Recombinant cFGF2 Protein and Culture Medium for Avian Primordial Germ Cells and Their Applications

Abstract

The present invention relates to a recombinant cFGF2 protein and a culture medium for avian primordial germ cells and its application, belonging to the technical field of molecular biology. The recombinant cFGF2 protein of the present invention has one or more mutations at the 8th, 31st, 34th, 51st, 55th, 57th, 62nd, 81st, 95th, 97th, 99th, 112th, and 124th positions of the amino acid sequence of the FGF2 protein; the amino acid sequence of the FGF2 protein is as shown in SEQ ID NO.1. The recombinant cFGF2 protein of the present invention has FGF2 biological activity and thermal stability, and can maintain the normal growth and proliferation, self-renewal of avian PGCs under in vitro culture conditions, as well as the development status in recipient embryos and the ability to colonize the genital ridges, and can be used for the long-term culture of avian PGCs and gene editing of birds.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly to recombinant cFGF2 protein, a culture medium for avian primordial germ cells, and their applications. Background Art

[0002] Gene editing technology refers to the technology of targeting and modifying the genes of organisms, thereby performing heritable modification of the traits of organisms, so that individuals can obtain new phenotypic characteristics or functions. Currently, it has been widely practiced in multiple fields and species. However, due to the unique reproductive and developmental patterns of birds, the practice of gene editing technology in birds has been progressing slowly. Primordial germ cells (PGCs) are the common precursor cells of both male and female gametes. They migrate to the genital ridge via blood vessels and colonize during the early stage of development, and can differentiate into sperm or eggs, with the potential for germline chimerism. Based on the maturity of the CRISPR / Cas9 gene editing technology, culturing and editing avian PGCs in vitro and then injecting them into recipient chicken embryos has become a feasible way of avian gene editing. However, there have always been problems such as great difficulty in culturing and low success rate of establishing cell lines in the in vitro culture of avian PGCs.

[0003] In the in vitro culture system of avian PGCs, the combined use of multiple growth factors can better maintain the cell proliferation and undifferentiated state. However, the binding ability and effect of the human-derived rhFGF2 protein selected in the prior art are different from those of chicken-derived proteins, which has potential adverse effects as a growth factor; the key growth factor fibroblast growth factor 2 (FGF2) in the in vitro culture system has very poor thermal stability, which is extremely likely to cause the differentiation or death of avian PGCs. Therefore, it is necessary to frequently replace the in vitro culture system, which not only affects the cell state of avian PGCs but also additionally increases the culture cost, thus limiting the establishment and popularization of avian gene editing technology. Summary of the Invention

[0004] The purpose of the present invention is to provide a recombinant cFGF2 protein, a culture medium for avian primordial germ cells, and their applications, so as to solve the problems of great difficulty in culturing avian PGCs in vitro, high culture cost, poor cell state of avian PGCs obtained by in vitro culture, and low success rate of establishing cell lines in the prior art.

[0005] To achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0006] The present invention provides a recombinant cFGF2 protein, wherein the recombinant cFGF2 protein comprises one or more mutations at the 8th, 31st, 34th, 51st, 55th, 57th, 62nd, 81st, 95th, 97th, 99th, 112th, and 124th positions of the amino acid sequence of the FGF2 protein;

[0007] The amino acid sequence of the FGF2 protein is as shown in SEQ ID NO.1.

[0008] Preferably, the recombinant cFGF2 protein has mutations at the 8th, 31st, 34th, 51st, 55th, 57th, 62nd, 81st, 95th, 97th, 99th, 112th, and 124th positions of the amino acid sequence of the FGF2 protein;

[0009] The amino acid sequence of the recombinant cFGF2 protein is as shown in SEQ ID NO.3.

[0010] Preferably, the recombinant cFGF2 protein has mutations at the 31st, 81st, 99th, and 112th positions of the amino acid sequence of the FGF2 protein;

[0011] The amino acid sequence of the recombinant cFGF2 protein is as shown in SEQ ID NO.5.

[0012] Preferably, the mutations include mutating the 8th amino acid from serine to glycine, the 31st amino acid from aspartic acid to glutamic acid, the 34th amino acid from arginine to leucine, the 51st amino acid from arginine to lysine, the 55th amino acid from valine to threonine, the 57th amino acid from glutamic acid to aspartic acid, the 62nd amino acid from histidine to phenylalanine, the 81st amino acid from threonine to isoleucine, the 95th amino acid from leucine to tyrosine, the 97th amino acid from leucine to isoleucine, the 99th amino acid from cysteine to isoleucine, the 112th amino acid from serine to glutamic acid, and the 124th amino acid from serine to proline.

[0013] The present invention also provides the use of the recombinant cFGF2 protein in in vitro culture and / or highly efficient proliferation of avian primordial germ cells.

[0014] The present invention also provides the use of the recombinant cFGF2 protein in the preparation of a product for in vitro culture and / or highly efficient proliferation of avian primordial germ cells.

[0015] The present invention also provides a cell culture composition, which comprises the recombinant cFGF2 protein and an acceptable carrier.

[0016] The present invention also provides a culture medium for avian primordial germ cells, and the culture medium for avian primordial germ cells comprises the recombinant cFGF2 protein described above.

[0017] Preferably, the solvent of the culture medium for avian primordial germ cells is water, and it comprises components with the following concentrations: 600 - 750 mL / L of DEME medium, 5 - 15 mL / L of alanyl-glutamine dipeptide, 8 - 12 mL / L of non-essential amino acids, 8 - 12 mL / L of penicillin / streptomycin, 0.05 - 0.3 mmol / L of β-mercaptoethanol, 5 - 15 mL / L of nucleosides, 0.1 - 0.4 mmol / L of sodium pyruvate, 100 - 300 mg / L of sodium heparin, 0.1 - 0.5 mmol / L of calcium chloride, 1 - 4 g / L of ovalbumin, 5 - 20 mg / L of ovotransferrin, 10 - 30 mL / L of B-27 supplement, 10 - 50 μg / L of human activin A, 4 - 50 μg / L of recombinant cFGF2 protein;

[0018] The recombinant cFGF2 protein is the recombinant cFGF2 protein described above.

[0019] The present invention also provides the use of the composition for cell culture and the culture medium for avian primordial germ cells described above in in vitro culturing and / or highly efficiently proliferating avian primordial germ cells.

[0020] The present invention has the following technical effects and advantages:

[0021] (1) The present invention uses a protein thermal stability prediction model of molecular simulation and artificial intelligence to perform site-directed mutagenesis on the FGF2 protein, and finds that only the D31E mutation can improve the thermal stability of the FGF2 protein, and the combined mutations of D31E, S81I, C99I, and S112E can further increase the thermal stability of the FGF2 protein, while the combined mutations of S8G, D31E, R34L, R51K, V55T, E57D, H62F, S81I, L95Y, L97I, C99I, S112E, and S124P can greatly improve the thermal stability of the FGF2 protein; thus, the present invention points out that any two or more mutations among S8G, D31E, R34L, R51K, V55T, E57D, H62F, S81I, L95Y, L97I, C99I, S112E, and S124P on the FGF2 protein can increase the thermal stability of the FGF2 protein, and D31E, S81I, C99I, and S112E are mutations closely related to the thermal stability of the FGF2 protein;

[0022] (2) The recombinant cFGF2 protein prepared and purified by the present invention using a prokaryotic protein expression system can act on the FGFR receptor of avian PGCs, activate the intracellular MAPK / ERK signaling pathway, indirectly promote the signal transduction of transforming growth factor β1 and activin, maintain the efficient proliferation, survival, self-renewal and pluripotency of avian PGCs, and at the same time does not affect the ability of avian PGCs to colonize the genital ridge, which is helpful for gene editing of birds. It still has long-term stable high activity after transportation, long-term low-temperature preservation and short-term room-temperature preservation, and can be used as a growth factor for in vitro culture of avian PGCs;

[0023] (3) Compared with rhFGF2 and FGF2 proteins, the recombinant cFGF2 protein of the present invention has stronger thermal stability and receptor affinity, stronger activation effect on the intracellular MAPK / ERK signaling pathway and more stable proliferation-promoting effect. Therefore, it can replace rhFGF2 and FGF2 proteins and be applied to the in vitro culture of avian PGCs. Brief Description of the Drawings

[0024] Figure 1 The SDS-PAGE electrophoresis detection results under different IPTG induction concentrations, where A is the SDS-PAGE electrophoresis detection results of the negative control under different IPTG induction concentrations, B is the SDS-PAGE electrophoresis detection results of the pET-28a-FGF2 bacterial solution under different IPTG induction concentrations, and C is the SDS-PAGE electrophoresis detection results of the pET-28a-cFGF2-M13 bacterial solution under different IPTG induction concentrations;

[0025] Figure 2 The SDS-PAGE electrophoresis detection results under different IPTG induction temperatures, where A is the SDS-PAGE electrophoresis detection results at 16 °C, B is the SDS-PAGE electrophoresis detection results at 25 °C, C is the SDS-PAGE electrophoresis detection results at 30 °C, and D is the SDS-PAGE electrophoresis detection results at 37 °C;

[0026] Figure 3 The preliminary purification results of each protein, where A is the preliminary purification result of the negative control, B is the preliminary purification result of the FGF2 protein, and C is the preliminary purification result of the cFGF2-M13 protein;

[0027] Figure 4 The activity measurement results of each protein, where A is the activity measurement result of rhFGF2, B is the activity measurement result of the FGF2 protein, and C is the activity measurement result of the cFGF2-M13 protein;

[0028] Figure 5The Western blot detection results of the thermal stabilities of various proteins are shown, where A is the Western blot detection result of rhFGF2, B is the Western blot detection result of FGF2 protein, and C is the Western blot detection result of cFGF2-M13 protein;

[0029] Figure 6 The results of the luciferase reporter system functional analysis of the thermal stabilities of various proteins are shown, where A is the result of the luciferase reporter system functional analysis of rhFGF2, and B is the result of the luciferase reporter system functional analysis of cFGF2-M13 protein;

[0030] Figure 7 The identification results of the in vitro proliferation effects of various proteins on male PGCs;

[0031] Figure 8 The identification results of the colonization ability of cFGF2-M13 protein on the PGC-mCherry cell line. Specific implementation mode

[0032] The present invention provides a recombinant cFGF2 protein, and the recombinant cFGF2 protein has mutations at one or more of the 8th, 31st, 34th, 51st, 55th, 57th, 62nd, 81st, 95th, 97th, 99th, 112th, and 124th positions of the amino acid sequence of FGF2 protein;

[0033] The amino acid sequence of the FGF2 protein is as shown in SEQ ID NO.1.

[0034] In the present invention, the FGF2 protein further includes an FGF2 polypeptide functional fragment having FGF2 activity;

[0035] The FGF2 polypeptide functional fragment is an FGF2 polypeptide functional fragment having a sequence identity of more than 85% with the amino acid sequence shown in SEQ ID NO.1.

[0036] In the present invention, the FGF2 protein further includes an FGF2 polypeptide functional fragment with a C-terminal deletion and / or an N-terminal deletion.

[0037] In the present invention, the recombinant cFGF2 protein has mutations at the 8th, 31st, 34th, 51st, 55th, 57th, 62nd, 81st, 95th, 97th, 99th, 112th, and 124th positions of the amino acid sequence of FGF2 protein;

[0038] The amino acid sequence of the recombinant cFGF2 protein is as shown in SEQ ID NO.3;

[0039] The recombinant cFGF2 protein is the cFGF2-M13 protein.

[0040] In the present invention, the recombinant cFGF2 protein has mutations at positions 31, 81, 99, and 112 of the amino acid sequence of the FGF2 protein;

[0041] The amino acid sequence of the recombinant cFGF2 protein is as shown in SEQ ID NO.5;

[0042] The recombinant cFGF2 protein is the cFGF2-M4 protein.

[0043] In the present invention, the mutations include the mutation of the 8th amino acid from serine to glycine (S8G), the 31st amino acid from aspartic acid to glutamic acid (D31E), the 34th amino acid from arginine to leucine (R34L), the 51st amino acid from arginine to lysine (R51K), the 55th amino acid from valine to threonine (V55T), the 57th amino acid from glutamic acid to aspartic acid (E57D), the 62nd amino acid from histidine to phenylalanine (H62F), the 81st amino acid from threonine to isoleucine (S81I), the 95th amino acid from leucine to tyrosine (L95Y), the 97th amino acid from leucine to isoleucine (L97I), the 99th amino acid from cysteine to isoleucine (C99I), the 112th amino acid from serine to glutamic acid (S112E), and the 124th amino acid from serine to proline (S124P).

[0044] The present invention also provides the use of the recombinant cFGF2 protein in culturing in vitro and / or highly proliferating avian primordial germ cells.

[0045] The present invention also provides the use of the recombinant cFGF2 protein in the preparation of a product for culturing in vitro and / or highly proliferating avian primordial germ cells.

[0046] The present invention also provides a cell culture composition, and the cell culture composition includes the recombinant cFGF2 protein and an acceptable carrier.

[0047] In the present invention, the cell culture composition further includes one or more of activin A, bone morphogenetic protein 4 (BMP4), and insulin.

[0048] The present invention also provides an avian primordial germ cell culture medium, and the avian primordial germ cell culture medium includes the recombinant cFGF2 protein.

[0049] In the present invention, the avian primordial germ cell culture medium uses water as a solvent and comprises components at the following concentrations: DEME medium 600 - 750 mL / L, preferably 700 mL / L; L-alanyl-L-glutamine (GlutaMAX) 5 - 15 mL / L, preferably 10 mL / L; non-essential amino acids (NEAA) 8 - 12 mL / L, preferably 10 mL / L; penicillin / streptomycin (Pen / Strep) 8 - 12 mL / L, preferably 10 mL / L; β-mercaptoethanol 0.05 - 0.3 mmol / L, preferably 0.1 mmol / L; nucleosides 5 - 15 mL / L, preferably 10 mL / L; sodium pyruvate 0.1 - 0.4 mmol / L, preferably 0.2 mmol / L; heparin sodium 100 - 300 mg / L, preferably 200 mg / L; calcium chloride 0.1 - 0.5 mmol / L, preferably 0.15 mmol / L; ovalbumin 1 - 4 g / L, preferably 2 g / L; ovotransferrin 5 - 20 mg / L, preferably 10 mg / L; B-27 supplement 10 - 30 mL / L, preferably 20 mL / L; human activin A 10 - 50 μg / L, preferably 25 μg / L; recombinant cFGF2 protein 4 - 50 μg / L, preferably 20 μg / L;

[0050] The recombinant cFGF2 protein is the aforementioned recombinant cFGF2 protein.

[0051] In the present invention, the initial concentration of L-alanyl-L-glutamine is 50 - 200 times, preferably 100 times; the initial concentration of non-essential amino acids is 50 - 200 times, preferably 100 times; the initial concentration of penicillin / streptomycin is 50 - 200 times, preferably 100 times; the initial concentration of nucleosides is 50 - 200 times, preferably 100 times; the initial concentration of B-27 supplement is 25 - 75 times, preferably 50 times.

[0052] The present invention also provides the use of the aforementioned cell culture composition and the aforementioned avian primordial germ cell culture medium in vitro culturing and / or highly efficiently proliferating avian primordial germ cells.

[0053] In the present invention, the birds include chickens, ducks, geese, zebra finches, parrots or pigeons.

[0054] In the present invention, the avian primordial germ cells (PGCs) are derived from the blood and / or genital ridges of avian embryos.

[0055] The following detailed description is given of the technical solutions provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0056] In the present invention, Escherichia coli BL21 strain (product number: B528419) was purchased from Sangon Biotech (Shanghai) Co., Ltd., HEK293 cell line (product number: STCC10305P) was purchased from Wuhan Zishan Biotechnology Co., Ltd., HEK293-cFGFR1 cell line, PGC-mCherry cell line, and male PGCs (isolated from male Roman chickens) were from Sichuan University. Camellia chicken embryos were purchased from Pengzhou Quanfang Poultry Industry Development Co., Ltd. The research-grade fluorescence stereomicroscope (ZEISS SteREO Discovery.V12) was purchased from Carl Zeiss AG in Germany;

[0057] In the present invention, the pET-28a vector was purchased from Novagen in Germany, the pGL3-SRE-luciferase reporter plasmid was purchased from Promega in the United States. His-tagged protein purification magnetic beads (HisSep Ni-NTA MagBeads, product number: 20561ES08), and BCA protein concentration assay kit (Enhanced BCA Protein Quantification Kit, product number: 20201ES86) were purchased from Yeasen Biotech Co., Ltd. in Shanghai;

[0058] In the present invention, rhFGF2 (Recombinant Human FGF2(157aa) Protein, product number: 234-FSE) was purchased from R&D Systems in the United States. DMEM medium (high glucose, without glutamine, without calcium), GlutaMAX, non-essential amino acids (NEAA), penicillin / streptomycin (Pen / Strep), and B-27 supplement were purchased from Gibco in the United States. β-mercaptoethanol, sodium pyruvate, heparin sodium, calcium chloride, ovalbumin, and ovotransferrin were purchased from Sigma in Germany. Nucleosides were purchased from Merck in Germany. Human activin A was purchased from Peprotech in the United States.

[0059] Example 1: Prokaryotic expression of FGF2 protein and cFGF2-M13 protein

[0060] (1) Plasmid construction: Based on the amino acid sequence of wild-type chicken (Gallus gallus) FGF2 protein obtained from NCBI (shown as SEQ ID NO.1, see NCBI GeneBank NO: NP990764.1), Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to perform combinatorial mutations of S8G, D31E, R34L, R51K, V55T, E57D, H62F, S81I, L95Y, L97I, C99I, S112E, and S124P on the FGF2 protein and gene synthesis to obtain the nucleotide sequence encoding the recombinant cFGF2 protein (shown as SEQ ID NO.4), and the recombinant cFGF2 protein translated therefrom was named cFGF2-M13 protein (shown as SEQ ID NO.3). Referring to the operation instructions of the pET-28a vector, recombinant plasmids pET-28a-FGF2 and pET-28a-cFGF2-M13 were constructed.

[0061] The amino acid sequence of the FGF2 protein is shown as SEQ ID NO.1.

[0062] SEQ ID NO.1:

[0063] MAAGAAGSITTLPALPDDGGGGAFPPGHFKDPKRLYCKNGGFFLRINPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVSANRFLAMKEDGRLLALKCATEECFFFERLESNNYNTYRSRKYSDWYVALKRTGQYKPGPKTGPGQKAILFLPMSAKS

[0064] The nucleotide sequence encoding the FGF2 protein is shown as SEQ ID NO.2.

[0065] SEQ ID NO.2:

[0066] ATGGCGGCCGGCGCCGCGGGTAGCATTACCACCTTACCGGCGCTGCCGGATGATGGCGGTGGCGGCGCGTTTCCGCCGGGCCATTTTAAAGATCCGAAACGCCTGTATTGCAAAAACGGCGGCTTTTTTCTGCGCATTAACCCGGATGGCCGCGTGGATGGCGTGCGCGAAAAAAGCGATCCGCATATTAAACTGCAGCTGCAAGCGGAAGAACGCGGCGTGGTGAGCATTAAAGGCGTGAGCGCGAACCGCTTTCTGGCGATGAAAGAAGATGGCCGCCTGCTGGCGCTGAAATGCGCGACCGAAGAATGCTTTTTCTTTGAACGCCTGGAAAGCAACAACTATAACACCTATCGCAGCCGCAAATATAGCGATTGGTATGTGGCGCTGAAACGCACCGGTCAGTATAAACCGGGCCCGAAAACCGGCCCGGGTCAGAAAGCGATTCTGTTTCTGCCGATGAGCGCGAAAAGCTAAcFGF2-M13 protein's amino acid sequence is shown in SEQ ID NO.3.

[0067] SEQ ID NO.3:

[0068] MAAGAAGGITTLPALPDDGGGGAFPPGHFKEPKLLYCKNGGFFLRINPDGKVDGTRDKSDPFIKLQLQAEERGVVSIKGVIANRFLAMKEDGRLYAIKIATEECFFFERLEENNYNTYRSRKYPDWYVALKRTGQYKPGPKTGPGQKAILFLPMSAKS

[0069] The nucleotide sequence encoding the cFGF2-M13 protein is shown in SEQ ID NO.4.

[0070] SEQ ID NO.4:

[0071] ATGGCGGCCGGCGCCGCGGGTGGCATTACCACCTTACCGGCGCTGCCGGATGATGGCGGTGGCGGCGCGTTTCCGCCGGGCCATTTTAAAGAACCGAAACTGCTGTATTGCAAAAACGGCGGCTTTTTTCTGCGCATTAACCCGGATGGCAAAGTGGATGGCACCCGCGATAAAAGCGATCCGTTTATTAAACTGCAGCTGCAAGCGGAAGAACGCGGCGTGGTGAGCATTAAAGGCGTGATTGCGAACCGCTTTCTGGCGATGAAAGAAGATGGCCGCCTGTATGCGATTAAAATTGCGACCGAAGAATGCTTTTTCTTTGAACGCCTGGAAGAAAACAACTATAACACCTATCGCAGCCGCAAATATCCGGATTGGTATGTGGCGCTGAAACGCACCGGTCAGTATAAACCGGGCCCGAAAACCGGCCCGGGTCAGAAAGCGATTCTGTTTCTGCCGATGAGCGCGAAAAGCTAA

[0072] (2) Transformation: Referring to the operation instructions of Escherichia coli BL21 strain, the recombinant plasmids pET-28a-FGF2 and pET-28a-cFGF2-M13 were respectively transformed into Escherichia coli BL21 strain. After detecting positive colonies by PCR using universal vector primers (synthesized by Sangon Biotech (Shanghai) Co., Ltd., as shown in Table 1), they were inoculated into LB culture medium containing 10 μg / mL Kan and cultured at 37 °C and 200 rpm for 16 h to obtain pET-28a-FGF2 bacterial solution and pET-28a-cFGF2-M13 bacterial solution respectively, and the Escherichia coli BL21 strain transformed with pET-28a vector was used as a negative control.

[0073] Table 1 Universal vector primer sequences

[0074] Name Sequence (5’~3’) SEQ ID NO. T7 F: TAATACGACTCACTATAGGG 7 T7 Term R: GCTAGTTATTGCTCAGCGG 8

[0075] (3) Optimal IPTG induction concentration for prokaryotic expression: At 37 °C, the negative control, pET-28a-FGF2 bacterial solution, and pET-28a-cFGF2-M13 bacterial solution were induced with IPTG at concentrations of 0 mmol / L, 0.2 mmol / L, 0.4 mmol / L, 0.6 mmol / L, 0.8 mmol / L, and 1.0 mmol / L for 6 h. After the expression ended, each bacterial solution was collected and the protein expression was detected by SDS-PAGE electrophoresis. The results are as Figure 1 shown.

[0076] (4) Optimal IPTG induction temperature for prokaryotic expression: At 16 °C, 25 °C, 30 °C, and 37 °C, the negative control, pET-28a-FGF2 bacterial solution, and pET-28a-cFGF2-M13 bacterial solution were induced with 0.6 mmol / L IPTG for 6 h. After the expression ended, each bacterial solution was collected and the protein expression was detected by SDS-PAGE electrophoresis. The results are as Figure 2 shown.

[0077] The experimental results of the optimal IPTG induction concentration showed that no corresponding specific bands were detected in the negative control before and after IPTG induction. After adding different concentrations of IPTG to induce the pET-28a-FGF2 bacterial solution and the pET-28a-cFGF2-M13 bacterial solution, specific bands with molecular weights consistent with expectations (about 21 kDa) were detected; among them, the expression concentration of the FGF2 protein was positively correlated with the IPTG concentration. When induced with 0.8 mmol / L IPTG, the expression concentration of the FGF2 protein was the deepest, and when induced with 1 mmol / L IPTG, the expression concentration did not further deepen, indicating that the expression level of the FGF2 protein could reach the highest with 0.8 mmol / L IPTG induction; similar to the FGF2 protein, the expression level of the cFGF2-M13 protein reached the highest when the IPTG concentration was 0.8 mmol / L. Considering that too fast protein expression speed may lead to incorrect folding of peptide chains to form inclusion bodies, thereby affecting the protein yield and activity, the optimal IPTG induction concentration was determined to be 0.6 mmol / L;

[0078] The experimental results of the optimal IPTG induction temperature showed that the expression levels of both the FGF2 protein and the cFGF2-M13 protein were the lowest at 16 °C and the highest at 37 °C, indicating that IPTG induction at 37 °C could make the expression levels of the FGF2 protein and the cFGF2-M13 protein reach the highest. Considering that the lower the IPTG induction temperature, the more conducive it is to maintaining protein activity, the optimal IPTG induction temperature was determined to be 25 °C, and the induction method was overnight induction.

[0079] Example 2: Purification of FGF2 protein and cFGF2-M13 protein

[0080] (1) Preliminary purification: The Escherichia coli BL21 bacterial solution transformed with the recombinant plasmids pET-28a-FGF2 and pET-28a-cFGF2-M13 carrying His-tag was induced overnight at 25 °C with 0.6 mmol / L IPTG. The Escherichia coli BL21 bacterial solution transformed with the pET-28a vector was used as a negative control. After ultrasonic lysis to obtain the supernatant, the His-tag protein purification magnetic beads were used to preliminarily purify each supernatant; then gradient elution was performed with imidazole solutions at concentrations of 20 mmol / L, 25 mmol / L, 30 mmol / L, 50 mmol / L, 100 mmol / L, 200 mmol / L, and 250 mmol / L respectively. The flow-through solutions eluted with 100 mmol / L and 200 mmol / L imidazole solutions were mixed to obtain the preliminarily purified solution, and SDS-PAGE electrophoresis was used to detect the preliminary purification results of FGF2 protein and cFGF2-M13 protein. The results are as Figure 3 shown.

[0081] The amino acid sequence of the His-tag is shown in SEQ ID NO.9.

[0082] SEQ ID NO.9: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGS

[0083] The nucleotide sequence of the His-tag is shown in SEQ ID NO.10.

[0084] SEQ ID NO.10:

[0085] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCC

[0086] (2) Concentration and purification: The preliminarily purified solutions of FGF2 protein and cFGF2-M13 protein were sequentially ultrafiltered by high-speed centrifugation using 30 kDa and 10 kDa ultrafiltration columns respectively to obtain the concentrated and purified solutions of each protein; the absorbance values of each protein concentrated and purified solution at A562nm were measured using a BCA protein concentration assay kit. At the same time, a standard curve was drawn using the BSA standard protein provided by the kit, and the concentrations of the concentrated and purified FGF2 protein and cFGF2-M13 protein were calculated according to the standard curve formula;

[0087] The steps for constructing the standard curve are as follows: Mix BCAReagentA and BCARengentB thoroughly at a volume ratio of 50:1 to obtain the BCA working solution. Referring to the addition amounts of each reagent shown in Table 2, dilute the BSA standard protein with deionized water to the corresponding concentrations to obtain samples. Take 20 μL of each sample and mix it with 200 μL of the BCA working solution to obtain a mixed solution, and add it to a 96-well microplate. After placing it at 37 °C for 30 min, use a microplate reader to measure the absorbance values of each mixed solution at A562nm, and use the mixed solution without BSA standard protein as a blank control to calculate the standard curve;

[0088] The standard curve formula is: y = 0.0221x + 0.1248 (R 2 = 0.9978).

[0089] Table 2 Reagent addition amounts in a 96-well microplate

[0090] Well Number 0 1 2 3 4 5 6 7 BSA Standard Protein (μL) 0 1 2 4 8 12 16 20 Deionized Water (μL) 20 19 18 16 12 8 4 0 BCA Working Solution (μL) 200 200 200 200 200 200 200 200 Corresponding Protein Content (μg) 0 1 2 4 8 12 16 20

[0091] The results of preliminary purification showed that no specific bands appeared in the negative control during the preliminary purification process; after ultrasonic lysis, the supernatant containing FGF2 protein basically had no detectable specific bands after incubation with His-tagged protein purification magnetic beads, and basically no impurity proteins were detected in the flow-through fractions eluted with 20 mmol / L, 25 mmol / L, 30 mmol / L, and 50 mmol / L imidazole solutions. After elution with 100 mmol / L imidazole solution, a large amount of FGF2 protein began to transfer to the flow-through fraction, and the elution efficiency of FGF2 protein was the highest with 200 mmol / L imidazole solution; after ultrasonic lysis, the supernatant containing cFGF2-M13 protein basically had no detectable impurity proteins in the flow-through fraction eluted with 100 mmol / L imidazole solution, and the color of the specific band deepened. The elution efficiency of cFGF2-M13 protein was the highest with 200 mmol / L imidazole solution. It shows that the optimal elution concentrations of FGF2 protein and cFGF2-M13 protein are 100 mmol / L and 200 mmol / L.

[0092] The results of concentration and purification showed that the molecular weights of both FGF2 protein and cFGF2-M13 protein met the expectations (about 21 kDa), there were no obvious impurity proteins, the concentration of FGF2 protein was 3.48 μg / μL, and the concentration of cFGF2-M13 protein was 5.18 μg / μL.

[0093] Example 3: Prokaryotic expression and purification of cFGF2-M4 protein

[0094] (1) Plasmid construction: The method described in Example 1 was used to perform a combined mutation of D31E, S81I, C99I, and S112E on the FGF2 protein and gene synthesis to obtain a nucleotide sequence encoding the recombinant cFGF2 protein (shown as SEQ ID NO.6), and the recombinant cFGF2 protein translated therefrom was named cFGF2-M4 protein (shown as SEQ ID NO.5), and the recombinant plasmid pET-28a-cFGF2-M4 was constructed.

[0095] The amino acid sequence of the cFGF2-M4 protein is shown as SEQ ID NO.5.

[0096] SEQ ID NO.5:

[0097] MAAGAAGSITTLPALPDDGGGGAFPPGHFKEPKRLYCKNGGFFLRINPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVIANRFLAMKEDGRLLALKIATEECFFFERLEENNYNTYRSRKYSDWYVALKRTGQYKPGPKTGPGQKAILFLPMSAKS

[0098] The nucleotide sequence encoding the cFGF2-M4 protein is shown as SEQ ID NO.6.

[0099] SEQ ID NO.6:

[0100] ATGGCGGCCGGCGCCGCGGGTAGCATTACCACCTTACCGGCGCTGCCGGATGATGGCGGTGGCGGCGCGTTTCCGCCGGGCCATTTTAAAGAACCGAAACGCCTGTATTGCAAAAACGGCGGCTTTTTTCTGCGCATTAACCCGGATGGCCGCGTGGATGGCGTGCGCGAAAAAAGCGATCCGCATATTAAACTGCAGCTGCAAGCGGAAGAACGCGGCGTGGTGAGCATTAAAGGCGTGATTGCGAACCGCTTTCTGGCGATGAAAGAAGATGGCCGCCTGCTGGCGCTGAAAATTGCGACCGAAGAATGCTTTTTCTTTGAACGCCTGGAAGAAAACAACTATAACACCTATCGCAGCCGCAAATATAGCGATTGGTATGTGGCGCTGAAACGCACCGGTCAGTATAAACCGGGCCCGAAAACCGGCCCGGGTCAGAAAGCGATTCTGTTTCTGCCGATGAGCGCGAAAAGCTAA

[0101] (2) Transformation: The recombinant plasmid pET-28a-cFGF2-M4 was transformed into Escherichia coli BL21 strain by the method described in Example 1 to obtain the pET-28a-cFGF2-M4 bacterial solution.

[0102] (3) Prokaryotic expression and purification: The Escherichia coli BL21 bacterial solution transformed with the recombinant plasmid pET-28a-cFGF2-M4 carrying the His tag was induced for expression and purified by the method described in Example 2 to obtain the concentrated and purified cFGF2-M4 protein solution.

[0103] Example 4: Activity assay of recombinant cFGF2 protein

[0104] HEK293 cell line overexpressing chicken cFGFR1 gene (HEK293-cFGFR1 cell line) was treated with rhFGF2, FGF2 protein and cFGF2-M13 protein at concentrations of 0 ng / mL, 5 ng / mL, 20 ng / mL and 40 ng / mL respectively. After 6 h of treatment, the HEK293-cFGFR1 cell line was lysed and the phosphorylation level of intracellular ERK1 / 2 protein (pERK1 / 2) was detected by Western blot. The expression level of intracellular ERK1 / 2 was used as an internal reference. The results are as Figure 4 shown.

[0105] The results showed that after the HEK293-cFGFR1 cell line was treated with 5 ng / mL rhFGF2, the phosphorylation level of intracellular ERK1 / 2 protein was up-regulated, but there was no significant difference compared with that without treatment. When the concentration of rhFGF2 was increased to 20 ng / mL and 40 ng / mL, there were significant differences in the phosphorylation level of intracellular ERK1 / 2 protein compared with that without treatment, indicating that rhFGF2 could act on the cFGFR1 receptor to promote the phosphorylation of intracellular ERK1 / 2 protein and activate the intracellular MAPK / ERK signaling pathway. After the HEK293-cFGFR1 cell line was treated with 5 ng / mL FGF2 protein, the phosphorylation level of intracellular ERK1 / 2 protein was significantly increased. As the concentration of FGF2 protein increased to 20 ng / mL and 40 ng / mL, its phosphorylation level decreased slightly, but there were still significant differences compared with that without treatment. Different concentrations of cFGF2-M13 protein could significantly enhance the phosphorylation level of intracellular ERK1 / 2 protein in the HEK293-cFGFR1 cell line, and it had strong activity after being treated with 5 ng / mL cFGF2-M13 protein, indicating that the recombinant cFGF2 protein of the present invention had FGF2 biological activity, could activate the intracellular MAPK / ERK signaling pathway and phosphorylate intracellular ERK1 / 2 protein.

[0106] Example 5: Determination of the thermal stability of the recombinant cFGF2 protein

[0107] (1) Western blot detection: After the rhFGF2, FGF2 protein and cFGF2-M13 protein with a concentration of 20 ng / mL were placed at 37 °C for 24 h, 48 h and 72 h respectively, the HEK293-cFGFR1 cell line was treated and lysed, and then the intracellular pERK1 / 2 level was detected by Western blot. The expression level of intracellular ERK1 / 2 was used as an internal reference. The results were as Figure 5 shown.

[0108] (2) Luciferase reporter system functional analysis: The pGL3-SRE-luciferase reporter plasmid was co-transfected into the HEK293-cFGFR1 cell line. The HEK293-cFGFR1 cell line was treated with rhFGF2 and cFGF2-M13 protein without heat treatment and placed at 37 °C for 3 d for 6 h and then lysed, and then the fluorescence intensity was measured. The results were as Figure 6 shown.

[0109] The results of Western blot detection showed that after the HEK293-cFGFR1 cell line was treated with rhFGF2, the phosphorylation level of intracellular ERK1 / 2 protein increased significantly. The strongest phosphorylation level was observed at 24 h, and a relatively strong phosphorylation level could still be maintained at 48 h. However, the phosphorylation level of intracellular ERK1 / 2 protein decreased significantly at 72 h, showing no significant difference compared with that without treatment. This indicated that the activity of rhFGF2 could be maintained for 48 - 72 h in in vitro culture. FGF2 protein could efficiently activate the intracellular MAPK / ERK signaling pathway of the HEK293-cFGFR1 cell line, promoting the phosphorylation level of intracellular ERK1 / 2 to remain at a relatively high level from 24 h to 72 h. Although the phosphorylation level decreased after 72 h, there was still a significant difference compared with that without treatment, indicating that FGF2 protein had good thermal stability. After the HEK293-cFGFR1 cell line was treated with cFGF2-M13 protein, strong intracellular ERK1 / 2 phosphorylation signals were detected at 24 h, 48 h, and 72 h, and did not decay with the extension of time. This indicated that mutating FGF2 protein into the recombinant cFGF2 protein of the present invention could significantly improve the thermal stability of FGF2 protein;

[0110] The results of the luciferase reporter system functional analysis showed that both rhFGF2 and cFGF2-M13 proteins could activate the cFGFR1 receptor in a dose-dependent manner; after rhFGF2 was treated at 37 °C for 3 days, its activity decreased significantly, and only showed a certain activation ability at high concentrations; cFGF2-M13 protein still had an activity similar to that without heat treatment after being treated at 37 °C for 3 days and showed unique thermal stability. This indicated that mutating FGF2 protein into the recombinant cFGF2 protein of the present invention could significantly improve the thermal stability of FGF2 protein.

[0111] Example 6: Identification of the effect of recombinant cFGF2 protein on the in vitro proliferation of PGCs

[0112] (1) Preparation of avian primordial germ cell culture medium: Mix 700 mL of DMEM medium (high glucose, without glutamine, without calcium), 10 mL of GlutaMAX 100-fold solution, 10 mL of NEAA 100-fold solution, 10 mL of Pen / Strep 100-fold solution, 0.1 mmol of β-mercaptoethanol, 10 mL of nucleoside 100-fold solution, 0.2 mmol of sodium pyruvate, 200 mg of heparin sodium, 0.15 mmol of calcium chloride, 2 g of ovalbumin, 10 mg of ovotransferrin, 20 mL of B-27 supplement 50-fold solution, 25 μg of human activin A with appropriate amount of ultrapure water. Then, add 20 μg of FGF2 protein, cFGF2-M13 protein, and cFGF2-M4 protein respectively thereto, and make up the volume to 1 L with ultrapure water to obtain three kinds of avian primordial germ cell culture media, namely, the culture medium containing FGF2 protein, the culture medium containing cFGF2-M13 protein, and the culture medium containing cFGF2-M4 protein.

[0113] (2) Preparation of negative control culture medium: Prepare the negative control culture medium according to the method described in step (1), except that in this step, rhFGF2, FGF2 protein, cFGF2-M13 protein, cFGF2-M4 protein, and other growth factors are not added.

[0114] (3) Preparation of positive control culture medium: Add 4 μg of rhFGF2 to the negative control culture medium described in step (2) to obtain the positive control culture medium.

[0115] (4) Cell culture and detection: Inoculate male PGCs into the negative control culture medium, positive control culture medium, culture medium containing FGF2 protein, culture medium containing cFGF2-M13 protein, and culture medium containing cFGF2-M4 protein respectively for culture. Then, take 20 μL of each culture medium at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h of culture, and count the male PGCs growing in suspension under a microscope using a hemocytometer. The results are as Figure 7 shown.

[0116] The results showed that in the negative control medium, most male PGCs attached to the surface of feeder cells after 24 h of culture, growth stagnated, and then began to differentiate or apoptose; in the medium containing FGF2 protein, male PGCs could maintain a normal suspended growth state and stable proliferation within 72 h of culture. However, after 72 h, a large number of male PGCs aggregated and proliferated slowly. Some male PGCs changed in morphology and showed a tendency to differentiate. After that, the number of male PGCs growing in suspension in the medium containing FGF2 protein gradually decreased. Only a small number of male PGCs maintained a suspended growth state when cultured to 120 h; the growth status of male PGCs in the positive control medium was similar to that in the medium containing FGF2 protein; in the medium containing cFGF2-M13 protein and the medium containing cFGF2-M4 protein, the growth rate of male PGCs was significantly higher than that in other media within 72 h. After 72 h of culture, it was still possible to observe that male PGCs maintained a relatively high proliferation rate. When cultured to 120 h, most male PGCs could still maintain good cell morphology and grow in strings. In addition, the cell proliferation state of male PGCs in the medium containing cFGF2-M13 protein was better than that in the medium containing cFGF2-M4 protein. This indicated that the recombinant cFGF2 protein of the present invention could still maintain the normal growth and proliferation of avian PGCs when the culture time exceeded 72 h, had higher thermal stability compared with rhFGF2 and FGF2 protein, and was suitable for the long-term culture of avian PGCs.

[0117] Example 7: Identification of the colonization ability of recombinant cFGF2 protein on PGCs

[0118] The PGC-mCherry cell line was inoculated in the medium containing cFGF2-M13 protein prepared in Example 6 for culture. Then, the PGC-mCherry cell line that could stably express the mCherry protein was selected and injected into 100 2.5-day-old Camellia chicken embryos. 90 of them were successfully injected, and then they were placed in an incubator and continued to be incubated at a temperature of 37.8 °C and a humidity of 64%. During the incubation process, the Camellia chicken embryos were turned over every 2 h, and the turning angle was 90°; when cultured to 6-day-old embryos, 10 Camellia chicken embryos were taken to isolate the gonads, and the colonization situation was observed under a research-grade fluorescence stereomicroscope. The results were as Figure 8 shown.

[0119] The results showed that the PGC-mCherry cell line cultured in the medium containing cFGF2-M13 protein had normal cell morphology, obvious proliferation phenomenon, and clear red fluorescence signal expression. After injecting the PGC-mCherry cell line into the embryos of Chahua chickens and continuing incubation, red fluorescent protein was detected in the gonads of 6 randomly selected Chahua chicken embryos, indicating that the recombinant cFGF2 protein of the present invention could support the self-renewal of avian PGCs under in vitro culture conditions, maintain the developmental status of avian PGCs in recipient embryos, and had the ability to colonize the genital ridges, and could be applied to gene editing of birds.

[0120] As can be seen from the above examples, the present invention provides a recombinant cFGF2 protein, a medium for avian primordial germ cells and its applications. The recombinant cFGF2 protein of the present invention has FGF2 biological activity and strong thermal stability, can activate the intracellular MAPK / ERK signaling pathway and phosphorylate the intracellular ERK1 / 2 protein, can maintain the normal growth, proliferation, self-renewal of avian PGCs, the developmental status in recipient embryos and the ability to colonize the genital ridges under in vitro culture conditions, and can be used for long-term culture of avian PGCs and gene editing of birds.

[0121] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant cFGF2 protein, characterized in that: The recombinant cFGF2 protein comprises mutations at one or more of positions 8, 31, 34, 51, 55, 57, 62, 81, 95, 97, 99, 112 and 124 of the amino acid sequence of the FGF2 protein; The amino acid sequence of the FGF2 protein is shown in SEQ ID NO.1; The recombinant cFGF2 protein is mutated at positions 8, 31, 34, 51, 55, 57, 62, 81, 95, 97, 99, 112 and 124 of the amino acid sequence of the FGF2 protein; The amino acid sequence of the recombinant cFGF2 protein is shown in SEQ ID NO.3; Or the recombinant cFGF2 protein is mutated at positions 31, 81, 99 and 112 of the amino acid sequence of the FGF2 protein; The amino acid sequence of the recombinant cFGF2 protein is shown in SEQ ID NO.

5.

2. Use of the recombinant cFGF2 protein according to claim 1 in in vitro culture and / or efficient proliferation of avian primordial germ cells.

3. Use of the recombinant cFGF2 protein according to claim 1 in preparing a product for in vitro culture and / or efficient proliferation of avian primordial germ cells.

4. A composition for cell culture, characterized in that The cell culture composition comprises the recombinant cFGF2 protein according to claim 1 and an acceptable carrier.

5. A culture medium for avian primordial germ cells, characterized in that: The avian primordial germ cell culture medium comprises the recombinant cFGF2 protein according to claim 1.

6. The avian primordial germ cell culture medium according to claim 5, characterized in that The avian primordial germ cell culture medium uses water as a solvent and includes the following components in concentrations: 600-750 mL / L of DEME culture medium, 5-15 mL / L of alanine, 8-12 mL / L of non-essential amino acids, 8-12 mL / L of penicillin / streptomycin, 0.05-0.3 mmol / L of β-mercaptoethanol, 5-15 mL / L of nucleosides, 0.1-0.4 mmol / L of sodium pyruvate, 100-300 mg / L of heparin sodium, 0.1-0.5 mmol / L of calcium chloride, 1-4 g / L of ovalbumin, 5-20 mg / L of ovotransferrin, 10-30 mL / L of B-27 supplement, 10-50 μg / L of human activin A, and 4-50 μg / L of recombinant cFGF2 protein; The recombinant cFGF2 protein is the recombinant cFGF2 protein according to claim 1.

7. Use of the cell culture composition according to claim 4 or the avian primordial germ cell culture medium according to claim 5 or 6 in in vitro culture and / or efficient proliferation of avian primordial germ cells.

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