CN117866068A - Pinctada martensii galactose-binding lectin protein PFL-96 for promoting wound healing and preparation method and application thereof - Google Patents

Abstract

The invention discloses a pinctada martensii galactose-binding lectin recombinant protein PFL-96 for promoting wound healing, a preparation method and application thereof, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2. Biological activity detection shows that the PFL-96 recombinant protein not only can inhibit and kill methicillin-resistant staphylococcus aureus, but also can remarkably promote the healing of rat MRSA infectious wound, so that the recombinant protein can be used for preparing antibacterial wound repair medicines.

Description

Pinctada martensii galactose-binding lectin protein PFL-96 for promoting wound healing and preparation method and application thereof

Technical Field

The invention belongs to the technical field of recombinant proteins, and particularly relates to a pinctada martensii galactose-binding lectin PFL-96 recombinant protein and application thereof in preparation of a medicament for promoting wound healing.

Background

Along with the acceleration of the aging process of population and the change of human disease spectrum, the number of acute wound surfaces caused by wounds, burns and the like and the number of chronic wound surface patients difficult to heal on the body surface caused by diseases such as diabetes and the like is increased year by year, thereby seriously affecting the physical and psychological health and the quality of life of people. The wound repair is a hot topic of research in the current clinical medicine field, and bacterial infection, especially in hospitals, is likely to occur on the wound, and along with the use of antibiotics, multi-drug resistant strains in the hospitals are generated and spread, especially methicillin-resistant staphylococcus aureus are caused, so that infection often occurs in the wound repair process, inflammatory reaction is aggravated, serious infection-related complications are even brought, the wound repair process of patients is reduced, the treatment difficulty in the treatment process is increased, the treatment time is prolonged, the related cost is increased, and meanwhile, the economic burden is also required to be borne while the disease pain is brought to the patients. Therefore, it is of great importance to develop new drugs that are effective against multi-drug resistant bacterial infections while promoting healing of infectious wounds.

Pinctada martensii (Pinctadafucata) is one of important seawater cultured shellfish in China and main shellfish species for producing seawater pearls, belongs to marine invertebrates, has a unique complex natural immune system, and lectin is one of important pattern recognition molecules in the natural immune system. The pinctada martensii produces various sugar-specific binding lectins in a complex marine environment with low temperature, high salt and high pressure, and has a plurality of physiological functions of regulating organism metabolism, participating in host defense through recognition and binding of sugar residues invading the surface of pathogens, and the like. Five lectin or lectin-like genes have been identified in pinctada martensii, including C-type lectin, F-type lectin, galactose-binding lectin, a-N-acetylgalactose-binding lectin and serotonin subtype-2, and play a role in host defense.

The applicant has found in previous studies that a Pinctada martensii galactose-binding lectin PFL-96 (patent publication No. CN114426571A, publication No. 2022-05-03) was prepared by using pET-28A vector expression to prepare a PFL-96 protein containing a partial vector fragment, so that the recombinant protein purified by expression was relatively large, and the molecular weight was 14.36kDa; in addition, experimental researches show that the recombinant protein has bactericidal activity on staphylococcus aureus, bacillus subtilis, candida albicans and vibrio alginolyticus, and has no remarkable antibacterial effect on escherichia coli, pseudomonas aeruginosa and salmonella typhimurium.

By searching the prior art at home and abroad, the biological activity of the pinctada martensii galactose-binding lectin for promoting wound healing has not been reported in the literature.

Disclosure of Invention

Aiming at the defects and actual demands of the prior art, the invention aims to provide a pinctada martensii galactose-binding lectin PFL-96 recombinant protein, and a preparation method and application thereof.

In order to achieve the technical purpose, the inventor performs more deep exploration through a large number of experiments on the basis of early research results, and finally obtains the following technical scheme: an antibacterial galactose-binding lectin recombinant protein of pinctada martensii, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1. The lectin was expressed in E.coli to obtain recombinant protein PFL-96 containing a 6 XHIS tag from which the putative signal peptide was removed.

Furthermore, the invention also provides a coding gene of the pinctada martensii antibacterial galactose binding lectin recombinant protein, and the coding gene codes protein with an amino acid sequence shown in SEQ ID NO. 1. Specifically, the nucleotide sequence of the pinctada martensii antibacterial galactose binding lectin recombinant protein is shown as SEQ ID NO. 2.

Further, the invention also provides a recombinant expression vector, which is obtained by recombining the gene and the expression vector; still further preferably, the expression vector is pET-30a.

Further, the invention also provides a preparation method of the pinctada martensii antibacterial galactose-binding lectin recombinant protein, which comprises the following steps:

(1) Constructing the nucleotide sequence of the encoding gene of the pinctada martensii antibacterial galactose binding lectin recombinant protein into an expression vector pET-30a to obtain a recombinant plasmid pET-30a-PFL-96;

(2) And (3) converting the recombinant plasmid pET-30a-PFL-96 into competent cells of escherichia coli, culturing in a liquid culture medium, carrying out induced expression, collecting bacterial liquid, extracting and purifying to obtain the antimicrobial galactose-binding lectin recombinant protein of pinctada martensii.

More specifically, the preparation method comprises the following steps: (1) Synthesizing and removing the galactose-binding lectin gene fragment of the pinctada martensii of the hypothetical signal peptide, and connecting the galactose-binding lectin gene fragment into a prokaryotic expression vector pET-30a to obtain a prokaryotic expression recombinant plasmid pET-30a-PFL-96; (2) Introducing competent cells of escherichia coli by a calcium chloride heat shock method to obtain a recombinant strain; (3) Culturing the recombinant strain and inducing the expression of recombinant protein PFL-96; (4) Purifying, dialyzing, removing endotoxin, filtering and sterilizing to obtain PFL-96 recombinant protein, and performing concentration measurement, SDS-PAGE detection, western blot verification, mass spectrum and molecular weight identification.

Preferably, in the preparation method of the pinctada martensii antibacterial galactose binding lectin recombinant protein, the escherichia coli competent cell is an escherichia coli BL21 (DE 3) competent cell.

In addition, the invention explores the antibacterial activity of the pinctada martensii galactose-binding lectin recombinant protein PFL-96 on drug-resistant bacteria, namely methicillin-resistant staphylococcus aureus (MRSA), and the result shows that the PFL-96 has remarkable antibacterial and bactericidal activity on the MRSA; in addition, the inventor constructs the rat MRSA infectious wound surface, and discovers that PFL-96 has remarkable promotion effect on the rat MRSA infectious wound surface repair through local administration. Therefore, the invention also provides the application of the pinctada martensii antibacterial galactose-binding lectin recombinant protein in preparing medicines for inhibiting or/and killing methicillin-resistant staphylococcus aureus; the application of the pinctada martensii antibacterial galactose-binding lectin recombinant protein in preparing medicaments for promoting wound healing. Still further preferably, the wound is a methicillin-resistant staphylococcus aureus infectious wound.

Compared with the prior art, the invention has the following advantages and remarkable progress:

(1) The invention optimizes the expression path, selects a prokaryotic expression vector pET-30a to replace pET-28a for PFL-96 expression, and results show that compared with pET-28a, the target protein purified by using pET-30a as the expression vector does not contain vector fragments, the molecular weight is reduced to 10.57kDa, and unexpected technical effects are obtained.

(2) The invention discovers that the recombinant protein PFL-96 not only can inhibit and kill methicillin-resistant staphylococcus aureus, but also has remarkable promoting effect on the repair of MRSA infectious wound, so that the recombinant protein PFL-96 can be used for preparing medicaments for resisting methicillin-resistant staphylococcus aureus, can also provide a new treatment path for the rapid healing treatment of human MRSA infectious wound, and has good application prospect.

Drawings

FIG. 1 shows the electrophoresis pattern of the prokaryotic expression recombinant plasmid pET-30a-PFL-96 and Apa I, hin d III double enzyme digestion products constructed by the invention.

FIG. 2A shows the result of SDS-PAGE gel electrophoresis of the target protein expressed by prokaryotes. M is a protein molecular mass standard; 0 is pET-30a-PFL-96, which is not induced; 1 is a whole bacterium after recombinant plasmid induction; 2, inducing and crushing the recombinant plasmid to obtain a supernatant; 3 precipitation after disruption for recombinant plasmid induction. FIG. 2B shows the results of SDS-PAGE identification of purified proteins. M is a protein molecular mass standard; 1 is sediment centrifugally collected after the whole bacteria are crushed; 2 is a purification process flow-through sample; 3. and 4 and 5 are elution samples in the purification process. FIG. 2C shows the SDS-PAGE identification of purified proteins. M is a protein molecular mass standard; 1 is 0.5mg/mL Bovine Serum Albumin (BSA); 2 is the target protein sample after purification. FIG. 2D is a Western blot verification.

FIG. 3 is a diagram showing the verification of prokaryotic expression of recombinant protein PFL-96; wherein: a is mass spectrum identification; b is molecular weight identification.

FIG. 4 shows the bacteriostatic activity of lectin PFL-96 against MRSA as measured by the Oxford cup method. Wherein: a is antibacterial activity of PFL-96 to MRSA; b is a schematic diagram of oxford cup placement; c is a data analysis chart of PFL-96 on the diameter of the MRSA bacteriostasis area.

FIG. 5 is a graph showing the determination of the minimum inhibitory concentration and the minimum bactericidal concentration of lectin PFL-96 against methicillin-resistant Staphylococcus aureus. Wherein: a is determination of minimum inhibitory concentration of lectin PFL-96 on MRSA, and MIC is 8 mug/mL; lectin PFL-96 final concentration in plate B was 16. Mu.g/mL, giving a total of 0 single colonies, less than 5; the final concentration of lectin PFL-96 in the C plate was 8. Mu.g/mL, and a plurality of single colonies appeared, indicating that the minimum bactericidal concentration of lectin PFL-96 against MRSA was 16. Mu.g/mL.

FIG. 6 shows the detection of the number of Staphylococcus aureus colonies in the wound surface of the blank group and the wound surface of the negative infection group by using mannitol sodium chloride agar plates.

Fig. 7 shows that lectin protein PFL-96 significantly promotes healing of rat MRSA infectious wound.

Fig. 8 is a graph showing the change of wound healing rate with treatment time for each group of rats.

Detailed Description

The invention will be further illustrated with reference to specific examples, to which the scope of protection is not limited. The experimental methods in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.

EXAMPLE 1 synthesis of galactose-binding lectin PFL-96 Gene of Pinctada martensii and construction of prokaryotic expression plasmid and prokaryotic expression

According to the nucleotide sequence shown in SEQ ID NO.2 in the sequence table, a PFL-96 prokaryotic expression gene sequence is chemically synthesized, then cloned and constructed on a pET-30a prokaryotic expression vector, and transferred into competent cells of escherichia coli BL21 (DE 3) for prokaryotic expression, wherein the specific process is as follows: 1) Single colony is selected and inoculated into LB culture solution containing 50 mug/mL kanamycin, and cultured overnight at 37 ℃ at 200 rpm; 2) Transferring into 100mL LB culture solution containing 50 μg/mL kanamycin at 1% inoculum size, shake culturing for 3-4 hr, and standing until OD ₆₀₀ Is 0 to6-0.8; 3) Taking out 1mL of bacterial liquid, centrifuging at 10000rpm at room temperature for 1min, discarding supernatant, and preserving thallus sediment at-20deg.C; 4) Adding IPTG with a final concentration of 0.2mM into the rest culture, shaking at 15 ℃ and 200rpm for overnight, and inducing the expression of the fusion protein; 5) Taking out 1mL of bacterial liquid, centrifuging at 10000rpm at room temperature for 1min, discarding the supernatant, preserving the bacterial cells, centrifuging at 4000rpm at 4 ℃ for 10min at the same time, collecting the residual bacterial liquid, discarding the supernatant, and re-suspending bacterial cell sediment by PBS; 6) Ultrasonic crushing (ultrasonic 4sec, intermittent 8sec, whole course time of 20min, power of 400W), centrifuging at 4deg.C and 10000rpm for 10min, collecting supernatant and precipitate; 7) The pellet from steps 3, 5, 6 was resuspended with 100. Mu.L of 1 Xloading buffer and the supernatant from step 6 was resuspended with 2 Xloading buffer; 8) The protein bands were visualized by Coomassie blue staining, as detected by 12% SDS-PAGE gel electrophoresis.

As can be seen from FIG. 1, the constructed recombinant plasmid is subjected to double enzyme digestion verification, lane 2 is the original plasmid, and is shown as three bands, which are in the form of open loop, linear and closed loop from top to bottom, so that the plasmid is complete and has high quality; lane 1 is the plasmid after double digestion of Apa I, hin d III, shown as two fragments and consistent in size with expectations, demonstrating successful construction of the recombinant plasmid.

As can be seen from FIG. 2A, the recombinant protein is mainly expressed in inclusion bodies, but there is also a small expression in the supernatant, indicating that the target protein needs to be purified from inclusion bodies later.

EXAMPLE 2 protein purification

1) The bacterial pellet obtained in step 6 of example 1 was resuspended in 20mL of lysate (20 mM Tris-HCl, pH 8.0), sonicated (4 sec, 8sec intermittently, 20min whole time, power 400W); 2) Centrifuging at 10000rpm at 4 ℃ for 10min, and collecting sediment inclusion bodies; after washing the inclusion bodies 3 times with washing solution (20 mM Tris (pH 8.0), 300mM NaCl, 1% Triton X-100,1% Triton X-114,2mM EDTA,5mM DTT), the washing was centrifuged at 12000rpm for 5min at 4℃after each washing, and finally the pellet was dissolved at 4℃for 24 hours with dissolving solution (20 mM Tris (pH 8.0), 300mM NaCl,8M urea, 20mM imidazole) while the Ni-IDAz column was equilibrated with dissolving solution, the inclusion body dissolving solution was applied to the column, the target protein was eluted with an equilibration solution containing 100mM imidazole, 500mM imidazole concentration, and each eluted fraction was collected for SDS-PAGE detection. 3) Adding the eluate containing the target protein into a dialysis bag, stirring and renaturation in renaturation buffer (1 XPBS (pH 7.4), 4mM GSH,0.4mM GSSG,0.4M L-Arginine,1M Urea) for overnight, and dialyzing in storage solution (1 XPBS (pH 7.4), 5% glycol) for 6-8h; 4) Adding Triton X-114 with final concentration of 1% into the dialyzed protein sample, mixing thoroughly, ice-bathing for 5min, incubating at 37deg.C for 40min, centrifuging at 12,000rpm for 5min at room temperature after the solution has obvious delamination, absorbing supernatant, repeating the above steps for 4 times, collecting supernatant, filtering with 0.22 μm filter membrane filter for sterilization, measuring concentration by BCA method, packaging, and storing at-80deg.C. 8) 12% SDS-PAGE gel electrophoresis identification, mass spectrum and molecular weight identification.

FIG. 2B shows the results, lane 1, inclusion bodies; lane 2 is the liquid flowing down after the inclusion body is dissolved, and part of target protein is also in the liquid, which shows that the target protein is lost; lanes 3, 4 and 5 are eluted target proteins with higher purity, and can be used for subsequent protein renaturation. The results of FIG. 2C show that SDS-PAGE detection of the target recombinant protein subjected to final renaturation, dialysis, endotoxin removal and sterilization has high purity and no foreign protein, and the obtained target protein has higher quality. FIG. 2D is a graph showing that further Western blot was used to verify that the target protein was able to hybridize specifically and was consistent with the expected size, indicating successful purification to a sterile, endotoxin-free PFL-96 recombinant protein. As can be seen from FIG. 3A, the PFL-96 peptide fragment sequence can be detected by mass spectrometry identification of the PFL-96 protein, which indicates that the target protein is purified to be PFL-96; as can be seen from FIG. 3B, the molecular weight of the purified recombinant protein PFL-96 was examined, and it was found that it was about 10kDa, which is consistent with the theoretical molecular weight of 10.57 kDa. Purification to the recombinant protein of interest PFL-96 was successfully demonstrated by SDS-PAGE, westernblot, mass spectrometry and molecular weight detection.

Example 3 oxford cup method for measuring PFL-96 bacteriostatic Activity

1) Preparing a bacterial suspension: scraping methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcus aureus, MRSA, ATCC 43300) on the inclined surface of test tube, inoculating to 4mL LB culture medium, shaking culturing at 37deg.C and 180rpm overnight, diluting the bacterial solution 6 times, and measuring OD ₆₀₀ To make the range between 0.3 and 0.8Then multiply the OD according to the dilution factor ₆₀₀ The value is the OD of the original bacterial liquid ₆₀₀ Then diluted to 1OD with PBS buffer ₆₀₀ Diluting to 1×10 by ten times ⁶ The CFU/mL bacterial suspension is used.

2) A double-layer flat transparent ring method is adopted, namely 10mL of autoclaved agar (1.5 percent) is poured into a sterile culture dish (lower layer) 90mm, after solidification, 3 sterilized oxford cups (with the inner diameter of 6 plus or minus 0.1 mm, the outer diameter of 7.8 plus or minus 0.1 mm and the height of 10 plus or minus 0.1 mm) are quickly placed on a flat plate, and 15mL of culture medium mixed with test bacteria is added under the sterile condition. After the culture medium is solidified, the oxford cup is taken out, 100 mu L of the prepared PFL-96 recombinant protein (0.5 mg/mL) is added into each hole, 100 mu L of PBS is used as a negative control, and ampicillin and PFL-96 experimental groups are used as positive controls. The flat plate is pre-diffused for 2-4 hours at 4 ℃, then is cultured for 16-20 hours in a constant temperature box at 37 ℃, the diameter of the inhibition zone is observed and measured, each inhibition zone is measured for 3 times, and 3 independent repeated experiments are carried out to obtain an average value.

FIG. 4 shows the bacteriostatic activity of lectin PFL-96 against MRSA as measured by the Oxford cup method. A is antibacterial activity of PFL-96 to MRSA; b is a schematic diagram of oxford cup placement; c is a data analysis chart of PFL-96 on the diameter of the MRSA bacteriostasis area. The result shows that the diameter of the PFL-96 recombinant protein on the MRSA inhibition zone is 16.84+/-2.06 mm, which proves that the PFL-96 recombinant protein has stronger antibacterial activity on the MRSA.

EXAMPLE 4 measurement of minimum inhibitory concentration MIC and minimum inhibitory concentration MBC of PFL-96 by 96 well method

1) Bacterial suspensions were prepared as in example 3;

2) Taking sterile 96-well plate, adding 100 μl of sterile LB medium+100 μl of LPBS as blank for each row of 1-well plates, and adding 100 μl of freshly prepared 10 for each row of 2-12-well plates ⁶ The CFU/mL test bacteria bacterial suspension, each of wells 2-11 is added with 100. Mu.L of PFL-96 recombinant protein of 128, 64, 32, 16, 8, 4,2, 1, 0.5 and 0.25. Mu.g/mL, and the final drug concentration in each well is 64, 32, 16, 8, 4,2, 1, 0.5, 0.25 and 0.125. Mu.g/mL. Well 12 was added 100 μlpbs as a negative control. Each group had 3 duplicate wells.

3) Culturing 96-well plate in 37 deg.C incubator for 16-24 hr, and enzyme labelingOD (optical density) measurement ₆₀₀ Absorbance, minimum dilution with no bacteria growth visible to the naked eye was taken as minimum inhibitory concentration (Minimal inhibitory concentration, MIC), 20 μl of culture was applied to the plate from wells with no bacteria growth visible to the naked eye, incubated at 37 ℃ for 24h, colonies grown in the plate were observed, and the minimum concentration of the corresponding drug with no colonies grown or a number of grown colonies below 5 was taken as minimum inhibitory concentration (Minimumbactericidal concentration, MBC), all experiments were independently repeated 3 times.

FIG. 5 is a graph showing the determination of the minimum inhibitory concentration and the minimum bactericidal concentration of lectin PFL-96 against methicillin-resistant Staphylococcus aureus. FIG. 5A is a graph showing the determination of minimum inhibitory concentration of lectin PFL-96 on MRSA, MIC 8. Mu.g/mL; the final lectin PFL-96 concentration in the plate of FIG. 5B was 16. Mu.g/mL, giving a total of 0 single colonies, less than 5; the final concentration of lectin PFL-96 in the plate of FIG. 5C was 8. Mu.g/mL, and a plurality of single colonies appeared, indicating that the minimum bactericidal concentration of lectin PFL-96 for MRSA was 16. Mu.g/mL. Taken together, the PFL-96 recombinant protein has higher antibacterial and bactericidal activity at a lower concentration, and the bactericidal concentration is 2 times of the antibacterial concentration.

Example 5 preparation of rat MRSA infectious wound model and PFL-96 wound healing promoting action

1) 6 SPF-grade SD male rats (200-250 g) were divided into two groups: blank and model groups of 3 rats. All experimental procedures and animal care were performed as prescribed by the animal ethics committee of the university of chinese medicine, guangxi. The blank control group is to smear sterile PBS on the wound surface, and the model group is to smear MRSA on the wound surface. The specific flow is as follows: rats were anesthetized by inhalation of isoflurane, the back hair was shaved off with electric clippers, and removed with vetting depilatory cream. Preparing round wounds (wound surface size: 2 x 2 cm) on two sides of the back of a rat (two sides of the spine of the back of the rat are respectively and conveniently separated by 1 cm from two sides of the spine of the animal, and 2cm below the scapula), cleaning marks of marker pens around the wounds with 75% alcohol, and performing iodine disinfection on the wounds to keep the wounds clean; after the wound surface is prepared for 30 minutes, after the wound part is clean, 100 mu L of sterile PBS is dripped on a blank group, and the application is uniform; mu.L of MRSA bacterial suspension (1X 10) ⁸ CFU/mL) is dripped onThe model group is uniformly smeared;

2) After the preparation of the MRSA infection wound surface is finished, continuously observing for 5 days, taking materials on the 5 th day, shearing the surface tissue of the wound surface on a sterile ultra-clean workbench, adding 1mL of physiological saline, homogenizing the sample by a 2mL sterile glass homogenizer, diluting the obtained homogenate according to a ten-fold dilution method, diluting for 6 gradients, taking 10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 These four gradients were plated, 100 μl of each gradient was plated on mannitol sodium chloride agar plates, each gradient was repeated three times, incubated at 37 ℃ inverted for 16-24h, plate colony counts were observed and gram weight units (CFU) per gram of tissue were calculated. Bacterial load (CFU/g) =plate colony count x dilution x 10/tissue weight.

3) PFL-96 recombinant protein has the effect of repairing the infectious wound surface of the rat MRSA: 18 SPF-grade SD male rats (280-350 g). All experimental procedures and animal care were performed as prescribed by the animal ethics committee of the university of chinese medicine, guangxi, numbering the rat tails, 3 each; the experimental groupings were as follows: the blank control group is a wound surface uninfected group; the negative control group is an infectious wound group, and PBS is smeared; the positive control group is an infectious wound group, and is smeared with a Baiduobang liquid medicine; the experimental group is an infectious wound group, and 8, 16 and 32 mug/mL of PFL-96 recombinant protein are smeared. Rats were anesthetized by inhalation of isoflurane, the back hair was shaved off with electric clippers, and removed with vetting depilatory cream. Preparing round wounds (wound surface size: 2 x 2 cm) on two sides of the back of a rat (two sides of the spine of the back of the rat are respectively and conveniently separated by 1 cm from two sides of the spine of the animal, and 2cm below the scapula), cleaning marks of marker pens around the wounds with 75% alcohol, and performing iodine disinfection on the wounds to keep the wounds clean; after 30 minutes of wound preparation, 100. Mu.L of MRSA bacterial suspension (1X 10) ⁸ CFU/mL) was applied dropwise to the wound surfaces of rats in the negative control group, the positive group and the experimental group, and the blank group was applied with 100 μlpbs; continuously observing for 21 days, applying medicine at fixed points every day, continuously photographing the wound surface, drawing the size of each wound surface, and calculating the wound healing rate.

4) Statistical analysis: statistical analysis was performed using SPSS20.0 software. All experiments were repeated 3 times and the quantitative results were expressed as mean ± standard deviation (X ± S). The quantitative numerical comparison between two groups adopts independent sample T test, the quantitative numerical comparison between multiple groups adopts single factor analysis of variance, the two-by-two comparison adopts S-N-K method, and the test level alpha=0.05.

FIG. 6 shows the detection of the number of Staphylococcus aureus colonies in the wound surface of the blank group and the wound surface of the negative infection group by using mannitol sodium chloride agar plates. The mannitol sodium chloride agar plate is specially used for separating and culturing staphylococcus aureus, and the result shows that staphylococcus aureus can be separated in a large amount on the wound surface infected by MRSA, and the blank group not infected by MRSA basically has no growth of staphylococcus aureus, thus indicating that the MRSA infectious wound surface is successfully manufactured.

Fig. 7 shows that lectin protein PFL-96 significantly promotes healing of rat MRSA infectious wound. The first column is the uninfected MRSA wound group and given PBS treatment, blank group; the second column is the wound group infected with MRSA, and the treatment with PBS is negative; the third column is the wound surface group infected with MRSA, and the wound surface group is positive by spraying treatment of the Baidobang mupirocin; fourth to sixth are affected MRSA wound groups, low, medium and high concentration PFL-96 (8, 16, 32 mug/mL) treatment is given, and the treatment is continuous for 21 days, and the wound change is observed by regular photographing, and the result shows that: compared with a blank group, a negative group and a positive group, the PFL-96 with high concentration has the effect of obviously promoting the healing of the MRSA infection wound surface, and the PFL-96 with low and medium concentrations has no obvious effect.

Fig. 8 is a graph showing the change of the wound healing rate of each group of rats with the treatment time, and can intuitively show that the high-concentration PFL-96 treatment group has a remarkable effect of promoting wound healing compared with the blank group, the negative group and the positive group from day 3.

According to the research of the embodiment, the invention adopts a chemical synthesis method to synthesize a gene fragment which codes the pinctada martensii galactose-binding lectin PFL-96 protein and is suitable for prokaryotic expression, constructs a prokaryotic expression vector pET-30a through a genetic engineering technology, induces expression, purifies the protein PFL-96, and carries out concentration detection, SDS-PAGE detection, westernblot verification, mass spectrum and molecular weight identification. Biological Activity detection PFL-96 is capable of inhibiting and killing methicillin-resistant Staphylococcus aureus in vitro. In addition, the rat MRSA infectious wound surface is constructed, and the PFL-96 is found to have obvious promotion effect on the rat MRSA infectious wound surface repair through local administration. Therefore, the recombinant protein PFL-96 can be used for preparing medicines for resisting methicillin-resistant staphylococcus aureus, and can also provide a new treatment path for the rapid healing treatment of human MRSA infectious wound surfaces.

Claims (10)

1. The pinctada martensii antibacterial galactose-binding lectin recombinant protein is characterized in that the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.

2. A gene encoding the pinctada martensii antibacterial galactose binding lectin recombinant protein of claim 1.

3. The pinctada martensii antibacterial galactose binding lectin recombinant protein gene according to claim 2, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

4. A recombinant expression vector, wherein the recombinant expression vector is obtained by recombining the gene of claim 3 with an expression vector.

5. The recombinant expression vector of claim 4, wherein the expression vector is pET-30a.

6. The preparation method of the pinctada martensii antibacterial galactose-binding lectin recombinant protein is characterized by comprising the following steps of:

(1) Constructing the nucleotide sequence of the gene in claim 3 into an expression vector pET-30a to obtain a recombinant plasmid pET-30a-PFL-96;

(2) And (3) converting the recombinant plasmid pET-30a-PFL-96 into competent cells of escherichia coli, culturing in a liquid culture medium, carrying out induced expression, collecting bacterial liquid, extracting and purifying to obtain the antimicrobial galactose-binding lectin recombinant protein of pinctada martensii.

7. The method for preparing the pinctada martensii antibacterial galactose binding lectin recombinant protein according to claim 6, wherein the escherichia coli competent cell is escherichia coli BL21 (DE 3) competent cell.

8. Use of the antimicrobial galactose binding lectin recombinant protein of pinctada martensii according to claim 1 for the preparation of a medicament for inhibiting or/and killing methicillin-resistant staphylococcus aureus.

9. Use of the antimicrobial galactose binding lectin recombinant protein of pinctada martensii according to claim 1 for the preparation of a medicament for promoting wound healing.

10. The use of the pinctada martensii antibacterial galactose binding lectin recombinant protein according to claim 9 for the preparation of a medicament for promoting wound healing, wherein the wound is a methicillin-resistant staphylococcus aureus infectious wound.