A recombinant globulin Globulin-HM with cell migration promoting performance and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a recombinant globulin Globulin-HM having the ability to promote cell migration and its application. The 40-100th amino acid sequence of the recombinant globulin provided by the present invention undergoes a hydrophobic mutation to obtain a recombinant globulin having the ability to promote cell migration.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a recombinant globulin Globulin-HM with the ability to promote cell migration and an application thereof. Background Art

[0002] Globulin is a serum protein existing in the human body. It has good biocompatibility and will not cause obvious toxicity to the body or cells if it is further used as a protein biomaterial. The basic structure of serum globulin is a monomer composed of four symmetrical polypeptide chains connected by disulfide bonds, including two heavy chains and two light chains. When the body's immune system encounters foreign body invasion, it will produce a certain amount of globulin according to the characteristics of the foreign body. Therefore, globulin has certain immune functions and is one of the indicators for the examination of many diseases. At the same time, globulin is also a medical product with therapeutic uses.

[0003] At present, globulins are used in a variety of medical scenarios. For example, globulins are often used to improve immunity in people with poor physical fitness and weakness after illness. They are also used to prevent polio, urticaria, hepatitis, infectious diseases, etc. In addition, globulins can also be prepared into a variety of pharmaceutical dosage forms, such as placental globulins, intravenous immunoglobulin G, etc. However, natural globulins mainly come from human plasma or other tissues of the body, with small quantities, high separation difficulties, and high prices. Therefore, it is often necessary to use bioengineering and other means to develop recombinant globulins or other substitutes for natural globulins.

[0004] Currently, human fibronectin is used as a substitute for natural globulin. However, human fibronectin is produced by human liver or vascular endothelial cells, and the source of raw materials is also limited and expensive. In addition, although bioengineering methods can be used to develop functional recombinant globulins, it is found in practice that the preparation of recombinant globulins is often uncertain. It is currently found that when certain sites of recombinant globulins are mutated, the mutated recombinant globulins cannot be expressed normally and cannot be used. Therefore, it is still difficult to prepare recombinant proteins that can be significantly expressed and have specific functions.

[0005] In summary, it is necessary to provide new methods or strategies to make up for the shortcomings of existing technologies. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a novel recombinant globulin Globulin-HM having the ability to promote cell migration, and the application of the recombinant globulin. The specific technical scheme is as follows.

[0007] A recombinant globulin with the ability to promote cell migration, wherein the amino acid sequence of positions 40-100 (non-α-helical segment) of the recombinant globulin undergoes a hydrophobic mutation, wherein the hydrophobic mutation includes hydrophobic mutations at continuous amino acid sites and discontinuous amino acid sites; the amino acid sequence of the recombinant globulin is shown in SEQ ID NO.1.

[0008] As a preferred embodiment, the recombinant globulin mutates the hydrophilic amino acids at positions 40 (tyrosine, Y), 46 (asparagine, N), 52 (asparagine, N), 54 (tyrosine, Y), 58 (threonine, T), 88 (threonine, T), 89 (glutamine, Q), 90 (threonine, T), and 95 (threonine, T) of the natural Globulin protein into hydrophobic phenylalanine (F), leucine (L), leucine (L), phenylalanine (F), valine (V), valine (V), leucine (L), valine (V), and valine (V), for a total of 9 hydrophilic amino acids.

[0009] A recombinant expression vector comprises the above-mentioned recombinant globulin with the ability to promote cell migration.

[0010] Furthermore, the types of the vector include plasmid vectors, phage vectors or animal and plant virus vectors, etc.

[0011] A recombinant bacterium expresses the above-mentioned recombinant globulin with the ability to promote cell migration.

[0012] Furthermore, the types of the recombinant bacteria include one or more selected from Escherichia coli, Bacillus subtilis, Pichia pastoris and / or Saccharomyces cerevisiae.

[0013] The use of the above-mentioned recombinant globulin with the ability to promote cell migration in the preparation of drugs or medical devices for promoting epidermal wound healing.

[0014] Furthermore, the medicine also includes a pharmaceutically acceptable carrier and / or adjuvant.

[0015] Furthermore, the medicine is for external use.

[0016] Furthermore, the medical device is a dressing.

[0017] The application of the above recombinant globulin with the ability to promote cell migration in the preparation of an in vitro cell migration promoting reagent.

[0018] Beneficial technical effects

[0019] The invention obtains a recombinant globulin which can effectively promote cell migration by carrying out site-directed amino acid mutation modification on natural globulin, and the recombinant globulin has good biocompatibility of natural serum globulin and low cytotoxicity.

[0020] In the previous experiments, the present invention verified the mutation of multiple site combinations of non-α-helical segments of natural globulins, and found that recombinant proteins with multiple mutations could not be effectively expressed, and thus could not be used for downstream research and application. The recombinant protein Globulin-HM prepared by the method of the present invention has a crystal structure similar to that of natural Globulin, and its overall hydrophilicity and hydrophobicity are also similar to those of natural Globulin, but has the performance of significantly promoting cell migration, and has good research application and clinical development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0022] Figure 1 The electrophoretic band diagram of multiple proteins prepared by the present invention;

[0023] Figure 2 This is the advanced result prediction result of the recombinant protein Globulin-HM prepared in one of the embodiments of the present invention;

[0024] Figure 3 The hydrophilicity prediction result of the recombinant protein Globulin-HM prepared in one of the embodiments of the present invention;

[0025] Figure 4 This is a cytotoxicity experiment of the recombinant protein Globulin-HM prepared in one of the embodiments of the present invention;

[0026] Figure 5 This is a cell scratch test of the recombinant protein Globulin-HM prepared in one of the embodiments of the present invention;

[0027] Figure 6 This is a cell migration experiment of the recombinant protein Globulin-HM prepared in one of the embodiments of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0030] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0031] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0032] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0033] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0034] Materials and Reagents:

[0035] Human embryonic kidney cells 293T were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-502, cell name: human embryonic kidney cells.

[0036] Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Solebow Technology Co., Ltd.

[0037] The pET-22b(+) plasmid is a commercially available E. coli expression vector purchased from Beijing Qingke Biotechnology Co., Ltd. The vector tags are N-pelB and C-His, and the vector resistance is Ampicillin.

[0038] Restriction endonucleases NdeI and XhoI were purchased from NEB (Beijing) Co., Ltd. His-tag protein purification resin (nickel column) was purchased from Shanghai Lianmai Bioengineering Co., Ltd., catalog number LM-616. IPTG, ampicillin, and DMSO (dimethyl sulfoxide) were all purchased from Beijing Solebow Technology Co., Ltd. DMEM medium was purchased from GIBCO. CCK8 reagent was purchased from Chongqing Baoguang Technology Co., Ltd. and used according to the reagent instructions.

[0039] Culture medium:

[0040] Each liter of LB medium contains: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and the pH is adjusted to 7.0.

[0041] Preparation method: Dissolve 5g yeast extract, 10g tryptone, and 10g sodium chloride in 950mL double distilled water, adjust the pH to 7.0 with sodium hydroxide solution, and dilute to 1L with double distilled water. If preparing solid culture medium, add agar at 1.5g / 100mL. Sterilize at 121℃ with high pressure steam for 30min.

[0042] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, and can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of relevant reagent manufacturers.

[0043] Example 1

[0044] 1. Globulin-HM gene design and protein expression

[0045] The gene was designed, and its nucleotide sequence was shown in SEQ ID NO.2, with a total length of 1275 bp. The gene encodes a protein, and its amino acid sequence was shown in SEQ ID NO.1, with a total length of 425 amino acids, and was named as the recombinant protein Globulin-HM. When synthesizing the gene, NdeI restriction site (CATATG) and XhoI restriction site (CTCGAG) were added to the 5' end and 3' end of the gene, respectively. The gene sequence with restriction sites is shown in SEQ ID NO.3. The synthesized gene was sequenced and verified, and the gene with the correct sequence was used for subsequent vector construction. The specific sequences involved are shown in Table 1.

[0046] Table 1

[0047]

[0048]

[0049] 2. Vector Construction

[0050] In this embodiment, pET-22b(+) plasmid is preferably used as an expression vector. The pET-22b(+) plasmid and the target gene (SEQ ID NO.1) are subjected to double restriction digestion reaction with restriction endonucleases NdeI and XhoI, respectively, and then the target gene is connected to the pET-22b(+) vector by ligation reaction. It is understood that this embodiment can also be implemented by using phage vectors or animal and plant virus vectors.

[0051] 3. Recombinant Bacterial Transformation

[0052] (1) Take out the competent Escherichia coli BL21 (DE3) cells (Beijing Solebow) from the -80°C freezer and place them on ice for 5 min.

[0053] (2) After the glycerol containing the BL21(DE3) competent cells has melted, add the competent cells to the ligation product, pipette and release the pipette tip 3-4 times to mix, and place on ice for 30 min.

[0054] (3) Quickly wipe the tube wall dry with absorbent paper, then heat shock at 42°C for 90 seconds, and immediately place on ice for 2 minutes.

[0055] (4) Add 800 μL of LB liquid culture medium under sterile conditions and culture at 37°C, 150 rpm for 45 min.

[0056] (5) Collect the cells by centrifugation at 8000 rpm for 5 min, discard part of the supernatant, and use the remaining 100 μL of supernatant to resuspend the E. coli. Then, spread it evenly on LB solid culture medium containing 100 μg / mL ampicillin, place it in a 37°C incubator, and invert and culture for 10-16 h.

[0057] (6) A single clone was picked and inoculated into liquid LB medium containing 100 μg / mL ampicillin. After culturing at 37°C for 10-16 h, positive clones were identified.

[0058] In this embodiment, Escherichia coli is preferably used as the recombinant bacteria. It is understandable that this embodiment can also be implemented using Bacillus subtilis, Pseudomonas aeruginosa or Saccharomyces cerevisiae strains.

[0059] 4. Protein Expression and Purification

[0060] (1) Inoculation: Prepare liquid LB medium and sterilize it. Place the sterilized liquid LB medium in a clean bench and cool it to room temperature. Add ampicillin to the LB medium in the clean bench and mix it to a final concentration of 100 μg / mL. Then, inoculate 200 μL / L of Escherichia coli (positive clone) containing the target gene plasmid into the LB medium. Place the LB medium in a shaker and culture it at a speed of 170 rpm and a temperature of 37°C for 8-10 h.

[0061] (2) Induction: After culturing on a shaking table for 8-10 h, 2 mL of the bacterial solution was taken out and its OD600 value was measured by a spectrophotometer. When the OD600 value of the bacterial solution reached 0.6-0.8, IPTG was added to the bacterial solution at a final concentration of 200 μL / mL, and then the culture was shaken at 37°C and 170 rpm for 8 h.

[0062] (3) Purification: After adding IPTG and culturing on a shaking platform for 8 h, the bacterial solution was removed and centrifuged at 8000 rpm for 5 min at 4°C. After centrifugation, the supernatant was removed and the precipitate was retained (the precipitate is Escherichia coli).

[0063] (4) Ultrasonic disruption: E. coli was ultrasonically disrupted and then centrifuged. The precipitate obtained by centrifugation contained the target protein. The precipitate obtained by centrifugation was first washed with washing solution I (50 mmol / L Tris-HCl, 1 mol / L urea, 10 mL / L Triton X-100), then washed with washing solution II (50 mmol / L Tris-HCl, 2 mol / L urea, 5 mL / L Triton X-100), and then dissolved with inclusion body dissolution solution (50 mmol / L Tris-HCl, 8 mol / L urea, 100 mmol / L NaCl).

[0064] (5) Finally, the inclusion body solution was subjected to gradient renaturation as follows: the inclusion body solution was placed in a dialysis bag (Beijing Solebow Technology Co., Ltd., catalog number: YA1071), and the dialysis bag was placed in 6M, 4M, 2M, 1M, and 0.5M urea solutions in sequence for gradient renaturation. The renaturation was performed for 2-4 hours at each urea concentration, and the renaturation was performed at a low temperature of 4°C. The renaturation solution was purified by His-tag protein purification resin (nickel column, Shanghai Lianmai) to obtain the recombinant protein Globulin-HM (the gene sequence of the target protein was added with a histidine tag during the design). After purification, polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify whether the purified target protein was successfully obtained. The results are as follows. Figure 1 As shown, a target protein of about 47.639 kDa was obtained.

[0065] The higher-order structure of the recombinant protein Globulin-HM prepared in this example was predicted, and the results are shown in Figure 2 The protein crystal structure of the new recombinant protein Globulin-HM is similar to that of natural Globulin, with no significant changes.

[0066] Furthermore, the hydrophilicity prediction of the recombinant protein Globulin-HM prepared in this example was performed, and the results are shown in Figure 3 . Figure 3 (The horizontal axis in the figure represents the amino acid number of the protein; the vertical axis represents hydrophilicity, and the larger the value, the more hydrophobic it is. If it is a negative value, it represents hydrophilicity.) The overall hydrophilicity of the new recombinant protein Globulin-HM is similar to that of natural Globulin, with no significant difference.

[0067] Example 2

[0068] Recombinant Globulin protein without hydrophilic amino acid mutation

[0069] This example provides another method for constructing a prokaryotic expression vector, transforming recombinant bacteria, expressing and purifying a recombinant Globulin protein without mutation of hydrophilic amino acids. The method steps are the same as those in Example 1, and a recombinant protein Globulin without mutation of amino acid sites (referred to as unmutated recombinant protein Globulin) is obtained. The amino acid sequence, nucleotide sequence and nucleotide sequence with restriction sites of the unmutated recombinant protein Globulin constructed in this example are shown in Table 2.

[0070] Table 2

[0071]

[0072]

[0073]

[0074] After purification, SDS-PAGE was performed to determine whether the purified target protein was successfully obtained. Figure 1 As shown, a target protein of about 47.696 kDa was obtained.

[0075] Example 3

[0076] Evaluation of cytotoxicity of recombinant protein Globulin-HM

[0077] The toxicity of the recombinant protein Globulin-HM prepared in Example 1 to human embryonic kidney cells 293T (cell bank of Type Culture Collection Committee of Chinese Academy of Sciences) was verified by CCK8 method to evaluate its biosafety.

[0078] Method steps:

[0079] (1) In a 96-well plate, 1 × 10 4 Human embryonic kidney 293T cells were cultured in DMEM medium, and 5 experimental groups and 1 control group were set up, with 3 replicate wells in each group.

[0080] (2) After culturing the cells at 37°C for 24 h, recombinant protein Globulin-HM was added to the wells of the experimental group (experimental wells), so that the final concentrations of recombinant protein Globulin-HM in the five experimental groups were 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively. No recombinant protein Globulin-HM was added to the cells in the wells of the control group (control wells).

[0081] (3) Continue to culture at 37°C for 24 hours, and use the CCK8 method to detect the number of live cells. The steps are as follows: add 10 μL of CCK8 reagent to each well and incubate at 37°C for 3 hours. Then use an ELISA reader to measure the absorbance of each well at 450 nm.

[0082] Results Figure 4 . Figure 4 The results showed that all concentrations of the recombinant protein Globulin-HM had no obvious cytotoxicity, and the cell viability of the experimental group with a recombinant protein Globulin-HM concentration of 200 μg / mL or 400 μg / mL was higher than that of the experimental group with a recombinant protein Globulin-HM concentration of 100 μg / mL, which further indicated that the recombinant protein could promote cell proliferation to a certain extent.

[0083] Example 4

[0084] Cell scratch assay with recombinant protein Globulin-HM

[0085] (1) Mark the back of a 12-well plate by evenly drawing horizontal lines across the wells, with at least three lines across each well.

[0086] (2) L929 cells in the logarithmic growth phase were obtained, digested with trypsin to form a single cell suspension, and inoculated into a 12-well plate for culture. 3×10 4 L929 cells were inoculated in such a way that the fusion rate reached 100% overnight, and the total amount of culture medium in each well was 2 mL. Then, the 12-well plate was placed in an incubator for 24 hours at 37°C and 5% CO2.

[0087] (3) After 24 hours, use a 200 μL gun tip to scratch the horizontal line on the back parallel to the ruler, and pay attention to the verticality of the gun tip. Then, rinse the cells 2-3 times with PBS to wash away the scratched cells, add complete culture medium containing 200 μg / ml of recombinant protein Globulin-HM, and set up a blank control group (complete culture medium culture group without recombinant protein Globulin-HM). In this example, only complete culture medium containing 200 μg / ml of recombinant protein Globulin-HM was added for investigation, mainly based on the fact that 200 μg / ml of recombinant protein Globulin-HM has no obvious cytotoxicity and can effectively promote the proliferation and migration of L929 cells. At the same time, since keratin has been shown to have a certain ability to promote fibroblast proliferation and migration (Chen, Liling et al. Bioinspired Robust Keratin Hydrogels for Biomedical Applications. Nanoletters vol. 22, 22 (2022): 8835-8844; Sun, Changfa et al. Photopolymerized keratin-PGLa hydrogels for antibiotic resistance reversal and enhancement of infectious wound healing. Materials today. Bio vol. 23 100807.), 200 μg / ml keratin was set as a positive control group in this embodiment.

[0088] (4) Take photos under an optical microscope, ensure that the scratch is centered and vertical, keep the background as consistent as possible, and collect corresponding pictures at the set time point.

[0089] (5) Analyze the images with the help of imageJ software, randomly select relevant scratches, and calculate the mean distance between cells. Calculate the cell migration rate according to the following formula:

[0090] Cell migration rate = (initial intercellular distance mean - intercellular distance mean at a certain moment) / initial intercellular distance mean

[0091] The results are as follows Figure 5 and Figure 6 shown. Figure 5 It can be seen that the number of migrating cells in the blank control group and the experimental group at 12h was not much different; at 24h, the number of migrating cells in the experimental group showed a more obvious trend than that in the blank control group; starting from 36h, the number of migrating cells in the experimental group was significantly more than that in the blank control group. This shows that the recombinant globulin Globulin-HM prepared by the present invention can effectively accelerate the healing of L929 cell scratches and the migration of L929 cells, showing a good ability to promote cell migration. Figure 6 The result data are shown in Table 3.

[0092] Table 3 Cell migration rate after 72h

[0093]

[0094]

[0095] The cell scratch assay is a laboratory technique that studies cell migration, repair ability and cell-to-cell interaction by scratching a cell monolayer and capturing images. The migration ability of cells can be judged by measuring the scratch spacing at different time points and calculating the difference. Because it is similar to the in vitro wound healing process, it is also called the wound healing assay. The cell scratch assay can be effectively used to observe the effects of experimental objects (such as exogenous factors such as drugs and genes) on cell migration, repair and interaction. Therefore, it can be foreseen that the recombinant protein Globulin-HM prepared by the present invention can be used to prepare externally applied topical drugs or dressings that promote skin epidermal wound healing.

[0096] Example 5

[0097] This example provides other recombinant Globulins obtained by other mutation methods, as shown in Table 4.

[0098] Table 4

[0099]

[0100]

[0101] The methods for constructing prokaryotic expression vectors, transforming, expressing proteins and purifying other recombinant Globulin obtained by other mutation methods provided in this example are the same as those in Example 1. After purification, polyacrylamide gel electrophoresis (SDS-PAGE) was performed to identify whether the purified target protein was successfully obtained. The results are shown in FIG. Figure 1 shown.

[0102] Depend on Figure 1 The SDS-PAGE results show that after hydrophilic amino acid mutation, the recombinant protein Globulin-HM can be significantly expressed, while the recombinant proteins Globulin-7, Globulin-8 and Globulin-10 have no obvious expression, and thus these recombinant proteins cannot be used for subsequent research applications. It is speculated that recombinant globulins that require mutations in a combination of specific sites can be expressed and have function.

[0103] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims

1. A recombinant globulin having the ability to promote cell migration, characterized in that: The amino acid sequence of positions 40-100 of the recombinant globulin undergoes a hydrophobic mutation, and the hydrophobic mutation includes hydrophobic mutations occurring simultaneously at both continuous and discontinuous amino acid sites; the amino acid sequence of the recombinant globulin is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: It comprises a gene encoding the recombinant globulin having the ability to promote cell migration as claimed in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that The types of the vector include plasmid vectors, phage vectors or animal and plant virus vectors.

4. A recombinant bacterium, characterized in that Expressing the recombinant globulin having the ability to promote cell migration as claimed in claim 1.

5. The recombinant bacterium according to claim 4, characterized in that The types of the recombinant bacteria include one or more selected from Escherichia coli, Bacillus subtilis, Pichia pastoris and / or Saccharomyces cerevisiae.

6. Use of the recombinant globulin with cell migration promoting property according to claim 1 in the preparation of a drug or medical device for promoting epidermal wound healing.

7. The use according to claim 6, characterized in that The medicine further comprises a pharmaceutically acceptable carrier and / or adjuvant.

8. The use according to claim 6, characterized in that The medicine is for external use.

9. The use according to claim 6, characterized in that The medical device is a dressing.

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