A method for in vitro proliferation of marine fish myoblasts

Abstract

The present invention discloses a method for in vitro proliferation of marine fish myoblasts, wherein the culture medium uses a basal culture medium as a background culture medium; the culture medium contains an IGF2 protein with a final concentration of 20-200 ng / mL; and the IGF2 protein is derived from leopard gill perch. The present invention uses the above culture medium to culture passage cells to obtain myoblasts that proliferate in vitro of leopard gill perch, successfully achieving in vitro culture of leopard gill perch myoblasts, and the number of myoblasts reaches 30,000, and the in vitro proliferation rate reaches 87%. Raw materials are provided to explore the functions of myoblasts themselves and the gene regulation mechanism, and the prerequisite for in vitro verification experiments is provided for further exploring the influence of marine fish myoblasts on the growth rate, and also lays a foundation for the breeding of marine fish.

Description

Technical Field

[0001] The invention belongs to the technical field of marine fish growth and development and cell culture, and particularly relates to an in vitro proliferation method of marine fish myoblasts. Background Art

[0002] Leopard gill perch (Plectropomus leopardus), commonly known as the eastern star grouper, belongs to the order Perciformes, family Serranidae, subfamily Epinephelinae, genus Plectropomus, species Blue-spotted gill perch. It mainly inhabits tropical or subtropical waters of the Indian Ocean and Pacific Ocean, and is highly sought after by the market for its delicious meat and extremely high application value. This fish is one of the most important fishery resources in the world. As a pillar commodity in the entire Indian-Western Pacific region, leopard gill perch has important commercial value. The retail price of live fish ranges from US$50 to US$75 per kilogram. This high profit has led to a huge market demand for leopard gill perch, and the total annual harvest has increased year by year. However, due to the destruction of coral reefs and illegal overfishing in recent years, the number of wild leopard gill perch has decreased sharply, so it is urgent to fill the large gap in market demand with artificial large-scale breeding. At present, due to the long growth and development cycle of leopard gill perch, artificially cultivated leopard gill perch need two to three years to reach the market standard, and there are generally problems of slow growth rate and huge differences in individual growth rate, which has led to a decrease in the survival rate of leopard gill perch and an increase in breeding costs, seriously affecting the healthy development of the aquaculture industry. Growth is a somatic development process that reflects the balance between an organism's feed intake and utilization. The growth rate of fish is a key factor in the development of the aquaculture industry. By increasing the growth rate of skeletal muscle and shortening the breeding period, it has important guiding significance for reducing the risk and cost of breeding.

[0003] At present, unlike embryonic cell cultivation, several methods for the isolation and in vitro culture of fish muscle cells are still imperfect, and both the success rate and efficiency need to be greatly improved. Previous researchers used the dorsal muscle of young turbot as the original tissue, adopted the tissue adherent culture method, and used L-15 culture medium as the basic culture medium for cell culture. They successfully obtained primitive muscle cells that can be cultured for a long time and have a stable physiological state, but the existing culture medium is not enough to support the rapid proliferation of muscle cells. The establishment of primary cell lines in fish is already a relatively mature technology. Although there are patents for the establishment of muscle cell lines, the muscle cells of leopard gill perch cultured by these methods cannot proliferate in vitro, which is not conducive to the analysis of the mechanism of fish growth regulation.

[0004] Insulin-like growth factor (IGF) is a member of the IGF family and a multifunctional cell regulatory factor. Most IGFs are synthesized in the liver and mainly exist in the blood circulation. Insulin-like growth factor-2 (IGF2) is a member of the IGF family and a key regulatory factor for multifunctional cell proliferation. It plays an important role in promoting the differentiation and proliferation of somatic cells, the growth and development of embryos, and the proliferation of tumor cells. Studies have found that mammalian IGF2 can interfere with the proliferation and differentiation of mammalian cells by regulating the MAPK and PI3K / Akt signaling pathways. However, whether IGF2 affects the proliferation of marine fish cells in vitro and the functions of different concentrations of IGF2 in the process of cell proliferation culture in vitro have not been reported.

[0005] Therefore, it is necessary to establish a standardized method for in vitro proliferation of marine fish myoblasts in order to solve the problems of existing technologies that cannot proliferate myoblasts in vitro, low cell survival rate or unstable proliferating cells, so as to obtain myoblasts with high activity, provide raw materials for exploring the functions and gene regulation mechanisms of myoblasts themselves, and provide in vitro verification experimental prerequisites for further exploring the effects of marine fish myoblasts on growth rate. At the same time, it also lays the foundation for the breeding of marine fish. Summary of the invention

[0006] The purpose of the present invention is to establish a standardized method for in vitro proliferation of seawater myoblasts to solve the technical problem:

[0007] The first aspect of the present invention provides a culture medium for in vitro proliferation of marine fish myoblasts, wherein the culture medium uses a basal culture medium as a background culture medium; the culture medium contains an IGF2 protein with a final concentration of 20-70 ng / mL, 70-90 ng / mL, 90-110 ng / mL, 110-120 ng / mL, 120-150 ng / mL or 150-200 ng / mL; and the IGF2 protein is derived from leopard gill perch.

[0008] Furthermore, the marine fish is a Perciformes fish; the Perciformes fish is a Pinnipedia fish; the Pinnipedia fish is the Leopard Gill Perch, Humpback Perch, Brown Spotted Grouper, Red Spotted Grouper, Clouded Grouper or Jewel Grouper.

[0009] Furthermore, the basal culture medium includes L-15 medium, fetal bovine serum, NAEE, 1× double antibody and β-mercaptoethanol; L-15 medium is 62-63%, 63-64% or 64-65% of the volume of the basal culture medium; fetal bovine serum is 14.0-14.5%, 14.5-15.0% or 15.0-15.5% of the volume of the basal culture medium; NAEE is 0.8-0.9%, 0.9-1.0% or 1.0-1.1% of the volume of the basal culture medium; 1× double antibody includes: 100 IU / mL penicillin, 100 μg / mL streptomycin; the final concentration of β-mercaptoethanol is 0.04-0.045 mM, 0.045-0.050 mM or 0.050-0.055 mM.

[0010] The second aspect of the present invention provides an in vitro proliferation method for marine fish myoblasts. The in vitro proliferation method for marine fish myoblasts uses the above-mentioned culture medium. The in vitro proliferation method for marine fish myoblasts comprises the following steps: step A: pre-treating the skeletal muscle tissue of leopard gill perch to obtain pre-treated muscle tissue; step B: primary culturing the pre-treated muscle tissue to obtain primary cells; step C: subculture the primary cells to obtain subculture cells; step D: culturing the subculture cells using IGF2 culture medium to obtain in vitro proliferated myoblasts of leopard gill perch.

[0011] Further, step A is specifically as follows: using a healthy 2-month-old, 3-month-old or 4-month-old leopard gill perch to disinfect the fish body surface, removing the skeletal muscle tissue of the second dorsal myometrium of the leopard gill perch under sterile conditions, namely the leopard gill perch skeletal muscle tissue; placing the leopard gill perch skeletal muscle tissue in a PBS buffer containing 5× double antibody and immersing and washing it three times, each washing time is 5 min; cutting the leopard gill perch skeletal muscle tissue into 2 mm 2 Tissue blocks of different sizes were added with PBS buffer containing 2× double antibody to wash away the blood, and the skin membrane and residual blood of the skeletal muscle tissue of the leopard gill perch were removed to obtain the pretreated muscle tissue.

[0012] Furthermore, the PBS buffer containing 5× double antibody includes: 500 IU / mL penicillin and 500 μg / mL streptomycin; the PBS buffer containing 2× double antibody includes: 200 IU / mL penicillin and 200 μg / mL streptomycin.

[0013] Furthermore, step B is specifically as follows: inoculating the pretreated muscle tissue into a 6-well plate, using a basal culture medium to perform primary culture on the pretreated muscle tissue, replacing the basal culture medium every 2 days, and culturing upright at 28° C. to obtain primary cells.

[0014] Furthermore, step C is specifically as follows: when the plating rate of primary cells reaches 70-80%, the primary cells are digested with trypsin to obtain primary myoblasts; the primary myoblasts are subcultured with basal culture medium to obtain subculture cells; the concentration of trypsin is 0.0625%; the temperature of the digestion reaction is 25-26°C, 26-27°C or 27-28°C; the time of the digestion reaction is 5.0-5.5 min, 5.5-6.0 min or 6.0-6.5 min.

[0015] Furthermore, step D is specifically as follows: when the passage cells are in a logarithmic growth cycle, the passage cells are proliferated and cultured using IGF2 medium to obtain in vitro proliferated myoblasts of leopard gill bass; the temperature of the proliferation culture is 28° C., and the time of the proliferation culture is 12-20 h, 20-40 h, 40-60 h, 60-80 h or 80-100 h.

[0016] Furthermore, the number of myoblasts proliferated in vitro by the leopard gill perch reached 30,000; the proliferation rate of myoblasts proliferated in vitro by the leopard gill perch was 87%.

[0017] The present invention obtains leopard gill perch myoblasts by separating leopard gill perch skeletal muscle tissue by trypsin digestion and performing primary culture and subculture, and then successfully performs in vitro proliferation of leopard gill perch myoblasts by adding leopard gill perch-derived IGF2 protein in the culture medium, thereby providing a large number of leopard gill perch-derived myoblasts, and the cells can maintain a good growth state. In addition, after 96 hours of in vitro proliferation culture of myoblasts cultured with leopard gill perch-derived IGF2 protein at a concentration of 100 ng / mL, the number of myoblasts reaches 30,000, and after EdU is used for detection, the in vitro proliferation rate of myoblasts can reach 87%, and the cells have a good growth state and a high survival rate. Therefore, the method for in vitro proliferation of leopard gill perch myoblasts provided by the present invention can provide raw materials for exploring the functions of myoblasts themselves and the gene regulation mechanism. At the same time, the culture medium for in vitro proliferation of myoblasts and the in vitro proliferation method of myoblasts provided by the present invention are also applicable to other marine fish, such as Perciformes fish, or Perciformes fin fish such as humpback perch, brown-spotted grouper, red-spotted grouper, clouded grouper or jewel grouper, etc., and also provide in vitro verification experimental prerequisites for further exploring the influence of other marine fish myoblasts on the growth rate, and also lay the foundation for the breeding of marine fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above content and the following specific embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only examples of the technical solutions claimed for protection.

[0019] Figure 1 Agarose gel electrophoresis of the first amplified product of IGF2 (lane 1: maker; lanes 2-6: IGF2 gene 642 bp);

[0020] Figure 2 Agarose gel electrophoresis of the second amplification product of IGF2 (lane 1: maker; lane 2: blank control; lanes 3 and 4: 642 bp of the second amplification product of IGF2 gene);

[0021] Figure 3 The expression of IGF2 protein from Leopard gill perch under different induction conditions (lane 1: maker; lane 2: bacterial solution after induction with 0.4 mM IPTG at 37°C for 18 h; lane 3: bacterial solution after induction with 0.7 mM IPTG at 37°C for 18 h);

[0022] Figure 4 The SDS-PAGE image of the IGF2 protein from Leopard gill perch after expression and purification (wherein, lane 1: whole bacteria; lane 2: supernatant after fragmentation; lane 3: precipitate after fragmentation; lane 4: flow-through 1; lane 5: flow-through 2; lane 6: protein washed with 20 mM imidazole of IGF2; lane 7: protein washed with 50 mM imidazole of IGF2; lane 8: protein washed with 100 mM imidazole of IGF2; lane 9: protein washed with 100 mM imidazole of IGF2; lane 10: protein eluted with 250 mM imidazole of IGF2; lane 11: protein eluted with 250 mM imidazole of IGF2; lane 12: protein eluted with 500 mM imidazole of IGF2; lane 13: protein eluted with Ni treatment; lane 14: protein eluted with Ni treatment);

[0023] Figure 5 The BCA concentration determination curve of IGF2 protein from Leopard Gill Perch (where the horizontal axis is the concentration of IGF2 protein at OD 562 nm The absorbance value of the vertical axis is the protein concentration of IGF2, the unit is (ng / mL));

[0024] Figure 6 This is a diagram of the cell status of the muscle tissue of leopard gill bass on the 4th day of primary culture (scale bar: 1mm);

[0025] Figure 7 It is a fitting curve diagram of the absorbance value at A450 nm of leopard gill perch myoblasts after CCK8 enhancement (wherein the abscissa is the number of cells, in units (pieces); the ordinate is the absorbance value at A450 nm of myoblasts after CCK8 enhancement);

[0026] Figure 8It is a line graph showing the change in the number of myoblasts treated with IGF2 protein at different concentration gradients (where the horizontal axis is different treatment times, in units of (h); the vertical axis is the number of myoblasts, in units of (pieces));

[0027] Fig. 9 This is a green fluorescence image of the effect of EdU detection of IGF2 protein from Leopard gill perch on the proliferation of myoblasts in vitro (A is the Hoechst33342 staining of the control group; B is the EdU detection of the control group; C is the Merge detection of the control group; D is the Hoechst33342 staining of the third experimental group; E is the EdU detection of the third experimental group; F is the Merge detection of the third experimental group; blue dots represent cell nuclei; green dots represent liposomes);

[0028] Fig.10 The bar graph of Pax7 gene fluorescence quantitative analysis (where the horizontal axis is IGF2 concentration; the vertical axis is relative expression; * indicates significant difference);

[0029] Fig.11 This is a bar graph of TCF-4 gene fluorescence quantitative analysis (where the horizontal axis is IGF2 concentration; the vertical axis is relative expression; ** represents a very significant difference);

[0030] Fig.12 This is a bar graph of MyoD1 gene fluorescence quantitative analysis (where the horizontal axis is IGF2 concentration; the vertical axis is relative expression; ** represents a very significant difference). DETAILED DESCRIPTION

[0031] The detailed features and advantages of the present invention are described in detail in the specific implementation modes below, and the contents are sufficient to enable any person skilled in the art to understand the technical contents of the present invention and implement them accordingly. Moreover, according to the description, claims and drawings disclosed in this specification, those skilled in the art can easily understand the relevant objects and advantages of the present invention.

[0032] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0034] (1) Source of sample materials

[0035] In this embodiment, 2-3 month old leopard gill perch were purchased from Hainan Chenhai Aquatic Products Co., Ltd., Gancheng Town, Dongfang City, Hainan Province, China.

[0036] (2) Sources of reagents and consumables

[0037] Table 1 Sources and item numbers of reagents and consumables required for the experiment

[0038] name Manufacturer Part Number Scissors Admiralty (China) J21070 tweezers Admiralty (China) J42035 Cell culture flasks Thermo Fisher (USA) 156367 L-15 medium Giboco 11415064 100× double antibody Giboco 15140122 Trypsin with EDTA Sigma T4049 PBS buffer Giboco 10010-049 Fetal bovine serum Giboco A5669401 Nonessential amino acids Giboco 11140050 β-Mercaptoethanol Giboco 31350010 CCK8 Kit Blue Sky C0041 EdU Cell Proliferation Detection Kit Blue Sky C0071S 96-well plate Blue Sky FCP962 6-well plate Blue Sky FCP060 pET32a Novagen (China) HG-VYN0178 ArcticExpress Competent Cells Blue Sky D1003S Plasmid Extraction Kit Tiangen DP103 IPTG Solebao 367-93-1 Ni-NTA Novizan HP202-02 Imidazole BBI A600277-0100 PD-10 Desalting Column Si Tuo Fan 17085101 5× protein loading buffer (containing DTT, referred to as 5× loading buffer) Blue Sky P0286 Non-prestained protein maker Biosharp BL712A SDS-PAGE gel Yamei PG113 SDS-PAGE electrophoresis buffer Yamei TF101 4% PFA solarbio P1110 0.5% TritonX-100 penetrant Blue Sky P0096-100mL 1× Hoechst33342 reaction solution Blue Sky C0071S BlastTaqTM2X qPCR MasterMix Kit abm G891 Enhanced Glue Recovery Kit Tiangen DP219 High-fidelity enzyme Novizan P505-d1

[0039] (3) Source of instruments and equipment

[0040] Table 2 Sources and models of instruments and equipment required for the experiment

[0041] name Manufacturer model Constant temperature incubator Smoothness Herocell240P Shaker Smoothness Stab M2 Fluorescence quantitative PCR instrument ThermoFisher Qubit 4.0 Fluorescence inverted microscope Leica DMi4 UV spectrophotometer Hitachi UH5300 Ultrasonic cell disruptor Ningbo Xinzhi SCIENTZ-950E

[0042] Example

[0043] A method for in vitro proliferation of marine fish myoblasts comprises the following steps:

[0044] S1. Preparation and purification of IGF2 protein from Leopard gill perch. The specific reference steps are as follows:

[0045] (1) The IGF2 fragment gene was synthesized based on the sequence of insulin-like growth factor-2 (IGF2, GeneBank: XM_042495438.1: 128-769, SEQ ID NO. 1) published on NCBI, and gene-specific primers were designed using Primer Premier 5.0 (wherein, the upstream primer IGF2-F of the IGF2 fragment gene is SEQ ID NO. 2: 5'-GCGTGGGATCGTAGAGGAGTGT-3'; the downstream primer IGF2-R is SEQ ID NO. 3: 5'-AGTTGTCCGTGGCAAGCAAGAC-3').

[0046] (2) Insert the IGF2 fragment gene obtained above into the expression vector pET32a to obtain a recombinant expression plasmid.

[0047] The IGF2 gene was amplified by the first round of PCR reaction, wherein the first round of PCR reaction system was: 2 μL of synthesized IGF2 gene template DNA, 1 μL each of IGF2-F / IGF2-R (10 μM), 25 μL of 2 × Phanta Max Master Mix (high-fidelity enzyme), and ddH2O to a total volume of 50 μL. The mixture was vortexed and briefly centrifuged before the first round of PCR reaction. The first round of PCR reaction program was: pre-deformation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 61°C for 15 s, and extension at 72°C for 30 s. The denaturation to extension process was repeated for 35 cycles, followed by full extension at 65°C for 5 min and storage at 4°C, which was the first amplified product of IGF2.

[0048] After the first round of PCR reaction, the first amplified product of IGF2 was separated by 1.5% agarose gel electrophoresis, and a band of about 642 bp was cut out using a UV gel exciter, and DNA was recovered using an enhanced gel recovery kit. This was the target gene IGF2. The specific agarose gel electrophoresis results are shown in Figure 1 .

[0049] The target gene IGF2 was subjected to enzyme ligation reaction with the pET32a vector. The enzyme ligation reaction system was: 2 μL of the target gene IGF2 and 1 μL of the backbone vector pET32a. The mixture was gently pipetted and mixed at 25°C for 10 min to obtain the IGF2 ligation product, which was stored at 4°C for later use.

[0050] Trans-T1 competent cells were placed on ice to thaw, and the IGF2 ligation product was added to 50 μL Trans-T1 competent cells. After gently mixing, the cells were quickly placed on ice for 30 min, heat-shocked in a 42°C metal bath / water bath for 30 s, and immediately placed on ice for 2 min. Under a sterile environment, the transformed bacterial solution was added to 1 mL of non-resistant LB liquid culture medium, and the shaker was incubated at 37°C, 220 rpm, and shaken for 1 h to obtain the IGF2 transformed bacterial solution. Subsequently, 200 μL of the IGF2 transformed bacterial solution was spread on the LB solid culture medium containing ampicillin resistance, and inverted and cultured overnight at 37°C in the incubator to obtain the first IGF2 plate.

[0051] The monoclonal plaques in the first plate of IGF2 were picked for expansion culture, which was the first monoclonal bacterial solution of IGF2. 1 μL of the first monoclonal bacterial solution of IGF2 was taken for the second round of PCR reaction. The second round of PCR reaction system was: 5 μL of 2 × Rapid TaqMaster Mix (rapid PCR mixture), 1 μL of the first monoclonal bacterial solution of IGF2, 0.5 μL of T7 (SEQ ID NO.4: 5'-TAATACGACTCACTATAGGG-3'), 0.5 μL of IGF2-F / IGF2-R (10 μM), and 0.5 μL of each, and ddH2O was added to make the volume 2.5 μL. The second round of PCR reaction program was: pre-denaturation at 95℃, 3 min, denaturation at 95℃, 15 s, annealing at 61℃, 15 s, and extension at 72℃, 30 s. Among them, the denaturation to extension process was 35 cycles in total, and then fully extended at 65℃ for 5 min, which was the second amplification product of IGF2, and stored at 4℃ for use. The second amplified product of IGF2 was tested by 1.5% agarose gel electrophoresis. The fragment size was about 642 bp. The specific agarose gel electrophoresis verification diagram is shown in Figure 2 .

[0052] Select the positive clone of the second amplification product of IGF2 and expand the culture, place it on a shaker, and culture it at 200 rpm and 37℃ for about 12 hours. After extracting the plasmid with a plasmid mini-extraction kit, take an appropriate amount of plasmid and send it to Shanghai Shenggong Company, and sequence it with T7 and T7 Ter (SEQID NO.5: 5'-TGCTAGTTATTGCTCAGCGG-3') universal primers. The sequencing results were compared and analyzed, and the similarity was 100%, indicating that the recombinant protein plasmid vector was successfully constructed, that is, the IGF2 recombinant plasmid, which was marked and stored in a -20℃ refrigerator.

[0053] (3) The IGF2 recombinant plasmid in step (2) was transformed into E. coli ArcticExpress competent cells. Except for the competent cells, the transformation method was the same as S1 (2) screening. After the coating was completed, it was inverted and cultured overnight in a constant temperature incubator at 37°C to form the second IGF2 plate. A single colony was picked from the second IGF2 plate and cultured overnight in 40 mL of ampicillin-resistant LB liquid culture medium to form the second IGF2 monoclonal bacterial solution. After overnight culture, 20 mL of the second IGF2 monoclonal bacterial solution was inoculated into 2 L of ampicillin-resistant LB liquid culture medium at a ratio of 1:100 to form the first IGF2 expansion bacterial solution. It was placed in a shaker at 37°C and 220 rpm for about 4 h. When the OD of the first expansion bacterial solution reached 0.04, the culture temperature was 0.04 °C and the temperature was 0.04 °C. 600 nm=0.5-0.7, add IPTG (Isopropyl β-D-Thiogalactoside) with a final concentration of 0.5-1mM, and induce at 25-40℃ for 10-20h. That is, the lower the induction temperature, the longer the induction time. This is the bacterial solution after the first induction of IGF2. The bacterial suspension after the first induction of IGF2 was transferred to large centrifuge bottles, centrifuged at 4°C, 4000 rpm for 15 min, the supernatant was discarded, and the bacterial pellet was retained as the first IGF2 pellet. Then 30 mL of 1×PBS (phosphate buffered saline) was added to resuspend the first IGF2 pellet to obtain the first IGF2 resuspension. Then, an ultrasonic cell disruptor was used at 30% power, ultrasonicated for 2 s, with an interval of 4 s, and a total ultrasonic time of 60 min to obtain the second IGF2 resuspension. The second IGF2 resuspension was dispensed into 2 mL centrifuge tubes and centrifuged at 13000 rpm at 4°C for 30 min to collect the supernatant sample after IGF2 disruption. The pellet was suspended in 30 μL of 1×PBS to obtain the IGF2 disruption pellet sample. The expression of IGF2 protein under different induction conditions is shown in Figure 2. Figure 3 .

[0054] (4) Take 2 mL of Ni-NTA (nickel NTA affinity chromatography medium, referred to as nickel column), equilibrate the nickel column with 20 mL of 15-30 mM imidazole, and repeatedly inject the IGF2 supernatant into the nickel column twice. The outflow is IGF2 flow-through 1 and IGF2 flow-through 2; wash the nickel column with 20 mL of 20 mM imidazole solution to remove impurities, which is IGF2 20 mM imidazole wash impurities; wash the nickel column with 20 mL of 50 mM imidazole solution to remove impurities, which is IGF2 50 mM imidazole wash impurities; wash the nickel column with 20 mL of 100 mM imidazole solution twice to remove impurities, which is IGF2 100 mM imidazole wash impurities 1 and IGF2 100 mM imidazole wash impurities 2; wash the nickel column with 20 mL of 250 mM imidazole solution twice to elute the protein, which is IGF2 250 mM imidazole was used to elute protein 1 and IGF2 250 mM imidazole was used to elute protein 2; the nickel column was washed with 20 mL of 500 mM imidazole solution to elute protein IGF2 with 500 mM imidazole; Ni-NTA treatment was performed twice, the first time the nickel column was washed with 10 mL of 1 M imidazole solution to elute protein 1 for Ni treatment; the second time the nickel column was washed with 20 mL of ultrapure water to elute protein 2 for Ni treatment; finally, the nickel column was stored in 20% ethanol in a 4°C refrigerator.

[0055] 40 μL of each of the above-mentioned IGF2 first expanded culture solution (referred to as whole bacteria), IGF2 supernatant after disruption (referred to as disruption supernatant), IGF2 precipitate after disruption (referred to as disruption precipitate), IGF2 flow-through 1 (referred to as flow-through 1), IGF2 flow-through 2 (referred to as flow-through 2), IGF2 20 mM imidazole washed protein, IGF2 50 mM imidazole washed protein, IGF2 100 mM imidazole washed protein 1, IGF2 100 mM imidazole washed protein 2, IGF2 250 mM imidazole eluted protein 1, IGF2 250 mM imidazole eluted protein 2, IGF2 500 mM imidazole eluted protein, Ni-treated eluted protein 1 and Ni-treated eluted protein 2 were mixed with 10 μL of 5× loading buffer, heated in a metal bath at 99°C for 10 min, centrifuged at 13,000 rpm for 10 min, and subjected to SDS-PAGE electrophoresis at 200 V, 35 min. After the electrophoresis was completed, the PAGE gel was stained with SDS-PAGE fast dye and decolorized with tap water. It was clearly observed that there was a clear band of IGF2 protein at around 35 kDa. The protein was successfully expressed and the purity was good. Subsequent experiments can be continued. The specific IGF2 protein expression and purification diagram is shown in Figure 4 .

[0056] The IGF2 protein was desalted by PD-10 column according to the instructions to remove imidazole and other salts, and concentrated by 10 kDa concentrator tube to obtain IGF2 concentrated protein. The concentration of IGF2 protein was determined by BCA kit. The IGF2 protein was expressed at OD 562 nm The absorbance value was 0.657, and the fitting equation y=1.031x+0.0221 (R 2 =0.9931), the IGF2 protein concentration was measured to be about 0.7 mg / mL, and the aliquots were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator. For specific BCA protein concentration determination standard curve, see Figure 5 .

[0057] S2. Preparation of culture medium. The specific steps are as follows:

[0058] (1) Preparation of basal culture medium: 64% L-15 basal culture medium by volume, 15% fetal bovine serum by volume, 1% NAEE (non-essential amino acid solution) by volume, 1× double antibody (100 IU / mL penicillin and 100 μg / mL streptomycin), and a final concentration of 0.05 mM β-mercaptoethanol;

[0059] (2) Preparation of culture medium for in vitro proliferation of myoblasts: 64% by volume of L-15 (Gibco) basal medium (basal medium is used as background medium): 15% fetal bovine serum, 1% NAEE, 1× double antibody (100 IU / mL penicillin and 100 μg / mL streptomycin), 0.05 mM β-mercaptoethanol, and the final concentration of IGF2 protein from Leopard gill perch is 20-200 ng / mL.

[0060] S3. Pretreatment of skeletal muscle tissue of leopard gill perch. The specific reference steps are as follows:

[0061] Healthy 2-3 month old leopard gill perch were selected, and after being wiped and disinfected with 75% alcohol, the skeletal muscle tissue of the second dorsal myometrium was removed under sterile conditions and placed in a culture dish. PBS buffer containing 5× double antibody (500 IU / mL penicillin and 500 μg / mL streptomycin) was added and soaked and washed three times, each washing for 15 min. The skeletal muscle tissue after soaking and washing three times was cut into 2 mm in the culture dish. 2 The shredded skeletal muscle tissue blocks were cut into pieces and transferred to a new culture dish using tweezers. The solution was changed several times with PBS buffer solution containing 2× double antibody (200 IU / mL penicillin and 200 μg / mL streptomycin) until the blood was washed away. The skin film and residual blood of the leopard gill perch muscle tissue were removed to obtain the pretreated leopard gill perch muscle tissue.

[0062] S4. Pretreatment of the in vitro culture of the muscle tissue of the leopard gill perch. The specific reference steps are as follows:

[0063] (1) The muscle tissue of the leopard gill perch obtained in step S3 above was inoculated into a 6-well plate, inverted and dried for 6 h at room temperature, and then 1 mL of basal culture medium was added to each well. The basal culture medium was replaced every 2 days and the plate was placed in a 28°C constant temperature incubator for culture. On the 4th day, the cells began to migrate out. Figure 6 As shown, primary cells of Leopard spiny bass were obtained.

[0064] (2) When the plating rate of the primary cells of the leopard gill perch in the above step S4 (1) reaches 70%-80%, the subculture can be carried out. Under a sterile environment, use 500 μL of 0.0625% trypsin digestion solution to preliminarily digest the cells. The digestion temperature is 28°C and the digestion time is 5-6 min. After the digestion is terminated, the digested leopard gill perch primary myoblasts are collected into a sterile centrifuge tube and centrifuged at 200 × g for 3 min to obtain a myoblast pellet. Add 5 mL of basal culture medium to resuspend the myoblast pellet and transfer it to a 25 cm 2The cells were placed in an airtight culture bottle at 28°C and placed upright in a constant temperature incubator for continued culture, which was called subculture to obtain the subculture cells of the leopard gill bass.

[0065] (3) When the leopard gill perch passage cells in the above step S4 (2) are in the logarithmic growth cycle, the leopard gill perch passage cells in the logarithmic growth cycle are inoculated into a 96-well plate, and IGF2 culture medium containing different concentration gradients of IGF2 protein is added to the 96-well plate. The final concentration gradient of IGF2 protein in IGF2 culture is set to 0, 20 ng / mL, 100 ng / mL, and 200 ng / mL, and each concentration gradient is set to be repeated three times. Among them, the control group: the culture medium with an IGF2 protein concentration of 0 is used as the basal culture medium; the first experimental group: the IGF2 protein concentration is 20 ng / mL; the second experimental group: the IGF2 protein concentration is 100 ng / mL; the third experimental group: the IGF2 protein concentration is 200 ng / mL. The above leopard gill perch passage cells are transferred to a 28°C constant temperature incubator and cultured for 6 h, 12 h, 24 h, 48 h and 96 h respectively, so as to obtain the leopard gill perch in vitro proliferated myoblasts.

[0066] Comparative Example 1

[0067] In the embodiment of the present invention, the basic culture medium in step S2 (1) is used to perform in vitro proliferation culture on myoblasts of marine fish.

[0068] Compared with the embodiment, the difference between this comparative example 1 is that the cell type is different. This comparative example 1 uses other types of somatic cells of leopard gill perch for culture.

[0069] The experimental results show that other types of somatic cells of the leopard gill perch in comparative example 1 can be proliferated and cultured in vitro in the basal culture medium, and the number of cells thereof gradually increases, while the number of myoblasts of marine fish in this embodiment gradually decreases.

[0070] Comparative Example 2

[0071] In the embodiment of the present invention, the culture medium in step S2 (2) is used to perform in vitro proliferation culture on myoblasts of marine fish.

[0072] Compared with the embodiment, the difference between this comparative example 2 is that the species is different. This comparative example 2 uses skeletal myoblasts of mammals (mouse) for in vitro proliferation culture.

[0073] The experimental results show that the survival rate of the skeletal myoblasts of mammals in the in vitro proliferation culture of Comparative Example 2 is low, and subsequent experiments cannot be carried out.

[0074] Comparative Example 3

[0075] In the embodiment of the present invention, the IGF2 protein derived from Leopard gill perch is added to the basal culture medium at a final concentration of 20-200 ng / mL to perform in vitro proliferation culture of myoblasts.

[0076] Compared with the embodiment, the difference between this comparative example 3 is that the source of IGF2 protein is different. This comparative example 3 uses human IGF2 protein to carry out in vitro proliferation culture of leopard gill bass myoblasts.

[0077] The experimental results show that the in vitro proliferation effect of the leopard gill perch myoblasts of Comparative Example 3 is poor, the number of in vitro proliferating cells is basically unchanged, and the number of myoblasts gradually decreases with the increase of in vitro proliferation culture time.

[0078] Test Example 1

[0079] The absorbance value of the in vitro proliferated myoblasts of the leopard gill perch obtained in step (3) of Example S4 is detected, comprising the following steps:

[0080] (1) After culturing the cultured cells of P. leopardii in a 96-well plate with myoblast proliferation medium for 6 h, 12 h, 24 h, 48 h, and 96 h, 10 μL of CCK8 enhancement reagent was added to each well and incubated at 28°C for 1 h.

[0081] (2) The standard curve of the absorbance at 450 nm after adding CCK8 enhancement reagent in (1) above is measured by ultraviolet spectrophotometer as shown in the following figure: Figure 7 shown.

[0082] (3) Based on the change in absorbance, the fitting curve equation in the standard curve is: y = 0.0001x - 0.1342 (R 2 =0.9974) to calculate the number of cells.

[0083] The results are as follows Figure 8As shown in the figure, different concentrations of IGF2 protein were added to the basal culture medium. Within the first 6 hours, except for the control group, the number of myoblasts of the leopard gill perch in the experimental groups showed an increasing trend. Among them, the myoblasts in the third experimental group increased the most. At 12 hours, the myoblasts in the third experimental group almost did not proliferate. The number of cells in the first and third experimental groups was not much different, both around 10,000, but the number of myoblasts in the second experimental group was about 18,000. When the culture was continued to be cultured for 24 hours, it was found that the number of myoblasts cultured in the second experimental group with the addition of 100 ng / mL of IGF2 protein from the leopard gill perch was still the largest, about 22,000, followed by the first and third experimental groups, while the number of myoblasts in the control group began to decrease. When the culture was continued to be cultured for 48 hours, it was detected again, and it was found that the myoblasts in the second experimental group had grown all over the cell culture bottle, and the number of cells was the largest, about 28,000, followed by the first and third experimental groups. When the culture was continued to be cultured for 96 hours, the number of myoblasts in the second experimental group was the largest, about 22,000, followed by the first and third experimental groups. Another test was conducted at 14h, and it was found that the number of myoblasts in the second experimental group had reached 30,000, the number of myoblasts in the first experimental group had reached 20,000, and the number of myoblasts in the third experimental group had begun to decrease to less than 15,000. In summary, after adding different concentrations of IGF2 protein to the basal culture medium, the number of myoblasts of Leopard gill perch showed an increasing trend, indicating that an appropriate amount of IGF2 protein derived from Leopard gill perch can promote the proliferation of Leopard gill perch myoblasts, and in the second experimental group, adding IGF2 protein derived from Leopard gill perch at a concentration of 100 ng / mL can make the number of Leopard gill perch myoblasts proliferate the most in vitro, and the proliferation-promoting effect is the most obvious; in the fourth experimental group, adding IGF2 protein derived from Leopard gill perch at a concentration of 200 ng / mL inhibited the proliferation of Leopard gill perch myoblasts when CCK8 enhanced treatment was performed for 96 hours, while the number of Leopard gill perch myoblasts in the control group without adding IGF2 protein derived from Leopard gill perch showed a gradually decreasing trend after CCK8 enhanced treatment for 24 hours, that is, the basal culture medium cannot maintain the in vitro proliferation of Leopard gill perch myoblasts.

[0084] Test Example 2

[0085] In order to further verify the effect of IGF2 protein from leopard gill perch on the proliferation of leopard gill perch myoblasts, EdU cell proliferation detection was performed using myoblasts after CCK8 enhanced culture for 96 h in the control group and the second experimental group in Test Example 1, including the following steps:

[0086] EdU solution (provided in the EdU cell proliferation detection kit) was diluted with IGF2 medium and prepared into 10 μM EdU medium. After incubating the myoblasts of the control group and the third experimental group in a constant temperature incubator at 28°C for 48 h, the 10 μM EdU medium was discarded, and the cells were washed with 1× PBS buffer and fixed with 4% PFA at room temperature for 15 min; 1× Apollo® staining reaction solution (provided in the EdU cell proliferation detection kit) was added, incubated in a decolorizing shaker for 30 min at room temperature in a dark place, and the staining reaction solution was discarded; 0.5% TritonX-100 permeabilizer was added, washed in a decolorizing shaker, and the permeabilizer was discarded; 1× Hoechst33342 reaction solution diluted with deionized water was added, incubated in a decolorizing shaker for 30 min in a dark place at room temperature, and myoblasts were stained, and the staining reaction solution was discarded. After washing with 1× PBS, the myoblasts were placed under a fluorescent inverted microscope for observation.

[0087] The results are as follows Fig. 9 As shown, in the same period of time, the number of cells containing EdU labeling in the myoblasts cultured in the second experimental group with the addition of 100 ng / mL IGF2 protein from leopard gill perch was the largest, indicating that the proliferation rate of myoblasts in the second experimental group was significantly higher than that in the control group, further indicating that 100 ng / mL IGF2 protein from leopard gill perch can significantly accelerate the in vitro proliferation of leopard gill perch myoblasts, and the in vitro proliferation rate reached 87%.

[0088] Test Example 3

[0089] Fluorescence quantitative analysis was performed using the myoblasts cultured for 96 hours in the control group and the experimental group in Test Example 1, including the following steps:

[0090] The myoblasts cultured for 96 h in the control group and the experimental group in Test Example 1 were used as cDNA templates, and gene-specific primers were designed using Primer Premier 5.0 based on the muscle stem cell genes Pax7 (GeneBank: XM_042506558.1 821-1847; SEQ ID NO.6), TCF-4 (GeneBank: XM_042509549.1 1905-2655; SEQ ID NO.7) and myogenic regulatory factor MyoD1 gene (GeneBank: XM_042491329.1 375-1181; SEQ ID NO.8) of Leopard gill perch published on NCBI. Among them, the β-actin gene was used as the internal reference gene (β-actin-F: SEQ ID NO.9: 5'ATCGCCGCACTGGTTGTTGAC-3'; β-actin-R: SEQ ID NO.10: 5'-GAGAAAAGCAGAAGTGGAATCA-3'); the upstream primer Pax7-F of the Pax7 gene was SEQ ID NO.11: 5'- CACAGGATGGCAGCAGCACATT-3'; the downstream primer Pax7-R was SEQ ID NO.12: 5'- GCCAGGTAGTCCACAGCAGTCT-3'; the upstream primer TCF-4-F of the TCF-4 gene was SEQ ID NO.13: 5'- TATGACAAGCGGGCACGGTGA-3'; the downstream primer TCF-4-R was SEQ ID NO.14: 5'-TTGCTGTCGGCGTCCTCCTT-3'; the upstream primer MyoD1 gene MyoD1-F was SEQ ID NO. NO.15: 5'-CTGCAAGCGGAAGACCACCAA-3'; the downstream primer MyoD1-R is SEQ ID NO.16: 5'-CCACGATGCTGGACAGACAGTC-3'. The BlastTaqTM2X qPCR MasterMix kit was used to detect the relative expression. Among them, the qRT-PCR system is: cDNA 2μL, BlastTaqTM2X qPCR MasterMix 10 μL, 10 ΜmF / R 0.5μl each, supplemented with DEPC water to 20μl; the qRT-PCR program is: pre-denaturation 95℃, 300 s; denaturation 95℃, 15 s, annealing 55℃, 20 s, extension 72℃, 30 s, wherein the denaturation to extension process is 35 cycles in total, and the formula for converting the Ct value into relative expression is: SPSS software was used for difference analysis, p < 0.05 indicated that the difference was significant, and then Graphpad Prism9 was used to draw the bar graph.

[0091] The results are as follows Fig.10 , Fig.11 and Fig.12 As shown, the relative expression of TCF-4 and MyoD1 genes in the second experimental group was significantly higher than that in the control group, while the relative expression of Pax7 gene in the second experimental group was significantly lower than that in the control group. This indicates that the addition of IGF2 protein from leopard gill perch can promote the further transformation of skeletal muscle cells of leopard gill perch into fibroblasts and myoblasts, and the proportion of myoblasts is higher.

[0092] It can be concluded that the leopard gill perch myoblasts obtained by the present invention are obtained by primary culture and subculture by using the skeletal muscle tissue of the leopard gill perch separated by trypsin digestion, and then the in vitro proliferation of the leopard gill perch myoblasts is successfully carried out by adding the IGF2 protein derived from the leopard gill perch to the culture medium, which can provide a large number of myoblasts derived from the leopard gill perch, and the cells can maintain a good growth state. In addition, after 96 hours of in vitro proliferation and culture of myoblasts cultured with the IGF2 protein derived from the leopard gill perch at a concentration of 100 ng / mL of the present invention, the number of myoblasts reaches 30,000, and after EdU detection, the in vitro proliferation rate of myoblasts can reach 87%, and the growth state of the cells is good, and the cell survival rate is high. Therefore, the method for in vitro proliferation of leopard gill perch myoblasts provided by the present invention can provide raw materials for exploring the functions of myoblasts themselves and the gene regulation mechanism. At the same time, the culture medium for in vitro proliferation of myoblasts and the in vitro proliferation method of myoblasts provided by the present invention are also applicable to other marine fish, such as Perciformes fish, or Perciformes fin fish such as humpback perch, brown-spotted grouper, red-spotted grouper, clouded grouper or jewel grouper, etc., and also provide in vitro verification experimental prerequisites for further exploring the influence of other marine fish myoblasts on the growth rate, and also lay the foundation for the breeding of marine fish.

[0093] The terms and expressions used herein are for descriptive purposes only and the present invention should not be limited to these terms and expressions. The use of these terms and expressions does not mean to exclude any equivalent features of the illustrations and descriptions (or parts thereof), and it should be recognized that various modifications that may exist should also be included in the scope of the claims. Other modifications, changes and substitutions may also exist. Accordingly, the claims should be deemed to cover all such equivalents.

[0094] Similarly, it should be pointed out that although the present invention has been described with reference to the current specific embodiments, ordinary technicians in this technical field should realize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions may be made without departing from the spirit of the present invention. Therefore, as long as the changes and modifications to the above embodiments are within the scope of the essential spirit of the present invention, they will fall within the scope of the claims of the present invention.

Claims

1. A method for in vitro proliferation of marine fish myoblasts, characterized in that: The in vitro proliferation method of marine fish myoblasts comprises the following steps: Step A: pre-treating the skeletal muscle tissue of the leopard gill perch to obtain pre-treated muscle tissue; Step B: performing primary culture on the pretreated muscle tissue to obtain primary cells; Step C: subculturing the primary cells to obtain subculture cells; Step D: culturing the passaged cells using a culture medium for in vitro proliferation of marine fish myoblasts to obtain in vitro proliferated myoblasts of leopard gill bass; The culture medium for in vitro proliferation of marine fish myoblasts uses a basal culture medium as a background culture medium, and the culture medium for in vitro proliferation of marine fish myoblasts contains an IGF2 protein with a final concentration of 90-110 ng / mL; the nucleotide sequence encoding the IGF2 protein is shown in SEQ ID NO.1; The basic culture medium includes L-15 culture medium, fetal bovine serum, NAEE, 1× double antibody and β-mercaptoethanol; The L-15 medium is 63-64% or 64-65% of the volume of the basal medium; The fetal bovine serum accounts for 14.5-15.0% or 15.0-15.5% of the volume of the basal culture medium; The NAEE is 0.9-1.0% or 1.0-1.1% of the volume of the basal medium; The 1× dual antibody includes: 100 IU / mL penicillin, 100 μg / mL streptomycin; The final concentration of the β-mercaptoethanol is 0.045-0.050 mM or 0.050-0.055 mM; The step A is specifically as follows: Using healthy 2-month-old, 3-month-old or 4-month-old leopard gill perch to disinfect the fish body surface, and taking out the skeletal muscle tissue of the second dorsal myometrium of the leopard gill perch under aseptic conditions to obtain the leopard gill perch skeletal muscle tissue; The skeletal muscle tissue of the leopard gill perch was immersed in a PBS buffer containing 5× double antibody and washed three times, each washing time was 5 min; the skeletal muscle tissue of the leopard gill perch was cut into pieces of 2 mm 2 2× double antibody was added to a tissue block of 100 μm in size, and the blood was washed away, and the skin membrane and residual blood of the skeletal muscle tissue of the leopard gill perch were removed to obtain the pretreated muscle tissue; The step B specifically comprises: inoculating the pretreated muscle tissue into a 6-well plate, performing primary culture on the pretreated muscle tissue using the basal culture medium, replacing the basal culture medium every 2 days, and culturing upright at 28° C. to obtain primary cells; The step C specifically comprises: when the plating rate of the primary cells reaches 70-80%, digesting the primary cells with trypsin to obtain primary myoblasts; subculturing the primary myoblasts with the basal culture medium to obtain subculture cells; The concentration of trypsin is 0.0625%; the temperature of the digestion reaction is 25-26°C, 26-27°C or 27-28°C; the time of the digestion reaction is 5.0-5.5 min, 5.5-6.0 min or 6.0-6.5 min; The step D specifically comprises: when the passage cells are in a logarithmic growth cycle, the passage cells are proliferated and cultured using the culture medium for in vitro proliferation of marine fish myoblasts to obtain the in vitro proliferated myoblasts of the leopard gill perch; the temperature of the proliferation culture is 28° C., and the time of the proliferation culture is 12-20 h, 20-40 h, 40-60 h, 60-80 h or 80-100 h.

2. The method for in vitro proliferation of marine fish myoblasts according to claim 1, characterized in that: The PBS buffer containing 5× double antibody includes: 500 IU / mL penicillin and 500 μg / mL streptomycin; The PBS buffer containing 2× double antibodies includes: 200 IU / mL penicillin and 200 μg / mL streptomycin.

3. The method for in vitro proliferation of marine fish myoblasts according to claim 1, characterized in that: The number of myoblasts proliferated in vitro by the leopard gill perch reaches 30,000; and the proliferation rate of myoblasts proliferated in vitro by the leopard gill perch is 87%.

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