Transgenic system of hematopoietic tissue cells of freshwater crayfish and application thereof

Abstract

The invention discloses a transgenic system of hematopoietic tissue cells of freshwater crayfish and application of the transgenic system, and the transgenic system comprises a promoter which is the same as a sequence SEQ ID NO.12, SEQ ID NO.16 or SEQ ID NO.20 or has 80%-98% of homology with the sequence SEQ ID NO.12, SEQ ID NO.16 or SEQ ID NO.20. The promoters are connected in sequence to form a hybrid promoter group, the hybrid promoter group is used for starting expression of the freshwater crayfish hematopoietic factor atakine gene, and overexpression of the atakine gene is achieved under the electroporation transfection conditions that the voltage is 200 V, the electric pulse time is 30 ms, the electric pulse times are two, the electric pulse interval is 10 s and an electrotransfection buffer solution is CytoMix or under the chemical transfection conditions that the transfection reagent / DNA ratio is 2: 1. The expression efficiency under the chemical transfection condition reaches about 18%, the proliferation rate of transgenic cells is improved by about 7%, healthy and stable growth of more than three months is maintained, and the transgenic expression vector can also be applied to other freshwater crayfish. A reliable transgenic vector is provided for shrimp gene function research, and a foundation is laid for development of shrimp immortalized cell lines.

Description

Technical Field

[0001] The invention belongs to the technical field of freshwater shrimp cell transgenesis, and in particular relates to a transgenic system of freshwater crayfish hematopoietic tissue cells and an application thereof. Background Art

[0002] Crustaceans, especially shrimp and crayfish, have extremely high economic value in global aquaculture and contribute greatly to nutrition and food safety. However, as market demand continues to grow, shrimp and crayfish products are in short supply. This is mainly due to the frequent outbreaks of viral diseases that have severely restricted intensive farming and caused huge losses to the aquaculture industry. Shrimp mainly rely on their innate immune system to resist pathogen invasion. With the advancement of omics technology, researchers have identified key pathways that regulate these immune responses, such as Toll, IMD and JAK / STAT. However, due to the lack of transgenic vector tools suitable for shrimp cells and their host cell lines, research on related gene functions and the interaction mechanism between pathogens and host cells is still very limited.

[0003] The main immune defense function of crustaceans such as shrimp and freshwater crayfish is jointly performed by humoral immunity mediated by blood cells and cellular immunity, including antimicrobial peptides, antiviral peptides, phenoloxidase activation system, coagulation system, lectin system, as well as phagocytosis, encapsulation or nodulation and release of cytokines such as cell adhesion proteins. When pathogenic microorganisms invade, the number of blood cells will experience a sharp decline and gradual recovery process, in which hematopoietic tissue and blood cell proliferation-related cytokines play an important role.

[0004] At present, most studies rely only on the expression of fluorescent protein reporter genes (such as EGFP) to verify the efficiency of transgenics, and some reports on functional gene expression also show that the transgenic effect is not ideal. For example, oncogenes E6 and E7, SV40-T and 12S E1A, and H-ras were immortalized in shrimp cells cultured in vitro through transgenic technology, but ultimately failed to successfully induce cell proliferation in vitro. This may be because these mammalian oncogenes and shrimp cells are incompatible, so they cannot effectively promote proliferation. Secondly, the promoter is the "power motor" that drives plasmid expression, which is very important for the construction of efficient expression vectors. Previous research results show that some commonly used CMV and MoMLV promoters derived from mammalian viruses and pH promoters derived from insect baculovirus have very low promoter efficiencies in shrimp cells, which may be due to species specificity, and transgenic shrimp cells can often only survive for 5-14 days. Due to the lack of shrimp virus vector tool development, scholars choose to use modified retrovirus, adeno-associated virus and lentivirus and other mammalian virus vectors and insect baculovirus vectors to carry out shrimp cell transgenic experiments, but due to species differences, the efficiency of recombinant virus infection is very low. Therefore, a mild and stable transgenic method is also very important.

[0005] In summary, although scholars have made many efforts to develop transgenic tools for shrimp cells, most studies rely only on reporter gene expression to verify transgenic efficiency, but cannot achieve effective expression and verification of functional genes, and the cells will die quickly after transgenic. At present, there is still a lack of shrimp cell transgenic systems that can efficiently and stably express exogenous genes. Therefore, it is necessary to establish standardized transgenic vectors suitable for freshwater crayfish hematopoietic tissue cells to lay the foundation for the development of shrimp immortalized cell lines, thereby obtaining shrimp hematopoietic tissue cells with high activity, providing raw materials for exploring the functions of shrimp hematopoietic tissue cells themselves and gene regulation mechanisms, and also laying the foundation for the breeding of freshwater crayfish. Summary of the invention

[0006] The purpose of the present invention is to establish a transgenic vector suitable for freshwater crayfish hematopoietic tissue cells to solve the technical problem: In a first aspect, the present invention provides a promoter which is identical to or has 80%, 85%, 90%, 95% or 98% homology with the sequence SEQ ID NO.12, SEQ ID NO.16 or SEQ ID NO.20.

[0007] The second aspect of the present invention provides an application of a promoter in constructing a transgenic vector for hematopoietic tissue cells of freshwater crayfish.

[0008] The third aspect of the present invention provides a transgenic vector for freshwater crayfish hematopoietic tissue cells, the transgenic vector comprises an expression cassette, the expression cassette comprises a hybrid promoter group; the hybrid promoter group is composed of SEQ ID NO.20, SEQ ID NO.12 and SEQ ID NO.16 sequences connected in sequence.

[0009] Furthermore, the expression cassette is composed of a hybrid promoter group connected to the Ast gene; the nucleotide sequence of the Ast gene is shown in SEQ ID NO.10.

[0010] Furthermore, the transgenic vector is pIZ-OpIE2-P2-WSV249 （-300 / -1） -Ast-P2A-mCherry.

[0011] The fourth aspect of the present invention provides a method for constructing a transgenic vector for freshwater crayfish hematopoietic tissue cells, comprising the following steps: step A: obtaining the freshwater crayfish hematopoietic factor Ast gene; step B: activating the P2 promoter and WSV249 （-300 / -1） The promoter was cloned; pIZ-V5-His was used as the basic vector, and the P2 promoter and P2A-mCherry were double-digested and ligated in sequence to obtain the intermediate vector pIZ-OpIE2-P2-P2A-mCherry; Step C: WSV249 （-300 / -1） The promoter and Ast gene were sequentially connected to the intermediate vector pIZ-OpIE2-P2-P2A-mCherry by homologous recombination to obtain the transgenic vector pIZ-OpIE2-P2-WSV249. （-300 / -1） -Ast-P2A-mCherry.

[0012] Furthermore, step A includes: step A-1: ​​using a 2D or 3D culture system to culture freshwater crayfish hematopoietic tissue cells to obtain freshwater crayfish hematopoietic tissue cells; step A-2: extracting RNA and preparing cDNA from the freshwater crayfish hematopoietic tissue cells, using the prepared cDNA as a template to clone the freshwater crayfish hematopoietic factor Ast gene to obtain the complete CDS sequence of the Ast gene.

[0013] A fifth aspect of the present invention provides an application of a transgenic vector in the culture of freshwater crayfish hematopoietic cells, the establishment of hematopoietic cell lines, the overexpression of exogenous genes in hematopoietic cells, the proliferation of hematopoietic cells, the research on shrimp antiviral genes, or the detection of shrimp viruses for non-diagnostic and therapeutic purposes.

[0014] Furthermore, the transgenic vector was transferred into the hematopoietic tissue cells of freshwater crayfish so that the hematopoietic tissue cells of freshwater crayfish could grow stably for no less than 3 months; the overexpression efficiency of exogenous genes in the hematopoietic tissue cells of freshwater crayfish was 18.01 ± 1.83%; and the proliferation rate of the hematopoietic tissue cells of freshwater crayfish was 25.69 ± 1.19%.

[0015] Furthermore, electrofection or chemical transfection was used to achieve overexpression of exogenous genes in freshwater crayfish hematopoietic tissue cells; the operating parameters of the electrofection method were: voltage 200 V, electric pulse time 30 ms, number of electric pulses 2 times, electric pulse interval 10 s, and electrotransfection buffer CytoMix; the operating parameters of the chemical transfection method were: the ratio of transfection reagent to plasmid DNA in the transfection complex was 2:1; the time for incubating the freshwater crayfish hematopoietic tissue cells with the transfection complex was ≤ 6 h.

[0016] Further, the freshwater crayfish is of the family Pseudocranidae or Cheracidae; the family Pseudocranidae includes the red claw crayfish, the giant crayfish, the hairy crayfish, the smooth crayfish or the destroyer crayfish; the family Cheracidae includes the Procambarus clarkii, the Northeastern crayfish, the Korean crayfish or the dwarf giant crayfish.

[0017] The present invention successfully developed a hybrid promoter group (OpIE2-P2-WSV249) suitable for 2D or 3D cultured red claw crayfish hematopoietic tissue cells by modifying shrimp virus promoters and optimizing transfection methods. （-300 / -1） ) and transgenic expression vector (pIZ-OpIE2-P2-WSV249 (-300 / -1) -Ast-P2A-mCherry). The coding sequence of the hematopoietic factor astakine of red claw crayfish was cloned and inserted into the transgenic expression vector. The overexpression of the astakine gene was achieved under the conditions of electroporation transfection with a voltage of 200 V, an electric pulse time of 30 ms, two electric pulses, an electric pulse interval of 10 s, and an electroporation buffer of CytoMix or under the conditions of chemical transfection with a transfection reagent / DNA ratio of 2:1. Among them, the expression efficiency under the chemical transfection conditions reached about 18%, the proliferation rate of the transgenic cells increased by about 7%, and the healthy and stable growth was maintained for more than three months. The transgenic expression vector can also be applied to other freshwater crayfish. Therefore, the present invention provides a reliable transgenic vector for the study of shrimp gene functions. The disclosed transgenic vector of freshwater crayfish hematopoietic tissue cells can not only support the expression and functional research of exogenous genes, but also ensure the healthy growth of crayfish hematopoietic tissue cells after transgenic operation, laying a foundation for the development of shrimp immortalized cell lines. 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 The agarose gel electrophoresis diagram of RNA in hemocytes of red claw crayfish in Example 1 (wherein the first lane is a marker; the second lane is RNA, the 28 S band is about 1800 bp, and the 18 S band is about 900 bp); Figure 2 is a sequence alignment diagram of the known shrimp and crab astakine proteins in Example 1 (wherein, Ast is Astakine; Pl is the cochlear crayfish; Lv is the whiteleg shrimp; Mj is the Japanese marsupial; Pm is the giant tiger prawn; Es is the Chinese mitten crab; Pc is the crayfish); Figure 3 is an agarose gel electrophoresis diagram of the first amplification product in Example 1 (wherein lane 1 is a marker; lanes 2-5 are the first amplification products at annealing temperatures of 48°C, 50°C, 53°C and 54°C, respectively); Figure 4 This is the agarose gel electrophoresis diagram of the second amplification product in Example 1 (wherein the first lane is a marker; the second lane is the cloned red claw crayfish astakine gene, with a band of about 450 bp); Figure 5 It is the agarose gel electrophoresis diagram of the third amplification product in Example 1 (wherein, the first lane is a marker; lanes 2-5 are all bacterial solutions of the red claw crayfish astakine gene connected to the pMD18-T vector, and the band is about 490 bp); Figure 6 The nucleic acid and amino acid sequence diagram of the coding frame (CDS) of the astakine gene of the red claw crayfish in Example 1; Figure 7 is the predicted domain structure of the coding frame (CDS) of the astakine gene of the red claw crayfish in Example 1; Figure 8 It is a multiple sequence alignment diagram of the coding frame (CDS) of the red claw crayfish astakine gene in Example 1 and other proteins containing the prokineticin domain; Fig. 9 It is a phylogenetic analysis diagram of the coding frame (CDS) of the astakine gene of the red claw crayfish in Example 1; Fig.10 The agarose gel electrophoresis diagram of the fourth amplification product in Example 1 (wherein the first lane is a marker; lanes 2-4 are all P2 promoters, and the band is about 110 bp); Fig.11The agarose gel electrophoresis diagram of the fifth and sixth amplification products in Example 1 (where A is the fifth amplification product WSV249 （-504 / -1） Promoter, band around 500 bp; B is the sixth amplification product WSV249 （-300 / -1） promoter, the band is around 330 bp); Fig.12 This is the basic vector map of pIZ-V5-His in Example 1; Fig.13 The agarose gel electrophoresis diagram of the seventh amplification product in Example 1 (wherein the first lane is a marker; lanes 2 to 5 are all insertion sequences P2-Ast-P2A-mCherry, and the band size is about 1300 bp); Fig.14 This is a map of the pIZ-OpIE2-P2-Ast-P2A-mCherry vector in Example 1; Fig.15 The agarose gel electrophoresis diagram of the eighth amplification product in Example 2 (wherein lanes 1-2 are all insertion sequence WSV249 （-504 / -1） -Ast, band size is about 900 bp; lane 3 is a marker); Fig.16 pIZ-OpIE2-P2-WSV249 in Example 2 （-504 / -1） -Ast-P2A-mCherry vector map; Fig.17 This is the agarose gel electrophoresis diagram of the ninth amplification product in Example 3 (wherein the first lane is a marker; lanes 2-3 are all insertion sequences WSV249 （-300 / -1） -Ast, band size is around 750 bp); Fig.18 pIZ-OpIE2-P2-WSV249 in Example 3 （-300 / -1） -Ast-P2A-mCherry vector map; Fig.19 The agarose gel electrophoresis diagrams of the three expression vector plasmids in Example 4 (wherein: the first lane in A is a marker, and the second lane is pIZ-OpIE2-P2-Ast-P2A-mCherry plasmid DNA (plasmid a); the first and fourth lanes in B are both markers, and the second lane is pIZ-OpIE2-P2-WSV249 （-504 / -1） -Ast-P2A-mCherry plasmid DNA (plasmidb); Lane 3 is blank; Lane 5 is pIZ-OpIE2-P2-WSV249 （-300 / -1）-Ast-P2A-mCherry plasmid (plasmid c); Fig. 20 The light microscopy and mCherry fluorescence images of the red claw crayfish astakine gene expression vector in Test Example 1 expressed in Sf9 cells (where A1-A2, B1-B2, and C1-C2 are light microscopy images (bright field) and mCherry fluorescence images of the pIZ-OpIE2-P2-Ast-P2A-mCherry plasmid in Sf9 cells after transfection for 24, 48, and 72 hours, respectively; D1-D2, E1-E2, and F1-F2 are pIZ-OpIE2-P2-WSV249 (-504 / -1) -Light microscopy images (bright field) and mCherry fluorescence images of Ast-P2A-mCherry plasmids in Sf9 cells 24, 48, and 72 hours after transfection; G1-G2, H1-H2, and I1-I2 are pIZ-OpIE2-P2-WSV249, respectively. (-300 / -1) -Light microscopy images (bright field) and mCherry fluorescence images of Sf9 cells 24, 48, and 72 h after Ast-P2A-mCherry plasmid transfection; scale bar, 100 μm); Fig.21 It is a statistical diagram of the expression efficiency of the red claw crayfish astakine gene expression vector in Sf9 cells in Test Example 1 (the horizontal axis is the transfection efficiency; the vertical axis is the transfection time, which is 24 h, 48 h and 72 h respectively; ** and *** represent significant differences and extremely significant differences respectively); Fig. 22 pIZ-OpIE2-P2-WSV249 in Test Example 1 (-300 / -1) - Annotation diagram of Ast-P2A-mCherry vector construction (blue is P2 promoter; green is WSV249 (-300 / -1) Promoter; orange is astakine gene; pink is P2A short peptide; red is mCherry gene fluorescent marker); Fig.23 The light microscopy and fluorescence images of HPT cell activity of red claw crayfish under different voltage conditions in test example 2 (wherein, A1-A2, B1-B2, C1-C2, D1-D2, E1-E2, F1-F2, G1-G2, H1-H2, I1-I2 and J1-J2 are light microscopy images (bright field) and Calcein-AM fluorescence images of HPT cells after electrotransfection at voltages of 0, 50, 100, 200, 300, 400, 500, 600, 800 and 1000 V, respectively; scale bar is 50 μm); Fig.24This is a statistical graph of the survival rate of red claw crayfish HPT cells under different voltage conditions in Test Example 2 (wherein the horizontal axis is the cell survival rate, unit (%); the vertical axis is the voltage, unit (V); *, ** and *** represent differences, significant differences and extremely significant differences, respectively); Fig.25 The light microscopy and fluorescence images of the cell activity of the HPT cells of red claw crayfish in test example 3 at different electric pulse durations (wherein, A1-A2, B1-B2, C1-C2, D1-D2, E1-E2, F1-F2, G1-G2, H1-H2 and I1-I2 are light microscopy images (bright field) and Calcein-AM fluorescence images of the cell activity of HPT cells after electrotransfection at electric pulse durations of 0, 1, 5, 10, 15, 20, 25, 30 and 40 ms, respectively; the scale bar is 50 μm); Fig.26 This is a statistical graph of the survival rate of HPT cells of red claw crayfish in test example 3 under different electric pulse durations (wherein the horizontal axis is the cell survival rate, unit (%); the vertical axis is the electric pulse duration, unit (ms); *** represents a very significant difference); Fig. 27 The light microscopy and fluorescence images of the cell activity of the red claw crayfish HPT cells in test example 4 under different electrotransfection buffers (wherein, A1-A2, B1-B2, C1-C2, D1-D2 and E1-E2 are light microscopy and fluorescence images of the Calcein-AM cell activity of the HPT cells after electrotransfection using the CytoMix electrotransfection buffer at electric pulse durations of 0, 10, 20, 30 and 40 ms, respectively; F1-F2, G1-G2, H1-H2, I1-I2 and J1-J2 are light microscopy and fluorescence images of the Calcein-AM cell activity of the HPT cells after electrotransfection using the HEPES electrotransfection buffer at electric pulse durations of 0, 10, 20, 30 and 40 ms, respectively; the scale bar is 50 μm); Fig.28 This is a statistical graph of HPT cell viability of red claw crayfish in different electroporation buffers in Test Example 4 (wherein the abscissa is cell viability, unit (%); the ordinate is electroporation buffer; * and *** represent differences and extremely significant differences, respectively); Fig.29The light microscopy and fluorescence images of the HPT cell activity of red claw crayfish using chemical transfection reagents in different ratios in Test Example 5 (wherein, A1-A2, B1-B2 and C1-C2 respectively show the light microscopy images (bright field) and mCherry fluorescence microscopy images of HPT cells transfected at a transfection reagent / DNA ratio of 1:1 for 24, 48 and 72 h, D1-D2, E1-E2 and F1-F2 respectively show the light microscopy images (bright field) and mCherry fluorescence microscopy images of HPT cells transfected at a transfection reagent / DNA ratio of 2:1 for 24, 48 and 72 h, G1-G2, H1-H2 and I1-I2 respectively show the light microscopy images (bright field) and mCherry fluorescence microscopy images of HPT cells transfected at a transfection reagent / DNA ratio of 3:1 for 24, 48 and 72 h, J1-J2, K1-K2, and L1-L2 show the light microscopy images (bright field) and mCherry fluorescence microscopy images of HPT cells 24, 48, and 72 h after transfection at a transfection reagent / DNA ratio of 4:1, respectively; scale bar, 50 μm); Fig.30 The figure is a statistical graph of transfection efficiency of red claw crayfish HPT cells using chemical transfection reagents in different ratios in Test Example 5 (wherein the horizontal axis is transfection efficiency, unit (%); the vertical axis is transfection time, which is 24 h, 48 h and 72 h respectively; ** and *** represent significant differences and extremely significant differences respectively); Fig.31 Fluorescence images of red claw crayfish HPT cells under different transfection methods in Test Example 6 (wherein, A1-A3, B1-B3 and C1-C3 are mCherry fluorescence images of astakine overexpressed in HPT cells by electrotransfection, A1, B1 and C1 are bright field, A2, B2 and C2 are mCherry fluorescence, A3, B3 and C3 are bright field-fluorescence merge; Figures D1-D3, E1-E3 and F1-F3 are mCherry fluorescence images of astakine overexpressed in HPT cells by chemical transfection, D1, E1 and F1 are bright field, D2, E2 and F2 are mCherry fluorescence, D3, E3 and F3 are bright field-fluorescence merge; scale bar is 50 μm); Fig.32 This is a statistical graph of the transfection efficiency of red claw crayfish HPT cells under different transfection methods in Test Example 6 (wherein the abscissa is the transfection time, unit (h); the ordinate is the transfection efficiency, unit (%); *** represents a significant difference); Fig.33The agarose gel electrophoresis diagram of the semi-quantitative RT-PCR amplification of the mCherry gene in the HPT cells of red claw crayfish in Test Example 6 (wherein, the first lane is the Marker; the second lane is the untransfected cell control; the third lane is 24 h; the fourth lane is 48 h; the fifth lane is 72 h; the band of about 1000 bp is β-actin; the band of about 750 bp is mCherry; A is electrotransfection; B is chemical transfection); Fig.34 This is a statistical result diagram of the grayscale value of the mCherry gene band of the HPT cells of red claw crayfish in test example 6 (wherein the horizontal axis is the transfection time, unit (h); the vertical axis is the grayscale value; ** and *** represent significant differences and extremely significant differences, respectively); Fig.35 Fluorescence images of the red claw crayfish HPT cell spheres in test example 7 under different transfection methods (wherein, A1-A3, B1-B3 and C1-C3 are mCherry fluorescence images of astakine overexpressed in HPT cell spheres by electrotransfection, A1, B1 and C1 are bright field, A2, B2 and C2 are mCherry fluorescence, A3, B3 and C3 are bright field-fluorescence merge; Figures D1-D3, E1-E3 and F1-F3 are mCherry fluorescence images of astakine overexpressed in HPT cell spheres by chemical transfection, D1, E1 and F1 are bright field, D2, E2 and F2 are mCherry fluorescence, D3, E3 and F3 are bright field-fluorescence merge; scale bar is 50 μm); Fig.36 This is a statistical chart of the transfection efficiency of the red claw crayfish HPT cell spheres under different transfection methods in Test Example 7 (wherein the horizontal axis is the transfection time, unit (h); the vertical axis is the transfection efficiency, unit (%); *** represents a significant difference); Fig.37 The agarose gel electrophoresis diagram of the semi-quantitative RT-PCR amplification of the mCherry gene in the HPT cell sphere of the red claw crayfish in Test Example 7 (wherein, the first lane is the Marker; the second lane is the untransfected cell control; the third lane is 24 h; the fourth lane is 48 h; the fifth lane is 72 h; the band of about 1000 bp is β-actin; the band of about 750 bp is mCherry; A is electrotransfection; B is chemical transfection); Fig.38 This is a statistical result graph of the grayscale value of the mCherry gene band of the red claw crayfish HPT cell sphere in Test Example 7 (wherein the horizontal axis is the transfection time, unit (h); the vertical axis is the grayscale value; * and ** represent differences and significant differences, respectively); Fig.39The light microscopy and fluorescence images of astakine transgenic red claw crayfish HPT cells in test example 8 after 72 hours of transfection treatment (wherein, A1-A5, B1-B5 and C1-C5 are light microscopy images and fluorescence images of EdU proliferation of crayfish HPT cells after different transfection treatments for 72 hours, respectively; A1, B1 and C1 are bright field; A2, B2 and C2 are Hoechst fluorescence; A3, B3 and C3 are EdU fluorescence; A4, B4 and C4 are mChreey fluorescence; A5, B5 and C5 are bright field-fluorescence merge; NT is negative control group; MT is conditional control group; PT is experimental group; scale bar is 50 μm); Fig.40 The figure is a graph of the transfection efficiency and proliferation rate of the astakine transgenic red claw crayfish HPT cells after 72 hours of transfection treatment in Test Example 8 (where mCherry represents transfection efficiency; EdU represents proliferation efficiency; *, ** and *** represent differences, significant differences and extremely significant differences, respectively); Fig.41 The agarose gel electrophoresis diagram of the semi-quantitative RT-PCR amplification of astakine and PCNA in the HPT cells of the astakine transgenic red claw crayfish in Test Example 8 (wherein, the first lane is the Marker; the second lane is the NT negative control; the third lane is the PT experimental group; A: the band around 1000 bp is β-actin; B: the band around 500 bp is astakine; C: the band around 600 bp is PCNA); Fig.42 This is a statistical result graph of the grayscale values ​​of astakine and PCNA gene bands in the HPT cells of astakine transgenic red claw crayfish in Test Example 8 (wherein the horizontal axis is the NT negative control group and the PT experimental group; the vertical axis is the grayscale value; * and *** represent differences and extremely significant differences, respectively). DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] 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.

[0023] (1) Source of sample materials The healthy red claw crayfish in this embodiment were purchased from Zhejiang Freshwater Fisheries Research Institute.

[0024] (2) Sources of reagents and consumables Table 1 Sources and item numbers of reagents and consumables required for the experiment name Manufacturer Collagenase I Sigma Fetal bovine serum (FBS) BI 400 mesh sieve silk Qingdao Shibei District Feimate Experimental Supplies Business Department Ophthalmic scissors and tweezers Jiangxi Yuyuan Medical Equipment Co., Ltd. Sterilizable syringe filter Qingdao Shibei District Feimate Experimental Supplies Business Department SIM-SF Insect Cell Culture Medium Beijing Sino Biological Technology Co., Ltd. Trypsin Beijing Solable Technology Co., Ltd. Enzyme-free cell digestion solution Beijing Prilai Gene Technology Co., Ltd. TIANamp Genomic DNA Kit Tiangen Biochemical Technology (Beijing) Co., Ltd. DNase I Merck KGaA Potassium chloride (KCl) Sinopharm Chemical Reagent Co., Ltd. Sodium chloride (NaCl) Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Potassium dihydrogen phosphate (KH2PO4)]]> Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Disodium hydrogen phosphate (Na2HPO4)]]> Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Sodium bicarbonate (NaHCO3)]]> Sinopharm Chemical Reagent Co., Ltd. Sodium hydroxide (NaOH) Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Magnesium chloride hexahydrate (MgCl2·6H2O)]]> Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Absolute ethanol (C2H5OH)]]> Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O)]]> Sinopharm Chemical Reagent Co., Ltd. 4-Hydroxyethylpiperazineethanesulfonic acid (HEPES) Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Manganese chloride (MnCl2)]]> Sinopharm Chemical Reagent Co., Ltd. Sorbitol Sinopharm Chemical Reagent Co., Ltd. Lysine Shanghai MacLean Biochemical Technology Co., Ltd. Taurine Shanghai Aladdin Biochemical Technology Co., Ltd. Proline Shanghai Aladdin Biochemical Technology Co., Ltd. Aspartic acid Shanghai Aladdin Biochemical Technology Co., Ltd. Glutamic acid Shanghai Aladdin Biochemical Technology Co., Ltd. Hydroxyproline Shanghai Aladdin Biochemical Technology Co., Ltd. Ornithine Shanghai Aladdin Biochemical Technology Co., Ltd. Cystine Shanghai Aladdin Biochemical Technology Co., Ltd. β-Alanine Shanghai MacLean Biochemical Technology Co., Ltd. Gamma-aminobutyric acid (γ-ABA) Shanghai MacLean Biochemical Technology Co., Ltd. Glutamine Shanghai Aladdin Biochemical Technology Co., Ltd. Glucose Sinopharm Chemical Reagent Co., Ltd. Trehalose Shanghai Aladdin Biochemical Technology Co., Ltd. Gentiobiose Bailingwei Technology Co., Ltd. Sucrose Shanghai Aladdin Biochemical Technology Co., Ltd. Ribose Shanghai Aladdin Biochemical Technology Co., Ltd. Arabinose Shanghai Aladdin Biochemical Technology Co., Ltd. Lactose Shanghai Aladdin Biochemical Technology Co., Ltd. Fucose Shanghai Aladdin Biochemical Technology Co., Ltd. Xylose Shanghai Aladdin Biochemical Technology Co., Ltd. Fructose Shanghai Aladdin Biochemical Technology Co., Ltd. ATP Shanghai Bio-Tech Co., Ltd. Trans IT®-Insect Transfection Reagent Mirus Bleomycin Lifesensors Restriction enzymes TaKaRa T4 Ligase TaKaRa PrimeScript™ RT reagent Kit TaKaRa Premix Taq™ TaKaRa pMD™ 18-T Vector TaKaRa Trans2K PLUS DNA Marker Beijing Quanshijin Biotechnology Co., Ltd. Trans2K PLUS II DNA Marker Beijing Quanshijin Biotechnology Co., Ltd. TransZol™ Up Plus RNA Kit Beijing Quanshijin Biotechnology Co., Ltd. EasyPure Plasmid MiniPrep Kit Beijing Quanshijin Biotechnology Co., Ltd. EasyPure HiPure Plasmid MaxiPrep Kit Beijing Quanshijin Biotechnology Co., Ltd. EZ-10 Column DNA Gel Extraction Kit Shanghai Bioengineering Co., Ltd. DH5α Chemically Competent Cells Nanjing Novozymes Biotech Co., Ltd. Leibovitz's L-15 Thermo Fisher Scientific Inc. (Gibco) Calcein-AM Reagent Shanghai Bio-Tech Biotechnology Co., Ltd. EdU Imaging Kits Thermo Fisher Scientific Ltd. Epidermal Growth Factor (EGF) Beijing Sino Biological Co., Ltd. Basic fibroblast growth factor (bFGF) Beijing Sino Biological Co., Ltd. pIZ-V5-His plasmid Beijing Huayueyang Biotechnology Co., Ltd. 50×TAE electrophoresis buffer Beijing Solable Technology Co., Ltd. 100× Penicillin-Streptomycin Antibiotic Stock Solution Beijing Solable Technology Co., Ltd. Cell fixative Shanghai Bio-Tech Biotechnology Co., Ltd. Cell Permeabilization Solution Shanghai Bio-Tech Biotechnology Co., Ltd. Cell washing solution Shanghai Bio-Tech Co., Ltd. Sf9 cells Qingdao Shunlejia Biotechnology Co., Ltd. Grace serum-free insect cell culture medium Thermo Fisher Scientific Inc. (Gibco) (3) Source of instruments and equipment Table 2 Sources and models of instruments and equipment required for the experiment name Manufacturer ECM830 Electroporator BTX Electric cup BTX Ultra-low attachment cell culture plates Corning Incorporated Vertical Pressure Steam Sterilizer Shanghai Shen'an Medical Equipment Factory Pipette Eppendorf Clean bench Suzhou Antai Air Technology Co., Ltd. pH Meter Shanghai Mettler-Toledo Instruments Co., Ltd. Ultrapure water machine Jinan Taipingma Equipment Co., Ltd. Electric constant temperature blast drying oven Shanghai Jinghong Experimental Equipment Co., Ltd. Diaphragm vacuum pump Tianjin Jinteng Equipment Co., Ltd. Magnetic stirrer Shanghai Huxi Instrument Factory Co., Ltd. Inverted fluorescence microscope Nikon Corporation Electronic balance Poland Redway Company refrigerator Qingdao Haier Co., Ltd. Constant temperature water bath Jiangsu Shuangjie Experimental Instrument Factory Snow ice machine Changshu Xueke Electric Appliance Co., Ltd. Refrigerated centrifuge Eppendorf High-speed centrifuge Xiangyi Centrifuge Co., Ltd. Double head oxygen pump Guangdong Minjiang Aquarium Industry Co., Ltd. High speed electric homogenizer Jiangsu Zhongda Instrument Factory Freezing point osmometer Shanghai Medical University Instrument Factory HF-90 CO2 Incubator Shanghai Lishen Scientific Instrument Co., Ltd. JY-C electrophoresis apparatus Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd. JY04S-3C Gel Imaging Analysis System Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd. 100 W heating rod Qingdao Kangsijia Experimental Equipment Sales Department ELISA reader Thermo Fisher Scientific WH-2 Micro Vortex Mixer Shanghai Huxi Analytical Instrument Factory Co., Ltd. Tabletop Constant Temperature Oscillator Shanghai Jinghong Experimental Equipment Co., Ltd. Biochemical incubator Shanghai Jinghong Experimental Equipment Co., Ltd. Laser confocal microscope (LSM510) Carl Zeiss metal bath Beijing Tiangen Biochemical Technology Co., Ltd. Micro low speed centrifuge Beijing Tiangen Biochemical Technology Co., Ltd. Cell culture flasks / plates Corning Incorporated Disposable Syringe Filters Sartorius AG Disposable syringe Shaanxi Longkangxin Medical Equipment Co., Ltd. (4) Preparation method of experimental reagents 1) Preparation of PBS PBS refers to phosphate buffer saline (PBS). The preparation method is as follows: weigh 4.0 g NaCl, 0.1 g KCl, 1.5 g Na2HPO4·12H2O and 0.1 g KH2PO4 in a volumetric flask, dissolve them fully with ddH2O, make up to 500 mL, sterilize at 121℃ under high temperature and high pressure for 20 min, then aseptically dispense the cooled PBS into 100 mL reagent bottles in a clean bench and store them in a 4℃ refrigerator for later use.

[0025] 2) Preparation of CPBS CPBS refers to phosphate buffer saline of Cheraxquadricarinatus (CPBS). The preparation method is as follows: weigh 8.8 g NaCl, 1.11 mg CaCl2, 1.26 mgMnCl2, 1.42 g Na2HPO4 and 1.36 g KH2PO4 in a volumetric flask, dissolve them fully with ddH2O, make up to 1000 mL, sterilize at 121℃ under high temperature and high pressure for 20 min, then aseptically dispense the cooled CPBS into 100 mL reagent bottles in a clean bench and store them in a 4℃ refrigerator for later use.

[0026] 3) Preparation of 0.25% pancreatin Weigh 0.25 g of trypsin into a volumetric flask, dissolve it fully with PBS, and make up to 100 mL. Then, filter and sterilize it in an ultra-clean workbench (0.22 μm), aseptically dispense it into 15 mL centrifuge tubes, and store it in a -20°C refrigerator for long-term use or in a 4°C refrigerator for short-term use.

[0027] 4) Preparation of 0.1% collagenase I tissue dissociation solution Dissolve 100 mg of collagenase I powder in 100 mL of L-15 culture medium to prepare 0.1% collagenase I tissue dissociation solution (100 mg mL -1 ), filter and sterilize the solution using a 0.22 μm syringe filter in a clean bench into a 100 mL reagent bottle, divide the solution into appropriate amounts and store in a -20°C refrigerator for later use to avoid repeated freezing and thawing.

[0028] 5) Preparation of Sf9 cell culture medium Aseptically measure 90 mL of SIM-SF insect cell culture medium into a sterile serum bottle and add 10% (v / v) fetal bovine serum (FBS) to a final concentration of 100 IU mL -1 of penicillin sodium, the final concentration was 100 μg mL -1 streptomycin sulfate and a final concentration of 0.25 mg L -1 After mixing well, place in a 4°C refrigerator and use within 1 month.

[0029] 6) Sterile preparation of red claw crayfish serum (CP) Before collecting hemolymph, the crayfish should be placed in crushed ice for anesthesia for about 10 minutes, and then the anesthetized crayfish should be immersed in 75% ethanol for 10 minutes for deep anesthesia and disinfection, and the crayfish should be fixed with alcohol cotton strips. Use a 10mL syringe pre-filled with about 5 mL of CPBS to pierce the area below the base of the fifth leg and above the first abdominal segment, and slowly aspirate the hemolymph. After mixing the hemolymph and CPBS in a ratio of 1:1, centrifuge at 1000 × g for 5 minutes at 4°C, and take the supernatant as a crude mixture of serum and CPBS. Incubate the crude mixture in a 56°C water bath for 30 minutes, and then centrifuge at 5000 × g for 20 minutes at 4°C to remove the precipitate. Finally, the prepared crayfish serum (CP) was divided into appropriate amounts and stored in a -20°C refrigerator for later use.

[0030] 7) Preparation of 60% glycerol 60 mL of glycerol was diluted to 100 mL with sterile ddH2O, filtered through a 0.45 μm filter membrane, and stored at 4°C.

[0031] 8) Preparation of cell conductivity solution - HEPES buffer HEPES buffer is a buffer for cell electroporation. The preparation method is as follows: weigh 57 mg KH2PO4, 20 mg MgCl2·6H2O, 246 mg K2HPO4·3H2O, 48 mg HEPES and 5.47 g sorbitol in a volumetric flask, dissolve them fully with ddH2O and make up to 100 mL. Then, filter and sterilize them into a 100 mL reagent bottle using a 0.22 μm syringe filter in an ultra-clean workbench and store them in a 4°C refrigerator for later use.

[0032] 9) Preparation of CytoMix buffer, a cell conductivity solution CytoMix buffer is a buffer for cell electroporation, and its preparation method is as follows: weigh 0.9 g KCl, 1.7 mg CaCl2, 174.2 mg K2HPO4·3H2O, 600 mg HEPES, 76.2 mg EGTA·2Na, 47.6 mgMgCl2, ATP with a final concentration of 200 mM and 153.8 mg glutathione in a volumetric flask, dissolve them fully with ddH2O and make up to 100 mL. Then, filter and sterilize them into 100 mL reagent bottles using a 0.22 μm syringe filter in an ultra-clean workbench, and divide them into appropriate amounts under sterile conditions and store them in a -20°C refrigerator for later use.

[0033] 10) Preparation of 1% agarose gel Weigh 0.2 g agarose powder into a 50 mL conical flask, add 20 mL 1× TAE solution and heat in a microwave until completely dissolved, then pour into a mold and wait for it to cool and solidify before use.

[0034] 11) Preparation of LB liquid medium Weigh 1.0 g of tryptone, 0.5 g of yeast extract, and 1.0 g of NaCl and dissolve them in ddH2O. Adjust the pH to 7.2 ± 0.2 and make up to 100 mL. Sterilize at 121 °C under high pressure and then cool to room temperature. Store at 4 °C for later use. Add appropriate amount of antibiotics according to different plasmid resistance before use.

[0035] 12) Preparation of LB solid medium Weigh 1.0 g of tryptone, 0.5 g of yeast extract, 1.0 g of NaCl and 1.5 g of agarose and dissolve them in ddH2O. Adjust the pH to 7.2 ± 0.2 and make up to 100 mL. After autoclaving at 121°C, cool to about 60°C and add appropriate amount of antibiotics according to the requirements of plasmid resistance. After mixing, pour the culture medium into the plate to obtain LB solid culture medium plates with different plasmid resistance.

[0036] 13) Preparation of Red Claw Cell Growth Medium (OCCM) As shown in Table 3, the formula of the red claw crayfish cell growth medium is adjusted to 7.2 ± 0.2. The formula is from the published literature: Zhao, Y., Xue, T., Wang, J. & Guo, H. Development of reliable in vitro long-term culture systems for hematopoietic cells of crayfish Cherax quadricarinatus and virus susceptibility assay. Aquaculture589, 741016, doi: 10.1016 / j.aquaculture.2024.741016 (2024).

[0037] Table 3 Formulation of Red Claw Cell Growth Medium (OCCM) Amino Acids <![CDATA[Final concentration (unit: mg L -1 ).]]> Sugar compounds <![CDATA[Final concentration (unit: mg L -1 ).]]> Glycine 200 fructose 5.91 L-Alanine 225 Arabinose 18.86 L-Arginine 500 Trehalose 233.79 L-Asparagine 250 Gentianobiose 88.09 L-Cysteine 120 D-Galactose 900 L-Glutamine 300 Vitamins <![CDATA[Final concentration (unit: mg L -1 ).]]> L-Histidine 250 Inositol 2 L-Isoleucine 250 Folic acid 1 L-Leucine 125 Niacinamide 1 L-Lysine 245.85 Choline chloride 1 L-Methionine 75 D-Calcium Pantothenate 1 L-Phenylalanine 125 Pyridoxine Hydrochloride 1 L-Serine 200 Thiamine phosphate 1 L-Threonine 300 Riboflavin-5-phosphate sodium 0.1 L-Tryptophan 20 Inorganic salt <![CDATA[Final concentration (unit: mg L -1 ).]]> L-Tyrosine 300 KCl (Potassium Chloride) 400 L-Valine 100 NaCl (Sodium Chloride) 8000 Taurine 829.12 <![CDATA[CaCl2 (Calcium Chloride)]]> 140 L-Proline 1518.28 <![CDATA[MgCl2 (Magnesium Chloride)]]> 93.7 L-Aspartic Acid 53.90 <![CDATA[MgSO4 (Magnesium Sulfate)]]> 97.7 L-Glutamic Acid 98.39 <![CDATA[KH2PO4 (Potassium Dihydrogen Phosphate)]]> 60 Hydroxyproline 62.55 <![CDATA[Na2HPO4 (Disodium hydrogen phosphate)]]> 190 Ornithine 18.40 <![CDATA[NaHCO3 (Sodium Bicarbonate)]]> 1000 L-Cystine 23.20 Other Ingredients <![CDATA[Final concentration (unit: L -1 ).]]> β-Alanine 4.56 Fetal bovine serum (FBS) 50 mL GABA 3.06 Crawfish serum (CP) 30 mL Sugar compounds <![CDATA[Final concentration (unit: mg L -1 ).]]> Phenol red 10 mg Xylose 5.52 Streptomycin sulfate <![CDATA[1.0 × 10 5 μg <!-- 11 -->]]> Fucose 7.63 Sodium pyruvate 550 mg Ribose 27.52 Penicillin Sodium <![CDATA[1.0 × 10 5 IU]]> glucose 2935.55 Epidermal Growth Factor (EGF) 20 μg sucrose 69.41 Basic Fibroblast Growth Factor (bFGF) 20 μg lactose 17.53

[0038] Example 1 A transgenic system for freshwater crayfish hematopoietic tissue cells, comprising the following steps: S1. Cell culture. The specific reference steps are as follows: (1) Cultivation of Fall Armyworm Ovarian (Sf9) Cells The revived Sf9 cells were cultured in SIM-SF insect cell culture medium, placed in a 28°C biochemical incubator, and subcultured every 3 days using 0.25% trypsin at a ratio of 1:3.

[0039] (2) Primary culture of red claw crayfish hematopoietic tissue (HPT) cells ① Collection of Hematopoietic Tissue (HPT) of Red Claw Place the red claw crayfish in crushed ice for anesthesia for about 10 minutes, and soak the anesthetized crayfish in 75% ethanol for 10 minutes for deep anesthesia and disinfection. Fix the red claw crayfish's pair of claws and abdomen with alcohol cotton strips and rubber bands, and place them in a clean bench for sampling.

[0040] Use sterile scissors to cut and carefully remove the shell on the gills and back of the red claw crayfish, exposing the hematopoietic tissue as much as possible. Use ophthalmic scissors to cut off the connection between the hematopoietic tissue and other tissues. Use sterile tweezers to carefully clip the hematopoietic tissue out and place it in a sterilized bottle containing CPBS. The sampling is now completed and the red claw crayfish hematopoietic tissue (HPT) is obtained.

[0041] ② Aseptic preparation of hematopoietic tissue cells of red claw crayfish and its 2D and 3D cell culture The red claw crayfish hematopoietic tissue was repeatedly rinsed with CPBS for 7 times, and then washed twice with basal culture medium (serum-free L-15 culture medium), and then the crayfish hematopoietic tissue was placed in a centrifuge tube containing 1 mL of 0.1% collagenase I and cut into pieces, and the cells were repeatedly ground with a sterile grinding pestle to dissociate the cells from the red claw crayfish hematopoietic tissue. The entire enzymatic hydrolysis process must be completed within 20 minutes to obtain the crayfish hematopoietic tissue cell dissociation solution; the red claw crayfish hematopoietic tissue cell dissociation solution was filtered with two layers of 400-mesh sterilized sieve silk, the filtrate was collected, and the filtrate was centrifuged at 600 × g for 5 minutes; the supernatant was discarded to obtain the first precipitate, 1 mL of basal culture medium was added to resuspend the first precipitate, and centrifuged again (conditions are the same as above) to obtain the second precipitate (i.e., the red claw crayfish HPT cell precipitate); the red claw crayfish HPT cell precipitate was resuspended in the red claw crayfish cell growth medium, inoculated in a cell culture plate or a low-adsorption cell culture plate, and placed at 28°C containing 3% 2D culture or 3D culture is carried out in a CO2 cell culture incubator, and half of the medium needs to be replaced once a day to obtain red claw crayfish hematopoietic tissue cells (crayfish HPT cells).

[0042] Among them, 2D culture or 3D culture system are derived from the published literature: Zhao, Y., Xue, T., Wang, J.& Guo, H. Development of reliable in vitro long-term culture systems for hematopoietic cells of crayfish Cherax quadricarinatus and virus susceptibility assay. Aquaculture 589, 741016, doi: 10.1016 / j.aquaculture.2024.741016 (2024). 2D culture refers to suspending cells in a flat culture dish; 3D culture is a cell culture method that simulates the three-dimensional growth environment in vivo, by allowing cells to aggregate into 3D spheres or allowing cells to adhere, stretch and grow on a three-dimensional structure carrier with a component structure similar to that of solid tissue, so as to jointly regulate cell proliferation and differentiation in time and space, so that the tissue structure and function can be preserved to a large extent.

[0043] S2. Cloning of the hematopoietic factor astakine gene (Ast gene for short) of the red claw crayfish There are many methods to obtain the Ast gene of red claw crayfish, such as gene cloning, artificial synthesis, gene library screening, gene sequencing, etc. This patent mainly obtains the Ast gene of red claw crayfish through gene cloning method.

[0044] (1) Preparation of cDNA from red claw crayfish blood cells RNA extraction: using TransZol TM Up Plus RNA Kit (full gold) (the following reagents are included in this kit) extracts RNA from the blood cells of red claw crayfish. The specific experimental method is as follows: Use a 10mL syringe pre-filled with about 5 mL of CPBS to pierce the crayfish below the base of the fifth walking leg and above the first abdominal segment, and slowly aspirate the blood lymph. After the blood lymph and CPBS are mixed in a 1:1 ratio, centrifuge at 1000 × g for 5 min at 4°C, remove the supernatant to obtain the third precipitate (blood cell precipitate). Add 1 mL of TransZol Up and 0.2 mL of RNA ExtractionAgent to the third precipitate, and repeatedly blow and aspirate with a pipette until there is no obvious precipitation to obtain a blood cell suspension. Vortex the blood cell suspension at room temperature for 5 min, and centrifuge at 10000 × g and 4°C for 15 min. At this time, the sample is divided into three layers, a colorless aqueous phase (upper layer), a middle layer, and a pink organic phase (lower layer). Among them, RNA is in the colorless aqueous phase. Transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of anhydrous ethanol, gently invert and mix, a white flocculent precipitate (i.e., the fourth precipitate) will appear, add the obtained solution and the white Xu Zhuang precipitate to the centrifuge column, centrifuge at 12,000 × g, 4°C for 30 s, discard the first effluent; add 500 μL CB9, centrifuge at 12,000 × g, 4°C for 30 s, discard the second effluent (repeat this step once, discard the third effluent); add 500 μL WB9, centrifuge at 12,000 × g, 4°C for 30 s, discard the fourth effluent (repeat this step once, discard the fifth effluent); centrifuge at 12,000 × g, 4°C for 2 min to completely remove the residual ethanol, i.e., the sixth effluent, place the centrifuge column corresponding to the discarded sixth effluent into an RNase-free Tube (RNase-free tube, already provided in the kit), add 50-200 μL RNase-free Water (RNase-free water) in the center of the centrifuge column, and let it stand on ice for 1 minute. min; centrifuge at 12000 × g and 4°C for 1 min to elute RNA, which is the red claw crayfish blood cell RNA, and store it at -80°C.

[0045] Take a small amount of extracted red claw crayfish blood cell RNA sample and use a nucleic acid quantifier to detect the concentration and purity of RNA to ensure RNA OD 260 / 280=1.8 ~ 2.0, and the integrity of RNA was checked by agarose gel electrophoresis, such as Figure 1 As shown, the RNA bands showed that the 28 s band was around 1800 bp and the 18 s band was around 900 bp.

[0046] cDNA preparation: Use PrimeScript™ RT reagent Kit (TaKaRa) (the following reagents are included in this kit) to reverse transcribe the red claw crayfish blood cell RNA into cDNA. The reverse transcription system is: add 2 μL 5× PrimeScript Buffer, 0.5 μL PrimeScript RT Enzyme Mix I, 0.5 μL Oligo dT Primer, 0.5 μL Random 6 mers and 1 μL red claw crayfish blood cell RNA (500 ng / μL) in the PCR tube in sequence, and finally add RNase FreedH2O to 10 μL. The reverse transcription program is: 42℃ 15 min, 85℃ 5 s, 4℃ storage, and the red claw crayfish blood cell cDNA is obtained and stored in a -80℃ refrigerator.

[0047] (2) Cloning of the hematopoietic factor astakine gene of red claw crayfish According to the sequence alignment of known shrimp and crab astakine proteins in the NCBI database ( Figure 2), primers were designed using a conserved region as a template: Ast-1: 5'-GCACGGACACATTCC-3' (SEQ ID NO.1) and Ast-2: 5'-GGAATGTGTCCGTGC-3' (SEQ ID NO.2); According to the red claw crayfish genome (GenBank: JAPQEV000000000.1) data uploaded to the NCBI database by Tan et al. (2020), the CDS of the astakine gene was predicted using the Augustus: job submission (uni-greifswald.de) online website, and the CDS sequence of the astakine gene (YC-astakine: SEQ ID NO.3) was finally predicted. Two primers were designed based on the YC-astakine gene sequence: Ast-3: 5'-ATGCAACCCAACGTACGAACCACAG-3' (SEQ ID NO.4) and Ast-4: 5'-TCAGAAGAATCCGTAGGAATTGTCC-3' (SEQ ID NO.5); The extracted cDNA of red claw crayfish blood cells was used as a template, and Ast-1 and RACE universal primer UPM (SEQ ID NO.6: 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3') were used as amplification primers to carry out the first PCR reaction. The PCR reaction system was 20 μL, including 1 μL of primers Ast-1 and UPM, 1 μL of template cDNA, 10 μL of Taq enzyme and 7 μL of ddH2O. The PCR reaction procedure was denaturation at 95℃ for 30s, annealing at 48-65℃ for 30s, and extension at 72℃ for 1 min, with 30 cycles of denaturation, annealing and extension, followed by 72℃ for 10min and storage at 4℃; the first half sequence of the astakine gene was obtained, which was the first amplification product. The first amplification product was verified by agarose gel electrophoresis, as shown in Figure 3 As shown, the predicted first half sequence of the astakine gene was obtained, and the band size was about 600 bp. According to the sequence of the first amplified product, the 5' end primer of the astakine gene was designed, Ast-5: 5'-ATGATGGTAATAGTTCGAAGTGTGT-3' (SEQ ID NO.7); the second PCR reaction was carried out with Ast-4 and Ast-5 as primers and cDNA as template. The second PCR reaction system and reaction procedure were the same as above, and the second amplified product was obtained, and the second amplified product was verified by agarose gel electrophoresis, as shown in FIG. Figure 4As shown, the complete CDS sequence of the predicted red claw crayfish astakine gene was obtained, that is, the predicted red claw crayfish astakine gene, and the band size was about 450 bp.

[0048] (3) Verification of gene transformation of red claw crayfish hematopoietic factor astakine The PCR product obtained after gel recovery was connected to the pMD18-T vector and transformed into DH5α competent cells. Specific experimental operations: ① Gel recovery and purification: Use the EZ-10 Column DNA Gel Extraction Kit (the following reagents are included in the kit) to perform gel recovery and purification on the second amplification product. Use a clean scalpel to cut the agarose gel at 450 bp of the red claw crayfish astakine gene in the second amplification product, and put it into a 1.5 mL centrifuge tube and weigh it. According to the weight of the gel block, add 100 µL of Binding Buffer II for every 100 mg of agarose. Place the centrifuge tube in a 55-65℃ water bath for 5-10 min, mixing occasionally until the gel block is completely dissolved. Transfer the dissolved gel solution to the EZ-10 Column, let it stand for 2 min, centrifuge at 8000 rpm for 1 min, and pour out the liquid in the collection tube. Add 500 μL of Wash Solution, cover the lid and mix by inversion 3-5 times, centrifuge at 10000 rpm for 1 min, pour out the liquid in the collection tube (repeat once); put the EZ-10 Column back into the collection tube, centrifuge at 10000 rpm for 30 s to remove the Wash Solution remaining on the tube wall; place the EZ-10 Column in a clean 1.5mL centrifuge tube, add 20~40 μL of Elution Buffer to the center of the adsorption membrane, warm at 50℃ for 2 min, and centrifuge at 10000 rpm for 1 min to elute the DNA, which is the gel-recovered DNA.

[0049] ② pMD18-T vector connection: Add 1 μL of pMD18-T vector and 4 μL of gel-recovered DNA into a microcentrifuge tube, add 5 μL of Solution I, react at 16°C for 1 h to obtain the DNA to be transformed.

[0050] ③ DH5α competent cell transformation: Take out the DH5α competent cells from -70℃ and quickly put them on ice to thaw; add the DNA to be transformed to 100 μL DH5α competent cells, flick the tube wall to mix, and let it stand on ice for 30 min; heat shock in a 42℃ water bath for 45 s, and quickly put it on ice for 2 min; add 900 μL LB liquid culture medium (without antibiotics) to the centrifuge tube, mix well, and then put it in a shaker at 37℃ and 200 rpm for 45 min to recover the first recovery bacterial solution; take 50 μL of the first recovery bacterial solution and evenly spread it on the plate containing amp r The resistant LB solid culture medium plate was placed upright at 37°C incubator for 10 min. After the first resuscitated bacterial solution was completely absorbed, the plate was inverted for overnight culture, which was the first plate.

[0051] ④ Pick a single clone on the first plate for shaking and use the pMD18-T vector universal primers RV-M (SEQ ID NO.8: 5'-GAGCGGATAACAATTTCACACAGG-3') and M13-47 (SEQ ID NO.9: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3') to perform bacterial solution PCR (i.e., the third PCR reaction). The third PCR reaction system and reaction procedure are the same as above to obtain the third amplification product, and the third amplification product is verified by agarose gel electrophoresis, as shown in FIG. Figure 5 As shown in Figure 1, the bacterial solution of the red claw crayfish astakine gene connected to the pMD18-T vector was obtained, and the band size was about 490 bp. The successfully transformed bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were shown in Figure 1. Figure 6 As shown, it indicates that the astakine gene that has a proliferation-promoting effect on HPT cells has been successfully cloned from the blood cells of red claw crayfish, including the complete CDS sequence of the red claw crayfish astakine gene (Ast) (nucleotide sequence SEQ ID NO.10: 447 bp; amino acid sequence (Ast-pro) SEQ ID NO.11: 148 aa).

[0052] (4) Bioinformatics feature analysis DNAMAN software was used to compare the sequenced cloned red claw crayfish astakine protein sequence with the protein sequence of other known shrimp astakine, and SMART (http: / / smart.emblheidelberg.de / ) online prediction website was used to predict the relevant properties of red claw crayfish astakine protein: functional domain and signal peptide, etc. Figure 7 , Figure 8As shown in the figure, the SMART online prediction website predicts that the N-terminus of astakine protein has an endogenous signal peptide consisting of 20 amino acids, and predicts that the protein has a prokineticin domain, which plays a role in promoting angiogenesis and blood vessel growth in vertebrates. By comparing the astakine protein sequences with those of Pacifastacusleniusculus (PlAst and PlAst 2a), Litopenaeus vannamei (LvAst), Marsupenaeus japonicus (MjAst) and Penaeus monodon (PmAst), it was found that they all contain 10 highly conserved cysteine ​​sites and two amino acid conserved regions (GMCPC and TCQLP).

[0053] The phylogenetic tree was constructed using MEGA 11 software. Fig. 9 As shown in the figure, the red claw crayfish, Chinese mitten crab (Eriocheir sinensis) and Macrobrachium rosenbergii are in different evolutionary roots, indicating that the red claw crayfish is relatively distantly related to the two; the red claw crayfish, Chinese shrimp (Penaeus chinensis), giant tiger prawn (P. monodon), white shrimp (L. vannamei), communication crayfish (P. leniusculus) and Japanese shell shrimp (M. japonicus) are in a common evolutionary root, indicating that the red claw crayfish is relatively closely related to them. In summary, astakine genes have a certain degree of conservatism in species evolution, but judging from their distribution in different evolutionary branches, the red claw crayfish astakine genes have a certain degree of species specificity.

[0054] S3. Construction of the gene expression vector of red claw crayfish (1) Promoter cloning P2 promoter: P2 promoter exists in the infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp. The cloning template comes from the IHHNV genome stored in the laboratory of our research group. According to the IHHNV genome data stored in the NCBI database (GenBank: AF273215.1), the PCR amplification primers P2-F (SEQ ID NO.13: 5'-CCCAAGCTTCTGCGAGCGCTTCGCAGAAA-3') and P2-R (SEQ ID NO.14: 5'-CGCGGATCCGGAATAGCCTCTTCACTCGT-3') of the P2 promoter (nucleotide sequence: SEQ ID NO.12) were designed. The fourth PCR reaction system and reaction procedure were the same as above, and the amplification product of the P2 promoter, i.e., the fourth amplification product, was obtained. The fourth amplification product was verified by agarose gel electrophoresis, as shown in FIG. Fig.10 As shown, a P2 promoter nucleotide sequence band was obtained, which was about 110 bp in size.

[0055] WSV249 promoter: WSV249 （-504 / -1） and WSV249 （-300 / -1） The promoter exists in the white spot syndrome virus (WSSV). The cloning template is derived from the WSSV viral genome. According to the WSSV genome data stored in the NCBI database (GenBank: AF332093.3) and experimental needs, WSV249 was designed. （-504 / -1） Promoter (nucleotide sequence: SEQ ID NO.15) and WSV249 （-300 / -1） Promoter (nucleotide sequence: SEQ ID NO.16) PCR amplification primer: WSV249 （-504 / -1） -F (SEQ ID NO.17: 5'-AGTCCAGTGTGGTGGAATTCTCTCAAAAACTTTTAAAAAATTTTT-3'), WSV249 （-300 / -1） -F (SEQ ID NO.18: 5'-AGTCCAGTGTGGTGGAATTCAGGCGAGTCATGTTTCTTGC-3'), the common downstream primer WSV249-R (SEQ ID NO.19: 5'-CTTCGAACTATTACCATCATGGCTGCGAGAATGGTTTG-3'). The PCR reaction system and reaction procedure were the same as above to obtain WSV249 （-504 / -1） The amplification product of the promoter, i.e., the fifth amplification product, WSV249 （-300 / -1） The amplification product of the promoter is the sixth amplification product. The fifth and sixth amplification products are verified by agarose gel electrophoresis. Fig.11As shown, WSV249 （-504 / -1） The amplified product band of the promoter is about 500 bp, WSV249 （-300 / -1） The amplified product band of the promoter is around 330 bp.

[0056] (2) Construction of pIZ-OpIE2-P2-Ast-P2A-mCherry expression vector pIZ-OpIE2-P2-Ast-P2A-mCherry expression vector: based on the purchased commercial plasmid pIZ-V5-His ( Fig.12 As shown in the figure, the pIZ-V5-His basic vector contains the OpIE2 promoter (SEQ ID NO.20); and the P2 promoter and P2A-mCherry (this fragment comes from the laboratory plasmid template) are double-digested and ligated. （-300 / -1） The promoters are linked in sequence to form a hybrid promoter group OpIE2-P2-WSV249 （-300 / -1）: SEQ ID NO.21. Here, the double restriction primers for P2 promoter are SEQ ID NO.13 and SEQ ID NO.14; the double restriction primers for P2A-mCherry are P2A-mCherry-F (SEQ ID NO.22: 5'-CTAGTCTAGAGGAAGCGGAGCTACTAACTT-3') and P2A-mCherry-R (SEQ ID NO.23: 5'-TCCCCGCGGTTACTTGTACAGCTCGTCCA-3'); the double restriction primers for Ast (astakine) fragment are Ast-6-F (SEQ ID NO.24: 5'-CGCGGATCCATGATGGTAAT-3') and Ast-6-R (SEQ ID NO.25: 5'-CCGGAATTCGAAGAATCCGTAGGAA-3'). The restriction endonucleases used to connect the P2 promoter are Hind Ⅲ and BamHI; the restriction endonucleases used to connect P2A-mCherry are Xba I and Sac Ⅱ. At this time, the intermediate plasmid pIZ-OpIE2-P2-P2A-mCherry is generated. When the intermediate plasmid is used to connect the Ast gene, the restriction endonucleases used to connect the Ast gene are BamH I and EcoR I. The enzymes used for all fragment connections are T4 ligase. Finally, the connection product of the obtained pIZ-OpIE2-P2-Ast-P2A-mCherry plasmid is transformed into DH5α competent cells, plated and selected for single clone shaking culture, and PCR reaction of bacterial solution is carried out in the same way as above to obtain the seventh amplification product. The seventh amplification product is verified by agarose gel electrophoresis, as shown in FIG. Fig.13 As shown in Figure 1, the insertion sequence P2-Ast-P2A-mCherry band is about 1300 bp. The successfully transformed bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing was correct. The pIZ-OpIE2-P2-Ast-P2A-mCherry expression vector is as follows Fig.14 shown.

[0057] Example 2 Using the pIZ-OpIE2-P2-P2A-mCherry intermediate plasmid constructed in Example 1 as a template, construct pIZ-OpIE2-P2-WSV249 （-504 / -1） -Ast-P2A-mCherry expression vector.

[0058] WSV249 （-504 / -1） The promoter and Ast gene were connected to the pIZ-OpIE2-P2-P2A-mCherry intermediate plasmid by homologous recombination, WSV249 （-504 / -1）-Ast The restriction endonucleases used for homologous recombination are EcoR I and XbaI. The primers used for homologous recombination are Ast-7-R (SEQ ID NO.26: 5'-GTAGCTCCGCTTCCTCTAGAGAAGAATCCGTAGGAATTGTCCCCG-3'), SEQ ID NO.17 and SEQ ID NO.19. Finally, the obtained pIZ-OpIE2-P2-WSV249 （-504 / -1） -Ast-P2A-mCherry plasmid homologous recombination product was transformed into DH5α competent cells, plated, single clone was picked for shaking culture, and PCR reaction was performed in the same manner as above to obtain the eighth amplification product. The eighth amplification product was verified by agarose gel electrophoresis, as shown in Fig.15 As shown, the insertion sequence WSV249 （-504 / -1） -Ast band is around 900 bp. The successfully transformed bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing was correct, pIZ-OpIE2-P2-WSV249 （-504 / -1） -Ast-P2A-mCherry expression vector as Fig.16 shown.

[0059] Example 3 Using the pIZ-OpIE2-P2-P2A-mCherry intermediate plasmid constructed in Example 1 as a template, construct pIZ-OpIE2-P2-WSV249 （-300 / -1） -Ast-P2A-mCherry expression vector: WSV249 （-300 / -1） The promoter and Ast gene were connected to the pIZ-OpIE2-P2-P2A-mCherry intermediate plasmid by homologous recombination, WSV249 （-300 / -1） -Ast The restriction endonucleases used for homologous recombination are EcoR I and XbaI, and the primers used for homologous recombination are SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.26. Finally, pIZ-OpIE2-P2-WSV249 was obtained. （-300 / -1） -Ast-P2A-mCherry plasmid, wherein the hybrid promoter group and the Ast gene are connected to form an expression cassette (SEQ ID NO.27), pIZ-OpIE2-P2-WSV249 （-300 / -1） -Ast-P2A-mCherry plasmid homologous recombination product was transformed into DH5α competent cells, plated, single clone was picked and cultured, and PCR reaction was performed in the same manner as above to obtain the ninth amplification product. The ninth amplification product was verified by agarose gel electrophoresis, as shown in Fig.17As shown, the insertion sequence WSV249 （-300 / -1） -Ast band is around 750 bp. The successfully transformed bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing was correct, pIZ-OpIE2-P2-WSV249 （-300 / -1） -Ast-P2A-mCherry expression vector as Fig.18 shown.

[0060] Example 4 The pIZ-OpIE2-P2-Ast-P2A-mCherry plasmids and pIZ-OpIE2-P2-WSV249 verified to be correct in Examples 1, 2 and 3 above were （-504 / -1） -Ast-P2A-mCherry plasmid and pIZ-OpIE2-P2-WSV249 （-300 / -1） -Ast-P2A-mCherry plasmid bacterial solution was mixed with 60% glycerol at a ratio of 2:1 and placed in a -80℃ refrigerator for bacterial solution preservation.

[0061] The plasmid extraction kit of Nanjing Novozyme Biotechnology Co., Ltd. extracts three kinds of plasmids respectively (the reagents used in the following plasmid extractions are all included in the plasmid extraction kit). The specific experimental steps are as follows: ① Place the plasmid containing pIZ-OpIE2-P2-Ast-P2A-mCherry, pIZ-OpIE2-P2-WSV249 （-504 / -1） -Ast-P2A-mCherry plasmid and pIZ-OpIE2-P2-WSV249 （-300 / -1） -Ast-P2A-mCherry plasmids were inoculated into 200 mL LB liquid medium (containing bleomycin 50 μg mL -1 ), culture at 37°C, 200 rpm / min overnight, centrifuge at 8000 × g for 4 min, and discard the supernatant; ② Add 5 mL of column activation solution AB to the center of the Maxi-Plasmid Spin Column with Collection Tube (Maxi-Plasmid Spin Column with Collection Tube), centrifuge at 8000 × g for 1 min, discard the effluent and let stand for later use; ③ Add 10 mL of colorless solution RB, oscillate the suspended bacteria thoroughly to mix the bacterial cells thoroughly; add 10 mL of blue solution LB, gently turn it upside down and mix it 5-8 times to fully lyse the bacteria to form a blue transparent solution, which is the lysis product; ④ Add 10 mL of solution NB1 to the lysate, mix gently for 6-8 times (the color of the supernatant changes from blue to colorless, indicating complete neutralization), until a compact agglomerate is formed, and let stand at room temperature for 5 min; centrifuge at 10,000 × g for 15 min, carefully avoiding the precipitate, pour the supernatant into the Push Filter and push it into a new 50 mL centrifuge tube; ⑤ Add 0.3 times the volume of isopropanol to the filtrate, mix by inversion, transfer the liquid into the centrifuge column several times, centrifuge at 8000×g for 1 min, discard the effluent, and wait until all the liquid passes through the centrifuge column; ⑥ Add 5 mL of WB solution, centrifuge at 8000 × g for 1 min, and discard the effluent (repeat once); centrifuge at 8000 × g for 3 min to completely remove the residual WB, place the centrifuge column in a new 50 mL Collection Tube, open the centrifuge column and place it at room temperature for 5 min to allow the ethanol to evaporate completely; ⑦ Add 1 mL of deionized water to the center of the centrifuge column and let it stand at room temperature for 5 min; centrifuge at 8000 × g for 2 min, elute the DNA and store it at -20°C. （-504 / -1） -Ast-P2A-mCherry plasmid and pIZ-OpIE2-P2-WSV249 （-300 / -1） -Ast-P2A-mCherry plasmid) DNA.

[0062] The above three plasmids were detected by 1.5% agarose gel electrophoresis. The results are as follows: Fig.19 As shown, A is the agarose gel electrophoresis verification of pIZ-OpIE2-P2-Ast-P2A-mCherry expression plasmid (plasmid a) after large-scale extraction, in which the band around 1900 bp indicates the target plasmid; B is pIZ-OpIE2-P2-WSV249 （-504 / -1） -Ast-P2A-mCherry (plasmid b) and pIZ-OpIE2-P2-WSV249 （-300 / -1） -Agarose gel electrophoresis verification after large-scale extraction of Ast-P2A-mCherry expression plasmid (plasmid c) showed that the target plasmid indicator band appeared around 2100 bp.

[0063] Test Example 1 Validation of pIZ-OpIE2-P2-Ast-P2A-mCherry and pIZ-OpIE2-P2-WSV249 in Sf9 cells using TransIT®-Insect Transfection Reagent （-504 / -1） -Ast-P2A-mCherry and pIZ-OpIE2-P2-WSV249 （-300 / -1） -Ast-P2A-mCherry expression efficiency of the three plasmids. The specific experimental steps are as follows: Sf9 cell plating: 10-24 h before transfection with the above three plasmids, discard the culture medium in the Sf9 cell culture flask, add 1 mL of 0.25% trypsin digestion solution to digest the Sf9 cells for 30 s to 1 min at room temperature, and discard the 0.25% trypsin digestion solution when the Sf9 cells shrink and fall off under a microscope, add fresh culture medium and resuspend the Sf9 cells at 2×10 5 The cells were seeded at a density of 100 cells / mL in a 48-well cell culture plate, and the 48-well cell culture plate was placed in a 28° C. incubator for 10-24 h.

[0064] Prepare transfection complexes: vortex TransIT®-Insect Transfection Reagent and let it cool to room temperature. Take 26 μL of Grace serum-free insect cell culture medium and add 260 ng of the above three plasmids respectively. Mix gently to form three DNA dilutions. Add 0.52 μL TransIT®-Insect Transfection Reagent to each of the three DNA dilutions, mix gently, and incubate at room temperature for 15-30 min.

[0065] Distribute the transfection complexes into Sf9 cells in Sf9 insect cell culture medium: add the transfection complexes dropwise to different areas of the cell culture wells to distribute them evenly.

[0066] The electroporated Sf9 cells were placed in a 28°C incubator for further culture, and the fluorescence of mCherry was observed and photographed at 24 h, 48 h, and 72 h after electroporation. The fluorescence observation and statistical results are shown in Fig. 20 and Fig.21 As shown, pIZ-OpIE2-P2-WSV249 （-300 / -1） -Ast-P2A-mCherry expression vector had the highest expression efficiency, with expression efficiencies of 34.87 ± 4.27%, 72.16 ± 2.07% and 77.84 ± 3.21% at 24, 48 and 72 h after plasmid transfection, respectively; followed by pIZ-OpIE2-P2-WSV249（-504 / -1） -Ast-P2A-mCherry expression vector, the expression efficiency at 24, 48 and 72 h after plasmid transfection was 27.22 ± 2.08%, 58.69 ± 4.21% and 61.35% ± 3.63%, respectively; the expression efficiency was the lowest in the pIZ-OpIE2-P2-Ast-P2A-mCherry expression vector, the expression efficiency at 24, 48 and 72 h after plasmid transfection was 25.74 ± 4.18%, 53.27 ± 2.91% and 54.61 ± 4.22%, respectively. In summary, the hybrid promoter OpIE2-P2-WSV249 was successfully constructed and screened based on the pIZ expression plasmid. (-300 / -1) The efficient expression vector driven by PCR is: pIZ-OpIE2-P2-WSV249 (-300 / -1) -Ast-P2A-mCherry, map and construction annotation as Fig. 22 As shown, blue is P2 promoter, green is WSV249 (-300 / -1) Promoter, orange is astakine gene, pink is P2A short peptide, and red is mCherry fluorescent marker gene for connection.

[0067] Test Example 2 The electroporation transfection conditions in Test Example 1 were further optimized, and a gradient setting experiment was conducted on the voltage conditions.

[0068] The constant conditions were set as electric pulse duration of 300 μs, 2 pulses, 10 s pulse interval, and serum-free and double-antibody-free OCCM medium as electroporation buffer. Nine voltage gradient experimental groups were set up with crayfish HPT cells that were not electroporated as controls. The voltages of these nine experimental groups were 50, 100, 200, 300, 400, 500, 600, 800, and 1000 V, respectively. Calcein-AM cell activity was performed on crayfish HPT cells cultured for 1 day after electroporation to compare the survival rate of crayfish HPT cells in each group.

[0069] The results are as follows Fig.23 , 24As shown in the figure, the cell survival rates at voltages of 0, 50, 100, 200, 300, 400, 500, 600, 800 and 1000 V were 99.18 ± 1.12%, 85 ± 7.23%, 86 ± 4.41%, 87.36 ± 4.45%, 56.28 ± 1.14%, 43.49 ± 3.29%, 40.47 ± 6.05%, 9.85 ± 5.48%, 8.55 ± 3.16% and 4.57 ± 1.41%, respectively. It can be seen that when the voltage is ≤ 200 V, the survival rate of crayfish HPT cells is about 87.36 ± 4.45%; when the voltage is > 200 V, the cell survival rate significantly drops to below 56.28 ± 1.14%. Therefore, 200 V is the optimal voltage condition for crayfish plasmid electrofection.

[0070] Test Example 3 The electroporation transfection conditions in Test Example 1 were further optimized, and a gradient setting experiment was conducted on the electric pulse conditions.

[0071] The constant conditions were set as 200 V voltage (optimal voltage obtained in Test Example 2), 2 pulses, 10 s pulse interval, and serum-free and double-antibody-free medium as electrotransfection buffer. Using crayfish HPT cells without electrotransfection as control, 8 electric pulse duration gradient experimental groups were set up, and the electric pulse durations of these 8 experimental groups were: 1, 5, 10, 15, 20, 25, 30 and 40 ms. Calcein-AM cell activity was performed on crayfish HPT cells cultured for 1 day after electrotransfection to compare the survival rate of crayfish HPT cells in each group.

[0072] The results are as follows Fig.25 , 26 As shown in the figure, the cell survival rates of the electric pulse durations of 0, 1, 5, 10, 15, 20, 25, 30 and 40 ms were 99.4 ± 0.61%, 98.27 ± 2.06%, 98.8 ± 0.87%, 95.14 ± 1.82%, 79.65 ± 3.54%, 54.4 ± 5.04%, 53.01 ± 2.75%, 52.3 ± 5.38% and 36.1 ± 3.42%, respectively. Taking 50% cell survival rate as the judgment standard, it can be seen that when the electric pulse duration is ≤ 30 ms, the cell survival rate is around 52.3 ± 5.38%; when >30 ms, the survival rate of crayfish HPT cells significantly drops to below 36.1 ± 3.42%, so 30 ms is selected as the optimal electric pulse duration for electrotransfection.

[0073] Test Example 4 The electroporation transfection conditions in Test Example 1 were further optimized, and experiments were performed using different electroporation buffers (CytoMix, HEPES).

[0074] Constant conditions were set to optimize the electrotransfection buffer in the electrotransfection experiment. The constant conditions were set as 200 V voltage, 2 pulses, and 10 s pulse interval. Under the conditions of 10, 20, 30, and 40 ms electric pulse duration, the effects of CytoMix and HEPES electrotransfection buffers were compared with crayfish HPT cells that were not electrotransfected as controls. Calcein-AM cell activity was performed on crayfish HPT cells cultured for 1 day after electrotransfection to compare the survival rate of crayfish HPT cells in each group.

[0075] The results are as follows Fig. 27 , 28 As shown, the cell viability using CytoMix buffer at electric pulse durations of 0, 10, 20, 30 and 40 ms was 99.23 ± 0.89%, 87.31 ± 5.22%, 75.48 ± 1.34%, 60.1 ± 6.57% and 25.78 ± 3.28%, respectively; the cell viability using HEPES buffer at electric pulse durations of 0, 10, 20, 30 and 40 ms was 98.97 ± 0.73%, 90.45 ± 7.91%, 79.25 ± 4.57%, 65.13 ± 2.13% and 51.1 ± 3.85%, respectively. Taking 50% cell survival rate as the judgment standard, it can be seen that when the CytoMix electroporation buffer is used, the cell survival rate is about 60.1 ± 6.57% when the electric pulse duration is 30 ms; when the HEPES electroporation buffer is used, the cell survival rate is about 51.1 ± 3.85% when the electric pulse duration is 40 ms. However, in the subsequent electroporation experiment of the crayfish gene efficient expression vector, it was found that the plasmid could not be transferred into the crayfish HPT cells (no fluorescence) using the HEPES electroporation buffer. Therefore, CytoMix was selected as the best electroporation buffer for electroporation.

[0076] Test Example 5 TransIT®-Insect Transfection Reagent was used to chemically transfect crayfish HPT cells, with the ratios of 4 transfection reagents / DNA being 1:1, 2:1, 3:1 and 4:1. The specific operation is as follows: crayfish HPT cells were evenly inoculated in a 48-well cell culture plate, placed in an environment of 28°C and 3% CO2, and cultured for 24 hours until the density of crayfish HPT cells reached about 90%. The transfection complex was prepared using the transfection method in Test Example 1 and added to the corresponding cell wells. After 6 hours, fresh culture medium was replaced to reduce the toxicity of the reagent, and fluorescent images were taken at 24 hours, 48 ​​hours and 72 hours after transfection using an inverted fluorescence microscope, and the transfection efficiency of transfection reagents with different ratios was counted.

[0077] The results are as follows Fig.29 , 30 As shown in the figure, at 24 h after chemical transfection, the transfection rates of the transfection reagent / DNA ratios of 1:1, 2:1, 3:1 and 4:1 were 3.27 ± 0.41%, 8.45 ± 0.45%, 1.69 ± 0.64% and 1.54 ± 0.43%, respectively; at 48 h after chemical transfection, the corresponding transfection rates were 7.72 ± 0.35%, 10.28 ± 0.42%, 1.74 ± 0.61% and 1.63 ± 0.40%, respectively; at 72 h after chemical transfection, the transfection rates were 6.3 ± 0.77%, 13.18 ± 0.31%, 1.94 ± 0.31% and 1.8 ± 0.42%, respectively. Therefore, when the transfection reagent / DNA ratio was 2:1, the transfection efficiency reached the highest, and increasing the ratio on this basis would be accompanied by an increase in cytotoxicity.

[0078] Test Example 6 According to the results of test examples 2, 3 and 4, it can be determined that the optimal electroporation conditions for crayfish HPT cells are: voltage 200 V, electric pulse time 30 ms, electric pulse number 2 times, electric pulse interval 10 s, and electroporation buffer CytoMix.

[0079] The pIZ-OpIE2-P2-WSV249 obtained in Example 3 was transfected by chemical transfection and the above-mentioned optimal electrofection conditions. (-300 / -1) -Ast-P2A-mCherry plasmid was transfected into crayfish HPT cells cultured in 2D in vitro, and the transfection method was the same as described in Test Example 1.

[0080] The results are as follows Fig.31 , 32As shown, electrofection method: the plasmid expression efficiency at 24, 48 and 72 h after plasmid transfection was 2.67 ± 0.52%, 5.21 ± 0.37% and 2.13 ± 0.24%, respectively; chemical transfection method: the plasmid expression efficiency at 24, 48 and 72 h after plasmid transfection was 8.45 ± 0.45%, 10.28 ± 0.33% and 13.18 ± 0.51%, respectively.

[0081] The mCherry gene was further subjected to semi-quantitative RT-PCR detection using PrimeScript™ RT reagentKit and Premix Taq™, and the mCherry primers were designed as mCherry-F: 5'-ATGGTGAGCAAGGGCGAG-3' (SEQ ID NO.28) and mCherry-R: 5'-TTACTTGTACAGCTCGTCCA-3' (SEQ ID NO.29); the internal reference gene β-actin primers used were Cq-β-actin-F: 5'-ATGTGTGACGAAGAGGAGCTG-3' (SEQ ID NO.30) and Cq-β-actin-R: 5'-TTAGAAGCACTTGCGGTGGAC-3' (SEQ ID NO.31), the number of cycles for semi-quantitative PCR amplification of mCherry was 26, and other PCR reaction systems and PCR reaction procedures were the same as described in Example 1.

[0082] The results are as follows Fig.31 , 33 As shown in Figures 34, electrotransfection method: the expression of mCherry gene can be detected at 24 h, the expression level gradually increases at 48 h, and the expression level no longer increases after 72 h. In addition, as the cell culture time after electrotransfection increases, the cell state becomes worse and worse, and the cells die in about a week. Chemical transfection method: the expression of mCherry gene can be detected at 24 h, and the expression level gradually increases at 48 and 72 h. The growth of cells after transfection is not affected and can still be cultured continuously for more than 3 months.

[0083] Test Example 7 The pIZ-OpIE2-P2-WSV249 obtained in Example 3 was transfected by electrofection and chemical transfection. (-300 / -1) -Ast-P2A-mCherry plasmid was transfected into crayfish HPT cell spheres cultured in vitro in 3D. Fig.35 , 36As shown, electrofection method: the plasmid expression efficiency at 24, 48 and 72 h after plasmid transfection was 1 ± 0.16%, 1.04 ± 0.08% and 1.68 ± 0.13%, respectively; chemical transfection method: the plasmid expression efficiency at 24, 48 and 72 h after plasmid transfection was 1.19 ± 0.11%, 2.12 ± 0.16% and 2.56 ± 0.19%, respectively.

[0084] The mCherry gene was further tested by semi-quantitative RT-PCR. The semi-quantitative RT-PCR test steps were the same as those in Test Example 6. The results were as follows: Fig.35 , 37 As shown in Figures 38, electrotransfection method: the expression of mCherry gene can be detected at 24 h, and the expression levels gradually increase at 48 and 72 h. Moreover, with the increase of cell culture time after electrotransfection, the cells cannot grow in clusters well, and the cell fragments increase in about a week. Chemical transfection method: the expression of mCherry gene can be detected at 24 h, and the expression levels gradually increase at 48 and 72 h.

[0085] Test Case 8 Astakine gene overexpression and HPT cell proliferation detection in red claw crayfish Semi-quantitative RT-PCR technique was used to evaluate the effect of astakine gene overexpression on the proliferation of crayfish HPT cells, using proliferating cell nuclear antigen (PCNA) gene expression as a marker for crayfish HPT cell proliferation combined with EdU proliferation detection experiments.

[0086] The detailed steps are as follows: HPT cells cultured in vitro for 4 days (the cell proliferation state is the best at this stage) were selected for plasmid chemical transfection. The specific primers for astakine were designed as CqAst-7-F: 5'-ATGATGGTAATAGTTCGAAGTGTGT-3' (SEQ ID NO.32) and CqAst-7-R: 5'-TCAGAAGAATCCGTAGGAATTGTCC-3' (SEQ ID NO.33); the specific primers for PCNA were CqPCNA-F: 5'-ATGTTTGAGGCTCGTCTT-3' (SEQ ID NO.34) and CqPCNA-R: 5'-AGGTGATAGTGGAGTGGC-3' (SEQ ID NO.35). 72 hours after chemical transfection of red claw crayfish HPT cells, the expression levels of astakine and PCNA genes were detected and the cell proliferation rate was calculated.

[0087] The results are as follows Fig.39 , 40As shown, the transfection efficiency of astakine gene in crayfish HPT cells reached 18.01 ± 1.83%. Compared with the control group (NT represents the non-transfected negative control group, i.e., cells that were not transfected), astakine transgenic HPT cells showed more vigorous mitotic activity 72 h after transfection. Most of the cells in the division state were not only the cells with high astakine expression themselves, but also the cells nearby. This local proliferation effect was particularly significant. Quantitative detection found that the cell proliferation rates of the non-transfected (NT) and mock-transfected (MT) control groups were 19.47 ± 2.07% and 18.76 ± 0.74%, respectively; in contrast, the cell proliferation rate of the transfected experimental group (PT) reached 25.69 ± 1.19%, showing a significant increase in cell proliferation activity, with the proliferation rate increasing by about 7% ( Fig.40 ). This was further verified by semi-quantitative PCR ( Fig.41 , 42 ), and found that on the third day after transfection, the expression levels of astakine and proliferating cell nuclear antigen (PCNA) genes in the experimental group were significantly higher than those in the control group.

[0088] It can be concluded that the present invention has successfully developed a hybrid promoter group (OpIE2-P2-WSV249) suitable for 2D and 3D cultured red claw crayfish hematopoietic tissue cells by modifying shrimp virus promoters and optimizing transfection methods. （-300 / -1） ) and transgenic expression vector (pIZ-OpIE2-P2-WSV249 (-300 / -1)-Ast-P2A-mCherry). The coding sequence of the hematopoietic factor astakine of red claw crayfish was cloned and inserted into the transgenic expression vector. The overexpression of astakine gene was achieved under the conditions of electroporation transfection with voltage of 200 V, electric pulse time of 30 ms, electric pulse number of 2 times, electric pulse interval of 10 s, electroporation buffer of CytoMix and chemical transfection with transfection reagent / DNA ratio of 2:1. Among them, the expression efficiency under chemical transfection conditions reached about 18%, the proliferation rate of transgenic cells increased by about 7%, and the healthy and stable growth was maintained for more than three months. For other freshwater crayfish, such as the family of Pseudocanidae or the family of Crayfish, the family of Pseudocanidae includes the giant crayfish of Gu, the hairy crayfish, the smooth crayfish or the destroyer crayfish, and the family of Crayfish includes the Procambarus clarkii, the Northeast crayfish, the Korean crayfish or the dwarf giant crayfish, which are also suitable for 2D or 3D cell culture systems, and are also suitable for the overexpression of astakine gene by this transgenic vector. Therefore, the present invention provides a reliable transgenic vector for shrimp gene function research. The disclosed transgenic vector of freshwater crayfish hematopoietic tissue cells can support the expression and function research of exogenous genes, provide experimental materials for shrimp virus detection for non-diagnostic purposes, and ensure the healthy growth of crayfish hematopoietic tissue cells after transgenic operation, laying a foundation for the development of shrimp immortalized cell lines.

[0089] 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.

[0090] 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 promoter, characterized in that The sequence is identical to SEQ ID NO.12, SEQ ID NO.16 or SEQ ID NO.20 or has 80%, 85%, 90%, 95% or 98% homology.

2. Use of the promoter as claimed in claim 1 in constructing a transgenic vector for hematopoietic tissue cells of freshwater crayfish.

3. A transgenic vector for freshwater crayfish hematopoietic tissue cells, characterized in that: The transgenic vector comprises an expression cassette, and the expression cassette comprises a hybrid promoter group; the hybrid promoter group is composed of SEQ ID NO.20, SEQ ID NO.12 and SEQ ID NO.16 sequences connected in sequence.

4. The transgenic vector of freshwater crayfish hematopoietic tissue cells according to claim 3, characterized in that: The expression cassette is composed of the hybrid promoter group and Ast Gene connection structure; Ast The nucleotide sequence of the gene is shown in SEQ ID NO.

10.

5. The transgenic vector of freshwater crayfish hematopoietic tissue cells according to claim 3, characterized in that: The transgenic vector is pIZ-OpIE2-P2-WSV249 （-300 / -1） - Ast -P2A- mCherry .

6. A method for constructing a transgenic vector for hematopoietic tissue cells of freshwater crayfish, characterized in that: The following steps are involved: Step A: Obtaining freshwater crayfish hematopoietic factors Ast Gene; Step B: P2 promoter and WSV249 （-300 / -1） promoter for cloning; using pIZ-V5-His as the basic vector, and sequentially cloning the P2 promoter and P2A- mCherry The intermediate vector pIZ-OpIE2-P2-P2A- mCherry ; Step C: Place the WSV249 （-300 / -1） Promoter and the freshwater crayfish hematopoietic factor Ast The gene was sequentially connected to the intermediate vector pIZ-OpIE2-P2-P2A- mCherry The transgenic vector pIZ-OpIE2-P2-WSV249 was obtained. （-300 / -1） - Ast -P2A- mCherry .

7. The method for constructing a freshwater crayfish hematopoietic tissue cell transgenic vector according to claim 6, characterized in that: The step A comprises: Step A-1: ​​culturing freshwater crayfish hematopoietic tissue cells using a 2D or 3D culture system to obtain freshwater crayfish hematopoietic tissue cells; Step A-2: Extract RNA and prepare cDNA from the hematopoietic tissue cells of the freshwater crayfish, and use the prepared cDNA as a template to perform freshwater crayfish hematopoietic factor Ast Gene cloning, obtaining the Ast The complete CDS sequence of the gene.

8. Use of the transgenic vector as claimed in any one of claims 3 to 5 in the culture of freshwater crayfish hematopoietic cells, the establishment of hematopoietic cell lines, the overexpression of exogenous genes in hematopoietic cells, the proliferation of hematopoietic cells, the research on shrimp antiviral genes, or the detection of shrimp viruses for non-diagnostic and therapeutic purposes.

9. The use according to claim 8, characterized in that: The transgenic vector is transferred into the hematopoietic tissue cells of freshwater crayfish so that the hematopoietic tissue cells of freshwater crayfish can grow stably for no less than 3 months; the exogenous gene overexpression efficiency of the hematopoietic tissue cells of freshwater crayfish is 18.01±1.83%; the proliferation rate of the hematopoietic tissue cells of freshwater crayfish is 25.69±1.19%.

10. The use according to claim 8, characterized in that: Using electrofection or chemical transfection to achieve overexpression of exogenous genes in freshwater crayfish hematopoietic tissue cells; The operating parameters of the electrotransfection method are: voltage 200 V, electric pulse time 30 ms, number of electric pulses 2 times, electric pulse interval 10 s, and electrotransfection buffer is CytoMix; The operating parameters of the chemical transfection method are: the ratio of transfection reagent to plasmid DNA in the transfection complex is 2:1; the time for incubating the freshwater crayfish hematopoietic tissue cells with the transfection complex is ≤ 6 h.

11. The use according to claim 8, characterized in that: The freshwater crayfish belongs to the family of Pseudocranidae or Cheracidia; the family of Pseudocranidae includes red claw crayfish, giant crayfish, hairy crayfish, smooth crayfish or destroyer crayfish; the family of Cheracidia includes Procambarus clarkii, Northeastern crayfish, Korean crayfish or dwarf giant crayfish.

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