Patent Publication Number: US-2022227828-A1

Title: Method for overexpressing il-15 in porcine skeletal myoblasts and use thereof

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This patent application claims the benefit and priority of Chinese Patent Application No. 202110066701.5 filed with China National Intellectual Property Administration on Jan. 19, 2021, the disclosure of which is incorporated by reference herein in its entirety as part of the present application. 
     TECHNICAL FIELD 
     The present disclosure relates to the field of genetic engineering, in particular to a method for overexpressing IL-15 in porcine skeletal myoblasts and use thereof. 
     BACKGROUND 
     With the increase in the intensification degree of China&#39;s animal husbandry industry, especially modern breeding industry, pigs are affected by more and more stress factors during growth, fattening and slaughter, causing changes in nutrient distribution and free radical metabolism in the pig body. Stress will break the balance of oxidation and anti-oxidation in the body, leading to a sharp increase of free radicals in the body, which causes irreversible oxidative damage to many important biological macromolecules in the body and further destruction of cell structure and function, and ultimately affects animal health and meat quality (Kerth, 2013). 
     Interleukin-15 (IL-15) , discovered in 1994, was a pleiotropic cytokine capable of regulating the proliferation, differentiation, maintenance and activity of a variety of cells [2]. IL-15 is also an important muscle factor secreted by skeletal muscle and plays an important role in regulating metabolism of skeletal muscle and energy balance. It can regulate the metabolism of muscle and adipose tissue in the modes of endocrine and paracrine, thereby maintaining the homeostasis of the whole body. There are more and more researches on IL-15. However, prior studies mainly involved the increased expression of IL-15 in the cells by using the method of directly adding IL-15. Because IL-15 has a short half-life, it is difficult to achieve the desired effect in a single treatment, there is need for multiple addition of IL-15, the operation is cumbersome, and the research cost is increased. And there is no report on the effect of IL-15 on porcine skeletal myoblasts. 
     SUMMARY 
     The present disclosure provides a method for overexpressing IL-15 in porcine skeletal myoblasts and use thereof to solve the above-mentioned problems in the prior art. Overexpressing IL-15 in porcine skeletal myoblasts not only reduces the research cost, but also simplifies the operation steps. The present disclosure proves that overexpression of IL-15 has a regulatory effect on among the others the cell cycle and apoptosis rate of porcine skeletal myoblasts, and lays a foundation for further research on the effect of IL-15 on livestock and poultry meat. 
     In order to achieve the above objective, the present disclosure provides the following technical solutions. 
     In a first aspect, there is provided a method for overexpressing IL-15 in porcine skeletal myoblasts, comprising transforming IL-15 into a lentiviral vector, then transforming the lentiviral vector into porcine skeletal myoblasts to promote the porcine skeletal myoblasts to overexpress IL-15. 
     In an embodiment of the present disclosure, the lentiviral vector is GV492 lentiviral expression vector. 
     In a second aspect, there is provided the use of overexpression of IL-15 in reducing apoptosis of porcine skeletal myoblasts. 
     In a third aspect, there is provided the use of overexpression of IL-15 in enhancing the ability of porcine skeletal myoblasts to resist oxidative damages. 
     In a fourth aspect, there is provided the use of overexpression of IL-15 in promoting division and proliferation of porcine skeletal myoblasts. 
     The present disclosure provides a method for overexpressing IL-15 in porcine skeletal myoblasts and its uses. In the present disclosure, lentiviral vector is used to transfer the gene of porcine IL-15 into porcine skeletal myoblasts, and the overexpressed of IL-15 in porcine skeletal myoblasts can be proven by detecting the mRNA and protein expression level. In the present disclosure, the gene of IL-15 is overexpressed directly in the porcine skeletal myoblasts, which addresses the problems of multiple additions and cumbersome operations in the treatment of IL-15 in prior studies, and lays a foundation for further study of the effect of IL-15 on meat. 
     In the present disclosure, it is proven through experiments that IL-15 is capable of promoting the division and proliferation of porcine skeletal muscle, enhancing its ability to resist oxidative damage and reducing the rate of cell apoptosis. There is also provided the use of overexpression of IL-15, which charts a course for further research. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a map of vector GV492 in Example 1. 
         FIG. 2  is an electrophoregram of the vector after digestion in Example 1, wherein Lane 1 is a 10 kb Marker (from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, and 250 bp, respectively), lane 2 is the digested product of the vector, and lane 3 is the undigested vector. 
         FIG. 3  is an electrophoregram of the target gene fragment in Example 1, wherein, the left band represents the Marker (5 kb, 3 kb, 2 kb, 1.5 kb, 1 Kb, 750 bp, 500 bp, 250 bp, and 100 bp, respectively), and the right bands represent PCR products. 
         FIG. 4  is an electrophoregram for PCR of the transformant in Example 1, wherein lane 1 represents the negative control (ddH 2 O), lane 2 represents the negative control (unloaded self-linked control group), lane 3 represents the positive control (GAPDH), lane 4 represents the Marker (from top to bottom: 5 kb, 3 kb, 2 kb, 1.5 kb, 1 Kb, 750 bp, 500 bp, 250 bp, and 100 bp, respectively), lanes 5-12 represent transformants 1-8, respectively. 
         FIG. 5  is a diagram showing the results for sequence alignment of positive clones in Example 1. 
         FIG. 6  is a diagram showing the results for fluorescence observation of cells after transfection in Example 1. 
         FIG. 7  is an electrophoregram of the cells transfected in Example 1 by Western Blot, wherein lane 1 represents the positive control, lane 2 represents the negative control, and lane 3 represents the sample obtained from transinfection of plasmid for the target gene by 293T cells. And the primary antibody used in the electrophoresis was a Flag protein that encodes a hydrophilic peptide consisting of eight amino acids (DYKDDDDK, SEQ ID NO: 9). The secondary antibody used in the electrophoresis was goat anti-mouse IgG (H+L), HRP. 
         FIG. 8  is a diagram of porcine skeletal muscle satellite cells isolated and cultured in Example 2. 
         FIG. 9  is a diagram of the induced and differentiated porcine skeletal myoblasts in Example 2. 
         FIG. 10  is diagram showing the results for immunofluorescence assay, wherein it is shown from top to bottom the porcine skeletal muscle satellite cells, differentiated porcine skeletal myoblasts, porcine skeletal myoblasts transfected with control lentivirus, and porcine skeletal myoblasts transfected with overexpression lentivirus. 
         FIG. 11  is a diagram of results for cell cycle detection in Example 2. 
         FIG. 12  is a diagram showing the detection of apoptosis by flow cytometry in Example 2. 
         FIG. 13  is a diagram of showing the detection of apoptosis rate of porcine skeletal myoblasts by flow cytometry in Example 2; 
         FIG. 14  is a diagram showing the comparison of gene expression levels of IL-15 in porcine skeletal myoblasts in Example 2. 
         FIG. 15  is a diagram showing the comparison of protein expression levels of IL-15 in porcine skeletal myoblasts in Example 2. 
         FIG. 16  shows the effect of H202 on cell viability in Example 3. 
         FIG. 17  is a diagram showing the detection results of cell cycle under oxidative stress conditions in Example 3. 
         FIG. 18  is a diagram of flow cytometry of cell apoptosis under oxidative stress conditions in Example 3. 
         FIG. 19  is a diagram showing the detection of cell apoptosis rate by flow cytometry under oxidative stress conditions in Example 3. 
         FIG. 20  is the results for detection of cell apoptosis rate by Western Blot under oxidative stress conditions in Example 3. 
         FIG. 21  is a diagram showing the results for ROS detection of the negative group by flow cytometry in Example 3. 
         FIG. 22  is a diagram showing the results for ROS detection of the blank cell plus H 2 O 2  group by flow cytometry in Example 3. 
         FIG. 23  is a diagram showing the results for ROS detection of virus transfected with the control group+hydrogen peroxide group by flow cytometry in Example 3. 
         FIG. 24  is a diagram showing the results for ROS detection of virus transfected overexpressing IL-15 with+hydrogen peroxide group in Example 3. 
         FIG. 25  is a diagram showing the results for ROS detection of the cells in Example 3. 
         FIG. 26  is a diagram showing the results of IL-15 secretion by cells in Example 3. 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     Various exemplary embodiments of the present disclosure will now be described in detail. The detailed description should not be considered as a limitation to the present disclosure, but should be understood as a more detailed description of certain aspects, characteristics, and embodiments of the present disclosure. 
     Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the field of the present disclosure. Although the present disclosure only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. 
     Without departing from the scope or spirit of the present disclosure, various modifications and variations can be made to the specific embodiments of the present disclosure description, which will be apparent to those skilled in the art. Other embodiments derived from the description of the present disclosure will be apparent to the skilled person. The specification and examples of this disclosure are merely exemplary. 
     Unless otherwise specified, the materials and reagents used in the following examples can be available from commercial sources. Unless otherwise specified, the experimental methods used are all conventional experimental methods in the field. 
     Example 1 
     Preparation of Lentivirus for Overexpressing Porcine Gene IL-15 
     The sequence of porcine gene IL-15 was queried in NCBI, and the accession number was NM_214390. 
     1. Construction of Overexpression Lentiviral Vector 
     1. Materials and Methods 
     1.1 Main Reagents 
     Main reagents are shown in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Main reagent information 
               
            
           
           
               
               
               
            
               
                 Reagent name 
                 Reagent source 
                 Model 
               
               
                   
               
               
                 1kp DNA ladder Marker 
                 Fermentas Inc. 
                 #SM0311 
               
               
                 250bp DNA ladder Marker 
                 Shanghai Generay 
                 DL250+, 100T 
               
               
                   
                 Biotech Co., Ltd 
                   
               
               
                 Agarose 
                 Beijing SBS  
                 GA4-100 
               
               
                   
                 Genetech Compay 
                   
               
               
                 In-Fusion ™ PCR  
                 clontech 
                 639626 
               
               
                 Cloning Kit 
                   
                   
               
               
                 Taq polymerase 
                 SinoBio 
                 E001-02B 
               
               
                 dNTP 
                 Takara 
                 D4030A 
               
               
                 Primer 
                 Shanghai Generay 
                   
               
               
                   
                 Biotech Co., Ltd. 
                   
               
               
                 Restriction endonuclease 
                 NEB 
                   
               
               
                 Plasmid extraction kit 
                 Promega 
                 A1460 
               
               
                 Agarose gel DNA  
                 TIANGEN BIOTECH 
                 DP209-03 
               
               
                 recovery kit 
                 (BEIJING) CO., LTD. 
               
               
                   
               
            
           
         
       
     
     1.3 Main Instruments And Equipment 
     Main instruments and equipment are shown in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Main instrument information 
               
            
           
           
               
               
               
            
               
                 Equipment name 
                 Reagent source 
                 Model 
               
               
                   
               
               
                 PCR instrument 
                 Applied Biosystems 
                 2720 thermal cycler 
               
               
                 Positive clone 
                 Shanghai Meiji Biotech 
                 ABI3730 
               
               
                 Sequencer 
                   
                   
               
               
                 Constant voltage DNA 
                 BioRad 
                   
               
               
                 electrophoresis 
                   
                   
               
               
                 apparatus 
                   
                   
               
               
                 Gel imager 
                 Shanghai Tanon Technology 
                   
               
               
                   
                 Co., Ltd. 
                   
               
               
                 Bacteria shaker 
                 Taicang Hualida Laboratory 
                 HI-9211K 
               
               
                   
                 Equipment Co., Ltd 
                   
               
               
                 Bacteria incubator 
                 Shanghai Yiheng Scientific 
                   
               
               
                   
                 Equipment Co., Ltd 
                   
               
               
                 Gilson pipette 
                 Gilson 
                   
               
               
                 High-speed centrifuge 
                 Hitachi 
                 TGL-16G-A 
               
               
                 Disposable disc 
                 Hunan Changsha Tiandiren 
                   
               
               
                   
                 biotechnology Co. Ltd. 
                   
               
               
                 1L flask 
                 Jintan Jingbo Experimental 
                 1111.1115 
               
               
                   
                 Instrument Factory 
                   
               
               
                 50 ml polypropylene 
                 Shanghai Wuhua Chemical 
                   
               
               
                 tube 
                 Co., Ltd. 
               
               
                   
               
            
           
         
       
     
     2. Digestion of Vector 
     2.1 Vector Information 
     The name of the vector used is GV492, and the sequence of elements: Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin, which contains two cloning sites BamHI/AgeI. The map of the vector is shown in  FIG. 1 . The control empty vector number is CON335. 
     2.2 Results of Restriction Digestion 1601 Plasmids extracted from bacteria have different migration rates due to different conformations such as supercoil, open loop, linearity, and present bands of different sizes in agarose gel electrophoresis. Therefore, the electrophoresis bands of plasmids can only be used as a reference for determining the molecular weight of plasmids, and it cannot be used as a basis for accurate determination. After the plasmids were subjected to single digestion, it showed a uniform electrophoretic band. At this time, comparison to the Marker could be used to determine its molecular mass. The PCR result of the vector after digestion is shown in  FIG. 2 . It can be seen that the vector after single digestion formed a uniform band. When compared with the Marker, the molecular weight of the band was the same as recorded in the vector map, which proved successful digestion of the vector. 
     3. Acquisition of Target Gene Fragments 
     3.1 Primer Information 
     Full gene synthesis was performed according to the sequence of the target gene, and the target gene was amplified by PCR according to the manual of the vector (Overexpression Lentiviral Vector Package and Construction Manual, https://www.genechem.com.cn/index/supports/download.html). Information on primers for PCR is shown in Table 3. It is shown that the primer contained a base pair exchange, and restriction digestion sites. The primer further contained partial sequence at the 5′-end of the target gene, which was used to tag the target gene by PCR. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 PCR primer information (SEQ ID NO.1-SEQ ID NO.2) 
               
            
           
           
               
               
            
               
                 ID 
                 Sequence 
               
               
                   
               
               
                 IL15(44657-6)-P1 
                 AGGTCGCTCTAGAGGATCCCGCCACCATGAGA 
               
               
                   
                 ATTTTGAAACCATGTTTG 
               
               
                   
               
               
                 IL15(44657-6)-P2 
                 TCCTTGTAGTCCATACCAGAAGGGTTGATGAA 
               
               
                   
                 CATTTGC 
               
               
                   
               
            
           
         
       
     
     3.2 PCR Results 
     The electrophoregram for PCR is shown in  FIG. 3 , and the product is about 530 bp in size. It was proven that the target gene fragment was successfully obtained. 
     4. Construction of Recombinant Plasmid 
     The resulting PCR product of the target gene was exchanged into the linearized expression vector for PCR identification, according to the vector specification (overexpression lentiviral vector package and construction manual, https://www.genechem.com.cn/index/supports/download.html). The information on primers for PCR identification is shown in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Primers for PCR identification 
               
               
                 (SEQ ID NO.3-SEQ ID NO.4) 
               
            
           
           
               
               
               
            
               
                   
                 ID 
                 Sequence 
               
               
                   
                   
               
               
                   
                 Ubi-F 
                 GGGTCAATATGTAATTTTCAGTG 
               
               
                   
                   
               
               
                   
                 FLAG-R-2 
                 CCTTATAGTCCTTATCATCGTC 
               
               
                   
                   
               
            
           
         
       
     
     The results for PCR identification is shown in  FIG. 4 , the PCR product of positive transformants have a size of 669 bp, and the PCR product of negative transformants has a size of 185 bp. 
     The positive transformants were subjected to sequencing and alignment, and the alignment results ( FIG. 5 ) showed that the overexpression lentiviral vector was successfully constructed. 
     2. Detection of IL-15 Expression 
     The successfully constructed lentiviral vector was used to transfect 293T cells, the cell fluorescence was observed with a fluorescence microscope, and the expression of IL-15 was detected by Western Blot. The size of the target gene fusion protein was about 20 kD. 
     1. Materials and methods 
     The target cells to be transfected were 293T cells cultured in DMEM medium (containing 10% fetal bovine serum). The antibody information used is shown in Table 5. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Antibody information 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Antibody  
                   
                 Product 
                 Dilution  
               
               
                 No. 
                 Antibody 
                 name 
                 Manufacturer 
                 No. 
                 ratio 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 Primary 
                 FLAG 
                 Sigma 
                 F1804 
                 1:2000  
               
               
                   
                 antibody 
                   
                   
                   
                   
               
               
                 2 
                 Secondary 
                 Mouse 
                 santa-cruz 
                 sc-2005 
                 1:10000 
               
               
                   
                 antibody 
               
               
                   
               
            
           
         
       
     
     The above information comes from the antibody instructions, including the antibody dilution ratio (recommended dilution ratio in the instructions), and positive control samples (recommended positive control in the instructions). 
     2. Experimental Method 
     2.1 Extraction of Cell Proteins 
     The RIPA protein extraction reagent was pre-cooled and protease inhibitors were added in the protein extraction reagent. 0.1M PMSF mother liquid was added before the start of protein extraction, the final concentration of PMSF was 1 mM. Cell count: The cells was added to 300 μL of lysis buffer in a number of 1×10 7  and blown with the pipette tip to suspend the cells. The cells were incubated on ice for 20 min after the completion of suspension. And centrifugation was conducted for 20 min at 4° C. and 13000 rpm. After the completion of the centrifugation, the supernatant was dispended for further measurement. 
     2.2 Quantification by BCA Protein Method 
     BCA working solution (solution A: solution B=50:1) was prepared, and respective BSA extraction standard. The sample was diluted with PBS. 
     The sample and the BCA working solution was mixed in a ratio of 1:8. After being mixed uniformly, the mixture was incubated at 37° C. for 30 min or at room temperature for 60 min. The OD value was measured with a 570 nm-wavelength filter in the enzyme-labeled instrument. 
     2.3 Adjustment of Protein Concentration 
     The protein concentration was adjusted with RIPA and 5× reducing sample buffer was added to a sample final concentration of 2-4 mg/ml (determined with respect to different samples). The mixture was boiled for denaturation for 5 min. 
     2.4 Western Blot Experiment 
     (1) 10% of separating gel was based on the molecular weight of the target protein, and the concentration of the concentrated gel was 5%. 
     (2) 20 μg of protein sample was loaded, a WB standard (SURVIVIN-3FLAG-GFP, molecular weight: 48 kD) was used as the positive control, and the 293T cells were used as the negative control. 
     (3) Electrophoresis conditions: concentrated gel, constant voltage at 90V for about 20 min; separating gel constant voltage 160V, and the time to stop the electrophoresis was determined by pre-stained the protein Marker. 
     (4) Wet transfer method: membrane transfer conditions: 300 mA of constant electric current; 0.45 μM pore size NC membrane, membrane transfer time 2.5 h. After completion of membrane transfer, the membrane was stained with the staining reagent Ponceau, membrane transfer effect was observed, and the lanes were marked at the same time. 
     (5) Blocking: the membrane was completely immersed in 3% BSA-TBST and gently shaken at room temperature for 30 minutes. 
     (6) Primary antibody incubation: the primary antibody was diluted with 3% BSA-TBST, incubated at room temperature for 10 minutes, and allowed to stand overnight at 4° C. And the membrane was taken out from 4° C. incubator the next day, and then incubated at room temperature for 30 minutes. Membrane washing: the membrane was washed 5 times with TBST, 3 min per wash. 
     (7) Secondary antibody incubation: the secondary antibody goat anti-mouse IgG (H+L) HRP was diluted with 5% skimmed milk powder-TBST in a ratio of 1:10000, and the diluted solution was gently shaken at room temperature for 40 minutes. Membrane wash: the membrane was washed with TBST 6 times, 3min per wash. 
     (8) Color development by ECL method combined with X-ray film: after ECL was added to the film, the reaction was carried out for 3-5 min, the film was exposed to light for 10 s-5 min (the exposure time was adjusted based on the light intensity), developed for 2 min, and fixed. 
     2.5 Detection of Virus Titer 
     The virus titer was measured using a fluorescence method, and the measured virus titer was 2.5E+9. 
     3. Experimental Results 
     The result of cell fluorescence observation after transfection is shown in  FIG. 6 . It can be seen that notable fluorescence could be observed in the cells after transfection, indicating that the target plasmid was normally transfected and the expression of the fluorescent marker gene in the target plasmid was normal. 
     The results of Western Blot electrophoresis are shown in  FIG. 7 . It can be seen that there was an obvious characteristic band at 20 kD, and the size was consistent with the target gene fusion protein, which proved the successful overexpression of the target gene. 
     Example 2 
     Overexpression of IL-15 in Porcine Skeletal Myoblasts 
     1. Materials and Equipment 
     The required experimental equipment is shown in Table 6, and the materials and reagents are shown in Table 7. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Experimental instrument information 
               
            
           
           
               
               
               
            
               
                   
                   
                 Specification  
               
               
                 Equipment name 
                 Manufacturer 
                 and model 
               
               
                   
               
               
                 Biological safety cabinet 
                 Jinan BIOBASE Co., Ltd. 
                 BSC-1500IIA2-X 
               
               
                 CO 2  cell incubator 
                 Shanghai Boxun Industry &amp; 
                 BC-J160S 
               
               
                   
                 Commerce Co., Ltd. 
                   
               
               
                 Fluorescence inverted 
                 Nikon 
                 DS-Ri2 
               
               
                 microscope 
                   
                   
               
               
                 High-speed refrigerated 
                 Thermo Fisher 
                 Multifuge X1R 
               
               
                 centrifuge 
                   
                   
               
               
                 Electric heating constant 
                 Shanghai Jing Hong  
                 DHG-9123A 
               
               
                 temperature blast drying 
                 Laboratory 
                   
               
               
                 oven 
                 Instrument Co., Ltd. 
                   
               
               
                 Electric heating constant 
                 Shanghai Jing  
                 DK-2B 
               
               
                 temperature oscillating 
                 Hong Laboratory 
                   
               
               
                 sink 
                 Instrument Co., Ltd. 
                   
               
               
                 Flow cytomerters 
                 Miltenyi Biotec 
                 130-092-197 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Experimental materials and reagent information 
               
            
           
           
               
               
               
            
               
                   
                   
                 Specification/ 
               
               
                   
                   
                 Product  
               
               
                 Reagents and consumables 
                 Manufacturer 
                 number 
               
               
                   
               
               
                 T25 cell culture flask 
                 Coming 
                 430639 
               
               
                 Hemocytometer 
                 Marienfeld 
                 Neubauer 
               
               
                   
                   
                 improved 
               
               
                 Special round coverslips  
                 Solar bio 
                 YA0350 
               
               
                 for 24-well plate 
                   
                   
               
               
                 Cell culture plate 
                 Shanghai WHB 
                   
               
               
                   
                 Scientific Co., Ltd. 
                   
               
               
                 Fetal bovine serum 
                 Gibico 
                 1414426 
               
               
                 Special culture medium  
                 Shanghai iCell  
                 Primed-iCell- 
               
               
                 for skeletal muscle cells 
                 Bioscience Inc. 
                 018 
               
               
                 0.25% trypsin (containing  
                 Gibico 
                 1734858 
               
               
                 0.02% EDTA) 
                   
                   
               
               
                 Paraformaldehyde (PFA) 
                 Solarbio 
                 P1110 
               
               
                 DAPI 
                 Solarbio 
                 C0060 
               
               
                 Triton X-100 
                 Solarbio 
                 T8200 
               
               
                 Goat serum 
                 Solarbio 
                 SL038 
               
               
                 α-SM 
                 Abeam 
                 Ab124964 
               
               
                 CoraLite594-conjugated  
                 Proteintech 
                 SA00013-4 
               
               
                 goat anti-Rabbit 
                   
                   
               
               
                 IgG (11 + L) 
                   
                   
               
               
                 Fluoromount-G  
                 SourthernBiotech 
                 0100-01 
               
               
                 fluorescence Mounting 
                   
                   
               
               
                 agent 
                   
                   
               
               
                 LV-IL15 
                 GeneChem Co., Ltd. 
                 44657-6 
               
               
                 Negative control virus 
                 GeneChem Co., Ltd. 
                 LVC0N335 
               
               
                 CON335(Ubi-MCS-3FLAG- 
                   
                   
               
               
                 CBh-gcGFP-IRES-puromycin) 
                   
                   
               
               
                 Cell Cryopreservation Solution 
                 Corning 
                 88-702-CB 
               
               
                 MTT 
                 Solarbio 
                 M8180 
               
               
                 H 2 O 2   
                 Jiangxi Caoshanhu 
                 20190103 
               
               
                   
                 disinfection supplies 
                   
               
               
                   
                 Co., Ltd. 
                   
               
               
                 ROS detection kit 
                 Beyotinne 
                 S0033 
               
               
                   
               
            
           
         
       
     
     2. Experimental Procedure and Results 
     1. Isolation, Culture and Induced Differentiation of Porcine Skeletal Muscle Satellite Cells 
     1.1 Isolation and Culture of Porcine Skeletal Muscle Satellite Cells 
     The piglets was bled to death at the neck artery, then the uterine tissue was taken out, placed in PBS containing P/S, and the piglet was disinfected with 75% alcohol. The thigh skeletal muscle was separated under aseptic conditions and placed in PBS containing a secondary antibody. The thigh skeletal muscle was rinsed repeatedly for 3-5 times, transferred to a petri dish, minced into 1 mm 3 -size tissue blocks with an ophthalmological scissor. The minced tissue blocks were transferred to a 50 mL centrifuge tube, 0.25% Trypsin was added and digested at 37° C. for 30 min in water bath and under vibration, and then a complete medium was used to neutralize the digestion fluids. The mixture of the digested tissue was filtered through a 100 μm mesh, the filtrate was collected and centrifuged at 1500 rpm for 10 min. After the completion of centrifugation, the supernatant was discarded; the precipitate was re-suspended with culture medium, transferred to a T-25 culture flask. The 1-25 culture flask was placed into an incubator, and the culture medium was changed every 24 hours. The cultured cells are shown in  FIG. 8 , which shows that the cell culture was successful. 
     1.2 Induced Differentiation of Porcine Skeletal Muscle Satellite Cells 
     The second-generation porcine skeletal muscle satellite cells were inoculated in a culture flask padded with L-polylysine. When the cell fusion degree was about 50-60%, differentiation medium DMEM (low sugar)+2% goat Serum was added and the medium was changed on the third day of the culturing. After culturing for a total of 5 days, growth of the cells was observed. The cultured cells are shown in  FIG. 9 , which shows that the cells had successfully differentiated and the porcine skeletal myoblasts were thus obtained. 
     1.3 Transfection with Lentivirus Overexpressing IL-15 
     The differentiated myoblasts were inoculated into a 6-well plate, the number of cells per well was about 1×10 5  cells. The medium was changed when cells were cultured to wall adherence. One milliliter of complete medium was added, and then 20 μL of control lentivirus and lentiviral overexpression (MOI=100) were added respectively, the resulting culture was mixed well and continued to culture for 72 h transfection. 
     1.4 Immunofluorescence Assay 
     Porcine skeletal muscle satellite cells, differentiated porcine skeletal myoblasts, porcine skeletal myoblasts transfected with control lentivirus, and porcine skeletal myoblasts transfected with overexpression lentivirus were measured. Specific steps are as follows: 
     (1) Round Coverslip 
     Three round coverslips in a 24-well plate, 1 mL of culture medium was added to each well, and 0.2×10 6  cells/well was added. The resulting round coverslips were placed in the incubator and incubated for 2 h or overnight. 
     (2) Immobilization 
     After the cells are attached to the round coverslips, the culture medium was aspirated, and the round coverslips were washed with PBS 1 time and 4% PFA was added at 4° C. and immobilization was carried out for 30 min. The round coverslips were washed with PBS 3 times, and 5 min each time. Alternatively, the round coverslips may be allowed to stand at 4° C. overnight without aspirating the PBS for the last time. 
     (3) Permeabilization and Blocking 
     The round coverslips was get rid of water and placed on a Petri dish support. 
     Preparation of blocking solution for the round coverslips: 0.5% Trition X-100 mixed with PBS in a ratio of 1:1, with additional 10% serum. 
     Fifty microliter of permeabilization wash buffer was dripped on a waterproof membrane, and the surface having cells was covered and kept for 2 hours. 
     (4) Incubation of Primary Antibody 
     Preparation of primary antibody (α-SMA): antibody was diluted with PBS in a ratio of 1:100. 
     After the permeabilization and blocking, 50 μL of primary antibody was applied to a waterproof membrane (in a humidified chamber), and the round coverslips (the surface having cells) was covered and kept at 4° C. (for up to one week). 
     (5) Incubation of Secondary Antibody 
     Preparation of secondary antibody (CoraLite594-conjugated Goat Anti-Rabbit IgG (H+L)): antibody was diluted with PBS in a ratio of 1:500. 
     After the secondary antibody was incubated at room temperature for 2 hours in the dark, the secondary antibody was washed with PBS 3 times and 5 min for each time. Then the secondary antibody was stained with DAPI (DAPI:PBS =1:1000) for 5 min and washed with PBS 3 times and 5 min for each time. 
     (6) Embedding 
     One drop of Fluoromount-G was dripped on each round coverslips, and the surface having cells was covered for observation. 
     The results of the fluorescence measurement are shown in  FIG. 10 . 
     1.5 Determination of Cell Cycle 
     The cells were digested with EDTA-free trypsin, and centrifuged at 1000 r/min for 5 min, cells were collected, washed with PBS, and immobilized overnight with at 75% pre-cooled ethanol. The resulting mixture was centrifuged at 1000 r/min for 10min and ethanol was discarded. The cells were incubated in a staining solution containing 100 μg/mL of PI and 200 μg/mL of RNAase and incubated for 30 min. The apoptotic cell peak was detected by flow cytometry, and the fluorescence signals were processed by cell cycle analysis software Multicycle in a flow cytometer. 
     The test results are shown in  FIG. 11 . After transfection of IL-15, the proportion of cells in G1 phase decreased significantly, and the proportion of cells in S phase and G2/M phase increased significantly. It is indicated that IL-15 could promote the division and proliferation of myoblasts. 
     1.6 Detection of Apoptosis 
     An Annexin V-FITC Apoptosis Detection Kit (Tongjin Institute of Chemistry, Japan, lot number VN665) was used for apoptosis detection. The cells were digested with trypsin without EDTA and centrifuged at 1000 r/min for 5 min. 1×AnnexinV binding buffer was added after the cells were resuspended. 5 μL of Annexin V-FITC, 5 μL of PI were added and mixed well, the resulting mixture was incubated at room temperature in dark for 15 min, and the cells were detected by flow cytometry, the detection results are shown in  FIG. 12  and  FIG. 13 . After the cells were transfected with IL-15, the apoptosis rate of myoblasts decreased significantly. It shows that IL-15 can inhibit the apoptosis of myoblasts. 
     1.7 Measurement of Cell Gene Expression 
     1.7.1 Materials and Reagents 
     The cells were digested with EDTA-free trypsin and centrifuged at 1000 r/min for 5 min. The cells were collected for detection of IL-15 gene expression. The instruments and reagents used are shown in Table 8. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Instruments and reagents for use 
               
            
           
           
               
               
               
            
               
                 Reagent/Instrument 
                 Manufacturer 
                 Product No. 
               
               
                   
               
               
                 TRNzol total RNA  
                 TIANGEN BIOTECH  
                 DP405-02 
               
               
                 extraction reagent 
                 (BEIJING) CO., LTD. 
                   
               
               
                 PrimeScript ™ RT reagent  
                 TaKaRa (Dalian) 
                 RR047B 
               
               
                 Kit with gDNA Eraser 
                   
                   
               
               
                 SYBR ® Premix Ex Taq ™  
                 TaKaRa (Dalian) 
                 RR82LR 
               
               
                 II (Tli RNaseH Plus), 
                   
                   
               
               
                 ROX plus 
                   
                   
               
               
                 DL2,000 DNA Marker 
                 TaKaRa (Dalian) 
                 3427Q 
               
               
                 primer synthesis 
                 Invitrogen 
                   
               
               
                 Vortex oscillator 
                 Haimen Kylin-Bell Lab 
                 QL-902 
               
               
                   
                 Instruments Co., Ltd. 
                   
               
               
                 Centrifuge 
                 Eppendorf 
                 Centrifuge 
               
               
                   
                   
                 5415D 
               
               
                 Spectrophotometer 
                 Therno scientific 
                 NANODROP 
               
               
                   
                   
                 2000 
               
               
                 Gel imaging system 
                 Shanghai Tanon  
                 Tanon 1600 
               
               
                   
                 Technology Co., Ltd. 
                   
               
               
                 Fluorescence Quantitative  
                 Applied Biosystems 
                 ABI7500 
               
               
                 PCR Instrument 
               
               
                   
               
            
           
         
       
     
     The primers used in Real Time PCR to detect the target gene are shown in Table 9 (SEQ ID NO.5-SEQ ID NO. 8). The following primers were synthesized by Beijing Invitrogen Company. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Primer information used to measure gene expression 
               
            
           
           
               
               
               
            
               
                   
                   
                 Product 
               
               
                   
                 Primer 
                 size 
               
               
                 Primer name 
                 sequence (5′ to 3′) 
                 (bp) 
               
               
                   
               
               
                 Upstream primer 
                 GCATCCAGTGCTACTTGTGTR 
                 118 
               
               
                 for IL15 
                   
                   
               
               
                   
               
               
                 Downstream primer 
                 TGCCAGGTTGCTTCTGTTTT 
                   
               
               
                 for IL15 
                   
                   
               
               
                   
               
               
                 Upstream primer 
                 TGCGGGACATCAAGGAGAAG 
                 216 
               
               
                 for ACTIN 
                   
                   
               
               
                   
               
               
                 Downstream primer 
                 AGTTGAAGGTGGTCTCGTGG 
                   
               
               
                 for ACTIN 
                   
                   
               
               
                   
               
            
           
         
       
     
     1.7.2 Experimental Method 
     1.7.2.1 Extraction and Quality Testing of Sample RNA 
     A TRNzol total RNA extraction reagent was use for extraction of sample RNA, and the experimental operation was carried out according to the product instructions. 
     The concentration and purity were measured by a UV absorption method. An ND-2000 ultraviolet-visible spectrophotometer was used to measure the concentration and purity of RNA, and the spectrophotometer was adjusted to zero with a DEPC solution which was used to dissolve RNA before measurement. The readings at 260 nm and 280 nm were measured to calculate the concentration and purity of the RNA sample. 
     RNA concentration was calculated as follows: a reading value of 1 at 260 nm was equivalent to 40 ng RNA/μL. The formula for calculating the sample RNA concentration was: A260×40 ng/μL. In this Example, it was measured A 260=65.003. Upon calculation, RNA concentration was 2600.12 ng/μL, the remaining total amount of RNA excluding the consumed test sample was 19 μL×2600.12 ng/UL=49.4 μg. 
     RNA purity was judged through the ratio of A260/A 280, and the ratio ranged from 1.8 to 2.1. Even if the ratio exceeded this range, RNA samples could also be used in some common experiments such as Northern hybridization, RT-PCR and RNase protection experiments. The ratio of A260/A280 measured in this embodiment was 2.0. 
     1.7.2.3 Denaturing Agarose Gel Electrophoresis 
     One gram of agarose was dissolved in 72 ml of water, cooled to 60° C., and 10 ml of 10×MOPS electrophoresis buffer and 18 ml of 37% formaldehyde solution (12.3 M) were added. 
     Calculated based on the final concentration, 10×MOPS electrophoresis buffer contained 0.2 M MOPS (pH 7.0), 0.02M sodium acetate, and 0.01M EDTA. 
     Gel plates were filled and sample wells reserved for addition of at least 25 μL of solution. After gelation, the combs were removed, and the gel plates were placed in the electrophoresis tank, and a sufficient amount of 1×MOPS electrophoresis buffer was added to cover the gel surface by a few millimeters. 
     Into 1 μg of RNA was added 1 volume of the formaldehyde loading stain and EB was added in the formaldehyde loading stain to a final concentration of 10 μg/ml. The mixture was heated to 70° C. and incubated for 5 minutes to denature the sample. The sample was loaded into the gel well, and electrophoresis was conducted at a voltage of 5-6V/cm until the bromophenol blue indicator entered the gel by at least 2-3 cm. Observation was performed under transmitted UV light, the results showed that the RNA sample had a high purity and could be used in subsequent experiments. 
     1.7.2.4 Sample Detection by Real Time PCR 
     The purified RNA samples were reverse transcribed to cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser. The resulting cDNA samples were tested by Real Time PCR, and the specific steps were included: 
     (1) Preparation of reaction system: Real Time PCR reaction systems were separately prepared for all cDNA samples. Realtime PCR reaction systems each had a total volume of 18 μL, specifically containing 10 μL of 2×Master Mix, 0.5 μL, of 10 μM PCR specific primers F, 0.5 μL of 10 μM of PCR specific primers R, and water was added to a total volume of 18 μL. The tube was flicked at the bottom to mix the solution, and centrifuged briefly at 5000 rpm. 
     (2) Sample loading: 18 μL of mixed solution was added to each corresponding well of a 96-well PCR plate, and 2 μL of cDNA was added. A sealing film was attached carefully and the tube was mixed by brief centrifugation. The well prepared PCR plate was placed on ice before setting the PCR procedure. 
     (3) PCR reaction: proceed as following procedure: 95° C., 30 s; 40 PCR cycles (95° C., 5 s; 60° C., 40 s (fluorescence was collected)). In order to establish the melting curve of the PCR product, the PCR product was slowly heat from 60° C. to 99° C. (the instrument automatically implemented a Ramp rate of 0.05° C./s) as per a procedure of 95° C., 10 s; 60° C., 60 s; and 95° C., 15 s) after the amplification reaction is completed. 
     1.7.3 Experimental Results 
     The target gene and internal reference gene of each sample were subjected to Real Time PCR reaction, and each sample was tested in 3 replicate wells. The data was analyzed by 2 −ΔΔ     CT    method. 
     The results are shown in  FIG. 14 . After transfection with lentivirus, expression of IL-15 gene in myoblasts increased significantly. It is showed that the lentivirus overexpressing IL-15 was successfully constructed. 
     1.8 Measurement of Cell Protein Expression 
     The cells were digested with EDTA-free trypsin and centrifuged at 1000 r/min for 5 min. The cells were collected and the IL-15 protein expression was measured. 
     The results are shown in  FIG. 15 . It is showed that protein expression from IL-15 in myoblasts increased significantly after transfecting lentivirus. 
     Example 3 
     Effect of IL-15 on oxidative damage of porcine skeletal myoblasts 
     1. Establishment of Oxidative Stress Model 
     The MTT method was used to measure cell viability. 
     (1) The porcine skeletal myoblasts was inoculated in a 96-well plate in a number of 1×10 4 /well and the plate was incubated at 37° C. for 24 hours in an incubator filled with 5% of CO 2.    
     (2) The culture liquid was removed and a culture medium containing 0.1, 0.2, 0.4 and 0.8 mM H 2 O 2  respectively, and a culture medium without addition of H 2 O 2  as control. Five replicates were set in each group, and the cells were continued to culture for further 1.5 hours. 
     (3) The culture medium was removed and washed with PBS 2 times. 
     (4) To each well was added 20 μL of 5 g/L of MTT solution, culturing was continued for another 4 hours. 
     (5) The supernatant was removed, and 150 μL, of DMSO was added to each well and shaken for 10min; 
     (6) OD values for each well were measured by an enzyme-labeled instrument at a wavelength of 570 nm. 
     The results are shown in  FIG. 16 . It is shown that when the oxidative stress model was established, the cell survival rate was usually in the range of 50% -70%. If the cell survival rate was too high, no significant oxidative damage was caused, and if the survival rate was too low, irreversible damage was caused. In both cases, it was not conducive to the establishment of an oxidative stress model. Therefore, 0.1 mM and 1.5 h were used as the concentration and time in the treatment of the H 2 O 2  oxidative stress model, respectively. 
     2. The Effect of IL-15 on the Oxidative Damage of Porcine Skeletal Myoblasts 
     Porcine skeletal muscle satellite cells were primarily cultured, when the cell fusion degree was about 50-60%, differentiation culture medium (low-sugar DMEM medium+2% goat serum) was added to induce differentiation of cells for 5 days. Then the differentiated myoblasts were transfected with an empty vector or GV492-IL-15 recombinant lentivirus for cell treatment. Seventy two hours later, cells were treated with 0.1 mM H 2 O 2  for 1.5 h to form three experimental treatment groups of blank cells+H 2 O 2  group, empty vector+H 2 O 2  group and GV492-IL-15 transfected recombinant lentivirus +H 2 O 2  group. 
     1. Measurement of Cell Cycle 
     Measurement cell cycle was performed according to the method of Example 2. As shown in  FIG. 17 , under oxidative stress conditions, the ratio of cells in G1 phase after IL-15 transfection significantly decreased, and the ratio of cells in G2/M phase increased significantly. Compared with the empty vector group, the proportion of S-phase cells decreased significantly, while compared with the blank cells, the difference was not significant. It is shown that IL-15 can promote the division and proliferation of myoblasts. 
     2. Measurement of Cell Apoptosis 
     2.1 Measurement by Flow Cytometry 
     The cells were digested with EDTA-free trypsin and centrifuged at 1000 r/min for 5 min. s1×Annexin V binding buffer was added after the cells were resuspended. Five microliter of Annexin V-FITC, 5 μL of PI were added and mixed well, the resulting mixture was incubated at room temperature in dark for 15 min, and then the cells were measured by flow cytometry. 
     The test results are shown in  FIG. 18  and  FIG. 19 . It can be seen that the proportion of apoptosis after transfection of IL-15 was significantly reduced. It is shown that IL-15 can reduce the apoptosis rate of cells under oxidative stress. 
     2.2 Measurement by Western Blot 
     The changes of cell apoptosis was reflected by measurement the expression profile of activated caspase-3 protein in the cells. Cleavedcaspase-3 antibody was purchased from Immunoway Company, catalog number YM3431, dilution ratio 1:500. 
     The results are shown in  FIG. 20 . Oxidative stress significantly increased the apoptotic rate of myoblasts, while transfection of IL-15 lentivirus could significantly decrease the apoptotic rate of cells, which confirmed that IL-15 can relieve oxidative damage by inhibiting cell apoptosis. 
     3. Measurement of ROS in Cell 
     The cells treated with H 2 O 2  were digested with cold Tyrode buffer and washed twice, and resuspended with 500 μL of Tyrodebuffer. Then 10 μM DCFH-DA (Sigma Corporation) was added, and cells were incubated at 37° C. for 30 min in dark, centrifuged, and the supernatant was discarded, and then washed with the buffer twice gently. The cells were resuspended after the supernatant was decanted, and the fluorescence intensity in each group was measured by flow cytometry, respectively. 
     The results shown in  FIG. 21 - FIG. 25 , it is shown that oxidative stress could significantly increase the ROS content in the cells. Treatment of lentivirus by transfection of IL-15 could significantly reduce the ROS content in cells. It is shown that IL-15 can inhibit the oxidative damage of myoblasts to a certain extent. 
     4. Secretion of IL-15 in Cells 
     The treated cell culture supernatant was collected, and the secretion of IL-15 was measured with an ELISA kit (R&amp;D Systems). 
     The results in  FIG. 26 , the under oxidative stress conditions, secretion of IL-15 in cells increased significantly after transfection of IL-15 in lentivirus, indicating that IL-15 could alleviate cell damage caused by oxidative stress. 
     The above-mentioned embodiments merely describe the preferred embodiments of the present disclosure, and are not intended to limit the scope of the present disclosure. Those of ordinary skill in the art can make various variations and improvements to the technical solutions of the present disclosure, without departing from the design spirit of the present disclosure. These variations and improvements should fall within the protection scope set forth by the appended claims of the present disclosure. 
     The application hereby incorporates by reference the Sequence Listing titled “BGI05-005-US_Sequence_Listing.txt”, created on Feb. 15, 2022, having a file size of 3 KB.