A rspo3 recombinant protein, a medium for in vitro proliferation culture of prawn cells and a culture method thereof

Abstract

The application discloses a kind of RSP03 recombinant proteins, culture medium for prawn cell in vitro proliferation culture and its culture method, proliferation is prawn cell in vivo proliferation or prawn cell in vitro proliferation;The nucleotide corresponding to RSP03 recombinant protein is as shown in sequence SEQ ID NO.1, and the amino acid sequence of RSP03 recombinant protein is as shown in SEQ ID NO.2.The endogenous growth factor RSP03 gene of South American white prawn is cloned, and expression vector is constructed to express and purify endogenous LvRSPO3 recombinant protein, and the in-vitro culture activity of prawn cell can be extremely significantly enhanced by adding 5~30 ng / mL LvRSPO3 recombinant protein on base medium, can more effectively promote the proliferation and division of prawn cell in vitro, and can be cultured for at least 30 days, to overcome the problem that prawn cell is difficult to divide when in vitro culture, and provide a train of thought for establishing prawn continuous cell line.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein expression and in vitro cell culture technology, specifically relating to an RSPO3 recombinant protein, a culture medium for in vitro proliferation culture of shrimp cells, and a culture method thereof. Background Technology

[0002] Litopenaeus vannamei, also known as the Pacific white shrimp, belongs to the family Penaeidae in the order Decapoda of the class Crustacea in the phylum Arthropoda. Due to its rapid growth, strong adaptability, and excellent meat quality, it has become one of the dominant aquaculture species worldwide. However, with the promotion of intensive farming methods, disease problems in Litopenaeus vannamei have become increasingly prominent. Because diseases spread rapidly in aquatic environments and are difficult to control, once an outbreak occurs, it can easily lead to high infectivity and high mortality rates, causing significant economic losses to the aquaculture industry.

[0003] Growth factors play a central role in regulating cell growth, division, differentiation, and maintaining normal cell function, and are key components in establishing in vitro cell culture systems. However, in current technologies, researchers generally use commercially available mammalian-derived growth factors (such as human or mouse-derived ones) to induce continuous division of shrimp cells and establish continuous cell lines. Because marine crustaceans and mammals are far removed in species relations, their cellular nutrient uptake, metabolic pathways, and the structure, receptor binding specificity, and downstream signal transduction mechanisms of growth factors differ significantly. This makes it difficult for these exogenous growth factors to effectively bind to the corresponding receptors on the surface of shrimp cells and activate downstream signaling pathways, thus often failing to achieve the expected proliferation-promoting effects in practical applications.

[0004] RSPO3 is a secretory protein widely expressed in various organisms and an important growth factor. The Fu domain within its protein structure is a key domain enabling it to play a crucial role. Numerous studies have reported a close relationship between RSPO3 and the Wnt signaling pathway, a core signaling pathway regulating cell fate and directly promoting cell proliferation and survival. RSPO3 acts as a key enhancer of the Wnt signaling pathway by binding to specific receptors on the cell membrane (such as LGR family receptors), thereby inhibiting the activity of the intracellular E3 ubiquitin ligase RNF43 / ZNRF3. The function of RNF43 / ZNRF3 is to persistently mediate the ubiquitination and degradation of the Wnt pathway receptor—Frizzled protein—thus negatively regulating Wnt signaling. By antagonizing the activity of RNF43 / ZNRF3, RSPO3 significantly stabilizes the level of Frizzled receptors on the cell membrane, thereby enhancing the cell's responsiveness to classical Wnt ligands. Therefore, RSPO3 can participate in the regulation of biological processes such as cell proliferation, division, differentiation and migration by activating the Wnt signaling pathway.

[0005] In summary, despite numerous efforts by scholars in culturing and establishing shrimp cell lines, the problem of shrimp cell division arrest during in vitro culture remains unsolved, and effective shrimp cell lines have not yet been established. The in vitro culture of shrimp immune cells is still in its early stages. Furthermore, the growth factors previously used in shrimp cell culture were all derived from exogenous species and could not exert their effective effects. The in vitro culture technology of shrimp immune cells remains an unsolved problem; the issue of slow cell division during in vitro culture remains unresolved. Moreover, there are no reports on the use of endogenous growth factors from Litopenaeus vannamei or the application of endogenous RSPO3 recombinant protein in shrimp cell culture media to promote in vitro proliferation and division of shrimp cells.

[0006] Therefore, cloning and expressing the endogenous RSPO3 recombinant protein of Litopenaeus vannamei to optimize in vitro cell culture technology and promote cell proliferation in Litopenaeus vannamei is of great value and significance. This research can provide key technical support for the long-term preservation of Litopenaeus vannamei germplasm resources and lay the foundation for germplasm bank construction and breeding. Summary of the Invention

[0007] The purpose of this invention is to clone and express the endogenous RSPO3 recombinant protein of Litopenaeus vannamei, thereby optimizing the in vitro culture technology of shrimp cells by adding the endogenous RSPO3 recombinant protein of Litopenaeus vannamei to the shrimp cell culture medium, promoting the in vitro cell culture and proliferation of Litopenaeus vannamei, and further providing key technical support for the long-term preservation of Litopenaeus vannamei germplasm resources, laying the foundation for the construction of germplasm banks and breeding.

[0008] To solve the above-mentioned technical problems, this application provides the following technical solution:

[0009] The application of an RSPO3 recombinant protein in promoting shrimp cell proliferation, wherein the proliferation is in vivo or in vitro proliferation of shrimp cells; the nucleotide sequence corresponding to the RSPO3 recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the RSPO3 recombinant protein is shown in SEQ ID NO.2.

[0010] Furthermore, the method for in vivo proliferation of shrimp cells includes: injecting recombinant RSPO3 protein into the base of the fifth walking leg of shrimp to induce the proliferation of shrimp Oka organs, shrimp hematopoietic tissues and shrimp hemolymphocytes in vivo.

[0011] Furthermore, the injection dosage of RSPO3 recombinant protein is 2-3 μg, 3-4 μg, 4-5 μg or 5-6 μg RSPO3 recombinant protein per gram of shrimp.

[0012] Another aspect of this patent provides a culture medium for the in vitro proliferation and culture of shrimp cells, the culture medium comprising a basal culture medium and RSPO3 recombinant protein; the RSPO3 recombinant protein is derived from Litopenaeus vannamei, and the final concentration of RSPO3 recombinant protein in the culture medium is 5~8 ng / mL, 8~12 ng / mL, 12~25 ng / mL, 25~30 ng / mL, 30~50 ng / mL or 50~100 ng / mL.

[0013] Furthermore, the basal culture medium includes a shrimp serum amino acid mixture at a concentration of 3-5× to replace the amino acid components in the 1.5× L⁻¹⁵ medium, and also includes a shrimp serum sugar mixture at a concentration of 10-50× and 10-15% shrimp ovarian extract; the osmotic pressure of the basal culture medium is 620 ± 20 mOsm / kg, and the pH of the basal culture medium is adjusted to 7.2 ± 0.2.

[0014] Furthermore, the basal culture medium formula is as follows: L-alanine 337 mg / L, L-arginine 750 mg / L, L-asparagine 375 mg / L, L-cysteine ​​180 mg / L, L-glutamine 450 mg / L, L-histidine 375 mg / L, L-isoleucine 375 mg / L, L-leucine 187.5 mg / L, L-methionine 112.5 mg / L, L-phenylalanine 187.5 mg / L, L-serine 300 mg / L, L-threonine 450 mg / L, L-tryptophan 30 mg / L, L-tyrosine 450 mg / L, L-valine 150 mg / L, glycine 300 mg / L, glycine 159.6 ± 4.30 mg / L, L-lysine 112.5 mg / L, L-lysine 395 ± 10.44 mg / L, L-proline 1229.85 ± 3.57 mg / L, taurine 537 ± 5.14 mg / L, L-ornithine 13.95 ± 0.42 mg / L, L-hydroxyproline 36.4 ± 0.54 mg / L, L-aspartic acid 28.3 ± 0.79 mg / L, L-citrulline 17.35 ± 0.21 mg / L, L-cysteine ​​13.1 ± 1.32 mg / L, β-alanine 11 ± 0.15 mg / L, L-homocytrulline 6.7 ± 0.24 mg / L, L-glutamic acid 6.55 ± 0.16 mg / L, L-homoserine 4.6 ± 0.55 mg / L, 2-aminoisobutyric acid 4.5 ± 0.15 mg / L, γ-aminobutyric acid 0.6 ± 0.01 mg / L, L-arginine 1.5 ± 0.83 mg / L, D-asparagine 4.35 ± 0.45 mg / L, D-lysine 6.6 ± 0.50 mg / L, D-aspartic acid 6.9 ± 0.35 mg / L, D-alanine 15.5 ± 5.20 mg / L, D-glutamic acid 25.3 ± 0.05 mg / L, glucose 1180.71 ± 2.42 mg / L, fructose 64.22 ± 6.46 mg / L, rhamnose 6.24 ± 0.20 mg / L, fucose 1.91 ± 0.26 mg / L, arabinose 1.75 ± 0.21 mg / L, xylose 1.68 ± 0.04 mg / L, galactose 900 mg / L, inositol 2 mg / L, folic acid 1 mg / L, nicotinamide 1 mg / L, choline chloride 1 mg / L, D-calcium pantothenate 1 mg / L, pyridoxine hydrochloride 1 mg / L, thiamine phosphate 1 mg / L, riboflavin-5-phosphate sodium 0.1 mg / L, fetal bovine serum (FBS) 150 mL / L, phenol red 10 mg / L, streptomycin sulfate 1 mg / L.0 × 10⁵ IU / L, sodium pyruvate 550 mg / L, sodium penicillin 1.0 × 10⁵ IU / L, shrimp ovary extract (OE) 150 mL / L, epidermal growth factor (EGF) 20 μg / L, basic fibroblast growth factor 20 μg / L, vascular endothelial growth factor 10 μg / L, insulin-like growth factor-1 5 μg / L, and RSPO growth factor 5 μg / L.

[0015] Furthermore, the cells are shrimp Oka organs, shrimp hematopoietic cells, or shrimp hemolymphocytes.

[0016] Furthermore, the shrimp include whiteleg shrimp, Chinese shrimp, tiger prawn, Japanese shrimp, long-haired shrimp, knife-edged shrimp, short-grooved shrimp, wide-grooved shrimp, blue shrimp, or black shrimp.

[0017] Another aspect of this patent provides a method for in vitro proliferation culture of shrimp cells using the above-mentioned culture medium, comprising the following steps:

[0018] Step A: Express the RSPO3 recombinant plasmid in prokaryotes to obtain the RSPO3 recombinant protein;

[0019] Step B: Add the RSPO3 recombinant protein prepared in Step A to the basal culture medium to obtain the culture medium;

[0020] Step C: Use culture medium to proliferate and culture the cells in vitro.

[0021] Furthermore, in step C, the conditions for in vitro proliferation culture are: the temperature inside the incubator is set to 27~29℃ and the CO2 concentration is 2.5~3.5%.

[0022] Furthermore, the in vitro proliferation culture is a long-term culture of at least 30 days.

[0023] Another aspect of this patent provides the application of shrimp cells cultured using the above-mentioned method of in vitro proliferation culture in shrimp virus isolation, shrimp disease resistance gene research, shrimp virus detection or breeding for non-diagnostic and therapeutic purposes.

[0024] Compared with existing technologies, this patent has the following beneficial technical effects:

[0025] This patent provides a recombinant RSPO3 protein, a culture medium for in vitro proliferation of shrimp cells, and a culture method thereof. By cloning the endogenous growth factor RSPO3 gene of Litopenaeus vannamei and constructing an expression vector for the expression and purification of the endogenous LvRSPO3 recombinant protein, adding 5-30 ng / mL of LvRSPO3 recombinant protein to the basal culture medium can significantly enhance the in vitro culture viability of shrimp cells, more effectively promote the proliferation and division of shrimp cells in vitro, and enable long-term culture for at least 30 days. This provides a solution to overcome the current problem of difficulty in dividing shrimp cells in vitro and to establish continuous shrimp cell lines. Attached Figure Description

[0026] The above description of the present invention and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0027] Figure 1 This is an agarose gel electrophoresis image of the first mixed amplification product in this patent;

[0028] Figure 2 This is a verification diagram of the second amplification product in this patent (where A is the agarose gel electrophoresis detection of the second amplification product, B is the predicted structural domain of the LvRSPO3 gene of Litopenaeus vannamei, C is the CDS sequence of the LvRSPO3 gene of Litopenaeus vannamei, the signal peptide is marked with a red underline, and the Furin-like structural domain is marked with a blue underline).

[0029] Figure 3 The image shows the verification diagram of the pET-32a-LvRSPO3 recombinant plasmid in this patent (where A is the pET-32a-LvRSPO3 recombinant plasmid vector image, and B is the agarose gel electrophoresis image of the pET-32a-LvRSPO3 recombinant plasmid extracted).

[0030] Figure 4 The diagram shows the protein expression at different IPTG induction concentrations in this patent (wherein, the control is pET-32a empty vector cells, test 1 shows the protein expression induced by pET-32a-LvRSPO3 cells at 16℃, 0.1 mM IPTG, 120 rpm, and 20 h; test 2 shows the protein expression induced by pET-32a-LvRSPO3 cells at 24℃, 0.2 mM IPTG, 150 rpm, and 16 h; test 3 shows the protein expression induced by pET-32a-LvRSPO3 cells at 37℃, 0.5 mM IPTG, 200 rpm, and 5 h; 1 and 2 refer to the supernatant and precipitate of the bacterial solution after cell lysis, respectively).

[0031] Figure 5The images are SDS-PAGE electrophoresis images of samples from various stages of the nickel column affinity chromatography purification process of the LvRSPO3 recombinant protein in this patent (where M is the marker, 1 is the original protein solution before column loading, 2 is the flow-through buffer, 3-5 are 20 mM imidazole wash buffer, 6 is 50 mM imidazole wash buffer, 7 is 150 mM imidazole wash buffer, and 8-13 are 250 mM imidazole elution buffer).

[0032] Figure 6 The Western blot results of the LvRSPO3 recombinant protein in this patent are shown (where M is the marker, and 1 and 2 are both LvRSPO3 recombinant proteins).

[0033] Figure 7 Standard curve for BCA protein concentration determination of LvRSPO3 recombinant protein (where the horizontal axis is the sample absorbance value; the vertical axis is the protein concentration, in mg / mL).

[0034] Figure 8 The figure shows the effect of in vivo injection of LvRSPO3 recombinant protein on the regulation of Wnt pathway gene expression in Litopenaeus vannamei (where A1-A2 are the expression changes of Wnt pathway-related genes in Oka organs 24 h and 72 h after injection of LvRSPO3 recombinant protein, respectively; B1-B2 are the expression changes of Wnt pathway-related genes in hematopoietic tissue 24 h and 72 h after injection of LvRSPO3 recombinant protein, respectively; C1-C2 are the expression changes of Wnt pathway-related genes in hemolymphocytes 24 h and 72 h after injection of LvRSPO3 recombinant protein, respectively).

[0035] Figure 9 The figure shows the effect of in vivo injection of LvRSPO3 recombinant protein on the regulation of cell proliferation-related gene expression in Litopenaeus vannamei (where A1-A2 represent the expression changes of cell proliferation-related genes in Oka organs 24 h and 72 h after injection of LvRSPO3 recombinant protein; B1-B2 represent the expression changes of cell proliferation-related genes in hematopoietic tissue 24 h and 72 h after injection of LvRSPO3 recombinant protein; and C1-C2 represent the expression changes of cell proliferation-related genes in hemolymphocytes 24 h and 72 h after injection of LvRSPO3 recombinant protein).

[0036] Figure 10 The graph shows the screening results of the working concentration of LvRSPO3 recombinant protein in cell culture in this patent (where the horizontal axis is time in days, the vertical axis is cell viability, and the letters a~d indicate significant differences, with the same letter indicating no significant difference and different letters indicating significant differences).

[0037] Figure 11The following figures illustrate the effect of the LvRSPO3 recombinant protein on the activity of shrimp tissue cells during long-term in vitro culture in this patent (A, B, and C are the Calcein-AM cell activity fluorescence detection images of shrimp hemolymphocytes, hematopoietic tissues, and Oka organs after 30 days of in vitro culture; D, E, and F are the corresponding statistical fluorescence intensity values ​​of shrimp hemolymphocytes, hematopoietic tissues, and Oka organ cells, with *** indicating significant differences).

[0038] Figure 12 The figure shows the effect of the LvRSPO3 recombinant protein on the proliferation of shrimp hemolymphocytes during long-term in vitro culture in this patent (where A is the EdU proliferation fluorescence detection image of shrimp hemolymphocytes cultured in the control group and experimental group culture medium for 3 days, 8 days and 30 days, scale bar is 100 μm; B is the quantitative statistical graph of cell proliferation fluorescence intensity, the horizontal axis is the number of days, the vertical axis is the fluorescence intensity value of cell viability, ns is no significant difference, *** represents significant difference).

[0039] Figure 13 The figure shows the effect of the LvRSPO3 recombinant protein on the proliferation of shrimp hematopoietic cells during long-term in vitro culture in this patent (where A is the EdU proliferation fluorescence detection image of shrimp hematopoietic cells cultured in the control group and experimental group culture medium for 3 days, 8 days and 30 days, scale bar is 100 μm; B is the quantitative statistical graph of cell proliferation fluorescence intensity, the horizontal axis is the number of days, the vertical axis is the fluorescence intensity value of cell viability, ns is no significant difference, *** represents significant difference).

[0040] Figure 14 The figure shows the effect of LvRSPO3 recombinant protein on the proliferation of shrimp Oka cells during long-term in vitro culture in this patent (where A is the EdU proliferation fluorescence detection image of Oka cells cultured in the control group and experimental group culture medium for 3 days, 8 days and 30 days, scale bar is 100 μm; B is the quantitative statistical graph of cell proliferation fluorescence intensity, the horizontal axis is the number of days, the vertical axis is the fluorescence intensity value of cell activity, ns is no significant difference, *** represents significant difference). Detailed Implementation

[0041] The following detailed description of the features and advantages of the present invention is sufficient to enable any person skilled in the art to understand the technical content of the present invention and implement it accordingly. Furthermore, based on the specification, claims and drawings disclosed herein, those skilled in the art can easily understand the related objects and advantages of the present invention.

[0042] It should be noted that in this specification, similar reference numerals and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0043] To make the objectives, technical solutions, 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0044] (1) Source of sample materials

[0045] Whiteleg shrimp: purchased from Hainan Zhongzheng Aquatic Technology Co., Ltd., with a body length of 12±1.2 cm and a weight of 15±1.5 g.

[0046] (2) Sources of reagents and consumables

[0047] Ultra-low adsorption cell culture plates and cell culture flasks / plates were purchased from Corning Incorporated, Inc., USA; fetal bovine serum (FBS) was purchased from BI; ophthalmic scissors and forceps were purchased from Jiangxi Yuyuan Medical Instrument Co., Ltd.; sterile needle filters were purchased from Qingdao Beiqu Feimate Laboratory Supplies Store; trypsin, agarose, ampicillin sodium, chloramphenicol, IPTG solution, 50×TAE electrophoresis buffer, and 100× penicillin-streptomycin antibiotic stock solution were purchased from Beijing Solarbio Technology Co., Ltd.; enzyme-free cell digestion solution was purchased from Beijing Pulilai Gene Technology Co., Ltd.; potassium chloride, sodium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, anhydrous ethanol, and glucose were purchased from Sinopharm Chemical Reagent Co., Ltd.; γ-aminobutyric acid, L-lysine, L-alanine, and methanol were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; L-serine, L-valine, L-cysteine, L-leucine, glycine, taurine, L-proline, L-tyrosine, ornithine, and L-arginine were purchased. L-histidine, L-isoleucine, L-threonine, β-alanine, L-methionine, L-asparagine, L-aspartic acid, L-glutamic acid, L-phenylalanine, hydroxyproline, L-glutamine, L-cysteine, L-tryptophan, xylose, fucose, ribose, sucrose, lactose, fructose, arabinose, trehalose, D-galactose, inositol, folic acid, nicotinamide, choline chloride, D-calcium pantothenate, pyridoxine hydrochloride, thiamine phosphate, riboflavin-5-phosphate sodium, chloride Calcium, magnesium chloride, magnesium sulfate, sodium bicarbonate, phenol red, streptomycin sulfate, amphotericin B, sodium pyruvate, sodium penicillin, tryptone, yeast extract, and agar powder were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; epidermal growth factor (EGF), vascular endothelial growth factor, insulin-like growth factor-1, RSPO growth factor, and basic fibroblast growth factor (bFGF) were purchased from Thermo Fisher Scientific; restriction endonucleases and PrimeScript™ were also purchased. RT reagent Kit, PrimeSTAR® Max DNA Polymerase, and pMD™ 18-T vector were purchased from TaKaRa; 10 × DNA Loading Buffer, ChamQ Universal SYBR qPCR Master Mix, and DH5α chemicompetent cells were purchased from Nanjing Novizan Biotechnology Co., Ltd.; Trans2K PLUS DNA Marker, Trans2K PLUS II DNA Marker, and TransZol™ Up Plus RNA Kit were purchased from Beijing TransGen Biotech Co., Ltd.; EZ-10 Column DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.; and Rosetta chemicompetent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.Western blot and IP cell lysis buffer, PMSF solution, BCA protein concentration assay kit, and BeyoClick™ EdU-488 cell proliferation assay kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Skim milk powder, PAGE gel rapid preparation kit, PVDF membrane, transfer buffer, SDS-PAGE electrophoresis buffer, TBST buffer, protein loading buffer, Omni-ECL™ enhanced chemiluminescence assay kit, and pre-stained protein marker were purchased from Shanghai Yamei Biomedical Technology Co., Ltd. Transfer filter paper was purchased from Bio-Rad Biotech (USA). Mouse anti-His-Tag mAb was purchased from Wuhan Aibote Biotechnology Co., Ltd. HRP-conjugated Goat anti-Mouse IgG (H+L) was purchased from Wuhan Aibote Biotechnology Co., Ltd. Endotoxin-free plasmid small-volume extraction kit was purchased from Beijing Tiangen Biotech Co., Ltd. Uniclone One Step Seamless Cloning Kit was purchased from Beijing Jinsha Biotechnology Co., Ltd.

[0048] (3) Source of instruments and equipment

[0049] The Ni-NTA 6FF (His-Tag) pre-loaded gravity column was purchased from Sangon Biotech (Shanghai) Co., Ltd.; the ultrasonic homogenizer was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; the ultrafiltration centrifuge tubes were purchased from Beijing Solarbio Technology Co., Ltd.; the microsyringe was purchased from Shanghai Gaoge Industry & Trade Co., Ltd.; the vertical pressure steam sterilizer was purchased from Shanghai Shenan Medical Instrument Factory; the pipettes and refrigerated centrifuge were purchased from Eppendorf GmbH, Germany; the clean bench was purchased from Suzhou Antai Air Technology Co., Ltd.; the centrifuge tubes were purchased from Shanghai Kejin Biotechnology Co., Ltd.; the pH test strips were purchased from Shanghai Sanaisi Reagent Co., Ltd.; the ultrapure water system was purchased from Jinan Taipingma Equipment Co., Ltd.; the electric thermostatic drying oven, WH-2 micro vortex mixer, biochemical incubator, and benchtop thermostatic shaker were purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.; the inverted fluorescence microscope was purchased from Leica Instruments GmbH, Germany; the electronic balance was purchased from Ruderweireld GmbH, Poland; the refrigerator was purchased from Qingdao Haier Co., Ltd.; and the thermostatic water bath was purchased from Jiangsu Shuangjie Experimental Instrument Factory. The following equipment was purchased: high-speed centrifuge from Xiangyi Centrifuge Co., Ltd.; dual-head oxygen pump from Guangdong Minjiang Aquarium Industry Co., Ltd.; high-speed electric homogenizer from Jiangsu Zhongda Instrument Factory; freezing point osmometer from Shanghai Medical University Instrument Factory; HF-90 carbon dioxide incubator from Shanghai Lishen Scientific Instrument Co., Ltd.; JY-C electrophoresis apparatus and JY04S-3C gel imaging analysis system from Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.; ELISA reader from Thermo Fisher Scientific; WH-2 miniature vortex mixer from Shanghai Huxi Analytical Instrument Factory Co., Ltd.; vertical electrophoresis transfer system from Bio-Rad (USA); cryopreservation tubes from Biosharp; metal bath and miniature low-speed centrifuge from Beijing Tiangen Biochemical Technology Co., Ltd.; microscope slides and microscope coverslips from Shanghai Beyotime Biotechnology Co., Ltd.; disposable needle filters from Sartorius (Germany); and disposable syringes from Shaanxi Longkangxin Medical Device Co., Ltd.

[0050] (4) Preparation method of experimental reagents

[0051] 1) 1× PBS: PBS refers to phosphate buffer saline (PBS). The preparation method is as follows: Weigh 4.0 g of sodium chloride, 0.1 g of potassium chloride, 1.5 g of disodium hydrogen phosphate and 0.1 g of potassium dihydrogen phosphate into volumetric flasks, dissolve them completely with ddH2O and bring the volume to 500 mL. Sterilize at 121℃ under high temperature and high pressure for 25 min. Then, in a clean bench, aseptically dispense the cooled PBS into 100 mL reagent bottles and store them in a refrigerator at 4℃ for later use.

[0052] 2) 2.5× PBS: Weigh 10.0 g sodium chloride, 0.25 g potassium chloride, 3.75 g disodium hydrogen phosphate and 0.25 g potassium dihydrogen phosphate into volumetric flasks, dissolve them completely with ddH2O and bring the volume up to 500 mL. Sterilize at 121℃ for 20 min. In a clean bench, aseptically dispense the cooled PBS into 100 mL reagent bottles and store at 4℃ for later use.

[0053] 3) 60% glycerol: Add 60 mL of glycerol to a final volume of 100 mL with sterile ddH2O, filter through a 0.45 μm filter membrane, and store at 4°C.

[0054] 4) 1% agarose gel: Weigh 0.2 g of agarose powder into a 50 mL Erlenmeyer flask, add 20 mL of 1× TAE solution, heat in a microwave oven until completely dissolved, pour into a mold, and use after cooling and solidification.

[0055] 5) LB liquid medium: Weigh 1.0 g tryptone, 0.5 g yeast extract and 1.0 g sodium chloride and dissolve them in ddH2O. Adjust the pH and bring the volume to 100 mL. Autoclave at 121℃ and cool to room temperature. Add an appropriate amount of antibiotic according to the plasmid resistance before use.

[0056] 6) LB solid medium: Based on the preparation method in 5) above, add 1.5 g of agarose per 100 mL, autoclave at 121℃, cool to about 60℃, and then add an appropriate amount of antibiotics according to the plasmid resistance requirements. After mixing well, pour the medium into plates.

[0057] 7) 10 mM imidazole buffer: Weigh 3.12 g sodium dihydrogen phosphate, 5.36 g disodium hydrogen phosphate, 29.22 g sodium chloride and 0.68 g imidazole and dissolve them in ddH2O. Adjust the pH to 7.4 and bring the volume to 1000 mL. Filter with a 0.45 μm filter membrane and store at 4℃ for later use.

[0058] 8) 250 mM imidazole eluent: The difference from the preparation method of 7) above is that the amount of imidazole added is 17.02g.

[0059] 9) Anticoagulant: Add 15 g sodium chloride, 0.2 g potassium chloride, 0.2 g KH2PO4, 3 g disodium hydrogen phosphate, 9.8 g trisodium citrate, 2.48 g EDTA·2Na and 0.8 g reduced glutathione (or 0.6 g cysteine) to 1 L of pure water. After dissolving completely, filter to sterilize, then dispense and store in a refrigerator at 4℃.

[0060] Example

[0061] A recombinant RSPO3 protein, a culture medium for in vitro proliferation of shrimp cells, and a culture method thereof, comprising the following steps:

[0062] S1. Cloning of the RSPO3 gene in Litopenaeus vannamei, the specific steps are as follows:

[0063] 1. RNA Extraction from Litopenaeus vannamei: RNA was extracted from the hematopoietic tissue, Oka organ, and hemolymphocytes of Litopenaeus vannamei using the TransZol Up Plus RNA Kit. The specific steps are as follows: The shrimp were disinfected with alcohol and iodine. Then, using autoclaved forceps and scissors, the Oka organ and hematopoietic tissue were collected and thoroughly ground into powder using a mortar and pestle. The powder was then transferred to centrifuge tubes. The hemolymphocytes were extracted using a 2 mL syringe and centrifuged at 800 g for 10 min at 4°C. Subsequently, 1 mL of TransZol Up and 0.2 mL of RNA Extraction Agent were added to the centrifuge tubes, mixed, vortexed at room temperature for 5 min, and centrifuged at 10000 g at 4°C for 15 min. At this point, the sample separated into three layers. The upper aqueous phase was taken into a new centrifuge tube, and an equal volume of anhydrous ethanol was added. The mixture was then inverted and mixed. The solution was then added to the centrifuge column, centrifuged at 12000 g at room temperature for 30 s, and the eluent was discarded. 500 μL of CB9 was added, centrifuged at 12000 g at room temperature for 30 s, and the eluent was discarded. This process was repeated once. Then, 500 μL of WB9 was added, centrifuged at 12000 g at room temperature for 30 s, and the eluent was discarded. This process was repeated once more, centrifuged at 12000 g at room temperature for 2 min. Finally, the centrifuge column was placed in an RNase-free tube, 60 μL of RNase-free water was added, and the mixture was allowed to stand for 1 min. It was then centrifuged at 12000 g at room temperature for 1 minute. After elution for 1 minute, the RNA solution was collected to obtain hematopoietic tissue RNA, Oka organ RNA, and blood lymphocyte RNA, respectively.

[0064] Small amounts of RNA from hematopoietic tissue, Oka organ, and blood lymphocytes were taken and their concentration and purity were detected using a nucleic acid quantification instrument to ensure that RNA OD260 / 280 = 1.8~2.0.

[0065] 2. Reverse Transcription: The RNA extracted in step 1 was reverse transcribed to synthesize cDNA using the Takara PrimeScript™ RT reagent Kit (all reagents mentioned below are included in this kit). The reverse transcription system was as follows: 2 μL of 5× PrimeScript Buffer, 0.5 μL of PrimeScript RTEnzyme Mix I, 0.5 μL of Oligo dT Primer, 0.5 μL of Random 6 mers, and 1 μL of a mixed sample of hematopoietic tissue RNA, Oka organ RNA, and blood lymphocyte RNA (500 ng / μL) were added to a PCR tube sequentially. Finally, RNase-free ddH2O was added to a final volume of 10 μL. The reverse transcription program was 37°C for 15 min, followed by 85°C for 5 s, yielding a mixed cDNA product from hematopoietic tissue, Oka organ, and blood lymphocytes. This product was then stored at -20°C.

[0066] 3. Cloning of the RSPO3 gene in Litopenaeus vannamei:

[0067] (1) According to the NCBI database, the gene ID number of the LvRSPO3 gene of Litopenaeus vannamei (hereinafter referred to as LvRSPO3 gene) is 113811189, the nucleotide sequence length is 330 bp (SEQ ID NO.1), and the encoded amino acid sequence is 109aa (SEQ ID NO.2).

[0068] (2) Using the cDNA mixture obtained in step 2 above as a template, perform the first PCR amplification reaction. The primer sequences for PCR amplification are as follows:

[0069] LvRSPO3-F: 5'-ATGGTGCCACAGGAGGCCACGATGCT-3' (SEQ ID NO.3);

[0070] LvRSPO3-R: 5'-CTAACCACCGGACAACTCACTGGCGCA-3' (SEQ ID NO. 4).

[0071] The first PCR amplification system was as follows: 25 µL Taq polymerase, 50 ng cDNA template, 1 µL LvRSPO3-F (10 µM), 1 µL LvRSPO3-R (10 µM), and ddH2O added to 50 μL.

[0072] The first PCR amplification program was as follows: pre-denaturation at 98℃ for 3 min, denaturation at 98℃ for 10 s, annealing at 55℃ for 5 s, extension at 72℃ for 5 s, and final extension at 72℃ for 5 min, for a total of 35 cycles from denaturation to extension.

[0073] The first amplification product was obtained, which was a mixed amplification product of hematopoietic tissue, Oka organs, and blood lymphocytes. The first amplification product was then verified by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, the CDS sequence of the RSPO3 gene in Litopenaeus vannamei was obtained, with a band size of 330 bp.

[0074] (3) The first amplification product from step (2) above was excised and purified by gel extraction. The PCR product obtained after gel extraction was ligated with the pMD18-T vector and transformed into DH5α competent cells. The specific experimental procedures are as follows:

[0075] ① Gel Extraction and Purification: The first amplification product was purified using the EZ-10 Column DNA Gel Extraction Kit (all reagents mentioned below are included in the kit). Using a clean scalpel, the agarose gel at the 330 bp region of the RSPO3 gene in *Litopenaeus vannamei* was cut and placed in a 1.5 mL centrifuge tube for weighing. Binding Buffer II was added at a ratio of 100 µL per 100 mg of agarose, based on the weight of the gel block. The centrifuge tube was placed in a 55-65°C water bath for 5-10 min, stirring occasionally, until the gel block was completely dissolved. The dissolved gel solution was transferred to an EZ-10 Column, allowed to stand for 2 min, centrifuged at 8000 rpm for 1 min, and the liquid in the collection tube was discarded. 500 μL of Wash Solution was added, the tube was capped, and the mixture was inverted and mixed 3-5 times. The tube was centrifuged at 10000 rpm for 1 min, and the liquid in the collection tube was discarded (repeat once). The EZ-10... Return the column to the collection tube and centrifuge at 10,000 rpm for 30 seconds to remove residual WashSolution from the tube wall. Place the EZ-10 column in a clean 1.5 mL centrifuge tube, add 20-40 μL of Elution Buffer to the center of the adsorption membrane, incubate at 50°C for 2 minutes, and centrifuge at 10,000 rpm for 1 minute to elute the DNA. This is the gel-recovered DNA.

[0076] ② pMD18-T vector ligation: Add 1 μL of pMD18-T vector and 4 μL of gel-recovered DNA to a microcentrifuge tube, add 5 μL of Solution I, and react at 16℃ for 1 h to obtain the DNA to be transformed.

[0077] ③ DH5α competent cell transformation: Remove DH5α competent cells from -70℃ and quickly place them on ice to thaw; add the DNA to be transformed to 100 μL of DH5α competent cells, gently tap the tube wall to mix, and incubate on ice for 30 min; after heat shock in a 42℃ water bath for 45 s, quickly place them on ice for 2 min; add 900 μL of LB liquid medium (antibiotic-free) to a centrifuge tube, mix well, and incubate at 37℃ and 200 rpm for 45 min to obtain the first revived bacterial solution; take 50 μL of the first revived bacterial solution and spread it evenly on an LB solid medium plate containing Amp resistance and incubate upright at 37℃ for 10 min. After the first revived bacterial solution is completely absorbed, invert the plate and incubate overnight to obtain the first plate.

[0078] ④ After picking single clones from the first plate and shaking them, perform colony PCR (the second PCR reaction) using the universal primers M13-47 (SEQ ID NO.5: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3') and M13-48 (SEQ ID NO.6: 5'-AGCGGATAACAATTTCACACAGGA-3') for the pMD18-T vector. The reaction system and procedure for the second PCR reaction are the same as above. The second amplification product is obtained and verified by agarose gel electrophoresis. Figure 2 As shown, a bacterial culture containing the LvRSPO3 gene linked to the pMD18-T vector was obtained, with a band size of 330 bp. The successfully transformed bacterial culture was sent to Qingke Biotechnology Co., Ltd. for sequencing, indicating that the LvRSPO3 gene was successfully cloned from Litopenaeus vannamei. The complete CDS sequence of the LvRSPO3 gene is shown (nucleotide sequence 330 bp, completely identical to SEQ ID NO.1; amino acid sequence (Ast-pro) 109 aa, completely identical to SEQ ID NO.2).

[0079] The SMART online prediction website predicts that the N-terminus of the LvRSPO3 protein contains an endogenous signal peptide consisting of 22 amino acids and a Furin-like domain. This domain is a key domain that enables RSPO3 to perform multiple functions such as promoting cell proliferation and division.

[0080] The construction of S2 and LvRSPO3 gene expression vectors is as follows:

[0081] Using pET-32a as the base plasmid, and the bacterial culture successfully transformed in step S1 (3) above as a template, the CDS sequence of the LvRSPO3 gene was ligated to the prokaryotic expression vector pET-32a plasmid via homologous recombination. The ligation product of the obtained pET-32a-LvRSPO3 plasmid was then transformed into DH5α competent cells, and single clones were picked and cultured in a plate. Then, bacterial PCR and sequencing verification were performed.

[0082] pET-32a was used as the basic plasmid. Double digestion with enzymes was performed, followed by homologous recombination to obtain the pET-32a-LvRSPO3 plasmid. The enzyme digestion primers are shown below:

[0083] pET-LvRSPO3-F: 5'-CGCGGATCCTTCTCTCACCTCCCCGCCGACC-3' (SEQ ID NO.7);

[0084] pET-LvRSPO3-R: 5'-CCCAAGCTTACCACCGGACAACTCACTGGCG-3' (SEQ ID NO. 8);

[0085] After enzyme digestion and ligation, 5 µL of the ligation product was added to 100 µL of DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 45 s, and then incubated on ice for 2 min. 900 µL of antibiotic-free LB broth was added to a centrifuge tube, mixed thoroughly, and incubated at 37°C on a shaker at 200 rpm for 45 min. 50 µL of the bacterial culture was evenly spread onto an LB agar plate containing Amp resistance and incubated overnight at 37°C. Positive clones were picked and sequenced for verification. For the correctly sequenced bacterial culture, 200 µL was added to 15 mL of LB agar containing Amp resistance and incubated overnight at 37°C on a shaker at 200 rpm. The plasmid was extracted using the Tiangen endotoxin-free plasmid mini-extraction kit and stored at -80°C, yielding the pET-32a-LvRSPO3 plasmid. An equal volume of 60% glycerol was added to the remaining bacterial culture as seed culture and stored at -80°C.

[0086] The results are as follows Figure 3 As shown, A is a diagram of the pET-32a-LvRSPO3 recombinant plasmid vector, and B is an agarose gel electrophoresis image of the pET-32a-LvRSPO3 recombinant plasmid extracted from the plasmid.

[0087] The expression and purification of S3 and LvRSPO3 recombinant proteins are detailed below:

[0088] 1. Add 10 ng of the pET-32a-LvRSPO3 plasmid extracted in step S2 above to the thawed Rosetta competent cells and incubate on ice for 30 min. After heat shock in a 42°C water bath for 45 s, quickly incubate on ice for 2 min. Add 900 µL of antibiotic-free LB liquid medium to a centrifuge tube, mix well by pipetting, and incubate at 37°C and 200 rpm for 45 min. Spread 50 µL of the bacterial culture evenly on an LB solid medium plate containing Amp resistance and incubate overnight at 37°C upside down. On the second day, single clones were picked and placed into 1 mL of LB liquid medium containing Amp and CAP resistance. The medium was shaken at 37°C and 200 rpm for 5 h. The culture system was then expanded by inoculation at a ratio of 1:50 and cultured at 37°C and 200 rpm until OD600 = 0.6~0.8. The optimal protein induction conditions were determined by setting three conditions (first: 37°C, 0.5 mM IPTG, 200 rpm, 5 h; second: 24°C, 0.2 mM IPTG, 150 rpm, 16 h; third: 16°C, 0.1 mM IPTG, 120 rpm, 20 h).

[0089] 2. The bacterial culture under the third induction experimental conditions described above was collected by precipitation. The culture was centrifuged at 5000 g for 10 min at 4℃ to obtain a bacterial precipitate. 10 mL of Beyotime's Western blot and IP cell lysis buffer was added to the precipitate to resuspend it. Before use, PMSF solution with a final concentration of 1 mM was added to the lysis buffer. The culture was then sonicated for 20 min under ice bath conditions at a power of 200 W, with 3 s sonication followed by a 5 s interval, to obtain the lysed bacterial cells. The lysed liquid was centrifuged at 12000 rpm for 15 min at 4℃, and the supernatant and precipitate were collected separately.

[0090] The results are as follows Figure 4 As shown, the Control group consisted of pET-32a empty vector cells. The Test-1, Test-2, and Test-3 groups showed the protein expression of pET-32a-LvRSPO3 cells under three induction conditions (16 ℃, 0.1 mM IPTG, 120 rpm, 20 h, 5 h; 24 ℃, 0.2 mM IPTG, 150 rpm, 16 h; 37 ℃, 0.5 mM IPTG, 200 rpm, 5 h). The protein expression levels induced by Test-1 and Test-2 were similar and higher than those of Test-3. Finally, the conditions of Test-1 were used to expand the culture system.

[0091] 3. Similarly, expand the single-clone bacterial culture from step S3.1 to 250 mL, and use the ultrasonic disruption method described in step S3.2 to disrupt the culture. Collect the supernatant containing the soluble target protein after ultrasonic disruption, and use a Sangon Ni-NTA 6FF (His-Tag) pre-packed gravity column to purify the His-tagged recombinant protein.

[0092] The specific procedures are as follows: Take a 5 mL Ni-NTA pre-packed column and allow the storage buffer to flow out by gravity; equilibrate the column with two column volumes of 10 mM imidazole buffer, allowing the buffer to slowly drain from the resin; mix the protein supernatant with 10 mM imidazole buffer to prepare the sample solution, add the sample solution to the column, and incubate on ice for 30 min. Take 30 μL of the sample as the unloaded stock solution; collect the flow-through in a centrifuge tube. If there is excess sample, it can be loaded again, and then flow-through repeated once. Take 30 μL of the flow-through after loading the column; add two column volumes of 20 mM imidazole to the purification column for washing three times, and collect the effluent to obtain 20 mM imidazole wash solution; add two column volumes of 50 mM imidazole to the purification column for washing once, and collect the effluent to obtain 50 mM imidazole wash solution; add two column volumes of 150 mM imidazole to the purification column for washing once, and collect the effluent to obtain 150 mM imidazole wash solution. 1 mM imidazole washing buffer; then add 250 mM imidazole elution buffer (20 mM NaH2PO4·2H2O, 20 mM Na2HPO4·7H2O, 0.5 M NaCl, 250 mM imidazole, pH 7.4) to the purification column to elute the target protein, and collect the eluent; elute the column material with 5 column volumes of 250 mM imidazole elution buffer, then equilibrate the column material with 5 column volumes of 10 mM imidazole buffer, and finally wash the column material with 5 volumes of ddH2O, add 20% ethanol protective solution, and store at 4°C.

[0093] 4. Prepare duplicate polyacrylamide gels. Mix the target protein solutions collected at each stage with heat denaturation buffer, denature in a 99℃ metal bath, and then load onto the gel wells for electrophoresis. The electrophoresis conditions are 200 V for 35 min. After electrophoresis, the PAGE gel is stained with SDS-PAGE fast staining solution and destained with tap water. A clear band of LvRSPO3 recombinant protein at approximately 35 kDa was observed, indicating successful protein expression and good purity. Subsequent experiments can proceed. See the specific LvRSPO3 recombinant protein expression and purification diagram below. Figure 5 .

[0094] 5. Further Western blot analysis was used to identify the band around 35 kDa as the target protein band. The specific steps were as follows: another gel sample was transferred to a 0.22 μm PVDF membrane using a transfer apparatus. The membrane was blocked with 5% skim milk powder for 90 min, then incubated with primary antibody (Mouse anti His-Tag mAb) overnight at 4°C. The membrane was then washed with TBST washing buffer, and incubated with secondary antibody (HRP-conjugated Goat anti-Mouse IgG) at 4°C for 60 min. After washing the membrane again, chromogenic solution was added to the membrane, and the membrane was developed and photographed using an optical imaging system to complete the identification of the target protein.

[0095] The results are as follows Figure 6 As shown, there is a distinct single band around 35 kDa, which is the LvRSPO3 recombinant protein.

[0096] 6. The purification effect of the eluted target protein was assessed by SDS-PAGE with polyacrylamide gel electrophoresis. The protein was then concentrated using Solarbio ultrafiltration centrifuge tubes (15 mL / 3 kDa) and the solution was replaced with PBS to remove salt. The concentration of LvRSPO3 recombinant protein was determined by the BCA method.

[0097] The specific steps are as follows: Dilute the protein standard solution to a final concentration of 0.5 mg / mL. Add 0, 1, 2, 4, 8, 12, 16, and 20 µL of the standard solution to the wells of a 96-well plate. Add standard diluent to bring the total to 20 µL. Separately, add 20 µL of protein sample solution to each well. Then, add 200 µL of BCA working solution to each well. Incubate at 37 ℃ for 25 min. Measure the absorbance at 562 nm using a Varioskan LUX multi-functional microplate reader. Plot a standard curve based on the concentration and absorbance of the standard protein. The fitting equation for the standard curve is y = ax + b, where y is the sample concentration and x is the sample absorbance. The measured concentration of LvRSPO3 recombinant protein is approximately 13.15 mg / mL. After aliquoting, flash-freeze in liquid nitrogen and store at -80 ℃. See the detailed BCA protein concentration determination standard curve for reference. Figure 7 .

[0098] The specific steps of the in vivo experiment on the expression of S4 and LvRSPO3 recombinant proteins in Litopenaeus vannamei-related pathways and cell proliferation genes are as follows:

[0099] 1. A total of three control groups and three experimental groups were set up. The control groups used 2.5×PBS buffer, and the experimental groups used LvRSPO3 recombinant protein. The 2.5×PBS buffer and LvRSPO3 recombinant protein were injected into the body from the base of the fifth walking leg of Litopenaeus vannamei using a microsyringe. The injection doses were 2 μg / g, 4 μg / g and 6 μg / g of shrimp body weight, respectively.

[0100] 2. At 24 h and 72 h post-injection, samples were collected from three tissues and cells: Oka organ, hematopoietic tissue, and hemolymphocytes of Litopenaeus vannamei. The sampling process employed a parallel design, with three replicates per group. RNA extraction and reverse transcription were performed, following the same experimental procedures as steps 1 and 2 in S1 above. Real-time quantitative PCR (qPCR) analysis was then conducted, using the LvEF1α gene as an internal control. The effects of overexpression of the LvRSPO3 recombinant protein on the Wnt pathway and the expression of cell proliferation-related genes were analyzed using qPCR. Specifically, in the analysis of Wnt signaling pathway-related gene expression, the RSPO3 receptor genes LGR4 / 5, the indirect-acting receptor (i.e., the Wnt receptor) genes Frizzled and LRP6 were selected as target genes for qPCR validation. In the analysis of cell proliferation gene expression, c-jun, CDK4, Survivin, MYC, and PCNA were selected as target genes for qPCR validation. Three biological replicates and three technical replicates were set up for each group. The qPCR reaction system (20 μL) contained 20 ng cDNA template, 0.4 μM forward and reverse primers, and 10 µL SYBR Green buffer. The amplification program was set as a two-step method: 95℃ pre-denaturation for 3 min, followed by 40 cycles, each cycle consisting of 95℃ denaturation for 15 s and 60℃ annealing extension for 1 min. Finally, the specificity of the amplified products was verified by melting curve analysis.

[0101] The primer information required for the qPCR reaction is shown below:

[0102] LvEF1α-qF: 5'-TGGCTACTCACCTGTGCTTG-3' (SEQ ID NO.9);

[0103] LvEF1α-qR: 5'-CCAGCTCCTTACCAGTACGC-3' (SEQ ID NO. 10);

[0104] LvLGR4-qF: 5'-CCATGCTGTTGCCACTTCCTGAT-3' (SEQ ID NO. 11);

[0105] LvLGR4-qR:5'-ATGTCCTGAGAGAGTCCGTCCTTC-3'(SEQ ID NO.12);

[0106] LvLGR5-qF:5'-TGTCCGTGGCGAACTCAAGTATGTG-3'(SEQ ID NO.13);

[0107] LvLGR5-qR:5'-AGAATGACCTGGCTGATGGAGTAGT-3'(SEQ ID NO.14);

[0108] LvC-jun-qF:5'-TGGAGGCAACCATGTACGAG-3'(SEQ ID NO.15);

[0109] LvC-jun-qR:5'-TCCAACTCGGGAGAGGCTAA-3'(SEQ ID NO.16);

[0110] LvFrizzled-qF:5'-CCCGAGAGGATGGATTGCTC-3'(SEQ ID NO.17);

[0111] LvFrizzled-qR:5'-GACTCGTTAGGATAGCGCCC-3'(SEQ ID NO.18);

[0112] LvLRP6-qF:5'-GTGCTTGTCCAGAGGGGAAA-3'(SEQ ID NO.19);

[0113] LvLRP6-qR:5'-GACTGCCCATCACACATCCA-3'(SEQ ID NO.20);

[0114] LvCDK4-qF:5'-CTCGAGACGTGTCCCTGATG-3'(SEQ ID NO.21);

[0115] LvCDK4-qR:5'-GGAAATAGGGGTGTCGGAGC-3'(SEQ ID NO.22);

[0116] LvMYC-qF:5'-ACTACCACTCCGAGGTGATGA-3'(SEQ ID NO.23);

[0117] LvMYC-qR:5'-TGGAGCTGCTTGCAGTAGAC-3'(SEQ ID NO.24);

[0118] LvPCNA-qF: 5'-TGCAAACATCAAGCTGGCAC-3' (SEQ ID NO. 25);

[0119] LvPCNA-qR: 5'-CAAGCAAAGGTGAGCGTGAC-3' (SEQ ID NO. 26);

[0120] LvSurvivin-qF: 5'-CGAAGATGACCCCTGGGAGG-3' (SEQ ID NO. 27);

[0121] LvSurvivin-qR: 5'-TCTCTCGTGTTTGCAGCTTGT-3' (SEQ ID NO. 28).

[0122] The results are as follows Figures 8-9 As shown, for the Oka organ, after injecting 2 μg / g, 4 μg / g, and 6 μg / g of shrimp body weight of recombinant LvRSPO3 protein into Litopenaeus vannamei, qPCR results were analyzed at 24 h and 72 h after injection. The results revealed that the optimal injection dose of 6 μg / g of shrimp body weight of recombinant LvRSPO3 protein was the most effective. At a dose of 6 μg / g of shrimp body weight of recombinant LvRSPO3 protein, the Wnt signaling pathway-related receptor genes LGR5, Frizzled, and LRP6 were significantly upregulated at 24 h; LGR4 / 5, Frizzled, and LRP6 were all significantly upregulated at 72 h. At a dose of 4 μg / g of shrimp body weight of recombinant LvRSPO3 protein, only LGR5 and Frizzled were significantly upregulated at both 24 h and 72 h. At a dose of 2 μg / g of shrimp body weight of recombinant LvRSPO3 protein, only Frizzled expression was upregulated. Regarding cell proliferation-related genes, injection doses of 4 μg / g and 6 μg / g significantly upregulated the expression of c-jun, MYC, and PCNA at 24 h and 72 h, respectively; while a dose of 2 μg / g only induced upregulation of MYC gene expression.

[0123] For hematopoietic tissue, after injecting 2 μg / g, 4 μg / g, and 6 μg / g of shrimp body weight of recombinant LvRSPO3 protein into Litopenaeus vannamei, qPCR results at 24 h and 72 h showed that the optimal injection dose of 2 μg / g of shrimp body weight was the most effective. Regarding the regulation of receptor genes related to the Wnt signaling pathway, at an injection dose of 6 μg / g of shrimp body weight of recombinant LvRSPO3 protein: at 24 h, LGR5, Frizzled, and LRP6 were all significantly upregulated; at 72 h, LGR4 / 5 and Frizzled were downregulated. At an injection dose of 4 μg / g of shrimp body weight of recombinant LvRSPO3 protein: at 24 h, only LRP6 was upregulated; at 72 h, only LGR5 was upregulated. When LvRSPO3 recombinant protein was injected at a dose of 2 μg / g shrimp body weight: only LRP6 was upregulated at 24 h; at 72 h, LGR5, Frizzled, and LRP6 were all upregulated. Regarding the regulation of cell proliferation-related genes, when LvRSPO3 recombinant protein was injected at a dose of 2 μg / g shrimp body weight, all five cell proliferation-related genes detected—c-jun, CDK4, Survivin, MYC, and PCNA—were upregulated at both 24 h and 72 h. When the injection dose was increased to 4 μg / g shrimp body weight: four cell proliferation-related genes—c-jun, Survivin, MYC, and PCNA—were upregulated at 24 h; at 72 h, three more cell proliferation-related genes—c-jun, Survivin, and PCNA—were upregulated. When the injection dose was increased to 6 μg / g shrimp body weight: at 24 h, upregulated expression of three cell proliferation-related genes, namely Survivin, MYC, and PCNA, was detected; at 72 h, only upregulated expression of the c-jun gene was detected, while CDK4 and MYC genes were downregulated.

[0124] For hemolymphocytes, after injecting 2 μg / g, 4 μg / g, and 6 μg / g of shrimp body weight of recombinant LvRSPO3 protein into Litopenaeus vannamei, qPCR results at 24 h and 72 h showed that the optimal injection dose of 6 μg / g of shrimp body weight was the most effective. Regarding the regulation of Wnt signaling pathway-related receptor genes, at injection doses of 4 μg / g and 6 μg / g of shrimp body weight of recombinant LvRSPO3 protein, upregulation of LGR4 / 5 was detected at both 24 h and 72 h for both doses; however, at an injection dose of 2 μg / g of shrimp body weight of recombinant LvRSPO3 protein, only upregulation of LGR5 was detected at both 24 h and 72 h. Regarding the regulation of genes related to cell proliferation, when LvRSPO3 recombinant protein was injected at a dose of 6 μg / g shrimp body weight, Survivin and PCNA were significantly upregulated at 24 h; at 72 h, c-jun, CDK4, Survivin, and PCNA were all upregulated. When LvRSPO3 recombinant protein was injected at doses of 2 μg / g and 4 μg / g shrimp body weight, only Survivin and PCNA were significantly upregulated at both 24 h and 72 h.

[0125] In summary, in the Oka organ and hemolymphocytes of Litopenaeus vannamei, the LvRSPO3 recombinant protein showed the most significant effect in promoting cell proliferation at an injection dose of 6 μg / g shrimp body weight; while in hematopoietic tissue, its optimal proliferation-promoting dose was 2 μg / g shrimp body weight. These results indicate that overexpression of the LvRSPO3 recombinant protein in vivo can effectively enhance the expression levels of genes related to cell proliferation regulation in different shrimp tissues, thereby promoting cell proliferation behavior in vivo. The dose-response of the LvRSPO3 recombinant protein and the involvement of related proliferation genes differ among different tissues and cells, indicating that the protein has tissue-specific effects on the proliferation of Litopenaeus vannamei cells. This provides in vivo experimental data to support the subsequent demonstration of differential proliferation effects of the LvRSPO3 recombinant protein on different tissues and cells in shrimp cell culture systems.

[0126] The specific steps of the experiment evaluating the in vitro cell proliferation-promoting ability of S5 and LvRSPO3 recombinant proteins in Litopenaeus vannamei are as follows:

[0127] 1. As shown in Table 1, the basic culture medium is prepared. Before use, the osmotic pressure of the basic culture medium needs to be adjusted to 620 ± 20 mOsm∙kg-1 and the pH value needs to be adjusted to 7.2 ± 0.2. The superscript b indicates that the amino acids and sugars are derived from shrimp serum.

[0128] Table 1. Basic Culture Medium Formulation

[0129] ;

[0130] 2. Sampling and culture of Oka organs, hematopoietic tissues and hemolymphocytes from Litopenaeus vannamei.

[0131] (1) For Oka organs and hematopoietic tissues, the shrimp were first disinfected with alcohol and povidone-iodine. Then, the disinfected shrimp were wrapped with alcohol swabs and placed in a clean bench for material collection. The shrimp shell was carefully cut open and removed using high-temperature and high-pressure sterilized scissors to expose the Oka and hematopoietic tissues. The connection between the Oka organs and hematopoietic tissues and other tissues was cut with ophthalmic scissors. The shrimp Oka organs and hematopoietic tissues were removed with sterile forceps and rinsed with 2.5× PBS. Then, the corresponding tissues were removed from the centrifuge tubes with forceps, washed three times in culture medium, and then placed in cell culture plates. The plates were inverted and dried in a 28℃, 3% CO2 incubator for 2-4 h. Shrimp cell culture medium was then added and the plates were cultured in an incubator.

[0132] (2) For hemolymphocytes, take a 2 mL disposable syringe, first draw 0.5 mL of anticoagulant, then quickly insert the syringe into the blood sinus from above the first foot of the shrimp after disinfection with alcohol and iodine. After locating the location, draw hemolymphocytes at a uniform speed. Immediately after drawing, shake the syringe up and down to ensure that the anticoagulant and hemolymph are mixed evenly to prevent blood clotting. After thorough mixing, remove the needle of the syringe and transfer the shrimp hemolymphocytes to a 15 mL centrifuge tube to obtain a mixture of shrimp hemolymph and anticoagulant. After mixing, centrifuge at 4℃ and 1000 ×g for 10 min. Discard the supernatant, wash once with 2.5×PBS, discard the supernatant, add shrimp cell culture medium, resuspend the cells to prepare a shrimp hemolymphocyte suspension, inoculate it into a cell culture plate, and incubate at 28℃ and 3% CO2.

[0133] (3) Optimization of the optimal working concentration of LvRSPO3 recombinant protein in shrimp cell culture

[0134] The optimal working concentration of LvRSPO3 recombinant protein in the culture medium was determined by in vitro culture of shrimp blood lymphocytes: The LvRSPO3 recombinant protein purified in step S3 above was added to the basal culture medium in step S5 (1) above at working concentrations of 0 μg / L (blank control group), 5 μg / L, 10 μg / L, 30 μg / L, 70 μg / L and 100 μg / L respectively, to prepare six LvRSPO3 recombinant protein working concentration test media.

[0135] Blood lymphocytes were cultured using different working concentration test media of LvRSPO3 recombinant protein as described above. The culture conditions were 28°C, 3% CO2, and the culture period was 7 days. Half of the culture medium was replaced every day during the culture process.

[0136] Cell viability was monitored using a CCK-8 cell viability assay. Cells were seeded in 96-well cell culture plates and cultured for 0, 1, 3, 5, and 7 days. 100 μL of fresh culture medium containing 10 μL of CCK-8 stock solution was added to each plate, and the cells were incubated at 28°C and 3% CO2 for 4 hours in the dark. After incubation, the absorbance (OD) values ​​at 450 nm and 650 nm were measured using a microplate reader. The values ​​were plotted with culture time as the x-axis and logOD = logo. [(实验组OD450 -对照组OD450)-(实验组OD650 -对照组OD650)] Plot the cell growth curve with the Y-axis value as the vertical axis.

[0137] The results are as follows Figure 10 As shown, compared with the blank control group, LvRSPO3 recombinant protein significantly promoted hemolymphocyte proliferation and improved cell viability within the concentration range of 5–30 ng / mL. However, cell culture viability was significantly inhibited when the protein concentration reached 70 ng / mL or higher. These results indicate that LvRSPO3 recombinant protein exhibits significant biological activity at low concentrations, and its effect is concentration-dependent; excessively high concentrations may have cytotoxic effects. 10 ng / mL was determined to be the optimal working concentration of this LvRSPO3 recombinant protein in the in vitro culture system.

[0138] (4) Long-term in vitro culture of shrimp hemolymphocytes, hematopoietic tissue cells and Oka organ cells.

[0139] The optimal concentration of the LvRSPO3 recombinant protein selected in step S5 (3) above is 10 ng / mL, which is added to the basal culture medium to obtain Litopenaeus vannamei cell culture medium.

[0140] The above-sampled hemolymphocytes, hematopoietic tissue cells and Oka organ cells were cultured in vitro for 30 days using Litopenaeus vannamei cell culture medium. At the same time, Litopenaeus vannamei cell culture medium without LvRSPO3 recombinant protein was set up as a control group. The culture conditions were the same as in step S5 (3) above.

[0141] ① Cell viability was assessed using the Calcein-AM cell viability assay. Calcein-AM stock solution was diluted in serum-free medium to a final concentration of 1 μM. 100 μL of the diluted Calcein-AM working solution was added to each well of a 96-well plate containing shrimp cells. The plate was then incubated at 28 °C in the dark for 45 min. After incubation, the cells were gently washed twice with PBS. Shrimp cell culture medium was added, and the cells were observed and photographed under a fluorescence microscope. Green fluorescence indicated the number of viable cells, thus assessing cell viability.

[0142] The results are as follows Figure 11 As shown, for hemolymphocytes and hematopoietic tissue cells, there was no significant difference in cell viability between the experimental group containing 10 ng / mL LvRSPO3 recombinant protein and the control group without LvRSPO3 recombinant protein. However, for Oka organ cells, significant cell viability was still detectable in the experimental group on day 30 of cell culture, while no cell viability was detected in the control group. Statistical analysis showed that the cell viability of the experimental group was 9.64 times that of the control group, indicating a significant difference between the two groups. These experimental results demonstrate that adding LvRSPO3 recombinant protein to the shrimp cell culture medium can effectively prolong the duration of activity of shrimp Oka organ cells during in vitro culture, thus proving that this LvRSPO3 recombinant protein can promote the long-term proliferation of shrimp Oka organ cells in vitro. This provides experimental evidence for further evaluating the proliferative effect of the optimized shrimp cell culture medium containing this LvRSPO3 recombinant protein in the long-term in vitro culture of shrimp Oka organ cells.

[0143] ② Cell proliferation energy was detected using the EdU cell proliferation assay. Culture medium containing 1x EdU (10 μM) working solution was added to the cells to be tested, and the cells were incubated at 28°C with 3% CO2 for 24 h. After incubation, the culture medium was aspirated, and the cells were washed three times with washing buffer (PBS containing 3% bovine serum albumin) for 3–5 min each time. Cell fixation buffer (PBS containing 3.7% paraformaldehyde) was then added, and the cells were fixed at room temperature for 15–30 min. Next, the cells were treated with cell permeation buffer (PBS containing 0.3% Triton X-100) and incubated at room temperature for 10–15 min. Then, the azide-labeled fluorescent dye AlexaFluor 488 azide was added to the cells, and the cells were incubated at room temperature in the dark for 30 min, followed by washing with PBS three times. To accurately calculate the percentage of proliferating cells in the test cells, cell nuclear staining was performed. Cells were incubated for 15 min in the dark using 1x Hoechst 33342 working solution, followed by washing with washing buffer three times. Finally, the processed cells were observed and photographed under an inverted fluorescence microscope.

[0144] The results are as follows Figures 12-14 As shown, in hemolymphocytes, the LvRSPO3 recombinant protein only exhibited a cell proliferation-promoting effect in the early stage of culture (day 3), with the proliferation rate of the experimental group being 1.21 times that of the control group. In hematopoietic tissue cells, there was no significant difference in cell proliferation capacity between the experimental and control groups throughout the entire culture period. In the long-term culture of Oka organocytes, the cell proliferation and division capacity of the experimental group were significantly higher than those of the control group. The proliferation rate of the experimental group was 2.42 times that of the control group on day 3 and 1.53 times that of the control group on day 8. Furthermore, the Oka cells in the experimental group remained active until day 30, while cell activity in the control group was undetectable. These results indicate that the optimized cell culture medium containing the LvRSPO3 recombinant protein can significantly improve the activity and proliferation capacity of Oka organocytes in vitro, while having no significant effect on the long-term culture of hemolymphocytes and hematopoietic tissue cells. Therefore, the shrimp cell culture medium containing LvRSPO3 recombinant protein provided by this invention can effectively promote the in vitro proliferation and division of Oka cells of Litopenaeus vannamei. This result provides key experimental support for the optimization of shrimp cell in vitro culture system, provides ideas for overcoming the current problem of shrimp cells being difficult to divide in vitro and establishing continuous shrimp cell lines, and reveals the biological function of RSPO3 gene in Litopenaeus vannamei.

[0145] Therefore, it can be concluded that this patent provides a recombinant RSPO3 protein, a culture medium for in vitro proliferation of shrimp cells, and a culture method thereof. By cloning the endogenous growth factor RSPO3 gene of Litopenaeus vannamei and constructing an expression vector for the expression and purification of the endogenous LvRSPO3 recombinant protein, adding 5-30 ng / mL of LvRSPO3 recombinant protein to the basal culture medium can significantly enhance the in vitro culture viability of shrimp cells, more effectively promote the proliferation and division of shrimp cells in vitro, and enable long-term culture for at least 30 days. This provides a solution to overcome the current problem of shrimp cells being difficult to divide in vitro and to establish continuous shrimp cell lines.

[0146] The terminology and expressions used herein are for descriptive purposes only, and the invention should not be limited to these terms and expressions. The use of these terms and expressions does not imply the exclusion of any illustrative and descriptive equivalents (or parts thereof), and it should be recognized that various modifications that may exist should also be included within the scope of the claims. Other modifications, variations, and substitutions may also exist. Accordingly, the claims should be considered to cover all such equivalents.

[0147] Similarly, it should be noted that although the present invention has been described with reference to the specific embodiments described above, those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions can be made without departing from the spirit of the present invention. Therefore, any changes or modifications to the above embodiments within the scope of the essential spirit of the present invention will fall within the scope of the claims of the present invention.

Claims

1. The application of a recombinant RSPO3 protein in promoting cell proliferation in Litopenaeus vannamei, characterized in that, The proliferation is either in vivo or in vitro proliferation of shrimp cells; the RSPO3 recombinant protein is derived from Litopenaeus vannamei, and the cells are Oka organs; The nucleotide sequence corresponding to the RSPO3 recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the RSPO3 recombinant protein is shown in SEQ ID NO.2; The concentration of the RSPO3 recombinant protein in the in vitro proliferation of shrimp cells was 10 ng / mL; The method for in vivo proliferation of shrimp cells is as follows: the RSPO3 recombinant protein is injected into the base of the fifth walking leg of the shrimp, and the injection dose is 4~5 μg of the RSPO3 recombinant protein per gram of shrimp.

2. The application of a recombinant RSPO3 protein in promoting cell proliferation in Litopenaeus vannamei, characterized in that, The proliferation is either in vivo or in vitro proliferation of shrimp cells; the RSPO3 recombinant protein is derived from Litopenaeus vannamei, and the cells are Oka organs; The nucleotide sequence corresponding to the RSPO3 recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the RSPO3 recombinant protein is shown in SEQ ID NO.2; The concentration of the RSPO3 recombinant protein in the in vitro proliferation of shrimp cells was 10 ng / mL; The method for in vivo proliferation of shrimp cells is as follows: the RSPO3 recombinant protein is injected into the base of the fifth walking leg of the shrimp, and the injection dose is 5-6 μg of the RSPO3 recombinant protein per gram of shrimp.

3. A culture medium for the in vitro proliferation and culture of Litopenaeus vannamei cells, characterized in that, The cells are Oka organs; the culture medium includes basal culture medium and RSPO3 recombinant protein. The RSPO3 recombinant protein is derived from Litopenaeus vannamei, and the final concentration of the RSPO3 recombinant protein in the culture medium is 10 ng / mL; The nucleotide sequence corresponding to the RSPO3 recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the RSPO3 recombinant protein is shown in SEQ ID NO.2; The basal culture medium comprises a shrimp serum amino acid mixture at a concentration of 3-5× to replace the amino acid components in the 1.5× L⁻¹⁵ medium, and simultaneously adds a shrimp serum carbohydrate mixture at a concentration of 10-50× and 10-15% shrimp ovarian extract; the osmotic pressure of the basal culture medium is 620±20 mOsm / kg, and the pH value of the basal culture medium is adjusted to 7.2±0.2; The basal culture medium formula is as follows: L-alanine 337 mg / L, L-arginine 750 mg / L, L-asparagine 375 mg / L, L-cysteine ​​180 mg / L, L-glutamine 450 mg / L, L-histidine 375 mg / L, L-isoleucine 375 mg / L, L-leucine 187.5 mg / L, L-methionine 112.5 mg / L, L-phenylalanine 187.5 mg / L, L-serine 300 mg / L, L-threonine 450 mg / L, L-tryptophan 30 mg / L, L-tyrosine 450 mg / L, L-valine 150 mg / L, glycine 300 mg / L, glycine b 159.6±4.30 mg / L, L-lysine 112.5 mg / L, L-lysine b 395±10.44 mg / L, L-proline b 1229.85±3.57 mg / L, taurine b 537 ± 5.14 mg / L, L-ornithine b 13.95±0.42 mg / L, L-hydroxyproline b 36.4±0.54 mg / L, L-aspartic acid b 28.3±0.79 mg / L, L-citrulline b 17.35±0.21 mg / L, L-cysteine b 13.1±1.32 mg / L, β-alanine b 11±0.15 mg / L, L-homocycubin b 6.7±0.24 mg / L, L-glutamic acid b 6.55±0.16 mg / L, L-homoserine b 4.6±0.55 mg / L, 2-aminoisobutyric acid 4.5±0.15 mg / L, γ-aminobutyric acid b 0.6±0.01 mg / L, L-homogeneous acid b 1.5±0.83 mg / L, D-asparagine b 4.35±0.45 mg / L, D-lysine b 6.6 ± 0.50 mg / L, D-aspartic acid b 6.9 ± 0.35 mg / L, D-alanine b 15.5 ± 5.20 mg / L, D-glutamic acid b 25.3 ± 0.05 mg / L, glucose b 1180.71 ± 2.42 mg / L, fructose b 64.22 ± 6.46 mg / L, rhamnose b 6.24 ± 0.20 mg / L, fucose b 1.91 ± 0.26 mg / L, arabinose b 1.75 ± 0.21 mg / L, xylose b 1.68 ± 0.04 mg / L, galactose 900 mg / L, inositol 2 mg / L, folic acid 1 mg / L, nicotinamide 1 mg / L, choline chloride 1 mg / L, D-calcium pantothenate 1 mg / L, pyridoxine hydrochloride 1 mg / L, thiamine phosphate 1 mg / L, riboflavin-5-phosphate sodium 0.1 mg / L, KCl 400 mg / L, NaCl 8000 mg / L, CaCl2 140 mg / L, MgCl2 93.7 mg / L, MgSO4 97.7 mg / L, KH2PO4 60 mg / L, Na2HPO4 190 mg / L, NaHCO3 1000 mg / L, fetal bovine serum 150 mL / L, phenol red 10 mg / L, streptomycin sulfate 1.0 × 10 5 IU / L, sodium pyruvate 550 mg / L, sodium penicillin 1.0 × 10 5 The extract contained IU / L of shrimp ovarian extract (150 mL / L), epidermal growth factor (20 μg / L), basic fibroblast growth factor (20 μg / L), vascular endothelial growth factor (10 μg / L), insulin-like growth factor-1 (5 μg / L), and RSPO growth factor (5 μg / L). The amino acids and sugars labeled with b were derived from shrimp serum.

4. A method for in vitro proliferation and culture of Litopenaeus vannamei cells using the culture medium as described in claim 3, characterized in that, The cells in question are Oka organs.

5. The method for in vitro proliferation and culture of Litopenaeus vannamei cells according to claim 4, characterized in that, The conditions for the in vitro proliferation culture are as follows: the temperature inside the incubator is set at 27~29℃ and the CO2 concentration is set at 2.5~3.5%.

6. The method for in vitro proliferation and culture of Litopenaeus vannamei cells according to claim 4, characterized in that, The in vitro proliferation culture is a long-term culture of at least 30 days.

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