A yak recombinant ddx6 protein and application thereof in regulating proliferation and cycle of myoblasts

Abstract

The application belongs to the field of livestock biotechnology and genetic engineering technology, and discloses a kind of yak recombinant DDX6 protein and its application in myoblast proliferation and cell cycle regulation. The CDS sequence of yak DDX6 gene is used as a coding template, and the protein N-terminal is fused with 6×His tag by preparing a prokaryotic expression system. It is applied to regulate myoblast proliferation and cell cycle progression, and solves the following technical defects existing in the prior art: 1. solving the technical problem of lacking stable preparation technology of yak DDX6 recombinant protein in the prior art; 2. solving the technical defect that the exogenous recombinant protein has significant limitations in cell entry and subcellular localization verification means in the prior art, and cannot provide reliable and high-resolution verification evidence; 3. solving the technical problem of lacking efficient exogenous protein regulation technology for yak skeletal muscle myoblast proliferation and cell cycle progression in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry biotechnology and genetic engineering technology, specifically relating to a recombinant yak DDX6 protein and its application in myoblast proliferation and cell cycle regulation. Background Technology

[0002] Yaks are rare ruminant livestock unique to the Qinghai-Tibet Plateau and a core pillar breed for livestock development in the region. Their meat production performance is a key economic trait determining the economic benefits of livestock farming. Skeletal muscle is a major component of livestock and poultry meat products, and its growth and development directly determine the meat production performance of livestock and poultry. Myoblasts, as the core functional cells in skeletal muscle development, play a crucial role in determining the number of muscle fibers and the growth rate of skeletal muscle through their proliferative activity and cell cycle progression. Therefore, identifying and regulating key functional proteins that control the proliferation of yak myoblasts and clarifying their regulatory mechanisms is of significant theoretical and industrial application value for the molecular genetic improvement of yak meat production traits, the breeding of new varieties, and the high-quality development of livestock farming in the plateau region.

[0003] DDX6 belongs to the DEAD-box RNA helicase family and is a highly conserved ATP-dependent RNA helicase widely involved in various cell biological processes, including mRNA transcription regulation, RNA splicing and transport, translation initiation, and ribosome assembly. It also plays a crucial regulatory role in life activities such as cell cycle progression, cell proliferation and differentiation, and cellular stress responses. Existing research indicates that DDX6 participates in the regulation of cell proliferation in human tumor cells and mouse embryonic stem cells by modulating RNA metabolism. However, research has primarily focused on human and mouse model animals, with extremely limited studies on the function of the DDX6 gene in high-altitude ruminant livestock such as yaks. Currently, there is no prokaryotic expression vector construction scheme for recombinant yak DDX6 protein, nor is there a mature system for induction expression, isolation, and purification. This makes it impossible to obtain high-purity, biologically active recombinant yak DDX6 protein, severely restricting the functional research and industrial application of this gene in the regulation of yak skeletal muscle development.

[0004] Whether exogenous recombinant proteins can effectively cross the membrane to enter target cells, and their subcellular localization characteristics after entry, are core prerequisites for elucidating their intracellular mechanisms of action and assessing their biological functions. Large protein molecules cannot freely diffuse across the cell membrane and mainly rely on pinocytosis for internalization. Previous studies have shown that Dex70 (dextran 70 kDa) mainly enters HeLa cells and coelomic cells of *C. elegans* via macropinocytosis, which is independent of clathrin and activator proteins and sensitive to amiloride. After 40 minutes, it accumulates in somatic structures within the cell and partially colocalizes with lysosomes, suggesting that it is internalized and transported via the endosome-lysosome pathway. Currently, studies on the localization of large molecules after entry mainly employ immunofluorescence colocalization techniques. While immunofluorescence staining can achieve preliminary intracellular localization of proteins, its optical resolution is limited, making it impossible to achieve precise localization of proteins in subcellular structures. Furthermore, it easily generates non-specific fluorescence signals, failing to conclusively confirm the cellular uptake efficiency and subcellular distribution characteristics of exogenous recombinant proteins. Furthermore, while Western blotting (WB) can qualitatively detect the presence of target recombinant proteins in cell samples, it cannot distinguish whether the protein is non-specifically adsorbed onto the cell membrane or truly transmembrane-bound to exert its function. Both of these techniques, used alone, have inherent limitations that cannot be overcome. Even a simple combination of the two cannot simultaneously confirm the actual cellular entry of exogenous recombinant proteins and achieve precise subcellular localization. Colloidal gold immunoelectron microscopy, with its nanoscale ultra-high resolution, can achieve precise visualization and localization of target proteins at the subcellular level, effectively overcoming the inherent limitations of the two aforementioned techniques. Currently, there is no existing technology that combines WB, immunofluorescence staining, and colloidal gold immunoelectron microscopy for the verification of exogenous recombinant protein subcellular localization systems. This combination of techniques has not been reported in studies of recombinant DDX6 protein, nor has it been applied in functional studies of other exogenous recombinant proteins, especially in the field of recombinant protein functional studies in livestock myoblasts, where this technological system is completely absent.

[0005] Existing research on DDX6 and cell proliferation regulation is limited to cell types such as tumor cells and stem cells in model animals ([1] Freimer JW, Hu TJ, Blelloch R. Decoupling the impact of microRNAs on translational repression versus RNA degradation in embryonicstem cells. Elife. 2018;7:e38014. Published 2018 Jul 25. doi:10.7554 / eLife.38014;[2] Chen Y, Mistry DS, Sen GL. Highly rapid and efficient conversion of human fibroblasts to keratinocyte-like cells. J InvestDermatol. 2014;134(2):335-344. (doi:10.1038 / jid.2013.327) No research reports have been found on the role of DDX6 in the proliferation and cell cycle regulation of livestock and poultry myoblasts. The biological function of yak DDX6 protein in regulating myoblast proliferation is completely unknown. Whether exogenous addition of yak recombinant DDX6 protein can regulate the proliferative activity of yak myoblasts and advance the G1 / S phase of the cell cycle is currently unknown.

[0006] Therefore, it is urgent to develop an efficient preparation method for yak recombinant DDX6 protein, establish a multi-technology combined precision verification system for subcellular localization of exogenous recombinant protein, and study the function and application of yak recombinant DDX6 protein in regulating myoblast proliferation and cell cycle progression. This will be beneficial for the breeding of superior yak breeds, regulation of skeletal muscle growth and development, and molecular breeding of meat traits. Summary of the Invention

[0007] Based on the current state of technology, this invention provides a yak recombinant DDX6 protein, which is applied to regulate myoblast proliferation and cell cycle progression, addressing the following technical deficiencies in existing technologies: 1. It solves the problem of lacking stable preparation technology for yak-derived recombinant DDX6 protein in existing technologies; 2. It addresses the technical deficiency that existing technologies have significant limitations in the verification methods for the entry of exogenous recombinant proteins into cells and their subcellular localization, failing to provide conclusive, high-resolution verification evidence; 3. It solves the problem of lacking efficient exogenous protein regulation technology for the proliferation and cell cycle progression of yak skeletal muscle myoblasts in existing technologies.

[0008] In order to achieve the objective of this invention and overcome the various technical defects existing in the prior art, the technical solution of this invention is as follows: On the one hand, the yak recombinant DDX6 protein of the present invention is prepared by a prokaryotic expression system using the CDS sequence of the yak DDX6 gene as a coding template, and the protein has a 6×His tag fused to its N-terminus. The 6×His tag provides a specific binding site for the purification of the recombinant protein, and the recombinant protein can be separated and purified efficiently and with high purity by nickel ion affinity chromatography. At the same time, the tag can also serve as a universal antigen recognition epitope, enabling the specific detection of the target recombinant yak DDX6 protein using commercially available anti-6×His tag antibodies, and providing a stable and reliable detection target for the subsequent colloidal gold immunoelectron microscopy subcellular localization experiment of the present invention.

[0009] The DNA sequence of the yak DDX6 gene CDS is as follows: Furthermore, this invention provides a method for preparing the above-mentioned recombinant yak DDX6 protein, the method comprising four core steps: prokaryotic vector construction, optimization of prokaryotic expression vector induction conditions, purification of recombinant DDX6 protein, and Western blot identification of recombinant DDX6 protein, as detailed below: (1) Construction of prokaryotic vectors Using the cloned yak DDX6 gene CDS sequence as a template, specific amplification primers with EcoRI restriction endonuclease sites and HindIII restriction endonuclease sites introduced at both ends were designed. After obtaining the target fragment of the yak DDX6 gene by PCR amplification, the target fragment was ligated to the EcoRI / HindIII multiple cloning site of the pET-28a prokaryotic expression vector by EcoRI / HindIII double digestion to construct the recombinant expression plasmid pET28a-DDX6. The recombinant plasmid was transformed into DH5α competent cells, plated on LB solid medium containing kanamycin sulfate, and cultured upside down. Single colonies were picked for PCR identification and EcoRI / HindIII double digestion identification. The recombinant plasmids that were identified positively were sent for sequencing verification. After sequencing confirmed that the sequence completely matched the yak DDX6 gene CDS sequence, the positive plasmid was transformed into E. coli. BL21(DE3) competent cells were used to obtain positive engineered strains, which were then preserved in an ultra-low temperature freezer. This step involved the design of specific restriction enzyme sites to achieve the directional ligation of the yak DDX6 gene with a prokaryotic expression vector, thereby constructing an engineered strain that can stably express the His-tagged yak DDX6 protein, providing a foundation for the subsequent induction of recombinant protein expression.

[0010] (2) Optimization of induction conditions for prokaryotic expression vectors The above-mentioned positive engineered strains were inoculated into LB liquid medium containing kanamycin sulfate and cultured with shaking until the bacterial culture reached OD. 600 The values ​​were set to 0.4-0.6; the final concentration gradients of isopropyl-β-D-thiogalactopyranoside (IPTG) were set to 0 mM, 0.1 mM, 0.5 mM, 1.0 mM, and 1.5 mM, the induction temperature gradients were set to 28 ℃ and 37 ℃, and the induction time gradients were set to 3 h, 6 h, and 12 h, to optimize the induction expression conditions. After induction, the bacterial cells were collected by centrifugation, washed with pre-cooled PBS, and resuspended. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to determine the optimal induction expression conditions for the recombinant protein. This step, through multi-gradient condition optimization, screened and obtained the most efficient induction expression conditions for the yak recombinant DDX6 protein, achieving efficient expression of the recombinant protein.

[0011] (3) Purification of recombinant DDX6 protein The engineered strain was induced to express large quantities of protein under the optimal induction conditions described above, and the bacterial cells were collected. The bacterial pellet was resuspended in pre-cooled PBS and then sonicated on ice. After sonication, the supernatant was collected by centrifugation. The recombinant DDX6 protein in the supernatant was purified using a nickel affinity chromatography column and a His-tagged protein purification kit. The purified protein solution was then concentrated by ultrafiltration using PALL ultrafiltration centrifuge tubes to obtain purified and concentrated yak recombinant DDX6 protein, which was stored at low temperature for later use. This step achieves efficient purification of the recombinant protein by specifically binding to the His tag of the recombinant protein through nickel affinity chromatography. Combined with ultrafiltration concentration, high-purity and high-concentration active recombinant protein is obtained, providing qualified experimental material for subsequent cell experiments.

[0012] (4) Western blot identification of recombinant DDX6 protein The purified and concentrated yak recombinant DDX6 protein was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane using a wet transfer method. The membrane was blocked with commercial blocking buffer, washed with commercial washing buffer, and then incubated overnight with anti-DDX6 primary antibody. After washing with washing buffer, the membrane was incubated at room temperature with the corresponding horseradish peroxidase (HRP)-labeled secondary antibody. After washing with washing buffer again, a chemiluminescent reagent was added, and the gel was developed and photographed using a gel imaging system to verify the expression and purification effect of the recombinant DDX6 protein. This step was confirmed by Western blotting to confirm that the purified protein was the target yak recombinant DDX6 protein, ensuring the accuracy of the protein used in subsequent experiments.

[0013] On the other hand, this invention provides a combined verification method for whether exogenous recombinant proteins have entered cells and their subcellular localization after entry. This method combines Western blotting, immunofluorescence staining, and colloidal gold immunoelectron microscopy to simultaneously confirm the actual entry of exogenous recombinant proteins into cells and verify their precise subcellular localization within target cells. This method is used to verify the localization of the aforementioned yak recombinant DDX6 protein in yak myoblasts, and the specific steps are as follows: When the cell confluence reaches 50%-60%, the culture medium for myoblasts is replaced with complete culture medium containing 2 μg / mL yak recombinant DDX6 protein, and cultured for another 24 h. The following three methods are used for joint verification: The complete culture medium for myoblasts is formulated as follows: 10% FBS, 1% penicillin-streptomycin-amphoteric B, 1% L-Alanyl-L-Glutamine and 88% DMEM basal medium, by volume percentage.

[0014] ① Immunofluorescence staining: Primary yak myoblasts were seeded into pre-prepared sterile cell spreaders in 6-well plates and cultured in a complete myoblast culture medium at 37 ℃ and 5% CO2 for 12 h. The medium was then replaced with complete myoblast culture medium containing 2 μg / mL recombinant DDX6 protein. After 12.5 h, the medium was discarded, and the cells were washed three times with pre-cooled PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton X-100 at room temperature for 10 min, washed with PBS, and blocked with commercial immunofluorescence blocking solution at room temperature for 10 min. Anti-His-Tag primary antibody was added, and the cells were incubated overnight at 4 ℃. The cells were washed three times with pre-cooled PBST, and the corresponding fluorescent secondary antibody was added. The cells were incubated at room temperature in the dark for 1 h. The cells were washed three times with pre-cooled PBST, and the nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI) at room temperature. After washing three times with PBS, the slides were mounted with anti-fluorescence quenching mounting medium and observed and photographed under a fluorescence microscope. This method can achieve preliminary observation of the distribution of recombinant proteins in cells and directly determine whether recombinant proteins are present near or inside yak myoblasts.

[0015] ②WB detection: After treating with 40 μg / mL recombinant protein as described above, cells were collected, commercially available cell lysis buffer was added, and the cells were lysed on ice for 20 min; centrifuged at 4℃ and 12000 rpm for 20 min, and the supernatant was the extracted total cell protein; after determining the protein concentration using the quinoline carboxylic acid (BCA) method, WB detection was performed using anti-His-Tag antibody as the primary antibody; this method can qualitatively verify whether there is His-tagged recombinant DDX6 protein in the total protein of yak myoblasts, confirming at the protein level that the recombinant protein can enter myoblasts, which is mutually verified with the immunofluorescence staining results.

[0016] ③ Colloidal gold immunoelectron microscopy: Cells were cultured in 10 cm culture dishes according to the above method. The myoblast complete culture medium containing a final concentration of 18 μg / mL recombinant DDX6 protein was replaced at 0, 2 and 3 h, respectively. The culture medium was removed at 3.5 h. Cells were collected after pre-cooled HBSS washing and added immunoelectron microscopy fixation solution and fixed at 4 ℃ for 8 h. After gradient dehydration, resin embedding and ultrathin sectioning, the samples were picked up and placed on nickel grids. Subsequently, blocking, primary antibody incubation, secondary blocking and colloidal gold secondary antibody incubation were performed in sequence, followed by washing with PBS. Uranium acetate and lead citrate were used for light counterstaining, followed by washing with double-distilled water and drying. The samples were observed and photographed using a transmission electron microscope (JEM-1400FLASH). This method utilizes the nanoscale ultra-high resolution of colloidal gold immunoelectron microscopy to achieve precise localization of recombinant DDX6 protein in the subcellular structure of yak myoblasts, clearly distinguishing whether the protein is adsorbed on the cell membrane surface or enters the cell, and determining its specific distribution location within the cell. This method overcomes the inherent defects of insufficient resolution of immunofluorescence staining and the inability of Western blotting to provide spatial localization information.

[0017] The method established in this invention, which combines Western blotting, immunofluorescence staining, and colloidal gold immunoelectron microscopy for verification, achieves comprehensive and accurate verification of the cell entry and subcellular localization of exogenous recombinant proteins from three dimensions: protein qualitative analysis, preliminary intracellular localization, and precise subcellular structural localization. This method solves the core technical problems of existing technologies, such as the inability to distinguish between protein adsorption to the cell membrane and intracellular uptake, and insufficient localization accuracy. It is not only applicable to the recombinant DDX6 protein of this invention, but can also be widely applied to the subcellular localization research of various exogenous recombinant proteins.

[0018] Furthermore, this invention provides the application of the aforementioned recombinant yak DDX6 protein in regulating the proliferation and cell cycle progression of yak myoblasts. By exogenously adding recombinant yak DDX6 protein, the proliferation activity of yak myoblasts is promoted, and the cell cycle progression of myoblasts in the G1 / S phase is advanced. The specific steps are as follows: (1) Detection of the regulatory effect of recombinant yak DDX6 protein on myoblast proliferation ① Cell viability assay: The effect of recombinant yak DDX6 protein on the proliferation activity of myoblasts was detected using the CCK-8 assay; yak myoblasts were inoculated at 1×10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 10 cells / well in 96-well plates. After 12 h of culture, the culture medium was replaced with complete myoblast culture medium containing 0 μg / mL, 0.1 μg / mL, 1 μg / mL, 5 μg / mL, and 10 μg / mL yak recombinant DDX6 protein, with 5 replicates per group. After culturing for another 72 h at 37 ℃ in a 5% CO2 incubator, cell viability was detected using a CCK-8 assay kit to screen for the optimal concentration of recombinant protein that promotes proliferation. Using the optimal concentration obtained above, cells were treated for 24 h, 48 h, and 72 h, respectively, and cell viability was detected in the same manner to clarify the time gradient effect of recombinant protein on myoblast proliferation regulation. This step, through concentration gradient and time gradient experiments, clarified the optimal concentration and optimal treatment time of yak recombinant DDX6 protein in promoting myoblast proliferation, and quantified its proliferative effect.

[0019] ② Detection of transcriptional levels of proliferation-related genes: After treating yak myoblasts with the optimal concentration of recombinant DDX6 protein for 72 h, the culture medium was discarded, and total RNA was extracted from the cells using the Trizol method. cDNA was synthesized using a reverse transcription kit. Using cDNA as a template, the mRNA transcriptional levels of proliferation-related marker genes PCNA (proliferating cell nuclear antigen gene) and MKI67 (proliferation marker Ki-67 gene) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The relative expression levels of the genes were calculated using β-Actin as an internal reference gene. This step verifies the regulatory effect of recombinant DDX6 protein on the expression of myoblast proliferation-related genes at the molecular level and elucidates the molecular mechanism by which it promotes myoblast proliferation.

[0020] (2) Detection of the regulatory effect of recombinant yak DDX6 protein on myoblast cell cycle ① Cell cycle detection: Propidium iodide (PI) staining combined with flow cytometry was used to evaluate the effect of recombinant yak DDX6 protein on the cell cycle of yak myoblasts. After starvation treatment, yak myoblasts were treated with the above-mentioned optimal concentration of recombinant yak DDX6 protein. When the cell confluence reached 70%-80%, the culture medium was removed, and the cells were washed once with PBS. Cells were then digested with 0.25% trypsin and collected. After washing with pre-cooled PBS, 75% pre-cooled anhydrous ethanol was added, and the cells were fixed overnight at 4 ℃. After centrifugation and discarding the fixative, the cells were washed twice with pre-cooled PBS, and PI staining solution was added. The cells were incubated at room temperature in the dark for 30 min. The cell cycle distribution was detected by flow cytometry, and the proportion of cells in the G0 / G1, S, and G2 / M phases was analyzed. This step clarified the effect of recombinant yak DDX6 protein on the distribution of different phases of the myoblast cell cycle and verified its core role in promoting the G1 / S phase of myoblasts.

[0021] ② Detection of transcriptional levels of cell cycle-related genes: Yak myoblasts were collected, total RNA was extracted and reverse transcribed to synthesize cDNA; the mRNA transcriptional levels of cell cycle-related genes CCNA2 (cyclin A2 gene), CCNB1 (cyclin B1 gene), CCNE1 (cyclin E1 gene), CDK1 (cyclin-dependent kinase 1 gene), CCND1 (cyclin D1 gene), and MCM6 (mini chromosome maintenance defect protein 6 gene) were detected by RT-qPCR, and the relative expression levels of the genes were calculated with β-Actin as an internal reference gene; this step elucidates the molecular mechanism by which recombinant yak DDX6 protein regulates the cell cycle process of myoblasts at the molecular level, providing theoretical support for its regulatory role.

[0022] Advantages of this invention: 1. It solves the core problem of the lack of stable preparation technology for yak-derived DDX6 recombinant protein in existing technologies. Existing technologies lack research on the role of yak recombinant DDX6 protein in myoblast proliferation and recombinant protein preparation schemes; while recombinant DDX6 proteins from heterologous species have low species homology matching with yak-derived cells, and have inherent defects such as weak biological activity, poor cell targeting, and low regulatory efficiency. They cannot meet the specific physiological regulation needs of yak skeletal muscle myoblasts in the hypoxic environment of high altitude, nor can they provide suitable dedicated protein tools for research and applications related to yak skeletal muscle development and muscle damage repair.

[0023] 2. This invention addresses the significant limitations of existing methods for verifying the cellular entry and subcellular localization of exogenous recombinant proteins, which fail to provide conclusive, high-resolution verification evidence. It achieves a direct and high-resolution visualization of the precise localization of exogenous recombinant proteins in intracellular subcellular structures, effectively eliminating interference from non-specific protein binding. This confirms the cellular entry efficiency and intracellular distribution characteristics of recombinant proteins, providing direct and reliable technical evidence for their intracellular mechanisms of action, and facilitating the optimization of protein delivery protocols and subsequent industrial applications.

[0024] 3. This invention addresses the technical challenge of lacking efficient exogenous protein regulation strategies for the proliferation and cell cycle progression of yak skeletal muscle myoblasts in existing technologies. Current research on the function of DDX6 primarily focuses on the basic molecular mechanisms of RNA helicases and the regulation of tumor cell proliferation, without clearly revealing the positive regulatory effect of recombinant yak DDX6 protein on the proliferation and cell cycle progression of yak skeletal muscle myoblasts. This invention experimentally demonstrates that recombinant yak DDX6 protein can efficiently advance the G1 / S cell cycle and significantly enhance myoblast proliferation, which is beneficial for yak breeding, skeletal muscle growth and development regulation, and muscle injury repair.

[0025] The DNA sequence of the yak DDX6 gene CDS involved in this invention and the DNA sequences of related primers are shown in Sequence Listing 1. Attached Figure Description

[0026] Figure 1 A schematic diagram of the construction of the prokaryotic expression vector for yak DDX6; the CDS region fragment of the yak DDX6 gene was cloned into the EcoRI / HindIII restriction site of the pET-28a vector to construct the recombinant plasmid pET28a-DDX6.

[0027] Figure 2SDS-PAGE images of recombinant yak DDX6 protein under different induction conditions are shown. The expression results of pET28a-DDX6 recombinant protein after 3 h of induction at different IPTG concentrations and temperatures are presented to determine the optimal induction conditions. In the images, M represents the protein molecular weight marker, and C represents the blank control without IPTG. Lanes 1–4 were induced at 37 ℃ with IPTG concentrations of 0.1 mmol / L, 0.5 mmol / L, 1.0 mmol / L, and 1.5 mmol / L, respectively; lanes 5–8 were induced at 28 ℃ with IPTG concentrations of 0.1 mmol / L, 0.5 mmol / L, 1.0 mmol / L, and 1.5 mmol / L, respectively. The results showed that a specific band appeared at approximately 60 kDa in the recombinant protein, indicating that pET28a-DDX6 was successfully induced to express the protein. The induction temperature and IPTG concentration had no significant effect on the expression level. The expression effect after 3 h of induction was better than that after 6 h and 12 h. The optimal conditions were determined to be 37 ℃, 0.5 mmol / L IPTG, and 3 h of induction.

[0028] Figure 3 The images show the purification and Western blot identification of recombinant yak DDX6 protein. The concentrated recombinant yak DDX6 protein was subjected to SDS-PAGE gel electrophoresis, membrane transfer, antibody incubation, and development before detection. The SDS-PAGE results showed a single, clear band at approximately 60 kDa, which was confirmed by Western blot identification as the target recombinant yak DDX6 protein. In the image, A represents the SDS-PAGE electrophoresis result of the recombinant protein after purification by nickel ion affinity chromatography, and B represents the Western blot identification result of the corresponding purified product.

[0029] Figure 4The images show the cellular and subcellular localization of recombinant yak DDX6 protein in yak myoblasts. Immunofluorescence, Western blot, and colloidal gold immunoelectron microscopy were used to verify the cellular and subcellular localization of recombinant yak DDX6 protein. A shows the immunofluorescence localization result of recombinant yak DDX6 protein in yak myoblasts (20×); B shows the Western blot quantitative detection result of recombinant DDX6 protein in yak myoblasts; C–H show the subcellular localization result of recombinant DDX6 protein using colloidal gold immunoelectron microscopy; C–D are electron micrographs of recombinant yak DDX6 protein entering yak myoblasts via endocytosis, showing obvious vesicle structures, at magnifications of 80,000× and 100,000× respectively; E shows the localization map of mitochondria and endoplasmic reticulum (40,000×); F shows the localization map of lysosomes (60,000×); G shows the localization map of the Golgi apparatus (40,000×); and H shows the localization map of the nucleus (25,000×). The results showed that the recombinant DDX6 protein can enter yak myoblasts through endocytosis, mainly accumulates around the nucleus, and is located in subcellular structures such as mitochondria, endoplasmic reticulum, lysosomes, Golgi apparatus, and nucleus.

[0030] Figure 5 To investigate the effect of recombinant yak DDX6 protein on the proliferation of yak myoblasts, the CCK-8 assay was used to detect the proliferative activity of yak myoblasts treated with recombinant yak DDX6 protein, and qRT-PCR was used to detect the transcriptional levels of genes related to yak myoblast proliferation. In the figures, A represents the results of screening for the optimal concentration of recombinant yak DDX6 protein in treating yak myoblasts; B represents the effect of the optimal concentration of recombinant DDX6 protein (1 μg / mL) on cell proliferation activity at different treatment times; and C–D represent the effects of the optimal concentration of recombinant DDX6 protein (1 μg / mL) on the transcriptional levels of PCNA, MKI67, and DDX6 genes in yak myoblasts. The results showed that 0.1 μg / mL and 1 μg / mL recombinant DDX6 protein significantly enhanced the proliferation activity of yak myoblasts (P<0.01), while 5 μg / mL and 10 μg / mL significantly inhibited cell proliferation (P<0.01). Based on the trend of proliferation activity, 1 μg / mL was determined to be the optimal concentration (A). The cell activity of yak myoblasts supplemented with 1 μg / mL recombinant DDX6 protein was significantly higher than that of the CON group from 1 to 3 days (P ≤ 0.01, B). The PCNA gene transcription level was significantly higher in the PCNA group from 1 to 2 days than in the CON group (P<0.01), while the PCNA gene transcription level was significantly lower in the PCNA group from 3 to 4 days (P<0.01, C). The MKI67 gene transcription level in the recombinant yak DDX6 protein group was significantly higher than that in the CON group (P<0.01), while the DDX6 gene transcription level tended to be lower in the PCNA group than in the CON group (P>0.05, D).

[0031] Figure 6 To investigate the effect of recombinant yak DDX6 protein on the cell cycle of yak myoblasts, flow cytometry was used to detect the cell cycle distribution of yak myoblasts treated with recombinant yak DDX6 protein, and qRT-PCR was used to detect the transcriptional levels of cell cycle-related genes in yak myoblasts. In the figures, A represents the effect of recombinant yak DDX6 protein on the cell cycle distribution of yak myoblasts, and B represents the effect of recombinant yak DDX6 protein on the transcriptional levels of cell cycle-related genes in yak myoblasts. The results showed that compared with the CON group, the proportion of cells in G0 / G1 phase was significantly decreased in the DDX6 group (P<0.05), the proportion of cells in S phase was significantly increased (P<0.01), and there was no significant difference in the proportion of cells in G2 / M phase (A). The transcriptional levels of CCNE1, CCNA2, MCM6, CCNB1, and CDK1 genes were significantly upregulated in the DDX6 group (P<0.05), while the transcriptional level of CCND1 gene showed no significant difference (B). Detailed Implementation

[0032] To better illustrate the present invention, the following embodiments are provided: Example 1

[0033] 1. Construction of prokaryotic vectors The obtained CDS region fragment of the yak DDX6 gene was cloned into the EcoRI / HindIII position of the pET-28a vector to construct the recombinant plasmid pET28a-DDX6. After identification by EcoRI and HindIII restriction enzyme digestion, positive plasmids were sequenced. The correctly sequenced recombinant plasmid was transformed into BL21(DE3) competent cells, and single colonies were picked, cultured, and preserved for later use.

[0034] 2. Optimization of induction conditions for prokaryotic expression vectors The bacterial strain with successfully constructed expression vector was inoculated into LB liquid medium containing kanamycin sulfate (final concentration 50 μg / mL) and cultured for 6 h (OD). 600 IPTG was added at final concentrations of 0, 0.1, 0.5, 1.0, and 1.5 mmol / L, respectively, and expression was induced at 28 ℃ and 37 ℃ for 3 h, 6 h, and 12 h, respectively, using a constant temperature shaker at 200 r / min. One mL of bacterial culture was centrifuged at 4000 r / min for 5 min, and the supernatant was discarded. The culture was resuspended in 100 μL of PBS buffer, and SDS-PAGE gel electrophoresis was used to detect protein expression and determine the optimal induction conditions.

[0035] 3. Purification of recombinant yak DDX6 protein Protein purification was performed using a nickel ion affinity column. Following the instructions for the His-tagged protein purification kit, the nickel ion column was first regenerated, as follows: (1) Drain the original ethanol from the column, and add 2 column volumes of 6 M guanidine hydrochloride, 3 column volumes of deionized water, and 1 column volume of 2% SDS in sequence. Drain the liquid from the column each time before adding the next liquid, and so on.

[0036] (2) Add 25%, 50% and 75% ethanol in sequence, then add 5 times column volume of anhydrous ethanol, and then add 75%, 50% and 25% ethanol in sequence.

[0037] (3) Add 1 column volume of deionized water, add 5 column volumes of 50 mM EDTA (pH 8.0), and add 3 column volumes of deionized water.

[0038] (4) Add 3 column volumes of 20% ethanol and 10 column volumes of deionized water.

[0039] (5) Add 5 times column volume of 50 mM NiSO4 and 3 times column volume of 8 M Bingding Buffer to purify the sample on the column.

[0040] The frozen bacterial strain was inoculated into 10 mL EP tubes containing 5 mL LB liquid medium (containing kanamycin sulfate), with an inoculation volume of 3%. After incubation at 37 °C and 200 r / min for 6 h on a shaker, the entire culture was passaged into Erlenmeyer flasks containing 300 mL LB liquid medium (containing kanamycin sulfate) and incubated at 37 °C and 200 r / min for 6 h on a shaker. 0.05% of 1 MIPTG (i.e., a final concentration of 0.5 mM) was added, and the culture was incubated at 37 °C and 200 r / min for 3 h on a shaker.

[0041] The bacterial culture was then centrifuged and the sample was purified by column loading, as follows: (1) Dispense the bacterial solution into 50 mL centrifuge tubes, centrifuge at 8000 r / min and 4 ℃ for 20 min, remove the supernatant and keep the precipitate.

[0042] (2) After resuspending the precipitate in 20 mL of PBS, transfer it to the same 50 mL centrifuge tube and mix well. Take 0.2 mL of the bacterial suspension for later use. The rest of the cells were sonicated for 45 min at 150 W on ice with the ultrasonic cell disruptor on for 2 s and off for 3 s.

[0043] (3) Centrifuge at 8000 r / min and 4 ℃ for 20 min, and retain all the supernatant.

[0044] (4) Add 10 mL of 8 M Bingding Buffer to the precipitate to resuspend the precipitate, centrifuge at 8000 r / min and 4 ℃ for 20 min, discard the precipitate and keep the supernatant. Take 0.2 mL of the supernatant for later use, and use the rest of the supernatant for column purification.

[0045] (5) After the supernatant is filtered through a 0.22 μm filter membrane, it is loaded onto the column. Take about 0.2 mL of the liquid that has passed through the column in the middle stage and keep it for later use.

[0046] (6) Add 3 times the cylinder volume of 8 M, 4 M, 2 M, 1 M and 0 M Bingding Buffer in sequence.

[0047] (7) Add 10 mL of Elution Buffer. Do not retain the first column volume of liquid, and retain all the liquid after passing through the column. This is the recombinant protein.

[0048] (8) Add 5 column volumes of 8 M Bingding Buffer, 10 column volumes of deionized water, and 3 column volumes of 20% ethanol in sequence. After draining 1 column volume of liquid, seal the column.

[0049] (9) Recombinant protein was concentrated using PALL ultrafiltration centrifuge tubes for subsequent experiments.

[0050] 4. Western blot identification of recombinant yak DDX6 protein The concentrated recombinant yak DDX6 protein was subjected to SDS-PAGE gel electrophoresis, membrane transfer, antibody incubation and development, and Western blotting was used to identify the recombinant yak DDX6 protein.

[0051] 5. Localization of recombinant DDX6 protein after entering yak myoblasts The entry of recombinant DDX6 protein into yak myoblast cells was observed using three methods: immunofluorescence, Western blotting, and immunoelectron microscopy.

[0052] (1) Immunofluorescence assay. Yak myoblasts were subjected to immunofluorescence at a concentration of 1 × 10⁻⁶. 5Cells were seeded at a density of 1 cell / mL into 6-well plates pre-filled with cell spreaders. After 12 h, the medium was replaced with serum-reduced medium and the cells were starved for another 12 h. The starvation period was then recorded as 0 h. At 0, 6, 9, 11 and 12 h, the medium was replaced with complete myoblast culture medium containing a final concentration of 2 μg / mL recombinant DDX6 protein. At 12.5 h, the medium was removed, and the cell spreaders were fixed and stained with fluorescent dye. Primary antibodies used were His-Tag Monoclonal antibody (mouse monoclonal antibody, dilution 1:500, 66005-1-Ig, Proteintech, China) and Recombinant Anti-GAPDH antibody (rabbit monoclonal antibody, dilution 1:500, GB15004-100, Servicebio, China). Secondary antibodies used were Alexa Fluor 488 goat anti-mouse IgG secondary antibody (dilution 1:500, A0428, Beyotime, China) and DyLight 594 goat anti-rabbit IgG secondary antibody (dilution 1:500, BA1142, BOSTER, China). Observation and photography were performed using an inverted fluorescence microscope.

[0053] (2) Western blot method. Yak myoblasts were subjected to a 1 × 10⁻⁶ WB assay. 5 Cells were seeded at a density of 1 cell / mL into 6 cm culture dishes. After 12 h, the culture medium was replaced with serum-reduced medium and the cells were starved for another 12 h. The starvation period was counted as 0 h. At 0, 6, 9, 11 and 12 h, the culture medium for myoblasts containing a final concentration of 40 μg / mL recombinant DDX6 protein was replaced. At 12.5 h, the culture medium was removed, and the cells were collected, lysed, subjected to SDS-PAGE gel electrophoresis, transferred to a membrane, incubated with antibodies and visualized. Primary antibodies used were His-Tag monoclonal antibody (mouse monoclonal antibody, dilution 1:5000, 66005-1-Ig, Proteintech, China) and β-Actin (rabbit polyclonal antibody, dilution 1:5000, 20536-1-AP, Proteintech, China). Secondary antibodies used were HRP-labeled goat anti-mouse IgG (dilution 1:10000, GB23301, Servicebio, China) and HRP-labeled goat anti-rabbit IgG (dilution 1:80000, A0545, SIGMA-ALDRICH, USA).

[0054] (3) Immunoelectron microscopy. Following the method described above (WB method), cells were cultured in 10 cm culture dishes. The culture medium was replaced with complete myoblast culture medium containing a final concentration of 18 μg / mL recombinant DDX6 protein at 0, 2, and 3 h, and removed at 3.5 h. Cells were washed once with HBSS, digested with 0.25% trypsin for approximately 1.5 min, and the digestion was terminated by adding an appropriate amount of FBS. The cells were then transferred to a 1.5 mL sterile conical centrifuge tube. Centrifuged at 1000 r / min for 6 min, the supernatant was removed, and the precipitate was retained. The cells were resuspended in diluted immunoelectron microscopy fixative and incubated at 4 ℃ for 5 min. Centrifuged at 4 ℃ and 12000 r / min for 10 min. The supernatant was gently removed, and undiluted immunoelectron microscopy fixative (without resuspending) was added. The cells were incubated at 4 ℃ for 8 h. After sample fixation, the cells were washed three times with PBS, dehydrated using an acetone gradient, and then embedded for polymerization. Sections approximately 70 nm thick were prepared using an ultramicrotome and placed on a 300-mesh nickel grid. The sections were etched with hydrogen peroxide for 30 min, followed by washing five times each with double-distilled water and PBS. Subsequently, the sections were sequentially blocked, incubated with primary antibody, then blocked again, and incubated with colloidal gold secondary antibody, followed by washing with PBS. The sections were lightly counterstained with uranium acetate and lead citrate, washed with double-distilled water, and dried. The samples were observed and photographed using a transmission electron microscope (JEM-1400FLASH).

[0055] 6. Effects of recombinant DDX6 protein on the proliferation of yak myoblasts Yak myoblasts were divided into 1 × 10 5 Cells were seeded at a density of 10 cells / mL in 96-well plates and cultured at 37 °C in a 5% CO2 incubator for 12 h. The culture medium was then removed, and myoblast complete culture medium containing 0, 0.1, 1, 5, and 10 μg / mL of recombinant DDX6 protein was added, with 5 biological replicates for each concentration. The culture medium was changed every 24 h, and cell viability was measured at 72 h using the CCK-8 assay. The optimal dose of recombinant DDX6 protein was selected, and cell viability was measured at 24, 48, and 72 h using the CCK-8 assay, with 4 biological replicates per day for each group.

[0056] This study also used RT-qPCR to perform relative quantification analysis of proliferation-related genes and DDX6 genes (Primer-1 to Primer-3). Changes in PCNA gene transcription levels in myoblasts supplemented with 1 μg / mL recombinant DDX6 protein were detected from 0 to 4 days, and the transcription levels of MKI67 and DDX6 genes were examined at 3 days. Results are shown below. Figure 5 .

[0057] 7. Effects of recombinant DDX6 protein on the cell cycle of yak myoblasts The effect of recombinant DDX6 protein on the cell cycle of yak myoblasts was assessed using a polymorphic inhibitor (PI). Two groups were set up: a control group (CON group) and a recombinant DDX6 protein group (DDX6 group), with three biological replicates in each group. Yak myoblasts were cultured at a rate of 1 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of [number] cells / mL in 6 cm culture dishes, gently agitated, and incubated at 37 °C in a 5% CO2 incubator for 24 h. The old culture medium was removed, replaced with serum-diluted medium, and incubated for another 24 h. After starvation, the old culture medium was removed and replaced with complete myoblast culture medium, changing the medium every 24 h. When cell confluence reached 70%-80%, the culture medium was removed, and the cells were washed once with PBS. Cells were digested with 0.25% trypsin for approximately 1 min, and the digestion was stopped by adding a small amount of serum. The cells were then transferred to a 1.5 mL sterile conical centrifuge tube. The cells were centrifuged at 1600 r / min for 6 min to remove the supernatant, and washed twice with PBS. The pellet was resuspended in 0.371 mL of pre-chilled PBS, and 1.129 mL of anhydrous ethanol was slowly added dropwise. After fixing the cell samples overnight at 4 °C, they were centrifuged at 300 ×g for 5 min. The supernatant was removed, and the cells were washed twice with PBS and then centrifuged again to remove the supernatant. Resuspend the cells in 0.5 mL of PI staining working solution, incubate at 37 °C in the dark for 30 min, and then use a flow cytometer to detect the cell cycle.

[0058] The complete myoblast culture medium formula described above is: 10% FBS, 1% penicillin-streptomycin-amphoteric B, 1% L-Alanyl-L-Glutamine and 88% DMEM basal medium.

[0059] Yak myoblasts were divided into 1 × 10 5 Cells were seeded at a density of [number] cells / mL in 10 cm culture dishes and cultured at 37 °C in a 5% CO2 incubator for 12 h, designated as day 0. The culture medium was then removed, and myoblast proliferation medium containing 0 and 1 μg / mL recombinant DDX6 protein were added, respectively. Three biological replicates were performed for each group, and the medium was changed every 24 h. After 72 h, cells were collected, total RNA was extracted using the Trizol method, and cDNA was synthesized. Relative quantification of cell cycle-related genes (Primer-4 to Primer-9) was performed using RT-qPCR. Results are shown below. Figure 6 .

[0060] Table 1 Gene Primer Sequences .

Claims

1. A recombinant DDX6 protein from yak, characterized in that, The protein was prepared using the CDS sequence of the yak DDX6 gene as a coding template through a prokaryotic expression system, and a 6×His tag was fused to the N-terminus of the protein; the DNA sequence of the CDS of the yak DDX6 gene is shown in SEQ ID NO.

1.

2. The method for verifying the localization of recombinant yak DDX6 protein in yak myoblasts as described in claim 1, characterized in that, First, the DDX6 polyclonal antibody and WB technology were used for identification: after the purified and concentrated yak recombinant DDX6 protein was subjected to SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane by wet transfer. Blocking with blocking buffer, washing with washing buffer, adding primary antibody against DDX6, and incubating overnight; washing with washing buffer, adding the corresponding horseradish peroxidase-labeled secondary antibody, and incubating at room temperature; washing with washing buffer, adding chemiluminescent reagent, developing and photographing with a gel imaging system to confirm that the purified protein is the target yak recombinant DDX6 protein; Then, Western blot, immunofluorescence staining, and colloidal gold immunoelectron microscopy were used to identify whether the exogenous recombinant protein had entered the cell and its subcellular localization after entry. The methods are as follows: ① Immunofluorescence staining: Primary yak myoblasts were seeded into well plates pre-filled with sterile cell slides and cultured in complete myoblast culture medium containing fetal bovine serum in an incubator. Then, the culture medium was replaced with complete myoblast culture medium containing recombinant DDX6 protein. The culture medium was discarded, and the cells were washed with pre-cooled PBS. The cells were fixed with paraformaldehyde at room temperature, permeabilized with Triton X-100 at room temperature, washed with PBS, and blocked with immunofluorescence blocking solution at room temperature. Anti-His-Tag primary antibody was added, and the cells were incubated overnight. The cells were washed with pre-cooled PBST, and the corresponding fluorescent secondary antibody was added. The cells were incubated at room temperature in the dark. The cells were washed with pre-cooled PBST, and the nuclei were stained with 4',6-diamidinyl-2-phenylindole at room temperature. After washing with PBS, the cells were mounted with anti-fluorescence quenching mounting medium, observed and photographed under a fluorescence microscope, and the presence of recombinant yak DDX6 protein near or inside the yak myoblasts was determined. ②WB detection: Yak recombinant DDX6 protein cells were treated with immunofluorescence staining, cell lysis buffer was added, and the cells were lysed on ice; after centrifugation, the supernatant was the extracted total cell protein; after determining the protein concentration using the diquinoline carboxylic acid method, WB detection was performed using anti-His-Tag antibody as the primary antibody; to qualitatively verify whether there is His-tagged recombinant DDX6 protein in the total protein of yak myoblasts; ③ Colloidal gold immunoelectron microscopy: Primary yak myoblasts were cultured in culture dishes, and the complete culture medium containing recombinant yak DDX6 protein was replaced at different time intervals. After culture, the culture medium was removed. Cells were collected after pre-cooled HBSS washing and fixed with immunoelectron microscopy-specific fixative. The samples were dehydrated in a gradient, embedded in resin, and ultra-thinly sectioned, then mounted on a nickel grid. Subsequently, the samples were blocked, incubated with primary antibody, blocked again, and incubated with colloidal gold secondary antibody, followed by washing with PBS. The samples were lightly counterstained with uranium acetate and lead citrate, washed with double-distilled water, and dried. The samples were observed and photographed using a transmission electron microscope to clearly distinguish whether the recombinant yak DDX6 protein was adsorbed on the cell membrane surface or entered the cell, and to determine its specific distribution location within the cell. The complete myoblast culture medium formula is: 10% FBS, 1% penicillin-streptomycin-amphoteric B, 1% L-Alanyl-L-Glutamine and 88% DMEM basal medium.

3. The application of the yak recombinant DDX6 protein as described in claim 1, characterized in that, It was used to regulate the proliferation of myoblasts and cell cycle progression in yak skeletal muscle.

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