Recombinant protein BAIWEN-1 with spermatogonial cell proliferation promoting performance and application

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a recombinant protein BAIWEN-1 with spermatogonial cell proliferation promoting performance and application. The invention firstly provides a recombinant protein BAIWEN-1 which is obtained by carrying out fusion design on key fragments of Collagen alpha-1 (VI) chain and Matrix metaproteinase-18 protein from African claw frogs, and the recombinant protein BAIWEN-1 is obtained by carrying out fusion design on the key fragments of the Collagen alpha-1 (VI) chain and the Matrix metaproteinase-18 protein from the African claw frogs. The recombinant protein BAIWEN-1 has the performance of remarkably promoting spermatogonial cell proliferation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a recombinant protein BAIWEN-1 having the ability to promote spermatogonia proliferation and its application. Background Art

[0002] Male infertility has become an important problem threatening human health, and it is usually characterized by abnormal spermatogenesis. The process of spermatogenesis includes a series of proliferation stages, differentiation and subsequent division to mitosis, meiosis and sperm maturation. Each stage involves different cell types, including spermatogonia, spermatocytes and spermatids. There are currently a variety of research and treatment methods to improve. For example, the patent with publication number CN113855659A and invention name "Application of 12-HEPE or its pharmaceutically acceptable fatty acid to improve spermatogenesis disorder" discloses that 12-HEPE or its pharmaceutically acceptable fatty acid is used to improve the occurrence disorder of experimental mouse sperm. However, 12-HEPE is currently used more as a biological reagent, and it is still a long way from being truly applied to the human body.

[0003] Arginine is one of the essential amino acids for sperm formation, and is also an essential amino acid for synthesizing proteins and cysteine. Arginine-rich proteins can significantly promote the occurrence and proliferation of sperm. The prior art also discloses a variety of schemes for improving male infertility using arginine. For example, the patent with publication number CN109172804A and invention name "a composition for improving sperm activity and improving infertility and its preparation method" discloses a composition composed of oligofructose, oligogalactose, ginseng, L-arginine, L-glutamine, wolfberry oyster peptide, taurine and zinc gluconate, which is used to improve sperm activity and improve infertility. For another example, the patent with publication number CN101167735A and invention name "a drug for improving sperm quality and treating male infertility" discloses a composition composed of L-carnitine, arginine, vitamin E, nucleotides, zinc gluconate, clomiphene, selenium citrate, and vitamin A acetate, which has a significant effect on improving sperm quality and treating male infertility. However, similar solutions using composite materials often have complex ingredients, and currently the arginine-rich proteins on the market are mainly natural proteins obtained by extraction, and their raw material sources are limited and relatively expensive.

[0004] In summary, it is necessary to propose new methods and strategies to alleviate the shortcomings of existing technologies. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant protein BAIWEN-1 having the ability to promote spermatogonia proliferation and its application, so as to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0006] A recombinant protein capable of promoting spermatogonia proliferation, wherein the amino acid sequence of the recombinant protein comprises key fragments of collagen alpha-1 (VI) chain protein and matrix metalloproteinase-18 protein; the amino acid sequence of the recombinant protein is shown in SEQ ID NO.1.

[0007] Furthermore, the recombinant protein is designed by fusing the key fragments of the collagen alpha-1(VI) chain and matrix metalloproteinase-18 protein from the African clawed frog. Specifically, it is composed of the amino acid sequence of positions 7 to 236 (fragment 1) and the amino acid sequence of positions 810 to 1038 (fragment 2) of the collagen alpha-1(VI) chain, and the amino acid sequence of positions 279 to 460 of the matrix metalloproteinase-18 (fragment 3). The three amino acid fragments are connected by two flexible linkers (GGGGSGGGGSGGGGS).

[0008] Furthermore, the hydrophilicity and hydrophobicity of the recombinant protein is hydrophilic.

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

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

[0011] A recombinant bacterium expresses the recombinant protein shown in SEQ ID NO.1.

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

[0013] The use of the recombinant protein shown in SEQ ID NO.1 in the preparation of a drug for promoting spermatogonia proliferation.

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

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

[0016] A reagent for promoting spermatogonia proliferation in vitro, comprising the recombinant protein shown in SEQ ID NO.1.

[0017] Furthermore, the reagent is provided in a unit dosage form, including 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL or 400 μg / mL.

[0018] A drug for promoting spermatogonia proliferation, comprising the recombinant protein as shown in SEQ ID NO.1 and other pharmaceutically acceptable carriers and / or adjuvants. Furthermore, the effective concentration of the recombinant protein in the drug is set to be no less than 200 μg / mL.

[0019] Furthermore, the dosage form of the drug includes oral preparation or injection.

[0020] A cell culture medium for promoting spermatogonia proliferation in vitro, wherein the cell culture medium contains a recombinant protein shown in SEQ ID NO.1.

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

[0022] Furthermore, 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: The present invention provides a recombinant protein BAIWEN-1. Experiments of the present invention prove that the recombinant protein BAIWEN-1 can significantly promote the proliferation of spermatogonia. In addition, the recombinant protein has no obvious cytotoxicity and has good biocompatibility, so it also has the potential to be subsequently developed as a drug for promoting spermatogonia proliferation. When prepared as a drug, it can be made into conventional oral protein dosage forms or injection forms such as tablets, pills, lyophilized powders or aqueous solutions. Furthermore, the recombinant protein BAIWEN-1 is hydrophilic and can also be used to develop into in vitro scientific research reagents or preparations for cell research and culture.

[0024] In summary, the recombinant protein BAIWEN-1 provided by the present invention has proven its effectiveness, and the preparation method is relatively simple, standardized, and quality-controlled, and it has the potential to become a means to replace natural arginine in the development of drugs for the treatment of infertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] Figure 1 This is the electrophoresis band diagram of the recombinant protein BAIWEN-1 prepared by the present invention; Figure 2 The high-level structure prediction result of the recombinant protein BAIWEN-1 prepared by the present invention; Figure 3 The hydrophilicity prediction result of the recombinant protein BAIWEN-1 prepared by the present invention; Figure 4 This is a cytotoxicity experiment of the recombinant protein BAIWEN-1 prepared by the present invention; Figure 5 These are the experimental results of the promotion of spermatogonia by the recombinant protein BAIWEN-1 prepared by the present invention and two natural proteins (Collagen alpha-1(VI) chain and Matrix metalloproteinase-18). DETAILED DESCRIPTION

[0027] 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.

[0028] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0029] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0030] 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.

[0031] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a 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 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 individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0032] Main materials and reagents: Human embryonic kidney cells 293T were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-502, cell name: human embryonic kidney cells. Mouse spermatogonia GC-1 spg were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-5254, cell name: mouse spermatogonia.

[0033] Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Solebow Technology Co., Ltd.

[0034] 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.

[0035] 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.

[0036] Culture medium: 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.

[0037] Preparation of the culture medium: 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 a solid culture medium, add agar at 1.5g / 100mL. Sterilize at 121℃ with high pressure steam for 30min.

[0038] All the above reagents are used in accordance with the instructions for use of the reagents. In addition, the experimental reagents not specifically described in the present invention are all conventional reagents in the art, which 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.

[0039] Example 1 This embodiment provides a method for recombinant gene design and protein expression.

[0040] 1. BAIWEN-1 gene design and protein expression The gene was designed, and its nucleotide sequence was shown in SEQ ID NO.2, with a total length of 2046 bp. The gene encodes a protein, and its amino acid sequence was shown in SEQ ID NO.1, with a total length of 682 amino acids, and was named as the recombinant protein BAIWEN-1. 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.

[0041] Table 1 2. Vector Construction In this embodiment, pET-22b(+) plasmid is preferably used as the 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.

[0042] It is understandable that this embodiment can also be implemented using bacteriophage vectors or animal and plant virus vectors.

[0043] 3. Recombinant Bacterial Transformation (1) Take out the competent Escherichia coli BL21(DE3) cells (Beijing Solebow) from the -80℃ freezer and place them on ice for 5 min.

[0044] (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.

[0045] (3) Quickly wipe the water off the tube wall with absorbent paper, then heat shock at 42℃ for 90 seconds, and immediately place in an ice bath for 2 minutes.

[0046] (4) Add 800 μL of LB liquid culture medium under sterile conditions and culture at 37°C, 150 rpm for 45 min.

[0047] (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.

[0048] (6) Pick a single clone and inoculate it into liquid LB medium containing 100 μg / mL ampicillin. After culturing at 37°C for 10-16 h, identify the positive clone.

[0049] 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.

[0050] 4. Protein Expression and Purification (1) Inoculation: Prepare liquid LB medium and sterilize it. Place the sterilized liquid LB medium in a clean bench to cool to room temperature. Add ampicillin to the LB medium in the clean bench and mix well to make the final concentration of ampicillin 100 μg / mL. Then inoculate Escherichia coli (positive clone) containing the target gene plasmid into the LB medium at a volume of 200 μL / L. Then 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.

[0051] (2) Induction: After 8-10 h of shaking culture, take out 2 mL of bacterial culture and measure its OD using a spectrophotometer. 600 When the OD of the bacterial solution is 600 When the value reached 0.6-0.8, IPTG was added to the bacterial solution at a final concentration of 200 μL / mL, and then cultured in a shaking incubator at 37°C and 170 rpm for 8 h.

[0052] (3) Purification: After adding IPTG and shaking culture for 8 h, remove the bacterial solution and centrifuge it at 4°C and 8000 rpm for 5 min. After centrifugation, remove the supernatant and retain the precipitate (the precipitate is Escherichia coli).

[0053] (4) Ultrasonic disruption: Ultrasonic disruption of E. coli was followed by centrifugation. The precipitate obtained by centrifugation contained the target protein. The precipitate was first washed with washing solution I (50mmol / L Tris-HCl, 1mol / L urea, 10mL / L Triton X-100), then washed with washing solution II (50mmol / L Tris-HCl, 2mol / L urea, 5mL / L Triton X-100), and then dissolved with inclusion body dissolution solution (50mmol / L Tris-HCl, 8mol / L urea, 100mmol / L NaCl).

[0054] (5) Finally, the inclusion body solution was subjected to gradient renaturation as follows: the inclusion body solution was placed in a dialysis bag (Beijing Solebold Technology Co., Ltd., catalog number: YA1071), and the dialysis bag was placed in 6M, 4M, 2M, 1M, and 0.5M urea solutions in turn 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 BAIWEN-1 (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, the target protein band obtained is between 72-100 kDa, which is consistent with the theoretical relative molecular mass (about 75.516 kDa).

[0055] The higher-order structure of the recombinant protein BAIWEN-1 prepared in this example was predicted, and the results are shown in Figure 2 As can be seen in the figure, the protein crystal structure of the recombinant protein BAIWEN-1 is generally composed of three parts, among which the secondary structure mainly composed of α-helix is ​​the majority.

[0056] Furthermore, the hydrophilicity and hydrophobicity of the recombinant protein BAIWEN-1 prepared in this example were predicted. Figure 3 . Figure 3 The horizontal axis represents the amino acid site number of the protein; the vertical axis represents the hydrophilicity, the larger the value, the more hydrophobic; if it is a negative value, it represents hydrophilicity. The total hydrophilicity value of the recombinant protein BAIWEN-1 is -0.312, so the recombinant protein is hydrophilic overall.

[0057] Example 2 This example provides a cytotoxicity evaluation of the recombinant protein BAIWEN-1.

[0058] In this example, the toxicity of the recombinant protein BAIWEN-1 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.

[0059] The specific experimental method steps are as follows.

[0060] (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.

[0061] (2) After culturing the cells at 37°C for 24 h, recombinant protein BAIWEN-1 was added to the wells of the experimental group (experimental wells), so that the final concentrations of recombinant protein BAIWEN-1 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 BAIWEN-1 was added to the cells in the wells of the control group (control wells).

[0062] (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 CCK8 reagent to each well and incubate at 37°C for 3 h. Then use an ELISA reader to measure the absorbance of each well at 450 nm, and calculate the cell survival rate of each experimental group.

[0063] Results Figure 4 . Figure 4 The results showed that the recombinant protein BAIWEN-1 had no obvious cytotoxicity at all concentrations. When the concentration of the recombinant protein BAIWEN-1 reached 200 μg / mL, the survival rate of 293T cells remained above 90%; when the concentration of the recombinant protein BAIWEN-1 reached 400 μg / mL, the survival rate of 293T cells remained above 85%. Based on this, the results of the CCK8 cytotoxicity experiment showed that the recombinant protein BAIWEN-1 had no obvious cytotoxicity to normal cells 293T and had good biocompatibility.

[0064] Example 3 This embodiment provides an experiment for evaluating the ability to promote the proliferation of mouse spermatogonia.

[0065] The recombinant protein BAIWEN-1 and natural proteins Collagen alpha-1(VI) chain and Matrix metalloproteinase-18 were dissolved in DMEM complete culture medium in turn to prepare DMEM culture medium containing recombinant protein BAIWEN-1 (referred to as BAIWEN-1), DMEM culture medium containing Collagen alpha-1(VI) chain protein (referred to as Collagenalpha-1(VI) chain) and DMEM culture medium containing Matrix metalloproteinase-18 (referred to as Matrix metalloproteinase-18). The final concentration of the three culture media was 200 μg / mL.

[0066] Mouse spermatogonia GC-1 spg were revived and cultured in 9 T25 culture flasks, so that the number of cells in each culture flask was about 1*10 6 Mouse spermatogonia GC-1 spg were cultured using the DMEM medium containing the recombinant protein BAIWEN-1, the DMEM medium containing the collagenalpha-1(VI) chain, and the DMEM medium containing the matrix metalloproteinase-18 prepared in the above steps. Three replicates were set for each culture medium experimental group (i.e., each group had 3 T25 culture flasks).

[0067] After 24 hours of culture, the number of mouse spermatogonia in the three experimental groups was measured in turn, and a bar graph of mouse spermatogonia proliferation rate was drawn based on the statistical results. The specific results are shown in Table 2.

[0068] Table 2 The above results show that neither Collagen alpha-1(VI) chain nor Matrix metalloproteinase-18 protein can significantly promote the growth of mouse spermatogonia. In the culture medium containing Matrix metalloproteinase-18 protein, the number of mouse spermatogonia is even reduced. However, the recombinant protein BAIWEN-1 significantly promotes the proliferation of spermatogonia. In the 24h culture, the number of experimental cells increased by about 20%.

[0069] 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.

[0070] 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 modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A recombinant protein having the ability to promote spermatogonia proliferation, characterized in that: The amino acid sequence of the recombinant protein includes a collagen alpha-1 (VI) chain protein fragment and a matrix metalloproteinase-18 protein fragment; the amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

2. The recombinant protein according to claim 1, characterized in that The hydrophilicity and hydrophobicity of the recombinant protein is hydrophilicity.

3. A recombinant expression vector, characterized in that: A gene comprising the recombinant protein according to claim 1.

4. A recombinant bacterium, characterized in that Expressing the recombinant protein according to claim 1.

5. Use of the recombinant protein according to claim 1 or 2 in the preparation of a drug for promoting spermatogonia proliferation.

6. The use according to claim 5, characterized in that The dosage form of the drug includes oral preparation or injection.

7. The use according to claim 6, characterized in that The dosage forms include tablets, pills, lyophilized powders or aqueous solutions.

8. A reagent for promoting spermatogonia proliferation in vitro, characterized in that: The reagent comprises the recombinant protein according to claim 1.

9. The reagent according to claim 8, characterized in that The reagent is provided in a unit dosage form, including 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL or 400 μg / mL.

10. A cell culture medium for promoting spermatogonia proliferation in vitro, characterized in that: The cell culture medium contains the recombinant protein according to claim 1.

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