Application of FNDC1 protein in promoting skeletal muscle regeneration and treating muscle development-related diseases

Abstract

This invention discloses the use of FNDC1 protein to promote skeletal muscle regeneration and treat muscle development-related diseases. Researchers found that when C2C12 cells were treated with FNDC1 protein, the ability of myotubes to fusion increased with increasing concentrations of the recombinant FNDC1 protein, indicating that FNDC1 protein can promote myogenic differentiation. Furthermore, treatment of CTX mice with FNDC1 protein revealed that it promoted muscle regeneration. This suggests that FNDC1 protein can be used to prepare drugs and feeds for treating skeletal muscle dysplasia.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a method for preparing a recombinant protein based on the mouse FNDC1 gene and an application thereof. Background Art

[0002] FNDC1, along with the secreted myogenic factors FNDC4 and FNDC5, are members of the fibronectin type III domain family (FNDC). Research indicates that this gene family's functions are primarily enriched in adipocyte browning, metabolic regulation, bone metabolism, and nervous system-related functions. FNDC1 is particularly involved in processes such as angiogenesis, energy metabolism, inflammation, and lipid metabolism, and has a positive impact on the treatment of various cancers. Summary of the Invention

[0003] The inventors used SignalP and DeepTMHMM online software to analyze the mouse FNDC1 protein sequence. The results showed that it contained a signal peptide structure but no transmembrane structure, suggesting that the protein is also a secretory protein.

[0004] Furthermore, the inventors discovered through cellular and in vivo studies that FNDC1 protein can be secreted by muscle cells and is a novel myogenic factor. Specifically, through cell experiments, C2C12 cells were treated with FNDC1 recombinant protein and found to promote myogenic differentiation of the cells. In mouse experiments, injection of FNDC1 recombinant protein into CTX mice was found to promote muscle growth and development in the body and treat diseases such as DMD muscle disease and muscular dystrophy, indicating that FNDC1 has the potential to be developed into a drug for the treatment of muscle development-related diseases. In addition, in animal husbandry, the growth and development of skeletal muscle directly affects the meat production and quality of livestock and poultry. Muscle growth traits are one of the key traits for breed selection and improvement. Therefore, at the molecular breeding level, the FNDC1 gene, as a gene that regulates muscle growth and development, can be studied as an important potential target.

[0005] Based on the inventors' research findings, the present invention provides, on one hand, the use of a protein encoded by the FNDC1 gene for preparing a feed for promoting skeletal muscle regeneration. The protein sequence encoded by the FNDC1 gene is shown in SEQ ID NO: 2.

[0006] The present invention also provides the use of the protein encoded by the FNDC1 gene for preparing a drug for treating diseases related to muscular dysplasia. Optionally, the diseases related to muscular dysplasia are DMD muscle disease and amyotrophic lateral sclerosis.

[0007] Furthermore, the FNDC1 gene is used as a target for breeding livestock varieties with improved meat yield or meat quality. The FNDC1 gene is shown in SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Schematic diagram of myofiber differentiation of C2C12 cells treated with recombinant FNDC1 protein in an embodiment of the present invention.

[0009] Figure 2 Schematic diagram of the therapeutic recovery after FNDC1 injection into CTX-injured mice in an embodiment of the present invention; Figure A is a HE staining image of CTX muscle tissue sections on the 5th and 14th days; Figure B is an immunofluorescence image of CTX muscle tissue sections on the fifth day.

[0010] Figure 3 Schematic diagram of the construction of the recombinant expression plasmid PET-28a-FNDC1 in the examples of the present invention (A) and the plasmid sequencing results (B).

[0011] Figure 4 The results of SDS-PAGE detection of the induced expression and purification of the recombinant protein FNDC1 in the embodiment of the present invention are shown; Lane 1: protein molecular weight standard (Marker); Lanes 2-3: purified FNDC1; Lane 4: protein molecular weight standard (Marker).

[0012] Figure 5 Figure 2 shows the protein spectrum of extracted FNDC1 (A) and liquid chromatography schematic diagram (B).

[0013] Figure 6 This is the specific identification result of the recombinant plasmid expression product using Western Blot technology in the embodiment of the present invention, lane 1: marker; lanes 2-4: purified FNDC1 protein. DETAILED DESCRIPTION

[0014] Unless otherwise specified, the scientific and technical terms used herein are understood according to the knowledge of ordinary technicians in the relevant fields.

[0015] The protein encoded by the FNDC1 gene of the present invention can be a commercially available product or synthesized by a relevant genetic engineering company, or prepared by itself using genetic engineering technology (Example 3 provides a method for preparing FNDC1 protein to ensure that those skilled in the art can implement the present invention. The reagents and materials used in this example are all commercially available products).

[0016] Example 1: FNDC1 protein promotes myogenic differentiation of C2C12 cells

[0017] C2C12 cells are mouse myoblasts that readily form retractable myotubes and produce characteristic myosin upon differentiation. This example uses C2C12 cells as a cell model for muscle growth and development, myocyte differentiation, and metabolism. The C2C12 cells used were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0018] Specific treatment method: FNDC1 protein was injected into the cytoplasm at different concentrations ( Figure 5 The concentrations shown were added to the corresponding C2C12 cell differentiation medium (AH30702871, purchased from HyClone); after four days of cell differentiation, immunofluorescence staining was used to observe the results. Figure 1 As shown, the myotube fusion ability of cells increased with the increase of protein concentration, indicating that FNDC1 protein can promote myogenic differentiation.

[0019] Example 2: FNDC1 protein promotes muscle regeneration in CTX mice

[0020] This example utilizes CTX-induced C57 / BL6J mice, which were purchased from the Xi'an branch of Chongqing Tengxin Biotechnology Co., Ltd.

[0021] CTX-Inducible Mouse Construction: 6-week-old C57 / BL6J mice were injected once intramuscularly with 50 μL of CTX (10 μM, Sigma-Aldrich, St. Louis, MO) into the tibialis anterior (TA) muscle.

[0022] Protocol: Recombinant FNDC1 protein was injected into CTX-induced mice every two days (0.0625 mg of FNDC1 protein per 25 g mouse body weight per injection). Binding buffer was used at a concentration of 25 mg / ml. Tissues were then sectioned and stained on days 5 and 14.

[0023] See also Figure 2 The results showed that muscle regeneration in CTX mice increased significantly with time.

[0024] The following example provides a method for preparing FNDC1 recombinant protein.

[0025] Example 3:

[0026] (1) Mouse FNDC1 gene cloning:

[0027] 1.1 Remove approximately 0.5 g of tissue sample from a -80°C freezer and place in a 2 ml centrifuge tube. Add 1 ml of Trizol and magnetic beads (clean the beads with DEPC water and add them to the centrifuge tube). Transfer to a tissue grinder and grind at 45 Hz for 1 minute. Incubate at room temperature for 5-10 minutes to allow for sufficient emulsification to separate the nucleic acid-protein complex. Centrifuge in a refrigerated centrifuge at 13,200 rpm for 10 minutes at 4°C to remove tissue debris. Carefully aspirate the supernatant into a new 1.5 ml tube.

[0028] 1.2 Add 200 μL of chloroform (1 / 5 of the volume of Trizol) to the centrifuge tube, cover the centrifuge tube tightly, shake vigorously for 15 seconds, wait until the liquid is fully emulsified, and let it stand on ice for 5 minutes; centrifuge at 12000 rpm at 4°C for 15 minutes; add an equal volume of isopropanol to the supernatant, mix by inversion, and let it stand on ice for 10 minutes; centrifuge at 12000 rpm at 4°C for 10 minutes. After centrifugation, a small amount of precipitate will appear at the bottom of the centrifuge tube (RNA precipitation is not common before centrifugation of the centrifuge tube); carefully discard the supernatant, slowly add 1 mL of 75% ethanol (prepared with 0.1% DEPC water) along the wall of the centrifuge tube, gently invert and wash the precipitate, centrifuge at 12000 rpm at 4°C for 5 minutes, carefully discard the ethanol, and wash twice; dry at room temperature, wait for the ethanol to evaporate fully, add ddH2O to dissolve, and gently pipette the precipitate; determine the RNA concentration by microspectrophotometer, and store at -80°C;

[0029] 1.3 Reverse transcription was performed using the ACCURATE Evo M-MLV RT for PCR Kit. The reaction system for the RNA template solution was as follows: 1 μL dNTP Mix (10 mM each), 1 μL OligodT Primer (2.5 μM), 1 μg Template RNA, and up to 10 μL RNase-free water. The reaction conditions were 65°C for 5 min. The reaction system for the reverse transcription solution was as follows: 10 μL RNA template solution, 4 μL 5X RTase Reaction Buffer, 0.5 μL RNase Inhibitor (40 U / μL), 0.5 μL Evo M-MLV RTase (200 U / μL), and up to 20 μL RNase-free water. The reaction conditions were 42°C for 15 min, followed by 95°C for 5 min. The cDNA was stored at -20°C.

[0030] 1.4 Primers were designed using the NCBI reference sequence for FNDC1 transcript 1 (XM_036160769.1), as shown in SEQ ID NO: 3 (upstream primer) and SEQ ID NO: 4 (downstream primer). PCR amplification was performed using TAKARA PrimeSTAR HSDNA Polymerase with GC Buffer. The reaction system consisted of 25 μL 2× PrimeSTAR GC Buffer, 4 μL dNTP Mixture, 0.2 μM Primer 1, 0.2 μM Primer 2, 500 ng template DNA, 0.5 μL PrimeSTAR HSDNA Polymerase, and up to 50 μL sterile water. The reaction conditions were 98°C for 10 sec, 68°C for 1 min / kb, for a total of 30 cycles. The product (amplified FNDC1 gene) was stored at 4°C.

[0031] (2) Construction and identification of recombinant expression vector:

[0032] 2.1 Double digest the PCR product obtained in step 1.4 and the prepared pET-28a plasmid vector using Thermo Scientific restriction endonucleases Xba I and Xho I. The PCR product digestion system is: 10X Tango Buffer, PCR product, 1 μL Xba I and 1 μL Xho I, and finally add nuclease-free water to 30 μL; the plasmid DNA digestion system is: 4 μL 10X Tango Buffer, 1 μg DNA, 1 μL Xba I and 1 μL Xho I, and finally add nuclease-free water to 20 μL. Products were detected on a 1% agarose gel and DNA fragments were recovered using the TIANGEN TIANgel Maxi Purification Kit.

[0033] 2.2 Use Thermo Scientific T4 ligase for enzyme digestion and ligation. The reaction system is: 100 ng vector DNA, 3:1 insert DNA, 0.2 μL T4 DNA Ligase (5 U / μL) and 2 μL 10X T4 DNA Ligase buffer. Finally, add RNase-free water to 20 μL. Place it in a 37°C water bath for 1 hour to obtain the ligation product.

[0034] 2.3 Remove one tube of BL21(DE3) competent cells from the -80°C freezer and place on ice for use; pour the melted LB solid medium into a plate and add 50μg / ml Kana antibiotics by volume; in an ice box on a clean bench, add 10μL of the ligation product to 100μL of BL21(DE3) competent cells and flick the tube wall three times, then place the tube on ice for 30 minutes; heat shock in a 42°C water bath for 90 seconds, then immediately place the tube on ice and let it stand for 2-3 minutes; add 900mL of LB antibiotic-free liquid medium to the tube and incubate it at 37°C in a shaker at 150rpm for 45 minutes; remove the transformation product from the shaker, pipette it, and gently apply it to the pre-prepared LB solid medium with a glass rod; incubate the culture dish upside down in a 37°C incubator overnight.

[0035] 2.4 Add 2 mL of LB liquid medium to each of three 15 mL centrifuge tubes, and add 4 μL of 50 mg / mL Kana solution to each tube and mix well. Remove the plate with overnight culture and colony growth, pick three monoclonal bacterial colonies with a disposable sterile white pipette tip, expand the monoclonal culture, and place it in a shaker at 37°C and 250× rpm overnight. Take 1 mL of the expanded bacterial solution and add 1 mL of 30% glycerol. Store it in a -20°C refrigerator for seed preservation. Use the TIANGEN Endo Free MiniPlasmid Kit II kit to extract the remaining 1 mL of plasmid and send it to the company for sequencing to confirm the correct plasmid. The schematic diagram of the construction of the recombinant expression plasmid PET-28a-FNDC1 is shown in the figure. Figure 3 As shown in A, the plasmid sequencing results are shown in Figure Figure 3 As shown in B, the sequencing results showed that the PET28a-FNDC1 recombinant plasmid was successfully constructed.

[0036] (3) Prokaryotic induced expression of recombinant proteins

[0037] 3.1 Thaw the glycerol stock containing the PET28a-FNDC1 recombinant plasmid, take 10 μL of the stock and add it to 5 mL of LB liquid medium containing 50 μg / mL Kana antibiotic. Incubate the culture in a shaker at 37°C, 250 × rpm, and shake overnight. Add 1 / 100 of the overnight seed solution to the LB liquid medium and shake at 37°C, 250 × rpm, until the OD600 value of the culture solution reaches approximately 5.5.

[0038] 3.2 In a clean bench, transfer 1 mL of the prepared bacterial solution to a 1.5 mL centrifuge tube, label it as uninduced, and place it in a 4°C refrigerator until ready for use. Add 1 mL of the bacterial solution to each of 15 15 mL centrifuge tubes and perform the following treatments: 9 tubes were induced with 0, 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mM IPTG for 5 h, respectively; the other 6 tubes were induced with 0.1 mM IPTG for 0.5, 2.5, 3, 4, 5, and 6 h, respectively. Shake the tubes at 30°C and 180 × pm. The collected bacterial solution was stored at 4°C until ready for use.

[0039] 3.3 Centrifuge the collected bacterial liquid sample at 10,000×rpm for 5 minutes, discard the supernatant, use PBS to resuspend the bacterial pellet and add 5× loading buffer, mix well and heat at 100℃ for 7 minutes, and immediately put the sample on ice after heating. Centrifuge the sample at 12,000×rpm for 5 minutes, take the supernatant and transfer it to a new 1.5mL EP tube; use 10% separation gel for protein electrophoresis; take out the run gel, boil the gel in distilled water, discard the water, add Coomassie brilliant blue dye, boil it, and then shake it slowly on a shaker for 15 minutes; recover the Coomassie brilliant blue dye, wash the remaining dye with tap water, add decolorization solution and boil it, and shake it slowly on a shaker for 20 minutes, repeat the decolorization step 3 times; collect the bacterial liquid induced at 37° for 5 hours.

[0040] (4) Crude purification and purification of protein

[0041] 4.1 Crude purification: The induced expression bacterial solution obtained in step (3) was divided into several 50 mL centrifuge tubes, with 45 mL of bacterial solution in each centrifuge tube, and centrifuged at 4°C, 5000×g for 15 min; after discarding the supernatant, the wet weight of the bacteria was weighed, and 3 mL of Cell Iysis Buffer (pre-cooled at 4°C) was added according to the amount of 1 g of bacteria, and the bacteria were suspended by pipetting; 100 mM PMSF and 10 mg / mL Lysozyme were added at a volume of 4 μL / g and mixed by pipetting, and placed on ice for 20 min; deoxycholic acid was added at a volume of 4 mg / g and placed on ice for 30 min, during which time the mixture was continuously pipetted and mixed; the bacteria were lysed using an ultrasonic disruptor, centrifuged at 4°C, 13000×rpm for 15 min, and the supernatant was discarded; centrifuged at 4°C, 13000×rpm for 15 min, and the supernatant was discarded. 8 mL of Binding Buffer and resuspend the pellet, incubate at 4°C for 30 min, pipetting intermittently to accelerate dissolution; centrifuge at 13,000 × rpm at 4°C for 15 min and collect the supernatant; filter the collected supernatant using a 0.45 μM filter and incubate at 4°C;

[0042] 4.2 Nickel column protein purification: exploration of purification conditions First, fix 1mL His Trap HP from GE, use an injector and syringe to load the sample, and set the injector forward speed to 1mL / min; use 10mL Binding Buffer to rinse the column, take 5mL sample of the crude protein to pass through the column, and at the same time use a 50mL centrifuge tube to collect the waste liquid flowing out of the end of the column (for subsequent detection); after the injection is completed, use about 5mL Binding Buffer to rinse the column; use 5mL 6mM imidazole elution buffer to elute the column, and use a 1.5mL EP tube to collect the outflowing liquid into several tubes; use 20mL ddH2O as the mobile phase to rinse the column; then use 10mL 20% ethanol as the mobile phase to rinse the column; finally, immerse the column in 20% ethanol and store it in a 4℃ refrigerator; SDS PAGE-Coomassie Brilliant Blue staining to identify the imidazole washing effect, such as Figure 4 As shown;

[0043] 4.3 Purify the target protein in large quantities: Use 6 mM imidazole elution buffer and the above experimental steps to purify all the crude protein solutions to obtain a large amount of purified protein. Liquid chromatography detection shows that the main component in the solution is FNDC1, and the mass spectrometry results show that the purity of FNDC1 is 92%, indicating that the prepared FNDC1 recombinant protein can be used for subsequent experiments, such as Figure 5 The purified protein was stored at 4°C until use.

[0044] (5) Protein refolding

[0045] Cut the dialysis bag into several small pieces of 11-21 cm in length; boil the dialysis bag in 50% ethanol solution for 1 hour; after the dialysis bag cools down, rinse it thoroughly with distilled water; place the dialysis bag in 30% ethanol (ensuring that the dialysis bag is immersed in the solution) and store it in a refrigerator at 4°C until ready to use; before use, remove the dialysis bag while wearing gloves and check for leaks; use only if there are no leaks;

[0046] Add the purified protein solution to the dialysis bag, leaving at least half of the space to prevent the bag from bursting due to excessive influx of the solution outside the bag. Expel bubbles and clamp the other end of the dialysis bag with a clamp. Because the protein exists in the form of inclusion bodies, place the dialysis bag containing the protein solution in 8M urea buffer (pre-cooled at 4°C in advance) and place the entire beaker in a chromatography cabinet at 4°C. Stir slowly with a magnetic stirrer for about 12 hours.

[0047] Remove the dialysis bag containing the protein solution and place it in 6M urea buffer (pre-cooled at 4°C in advance). Place the entire beaker in a chromatography cabinet at 4°C and stir slowly with a magnetic stirrer for about 12 hours.

[0048] Then take out the dialysis bag containing the protein solution and place it in 4M urea buffer (pre-cooled at 4°C in advance), and place the entire beaker in a chromatography cabinet at 4°C and stir slowly with a magnetic stirrer for about 12 hours;

[0049] Take out the dialysis bag containing the protein solution again and place it in 2M urea buffer (pre-cooled at 4°C in advance), and place the entire beaker in a chromatography cabinet at 4°C and stir slowly with a magnetic stirrer for about 12 hours;

[0050] The dialysis bag containing the protein solution was taken out again and placed in 0 M urea buffer (pre-cooled at 4°C in advance), and the entire beaker was placed in a 4°C chromatography cabinet and slowly stirred using a magnetic stirrer for about 12 hours.

[0051] The dialyzed protein was aliquoted, snap-frozen in liquid nitrogen, and stored at -80°C.

[0052] (6) Detection of the specificity of recombinant proteins

[0053] Electrophoresis: Take 12 μL of the protein solution collected after renaturation and dialysis in the previous step and add 3 μL of 5× loading buffer to each sample; mix well and heat at 100°C for 5 min. After heating, immediately place the sample on ice.

[0054] Use 10% separating gel. After adding the separating gel, seal the liquid surface with anhydrous ethanol and let it solidify for 1 hour. Then add the stacking gel, insert the comb, and let it solidify for 1 hour. Fix the electrophoresis tank, add the electrophoresis buffer, slowly remove the comb, and add 15 μg of protein sample to the well. Repeat for each sample three times.

[0055] Adjust the electrophoresis instrument to 80V and use constant voltage electrophoresis; after bromophenol blue enters the separation gel, adjust the voltage to 120V and continue electrophoresis until bromophenol blue runs to the bottom of the separation gel, then stop electrophoresis; cut the NC membrane of appropriate size for transfer, immerse the NC membrane in methanol for activation, and transfer it at a steady flow of 200mA for 1.5-2h. Remove the PVDF membrane and place it in 5% BSA and block it at room temperature for 2h; add PRMT1 primary antibody to the blocked membrane and incubate it at 4℃ overnight, then wash it three times with TBST, each for 10min; add secondary antibody and incubate it at room temperature for 2h, then wash it three times with TBST, each for 10min; develop it with ECL chemiluminescence solution, and take pictures and analyze it with a gel imaging system, as shown in Figure 2. Figure 6 As shown in FIG (Western blot results of purified recombinant FNDC1 protein), the figure shows that the size of the purified recombinant FNDC1 protein is consistent with the expected size, which is 35KD.

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Claims

1. Use of the protein encoded by the FNDC1 gene for preparing a feed for promoting skeletal muscle regeneration in an organism. The protein sequence encoded by the FNDC1 gene is shown in SEQ ID NO:

2.

2. Use of the protein encoded by the FNDC1 gene for preparing a drug for treating diseases related to muscular dysplasia. The protein sequence encoded by the FNDC1 gene is shown in SEQ ID NO:

2.

3. The use according to claim 2, characterized in that The muscle dysplasia-related disease is DMD muscle disease or muscle atrophy disease.

4. Application of the FNDC1 gene as a target for breeding livestock varieties with improved meat yield or meat quality, wherein the FNDC1 gene is represented by SEQ ID NO: 1.

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