. The amino acid sequence of the western bee MRJP 3C-terminal recombinant protein is shown as SEQ ID NO.1, and the polynucleotide for encoding the western bee MRJP 3C-terminal recombinant protein is shown as SEQ ID NO. 3. The invention also constructs plasmid for efficiently expressing the western bee MRJP 3C-terminal recombinant protein, and the western bee MRJP 3C-terminal recombinant protein produced by using 293F cells can promote the healing of in vitro wound models constructed by human immortalized keratinocytes, so that the western bee MRJP 3C-terminal recombinant protein provided by the invention has good application prospect as a wound healing agent.

Description

Western bee MRJP 3C-terminal recombinant protein and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a western bee MRJP 3C-terminal recombinant protein, a preparation method and application thereof.

Background

Wound healing is a complex process involving the combined action of multiple cellular and matrix components, where keratinocytes play a critical role in skin repair. After the skin is damaged, keratinocytes migrate to the wound site under the action of cytokines released by macrophages, migrate 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 the protective function. While keratinocytes secrete a variety of factors to resist pathogen invasion. Because of their important functions, keratinocytes proliferate and migrate, which are important contributors to wound healing. It is of great clinical importance to explore new substances that promote keratinocyte proliferation and migration and to elucidate their mechanism of action.

Royal Jelly (Royal Jelly) is a substance secreted by the feeding bees' hypopharynx and palate glands, and is consumed by three day-old larvae and queen bees in the colony. Many researches find that the royal jelly has various biological activities of resisting bacteria, resisting inflammation, resisting tumors, protecting liver and the like. In folk records, royal jelly has long been used for wound therapy. The laboratory has found that the mixture containing the components of the main proteins MRJP2, MRJP3, MRJP7 and the like of the royal jelly can obviously promote the healing of an in-vitro wound model, but the specific functional components are not yet determined.

Disclosure of Invention

The invention aims at providing a western bee MRJP 3C-terminal recombinant protein and a high-efficiency preparation method thereof.

The second purpose of the invention is to provide the application of the western bee MRJP 3C-terminal recombinant protein as a functional ingredient in promoting wound healing.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a recombinant protein at MRJP 3C end of Western bee comprises 113 amino acids, and has a molecular weight of 13.0 kDa, an isoelectric point of 10.65, and an amino acid sequence shown in SEQ ID NO. 1.

Furthermore, the MRJP 3C-terminal recombinant protein of the western bee is encoded by a polynucleotide with a nucleotide sequence shown as SEQ ID NO. 3.

A polynucleotide for coding the MRJP 3C-terminal recombinant protein of the Western bee has a sequence shown as SEQ ID NO. 3.

A vector comprising the polynucleotide shown in SEQ ID NO. 3.

A host cell comprising the above vector.

Further, the host cell is a 293F cell.

A method for preparing the western bee MRJP 3C-terminal recombinant protein, which comprises the following steps: constructing an expression vector containing polynucleotide for encoding the MRJP 3C-end recombinant Protein of the western bee, transfecting the obtained expression vector into a host cell, culturing the recombinant host cell, collecting a culture solution, and purifying by adopting a Protein A gel balance liquid chromatographic column to obtain the MRJP 3C-end recombinant Protein of the western bee.

Further, the method for preparing the western bee MRJP 3C-terminal recombinant protein specifically comprises the following steps:

(1) Obtaining MRJP3 protein sequence from NCBI database, through bioinformatics analysis, selecting 113 amino acids at C terminal as target protein, optimizing nucleotide sequence corresponding to target protein, adding human immunoglobulin Fc fragment at C terminal to obtain western bee MRJP 3C terminal recombinant protein nucleotide sequence, as shown in SEQ ID NO.3, then connecting to vector pcDNA3.1, and transforming E.coli;

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

(3) Culturing the 293F cells obtained in the step (2), collecting culture solution supernatant, and purifying by using a Protein A gel balance liquid chromatographic column to obtain the western bee MRJP 3C-terminal recombinant Protein.

The invention has the remarkable advantages that:

the invention successfully constructs a 293F cell expression western bee MRJP 3C-terminal recombinant protein system, and the western bee MRJP 3C-terminal recombinant protein expressed by the system can be used as a wound healing agent, and can obviously promote wound healing in an in vitro model.

Drawings

FIG. 1 is an electrophoresis chart of a recombinant protein at the MRJP 3C end of Western bees provided by the embodiment of the invention. Lane M is a Marker of the molecular weight of the pre-stained protein, and lane 1 is a purified recombinant protein at the MRJP 3C end of western bees.

FIG. 2 shows the proliferation activity of the recombinant protein at the MRJP 3C end of Western bee on HaCaT. Adding different concentrations of MRJP 3C-terminal recombinant proteins of western bees into equal amount of HaCaT cells, and performing a 48 h cell proliferation diagram; * P <0.05, < P <0.01, < P <0.001; serum-free medium was used as control.

FIG. 3 shows the healing promoting activity of the recombinant protein at the MRJP 3C end of the Western bee on an in vitro scratch model provided by the embodiment of the invention. Wherein A is a microscopic drawing of healing of HaCaT scratches at different time points after adding different concentrations of western bee MRJP 3C-terminal recombinant proteins into a HaCaT scratch model; b is a scratch healing rate analysis chart after HaCaT is added with 18 h; * P <0.05; serum-free medium was used as control.

FIG. 4 shows the HaCaT cell migration promoting activity of the recombinant protein at the MRJP 3C end of the Western bee provided by the embodiment of the invention. Wherein A is a HaCaT cell migration micrograph after a Transwell lower chamber is added with different concentrations of western bee MRJP 3C-terminal recombinant protein and 18 h; b is a cell mobility analysis chart after HaCaT dosing 18 h; * P <0.05, < P <0.01, < P <0.001; serum-free medium was used as control.

Detailed Description

In order to make the content of the invention more easily understood, the technical scheme of the invention is further described below with reference to specific embodiments. The examples are intended to illustrate the invention, but not to limit it in any way.

EXAMPLE 1 construction of System for efficient expression of the recombinant protein at the C-terminus of MRJP3 of Western bee

The recombinant protein at the MRJP 3C end of the western bee consists of 113 amino acids at the C end of the MRJP3 protein of the bee, and the sequence of the recombinant protein is shown as SEQ ID NO. 1. The sequence of MRJP3 of Western bee is published (GenBank: AY 663104.1). And (3) performing humanized optimization by taking the selected MRJP 3C-terminal protein of the Western bee as a template to obtain an optimized nucleotide sequence (shown as SEQ ID NO. 2). And adding a signal peptide and an Fc tag before and after the optimized nucleotide sequence to finally obtain the nucleotide sequence (shown as SEQ ID NO. 3) of the western bee MRJP 3C-terminal recombinant protein. The nucleotide sequence shown in SEQ ID NO.3 was ligated to vector pcDNA3.1 using pEASY-Uni Seamless Cloning and Assembly Kit (available from Transgen, CU 101-01) to obtain recombinant plasmid pcDNA3.1-C113-Fc. The recombinant plasmid pcDNA3.1-C113-Fc was transformed into competent cells of E.coli TOP10, and the transformed bacterial solution was spread on LB agar medium containing 1. Mu.g/mL ampicillin, and subjected to stationary culture at 37℃for 12 h. Single colonies were picked and transferred to 1 mL of LB liquid medium, shaking culture was performed at 37℃for 6 h, and then the culture medium was added to 300 mL of LB liquid medium, and cultured at 37℃for 14 h. After the incubation was completed, the plasmid was extracted using the FastPure EndoFree Plasmid Maxi Kit plasmid megaextraction kit (available from Vazyme, cat. DC 202-01) and the plasmid concentration was adjusted to 1 mg/mL.

Wherein the amino acid sequence of the signal peptide is MTKWLLLVVCLGIACQDVTS;

the amino acid sequence of the Fc tag is EPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；

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

the formula of the LB culture medium is as follows: 1% peptone, 0.5% yeast extract, 1% sodium chloride.

EXAMPLE 2 expression and purification of the Western bee MRJP 3C-terminal recombinant protein

(1) Expression analysis of western bee MRJP 3C-terminal recombinant protein

Sucking the mixture containing 8×10 ⁷ SMM 293-TII cell culture medium of 293F cells, centrifuging at 1000 rpm for 5 min, collecting cell pellet and supernatant, re-suspending cell pellet with 10 mL SMM 293-TII culture medium, inoculating into cell shake flask, adding supernatant and 80 mL SMM 293-TII culture medium after centrifuging 10 mL 293F cells, placing into CO ₂ The incubator was shake-cultured at 37℃and 160 rpm. 12 After h, 6 mL serum-free medium OptiPRO was taken ^TM SFM, 0.5 mL PEI and 150. Mu.g of the plasmid extracted in example 1 were mixed well, placed for 15 min and added to 293F cell shake flasks for transfection, after which 3.5 mL SMS M293-SUPI (containing 2.5% L-glutamine) was added to the shake flasks every 48. 48 h and samples were collected on day 7 after transfection.

(2) Purification of MRJP 3C-terminal recombinant protein of Western bee

1) The 293F cell culture broth after transfection was collected and centrifuged at 1000 rpm for 5 min. The supernatant was collected and centrifuged at 12000 rpm and 4℃for 30 min. The supernatant was collected again and filtered using a 0.45 μm filter membrane for subsequent protein purification.

2) Protein a gel equilibrium liquid chromatography column (available from Sangon Biotech, cat: c600951 Sequentially Washing with 5 times of sterile deionized water and 5 times of sterile Banding/Washing Buffer, adding the supernatant of the 293F cell culture fluid filtered in 1) into a Protein A gel balance liquid chromatographic column after the sterile Banding/Washing Buffer is drained, collecting effluent, repeatedly passing through the column for 3 times, washing with 15 times of sterile Banding/Washing Buffer, eluting target Protein in batches with 35 mL sterile Washing Buffer, adjusting the pH of the eluted Protein solution to 7.0 with Neutralizing Buffer, and concentrating with a 0.22 mu m regenerated cellulose ultrafiltration membrane to obtain purified MRJP 3C-end recombinant Protein (figure 1);

the formula of the band/Washing Buffer is as follows: 0.15 M NaCl,20 mM Na ₂ HPO ₄ ，pH 7.4；

The formula of the said solution Buffer is: 0.1 to M citric acid, and the pH value is 2.5 to 3.0;

the formula of Neutralizing buffer is as follows: 1M Tris-HCl (pH 9.0).

EXAMPLE 3 biological Activity of the recombinant protein at the C-terminus of MRJP3 of Western bee

(1) Proliferative Activity on HaCaT cells

At 4.0X10 ⁴ Cell density per well HaCaT cells were seeded uniformly in 96-well plates at 100 μl per well. After about 24 h a (after the cells have completely adhered), the culture broth is aspirated and starved with serum-free medium. When the cell growth state is consistent, 100 mu L of MRJP 3C-terminal recombinant proteins (0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, 200 and nM) with different concentrations are added into each well, and the culture is continued for 48 h for subsequent detection. Before detection, 10. Mu.L of 5% MTT solution was added to each well to incubate 4. 4 h in the dark, the culture medium was aspirated, 100. Mu.L of DMSO was added to each well, incubated for 10 min on a shaker, and finally absorbance was detected at 550. 550 nm. The result shows that the MRJP 3C-terminal recombinant protein of western bees has remarkable promotion effect on the proliferation of HaCaT cells at the concentrations of 12.5 nM, 6.25 nM and 3.13 nM by taking the added 0 nM recombinant protein group as a control (figure 2).

(2) Healing promoting Activity on HaCaT cell scratch model

Placing clean silica gel inserts into 24-hole cell culture plate, inoculating 3.5X10 of each insert cell ⁴ After culturing 12. 12 h HaCaT cells, inserts were removed, PBS (0.01M, pH 7.4) was washed 2 times to form 8.4 mm X0.5 mm regular scratch areas, then 1 mL different concentrations of MRJP 3C-terminal recombinant protein (0, 3.12, 6.25, 12.5, 25 nM) were added, and the scratch areas were microscopically recorded at 0, 12 and 18 h after addition of MRJP 3C-terminal recombinant protein, respectively. The results of the comparison with the added 0 nM recombinant protein group show that the western bee MRJP 3C-terminal recombinant protein has a remarkable promoting effect on the healing of the cell scratch model at the concentrations of 6.25 nM and 12.5 nM (figure 3).

(3) Migration promoting Activity on HaCaT cells

Inoculating 8×10 cells in each Transwell ⁴ The HaCaT cells were added with 600. Mu.L of MRJP 3C-terminal recombinant proteins (0, 3.12, 6.25, 12.5, 25 and nM) at different concentrations in the lower chamber, cultured in an incubator for 18 h and removed. Cells were fixed with polymethine and stained with crystal violet solution. Will beThe crystal violet in the chamber was aspirated, the non-migrated cells in the upper chamber were erased, placed under a microscope to observe the bottom of the chamber, and photographed. The results, with the addition of the 0 nM recombinant protein group as a control, show that the western bee MRJP 3C-terminal recombinant protein has a significant promoting effect on cell migration at concentrations of 6.25 nM and 12.5 nM (fig. 4).

Claims (10)

1. A western bee MRJP 3C-terminal recombinant protein, characterized in that: it consists of 113 amino acids, and has a molecular weight of 13.0 kDa, an isoelectric point of 10.65, and an amino acid sequence shown as SEQ ID NO. 1.

2. The western bee MRJP 3C-terminal recombinant protein of claim 1, wherein: the MRJP 3C-terminal recombinant protein of the western bee is encoded by a polynucleotide with a nucleotide sequence shown as SEQ ID NO. 3.

3. A polynucleotide encoding the western bee MRJP 3C-terminal recombinant protein of claim 1, characterized in that: the sequence of the polynucleotide is shown as SEQ ID NO. 3.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell comprising the vector of claim 4.

6. The host cell of claim 5, wherein: the host cell is a 293F cell.

7. A method for preparing the western bee MRJP 3C-terminal recombinant protein of claim 1, characterized in that: constructing an expression vector containing polynucleotide for encoding the MRJP 3C-end recombinant Protein of the western bee, transfecting the obtained expression vector into a host cell, culturing the recombinant host cell, collecting a culture solution, and purifying by adopting a Protein A gel balance liquid chromatographic column to obtain the MRJP 3C-end recombinant Protein of the western bee.

8. Use of a western bee MRJP 3C-terminal recombinant protein according to claim 1, a polynucleotide according to claim 3, a vector according to claim 4, or a host cell according to claim 5 for promoting skin cell proliferation and migration.

9. The use according to claim 8, characterized in that: the skin cells are human immortalized epidermis Hacat cells.

10. Use of a western bee MRJP 3C-terminal recombinant protein according to claim 1, a polynucleotide according to claim 3, a vector according to claim 4, or a host cell according to claim 5 for the preparation of a formulation for promoting wound healing.

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