Recombinant protein BAIWEN-1 with the property of promoting spermatogonial cell proliferation and its application

Abstract

The present invention belongs to the technical field of biomedicine, and particularly relates to a recombinant protein BAIWEN-1 with the performance of promoting spermatogonial cell proliferation and its application. The present invention first provides a recombinant protein BAIWEN-1 obtained by fusing key fragments of Collagen alpha-1(VI) chain and Matrix metalloproteinase-18 protein derived from Xenopus laevis. The recombinant protein BAIWEN-1 has the performance of significantly promoting spermatogonial cell proliferation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a recombinant protein BAIWEN-1 with the performance of promoting spermatogonial cell proliferation and its application. Background Art

[0002] Male infertility has become an important problem threatening human health, which is usually characterized by abnormal spermatogenesis. The process of spermatogenesis includes a series of proliferation stages, differentiation and subsequent division into mitosis, meiosis and sperm maturation. Each stage involves different cell types, including spermatogonia, spermatocytes and spermatids. At present, there are various research and treatment methods to improve it. For example, the patent with publication number CN113855659A and invention name "Application of 12-HEPE or its pharmaceutically acceptable fatty acid in improving spermatogenesis disorders" discloses that 12-HEPE or its pharmaceutically acceptable fatty acid is used to improve spermatogenesis disorders in experimental mice. However, 12-HEPE is currently more used as a biological reagent, and there is still a long way to go before it can be truly applied to humans.

[0003] Arginine is one of the essential amino acids for sperm formation, and it is also an essential amino acid for synthesizing proteins and cysteine. Proteins rich in arginine can significantly promote spermatogenesis and proliferation. Multiple solutions using arginine to improve male infertility have also been disclosed in the prior art. For example, the patent with publication number CN109172804A and invention name "A composition for improving sperm activity and treating infertility and its preparation method" discloses a composition composed of fructooligosaccharide, galactooligosaccharide, ginseng, L-arginine, L-glutamine, wolfberry oyster peptide, taurine and zinc gluconate, which is used to improve sperm activity and treat infertility. Another example is the patent with publication number CN101167735A and invention name "A drug for improving sperm quality and treating male infertility", which discloses a composition composed of L-carnitine, arginine, vitamin E, nucleotides, zinc gluconate, clomiphene citrate, selenium citrate and vitamin A acetate, which has a significant effect on improving sperm quality and treating male infertility. However, such similar solutions using compositions often have relatively complex components, and currently, proteins rich in arginine on the market are mainly natural proteins obtained by extraction, with limited raw material sources and relatively high prices.

[0004] In summary, it is necessary to propose new method strategies to alleviate the deficiencies of the prior art. Summary of the Invention

[0005] The purpose of the present invention is to provide a recombinant protein BAIWEN-1 with the performance of promoting spermatogonial cell proliferation and its application, to partially solve or alleviate the above deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0006] A recombinant protein with the property of promoting the proliferation of spermatogonial cells, wherein the amino acid sequence of the recombinant protein contains key fragments of Collagen alpha-1(VI) chain protein and Matrix metalloproteinase-18 protein; the amino acid sequence of the recombinant protein is as shown in SEQ ID NO.1.

[0007] Furthermore, the recombinant protein is obtained by fusing and designing key fragments of Collagen alpha-1(VI) chain and Matrix metalloproteinase-18 protein derived from Xenopus laevis. Specifically, it consists of the amino acid sequence at positions 7 - 236 (fragment 1) and positions 810 - 1038 (fragment 2) of Collagen alpha-1(VI) chain, and the amino acid sequence at positions 279 - 460 (fragment 3) of Matrix metalloproteinase-18. The three amino acid fragments are connected by two flexible linkers (GGGGSGGGGSGGGGS).

[0008] Furthermore, the hydrophilic-hydrophobic property of the recombinant protein is hydrophilicity.

[0009] A recombinant expression vector containing the gene of the recombinant protein as shown in SEQ ID NO.1 above.

[0010] Furthermore, the expression vector includes plasmid vectors, phage vectors or animal and plant virus vectors.

[0011] A recombinant bacterium expressing the recombinant protein as shown in SEQ ID NO.1 above.

[0012] Furthermore, the recombinant bacterium includes Escherichia coli, Bacillus subtilis, Pichia pastoris or Saccharomyces cerevisiae.

[0013] Use of the recombinant protein as shown in SEQ ID NO.1 in the preparation of a drug for promoting the proliferation of spermatogonial cells.

[0014] Furthermore, the dosage form of the drug includes oral dosage forms or injection dosage forms.

[0015] Furthermore, the dosage form includes tablets, pills, lyophilized powder or aqueous solutions.

[0016] A reagent for promoting the proliferation of spermatogonial cells in vitro, the reagent containing the recombinant protein as shown in SEQ ID NO.1.

[0017] Further, the reagent is provided in unit doses, including 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL or 400 μg / mL.

[0018] A drug for promoting the proliferation of spermatogonial cells, the drug comprising the recombinant protein shown in SEQ ID NO.1 and other pharmaceutically acceptable carriers and / or adjuvants. Further, the effective concentration of the recombinant protein in the drug is set to be not less than 200 μg / mL.

[0019] Further, the dosage form of the drug includes oral preparations or injections.

[0020] A cell culture medium for promoting the proliferation of spermatogonial cells in vitro, the cell culture medium comprising the recombinant protein shown in SEQ ID NO.1.

[0021] Further, the cell culture medium also includes DMEM complete medium.

[0022] Further, the concentration of the recombinant protein in the cell culture medium can be set to 100 μg / mL, 200 μg / mL or 400 μg / mL.

[0023] Beneficial technical effects:

[0024] The present invention provides a recombinant protein BAIWEN-1. Experiments of the present invention have proved that the recombinant protein BAIWEN-1 can significantly promote the proliferation of spermatogonial cells. In addition, the recombinant protein has no obvious cytotoxicity and has good biocompatibility, so it also has the potential for subsequent development into a drug for promoting the proliferation of spermatogonial cells. When prepared as a drug, it can be made into conventional oral protein dosage forms such as tablets, pills, lyophilized powders or aqueous solutions, or injection dosage forms. Further, the recombinant protein BAIWEN-1 is hydrophilic and can also be developed into an in vitro scientific research reagent or preparation for cell research and culture.

[0025] In summary, the recombinant protein BAIWEN-1 provided by the present invention has proved its effectiveness, and the preparation method is relatively simple, standardized and quality controllable, and has the potential to become a means to replace natural arginine in the development of drugs for treating infertility. Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to actual scale. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 Electrophoresis band pattern of the recombinant protein BAIWEN-1 prepared by the present invention;

[0028] Figure 2 Prediction result of the higher-order structure of the recombinant protein BAIWEN-1 prepared by the present invention;

[0029] Figure 3 Prediction result of the hydrophilicity and hydrophobicity of the recombinant protein BAIWEN-1 prepared by the present invention;

[0030] Figure 4 Cytotoxicity experiment of the recombinant protein BAIWEN-1 prepared by the present invention;

[0031] Figure 5 Proliferation promotion experiment results of the recombinant protein BAIWEN-1 prepared by the present invention and two natural proteins (Collagen alpha-1(VI) chain and Matrix metalloproteinase-18) on spermatogonial cells. Detailed implementation manners

[0032] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0033] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0034] As used herein, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0035] 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.

[0036] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is for convenience and brevity only and should not be construed as a rigid limitation on the disclosed range. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within that range. For example, the description of the range 1 - 6 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 the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0037] Main materials and reagents:

[0038] Human embryonic kidney cells 293T were purchased from the Cell Bank of the Chinese Academy of Sciences' Type Culture Collection, catalog number SCSP - 502, cell name: Human embryonic kidney cells. Mouse spermatogonial cells GC - 1 spg were purchased from the Cell Bank of the Chinese Academy of Sciences' Type Culture Collection, catalog number SCSP - 5254, cell name: Mouse spermatogonial cells.

[0039] Escherichia coli BL21(DE3) competent cells were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0040] The pET - 22b(+) plasmid is a commercially available Escherichia coli expression vector, purchased from Beijing Tsingke Biotechnology Co., Ltd. The vector tags are N - pelB and C - His, and the vector resistance is Ampicillin.

[0041] The restriction endonucleases NdeI and XhoI were purchased from NEB (Beijing) Co., Ltd. His - tag protein purification resin (nickel column) was purchased from Shanghai Lianmai Biotechnology Co., Ltd., product number LM - 616. IPTG, Ampicillin, DMSO (dimethyl sulfoxide) were all purchased from Beijing Solarbio Science & Technology Co., Ltd. DMEM medium was purchased from GIBCO. CCK8 reagent was purchased from Chongqing Baoguang Technology Co., Ltd. and was used according to the reagent instructions.

[0042] Medium: Each liter of LB medium contains: 5 g of yeast extract, 10 g of tryptone, 10 g of sodium chloride, and the pH is adjusted to 7.0.

[0043] Culture medium preparation method: Dissolve 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride in 950 mL of double-distilled water, adjust the pH to 7.0 with sodium hydroxide solution, and make up the volume to 1 L with double-distilled water. If preparing a solid medium, add agar at 1.5 g / 100 mL. Autoclave at 121 °C for 30 min.

[0044] All of the above reagents are used according to the reagent instructions. In addition, the experimental reagents not specifically described in the present invention are conventional reagents in the art, and can be prepared by conventional methods in the art or obtained from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and relevant experimental manuals can be referred to, such as the Molecular Cloning Experiment Manual or the instructions of relevant reagent manufacturers.

[0045] Example 1

[0046] This example provides a method for recombinant gene design and protein expression.

[0047] 1. Design and protein expression of the BAIWEN-1 gene

[0048] Design a gene, the nucleotide sequence of which is shown in SEQ ID NO.2, with a full length of 2046 bp. This gene encodes a protein, the amino acid sequence of which is shown in SEQ ID NO.1, with a full length of 682 amino acids, named recombinant protein BAIWEN-1. When synthesizing this gene, add NdeI restriction site (CATATG) and XhoI restriction site (CTCGAG) at 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 is sequenced and verified, and the gene with correct sequence is used for subsequent vector construction. The specific sequences involved are shown in Table 1.

[0049] Table 1

[0050]

[0051]

[0052] 2. Vector construction

[0053] In this example, the pET-22b(+) plasmid is preferably used as the expression vector. The pET-22b(+) plasmid and the target gene (SEQ ID NO.1) are digested with restriction endonucleases NdeI and XhoI respectively, and then the target gene is ligated into the pET-22b(+) vector through a ligation reaction.

[0054] It can be understood that in this example, phage vectors or animal and plant virus vectors can also be used to achieve this.

[0055] 3. Recombinant Bacterial Transformation

[0056] (1)Take out the Escherichia coli BL21(DE3) competent cells (Beijing Solarbio) from the -80°C refrigerator and place them on ice for 5 min.

[0057] (2)After the glycerol storing the BL21(DE3) competent cells melts, add the competent cells to the ligation product, pipette up and down 3 - 4 times to mix well, and let it stand on ice for 30 min.

[0058] (3)Quickly dry the moisture on the tube wall with absorbent paper, then perform heat shock at 42°C for 90 s and immediately place it on ice for 2 min.

[0059] (4)Under sterile conditions, add 800 μL of LB liquid medium, and culture at 37°C and 150 rpm for 45 min.

[0060] (5)Centrifuge at 8000 rpm for 5 min to collect the bacteria, discard part of the supernatant, resuspend the Escherichia coli with the remaining about 100 μL of supernatant, and then evenly spread it on the LB solid medium containing 100 μg / mL ampicillin, and place it in a 37°C incubator for inverted culture for 10 - 16 h.

[0061] (6)Pick a single clone and inoculate it into the liquid LB medium containing 100 μg / mL ampicillin. After culturing at 37°C for 10 - 16 h, perform the identification of positive clones.

[0062] In this example, Escherichia coli is preferably used as the recombinant bacterium. It can be understood that this example can also be achieved by using Bacillus subtilis, Pichia pastoris or Saccharomyces cerevisiae strains, etc.

[0063] 4. Protein Expression and Purification

[0064] (1)Inoculation: Prepare the liquid LB medium and sterilize it. After sterilization, place the liquid LB medium in the laminar flow hood to cool to room temperature. Add ampicillin to the LB medium in the laminar flow hood and mix well so that the final concentration of ampicillin is 100 μg / mL. Then inoculate the Escherichia coli (positive clone) containing the plasmid with the target gene into the LB medium at a ratio of 200 μL / L, and then place this LB medium in a shaker and culture it under the conditions of a rotation speed of 170 rpm and a temperature of 37°C for 8 - 10 h.

[0065] (2)Induction: After culturing in the shaker for 8 - 10 h, take out 2 mL of the bacterial solution and measure its OD 600 value. When the OD 600When the value reaches 0.6 - 0.8, IPTG with a final concentration of 200 μL / mL is added to the bacterial solution, and then the culture is shaken at 37 °C and 170 rpm for 8 h.

[0066] (3)Purification: After adding IPTG and culturing with shaking for 8 h, the bacterial solution is taken out, centrifuged at 4 °C and 8000 rpm for 5 min. After centrifugation, the supernatant is removed and the precipitate (the precipitate is Escherichia coli) is retained.

[0067] (4)Ultrasonic disruption: The Escherichia coli is ultrasonically disrupted and then centrifuged. The precipitate obtained by centrifugation contains the target protein. The precipitate obtained by centrifugation is 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).

[0068] (5)Finally, gradient renaturation is performed on the above inclusion body dissolution solution as follows: The inclusion body dissolution solution is filled in a dialysis bag (Solarbio Science & Technology Co., Ltd., Beijing, product number: YA1071), and then the dialysis bag is successively placed in urea solutions of 6 M, 4 M, 2 M, 1 M, and 0.5 M for gradient renaturation. Renaturation is carried out for 2 - 4 hours at each urea concentration, and renaturation needs to be carried out at a low temperature of 4 °C. The solution after renaturation is purified through His-tag protein purification resin (nickel column, Shanghai Lianmai) to obtain the recombinant protein BAIWEN-1 (the gene sequence of the target protein is added with a histidine tag during design). After purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to identify whether the purified target protein is successfully obtained. The results are as Figure 1 shown. The obtained target protein band is between 72 - 100 kDa, which is consistent with the theoretical relative molecular mass (about 75.516 kDa).

[0069] The higher-order structure of the recombinant protein BAIWEN-1 prepared in this example is predicted, and the results are shown in Figure 2 . It can be seen from the figure that the protein crystal structure of the recombinant protein BAIWEN-1 is generally composed of three parts, and the secondary structure mainly composed of α-helices is more.

[0070] Furthermore, the hydrophilicity and hydrophobicity of the recombinant protein BAIWEN-1 prepared in this example are predicted, and the results are shown in Figure 3 . Figure 3The abscissa represents the amino acid site number of the protein; the ordinate represents the hydrophilicity-hydrophobicity, and the larger the value, the more hydrophobic it is; if it is negative, it represents hydrophilicity. The total hydrophilicity-hydrophobicity value of the recombinant protein BAIWEN-1 is -0.312. Therefore, generally, this recombinant protein shows hydrophilic properties.

[0071] Example 2

[0072] This example provides the cytotoxicity evaluation of the recombinant protein BAIWEN-1.

[0073] In this example, the CCK8 method was used to verify the toxicity of the recombinant protein BAIWEN-1 prepared in Example 1 to human embryonic kidney cells 293T (Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection) to evaluate its biosafety.

[0074] The specific experimental method steps are as follows.

[0075] (1) In a 96-well plate, inoculate 1×10 4 human embryonic kidney cells 293T into DMEM medium, set up 5 experimental groups and 1 control group, and set 3 replicates for each group.

[0076] (2) After culturing the cells at 37 °C for 24 h, add the recombinant protein BAIWEN-1 to the wells of the experimental groups (experimental wells) so that the final concentrations of the recombinant protein BAIWEN-1 in the 5 experimental groups are 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL respectively. No recombinant protein BAIWEN-1 is added to the cells in the wells of the control group (control wells).

[0077] (3) Continue to culture at 37 °C for 24 h, 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 h. Then use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at 450 nm and calculate the cell survival rate of each experimental group.

[0078] The results are shown in Figure 4 . Figure 4 It shows that the recombinant protein BAIWEN-1 at each concentration has no obvious cytotoxicity. When the concentration of the recombinant protein BAIWEN-1 reaches 200 μg / mL, the survival rate of 293T cells remains above 90%; when the concentration of the recombinant protein BAIWEN-1 reaches 400 μg / mL, the survival rate of 293T cells remains above 85%. Based on this, the results of the CCK8 cytotoxicity experiment show that the recombinant protein BAIWEN-1 has no obvious cytotoxicity to normal cells 293T and has good biocompatibility.

[0079] Example 3

[0080] This example provides an experiment for evaluating the proliferation ability of mouse spermatogonial cells.

[0081] Recombinant protein BAIWEN-1, natural protein Collagen alpha-1(VI) chain, and Matrixmetalloproteinase-18 were successively dissolved in DMEM complete medium to prepare DMEM medium containing recombinant protein BAIWEN-1 (abbreviated as BAIWEN-1), DMEM medium containing Collagen alpha-1(VI) chain protein (abbreviated as Collagenalpha-1(VI) chain), and DMEM medium containing Matrix metalloproteinase-18 (matrix metalloproteinase 18) (abbreviated as Matrix metalloproteinase-18). The final concentration of all three media was 200 μg / mL.

[0082] Resuscitate mouse spermatogonial cells GC-1 spg and culture them in 9 T25 culture flasks so that the cell amount in each flask is approximately 1*10 6 cells. Culture mouse spermatogonial cells GC-1 spg using the DMEM medium containing recombinant protein BAIWEN-1, the DMEM medium containing Collagenalpha-1(VI) chain, and the DMEM medium containing Matrix metalloproteinase-18 prepared in the above steps. Set 3 replicates for each medium experimental group (i.e., 3 T25 culture flasks for each group).

[0083] After culturing for 24 hours, measure the number of mouse spermatogonial cells in the 3 experimental groups in sequence, and draw a bar chart of the proliferation rate of mouse spermatogonial cells according to the statistical results. The specific results are shown in Table 2.

[0084] Table 2

[0085]

[0086] The above results show that neither Collagen alpha-1(VI) chain nor Matrix metalloproteinase-18 protein can significantly promote the growth of mouse spermatogonial cells. Even in the medium containing Matrix metalloproteinase-18 protein, the number of mouse spermatogonial cells decreased slightly. However, recombinant protein BAIWEN-1 significantly promoted the proliferation of spermatogonial cells. During the 24-hour culture, the number of experimental cells increased by approximately 20%.

[0087] It should be noted that, in this document, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device that includes a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or device that includes such element.

[0088] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit of the present invention and the scope protected by the claims. All of these are within the protection scope of the present invention.

Claims

1. A recombinant protein with the property of promoting the proliferation of spermatogonial cells, characterized in that, The amino acid sequence of the recombinant protein contains a Collagen alpha-1(VI) chain protein fragment and a Matrix metalloproteinase-18 protein fragment; the amino acid sequence of the recombinant protein is as shown in SEQ ID NO.

1.

2. The recombinant protein according to claim 1, wherein The hydrophilic-hydrophobic property of the recombinant protein is hydrophilicity.

3. A recombinant expression vector, characterized in that, It contains a gene encoding the recombinant protein described in claim 1.

4. A recombinant bacterium, characterized in that, Express the recombinant protein described in claim 1.

5. A reagent for promoting the proliferation of spermatogonial cells in vitro, characterized in that, The reagent contains the recombinant protein described in claim 1.

6. The reagent according to claim 5, wherein The reagent is set in unit doses, including being set at 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL or 400 μg / mL.

7. A cell culture medium for promoting the proliferation of spermatogonial cells in vitro, characterized in that, The cell culture medium contains the recombinant protein described in claim 1.

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