Duck-source IGF2 recombinant protein as well as preparation method and application thereof

Abstract

The invention relates to a duck-origin IGF2 recombinant protein and a preparation method and application thereof, the amino acid sequence of the duck-origin IGF2 recombinant protein is shown as SEQ ID NO: 2, the duck-origin IGF2 recombinant protein is subjected to duck IGF2 gene coding region PCR amplification to obtain a duck IGF2 amplification product, then the duck IGF2 amplification product is subjected to recombination, inducible expression and purification to obtain the duck-origin IGF2 recombinant protein, and the sequence used for amplification is shown as SEQ ID NO: 3-4. The invention also discloses the application of the duck-source IGF2 recombinant protein in improving the in-vitro proliferation capacity of duck ovarian granular cells. The invention provides a simple, accurate and mature method for inducing the expression of the duck-derived IGF2 protein, and successfully prepares the duck-derived IGF2 recombinant protein. The obtained duck-source IGF2 recombinant protein is used for researching the influence of the IGF2 on the proliferation of duck granular cells and the action mechanism of the IGF2, and a foundation is laid for researching the regulation and control mechanism of the duck ovarian function.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology, and specifically relates to a duck-derived IGF2 recombinant protein and a preparation method and application thereof. Background Art

[0002] In recent years, duck eggs have been loved by people for their rich nutritional value, and the demand for duck eggs has increased year by year, so geneticists have focused their research on improving the egg production of laying ducks. In poultry, egg production is an important economic trait and an important indicator for evaluating reproductive performance, but the heritability of this trait is low (Zhu et al., 2016). The egg-laying performance of laying ducks is inseparable from the growth and development of ovarian follicles. Follicles are the functional units of the ovaries. They are multicellular functional units composed of oocytes and the surrounding granulosa cells and theca cells. They are the life source of the ovaries. The number and quality of follicles directly affect the vitality of the ovaries. The selection and development of poultry follicles will determine the number of follicles and the final number of eggs laid (Johnson, 2012). Granulosa cells (GCs) in the ovaries are the main functional cells that regulate follicle development and are involved in regulating the initiation, development, follicle selection, maturation and ovulation of primordial follicles (Cuiling et al., 2005). The growth of oocytes depends on granulosa cells. The apoptosis of granulosa cells will lead to follicle atresia, which in turn affects the growth state of oocytes. The growth rate and quality of granulosa cells determine the speed and quality of follicle development, thus affecting egg production.

[0003] The insulin-like growth factors (IGFs) family is a class of multifunctional cell regulatory factors, including insulin-like growth factor 1 (IGF1) IGF1 and insulin-like growth factor 2 (IGF2), with corresponding receptors IGF1R and IGF2R. IGFs can mediate the stimulation of growth hormone, regulate tissue growth and development, and play an important role in the maintenance of chicken muscle volume and strength, body composition and nutritional metabolism (Yang et al., 2022). IGF2 is the main growth factor in humans among IGFs and plays an important role throughout pregnancy. When IGF2 is destroyed, it will cause delayed fetal growth (Gicqueland Le Bouc, 2006). IGF2 is considered to be a mouse embryonic growth and development factor. It is highly expressed in mouse embryos and placentas, and the expression level of IGF2 drops sharply after birth (Becker et al., 2018). However, there are no reports on the biological functions of IGF2 gene and its protein in ducks in this field, and it is still unknown whether it can affect the proliferation of duck ovarian granulosa and follicle development, and through what pathway it exerts its effects.

[0004] In addition, the in vitro recombinant protein can not only be used as a growth promoter or additive for breeding, but also provide new ideas and methods for improving the reproductive performance of ducks. In the prior art, the detection methods of IGF2 protein are mostly based on monoclonal antibodies or polyclonal antibodies, which affects the research on the biological function of duck IGF2. Therefore, it is urgent to develop a simple, accurate and mature method to induce the expression of duck IGF2 protein and prepare duck IGF2 recombinant protein, so as to lay a foundation for studying the biological function of duck IGF2 and preparing monoclonal antibodies or polyclonal antibodies of duck IGF2 protein. Summary of the invention

[0005] The purpose of the present invention is to provide a duck-derived IGF2 recombinant protein and a preparation method thereof, as well as the application of the duck-derived IGF2 recombinant protein in improving the proliferation ability of duck ovarian granulosa cells, so as to lay a foundation for further studying the biological function of duck IGF2 and preparing monoclonal antibodies or polyclonal antibodies of duck-derived IGF2 protein.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A duck-derived IGF2 recombinant protein, whose amino acid sequence is shown in SEQ ID NO:2.

[0008] A method for preparing the duck-derived IGF2 recombinant protein comprises the following steps:

[0009] (1) PCR amplification of duck IGF2 gene coding region: using duck cDNA as template, using primers IGF2-F: GACAAGGCCATGGCT GATATC GCGTACGGCACGGCGGAGA and primer IGF2-R: ACGGAGCTCGAATTC GGATCC CTACTCAGACTTGACGGACTTGG, perform PCR amplification on it, and then perform gel excision and recovery on the PCR amplification product for subsequent fusion reaction;

[0010] (2) In Fusion for recombination of vector and target fragment: Seamless Cloning and Assembly Enzyme Mix was used to construct the recombinant plasmid; the reaction system and conditions for the fusion reaction of duck IGF2 amplification product and pET32 were as follows:

[0011]

[0012] E. coli BL21 (DE3) carrying pET32-IGF2 was obtained;

[0013] (3) Sequencing identification: 3-5 grown colonies were selected for sequencing;

[0014] (4) Induced expression of pET32-IGF2 protein: E. coli BL21 (DE3) carrying pET32-IGF2 after sequencing analysis was cultured, and then induced with IPTG. The bacterial precipitate was collected by centrifugation, protein loading buffer was added, boiled, and cooled to room temperature; then centrifuged, and the supernatant was taken for SDS-PAGE electrophoresis analysis;

[0015] (5) Identification of expression products by Western Blotting;

[0016] (6) Purification of pET32-IGF2 expression product:

[0017] Using HisPur TM The expressed product was purified by Ni-NTA Magnetic Beads (Thermo Fisher) to obtain duck IGF2 recombinant protein.

[0018] Wherein, the reaction system of PCR amplification in step (1) is:

[0019]

[0020] The reaction conditions for amplification are:

[0021]

[0022] The specific method of step (4) is:

[0023] S1 E. coli BL21 (DE3) carrying pET32-IGF2 after sequencing analysis was inoculated into 5 mL of LB liquid medium containing 50 μg / mL Ampicillin at a final concentration of 50 μg / mL and cultured overnight at 37°C and 200 rpm;

[0024] S2 was transferred to a new SOC liquid medium containing 50 μg / mL Ampicillin at a dilution of 1:100 and cultured at 37°C, 200 rpm for 2-3 hr. OD 600 When the concentration of pET32 protein was 0.5-0.6, IPTG was added to a final concentration of 0.5 mM and induced at 37°C for 1-5 hours. The bacterial solution was collected, and the pET32 empty vector IPTG-induced group and the pET32-IGF2 IPTG-uninduced group were used as controls.

[0025] S3 was centrifuged at 12000×g for 1 min, the bacterial precipitate was collected, 80 μL of 1× protein loading buffer was added, boiled for 5 min, and cooled to room temperature; then centrifuged at 12000×g for 5 min, and 5 μL of supernatant was taken for SDS-PAGE electrophoresis analysis.

[0026] The specific method of step (6) is:

[0027] Using HisPur TM The expression product was purified by Ni-NTA Magnetic Beads (Thermo Fisher). The specific scheme is as follows:

[0028] S1 bacterial liquid collection: 100 ml of bacterial liquid induced by 0.5 mM IPTG was centrifuged and resuspended in 10 ml PBS containing 1% Tritox-100. After ultrasonic disruption, centrifuged at 12,000 g for 20 min, and the supernatant was discarded and the precipitate was retained;

[0029] S2 Inclusion body resuspension and dissolution: Add 5 ml Eqμlibration Buffer to resuspend the inclusion body precipitate, rotate at 4℃ for 1 hour to dissolve the precipitate;

[0030] S3 magnetic bead pretreatment: Take 2 ml of HisPurNi-NTA Magnetic Beads, mix well, perform magnetic separation, and discard the supernatant; then add Eqμlibration Buffer to resuspend, discard the supernatant, and repeat this step twice;

[0031] S4 Binding of magnetic beads and proteins: Add the protein in step 5.2 to the magnetic beads pretreated in step 5.3, bind at room temperature for 30 minutes, then perform magnetic separation, discard the supernatant, and retain the magnetic beads;

[0032] S5 washing: add 5 ml of washing buffer to the magnetic beads to resuspend, then perform magnetic separation, discard the supernatant, retain the magnetic beads, and repeat this step 2 more times;

[0033] S6 elution: Add 250 μl of imidazole elution buffer with different concentration gradients to elute the target protein, then perform magnetic separation and pipette the supernatant into a new 1.5 ml centrifuge tube;

[0034] S7 electrophoresis: Take 5 μl of each eluted product, add an equal volume of 2×SDS-PAGE loading buffer, boil and load all of it for electrophoresis analysis.

[0035] In step S6, the imidazole eluent is: a PBS buffer solution with a pH of 8.0, and the PBS buffer solution contains 8M urea and 100-500mM imidazole.

[0036] Preferably, the imidazole eluent is a PBS buffer solution with a pH of 8.0, and the PBS buffer solution contains 8M urea and 250mM imidazole.

[0037] The duck-derived IGF2 recombinant protein is used in improving the in vitro proliferation ability of duck ovarian granulosa cells, and is further used in preparing a promoter for promoting the proliferation of duck ovarian granulosa cells.

[0038] Compared with the prior art, the advantages of the present invention are: the present invention provides a simple, accurate and mature method for inducing the expression of duck-derived IGF2 protein, and successfully prepares a duck-derived IGF2 recombinant protein. The obtained duck-derived IGF2 recombinant protein is used to study the effect of IGF2 on the proliferation of duck granulosa cells and its mechanism of action, laying the foundation for the study of the regulatory mechanism of duck ovarian function. By inducing duck ovarian granulosa cells cultured in vitro with IGF2, it was found that IGF2 can increase the proliferation level of duck granulosa cells cultured in vitro. IGF2 significantly upregulates the expression of marker gene proteins related to the PI3K-AKT signaling pathway and the expression level of IGF1R protein. When IGF2 and PI3K inhibitors are added at the same time, the expression of marker gene proteins related to the PI3K-AKT signaling pathway is upregulated, but the expression level is lower than that of the IGF2 addition group. These results show that IGF2 can regulate the level of duck ovarian granulosa cells through the PI3K-AKT signaling pathway and affect pathways such as Steroid hormone biosynthesis. The present invention provides new evidence for the function of IGF2 in ducks, lays an important experimental foundation for understanding the regulatory mechanism of duck granulosa cell proliferation, and provides new ideas for studying duck follicle development and improving duck egg-laying performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The figure is a graph of the duck IGF2CDS PCR amplification result of the present invention, wherein, M: DL5K DNA marker (100, 250, 500, 750, 1000, 2000, 3000, 5000 bp), 1: IGF2CDS PCR amplification product (about 207 bp);

[0040] Figure 2 The figure is the SDS-PAGE electrophoresis result of the total protein of the prokaryotic expression of pET32-IGF2 of the present invention, wherein M: PageRuler Protein Ladder, 1: empty vector (0.5mM IPTG induced for 3h), 2: ghrelin-pET32 vector (no IPTG added for 3h), 3: ghrelin-pET32 vector (0.5mM IPTG induced for 3h);

[0041] Figure 3 This is a Western Blotting identification diagram of the prokaryotic expression product of the pET32-IGF2 gene, where Marker: ECL DualVue WB Marker (GE Company), 1: -IPTG; 2: +IPTG (0.5mM) induced for 3h;

[0042] Figure 4 This is the SDS-PAGE electrophoresis result of total protein at different time points of pET32-IGF2 IPTG induction, where M: PageRulerProtein Ladder; Lanes 1-6: IPTG induction 0 to 5h;

[0043] Figure 5 This is the result of SDS-PAGE test of the existence form of the prokaryotic expression product of the pET32-IGF2 gene, where Marker: PageRulerProtein Ladder, 1: supernatant (soluble component); 2: inclusion body precipitate;

[0044] Figure 6 It is the result of SDS-PAGE electrophoresis after Ni-NTA purification of the prokaryotic expression product of pET32-IGF2 gene, where M: PageRulerProtein Ladder;

[0045] Figure 7 This is the result of EdU detection of IGF2 on the proliferation ability of follicular granulosa cells;

[0046] Figure 8 This is a Western Blot expression difference diagram.

[0047] Fig. 9 Transcriptome sequencing results. DETAILED DESCRIPTION

[0048] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0049] Example 1: Prokaryotic expression and protein purification of duck pET32-IGF2

[0050] The main reagents and consumables are shown in Table 1, and the main instruments are shown in Table 2

[0051] Table 1 Main reagents and production companies

[0052]

[0053] Table 2 Main instruments used in the experiment

[0054]

[0055] The specific operation process is as follows:

[0056] 1.1 Construction of duck pET32-IGF2 recombinant expression vector

[0057] 1.1.1 PCR amplification of duck IGF2 gene coding region

[0058] 1.1.1.1 Gene sequences based on coding region amplification

[0059] IGF2 full length cDNA:

[0060] GTCTCCTCGCTCGTGGGGCAGCCAGCCGGGAGCAGCGGGCAGCCAGGCAGCCGGCCCCTCCGCCCC

[0061] GGCCAGGGGGACGCAGTCAGTAAACACCACCACAACAAAAAGTGAGAGCCACCCAAGGGGCAGCC

[0062] AAAGCAGAAGGAGAGAAAGAGGAGGAGGAAAAAAAGGCAAAAAAAAAAAAAAGGGGGAACAAG

[0063] GAGAAAAAAATAACAAAAGAAAAAAAGAAAAAAGAAAAGGAAAAAAAAAAAAAGAAAAGGA

[0064] AAAAATAGAGAGGAGAAGGAAGGAAAGCGCTCGCTCGCTCTCTAGAATAACAAAATACCTAATCGT

[0065] AACAAAATAACATCCTCCAGACAAATTCAAGCAGGGACTTGACATACCAAAAAGGCAAAGCGAGG

[0066] AGAGCAAGCCAGGGCGCGGGGAAGGCGAGCCAAAGCCGCGATCGCTGCCAGGGCCGGACAGCAGC

[0067] ATGGCAAGCGCCGGGGCACACACGGATGAGCGCTGCCGGCAGCCTGCCTTCCTCCCCGGCCCGCCG

[0068] CCGCCAGAAGTTGAGAGTAGCAGCGGCAGCGCCAAGGTGCAGAGGATGTGTGCGGCCCGGCGGCTG

[0069] CTGCTGCTGCTGCTGGCCTTCCTGGCCTACGCGTTGGATTCGGCCGCGGCGTACGGCACGGCGGAGA

[0070] CCCTCTGCGGCGGGGAGCTGGTGGACACACTGCAGTTCGTCTGCGGGGACAGGGGCTTCTACTTCAG

[0071] TAGGCCAGTGGGACGAAATAACAGGAGGATCAACCGCGGCATCGTGGAGGAGTGCTGCTTTCGGAG

[0072] CTGCGACCTGGCTCTGCTGGAAACCTACTGCGCCAAGTCCGTCAAGTCTGAGCGCGACCTCTCCGCC

[0073] ACCTCCCTTGTGGGCCTCCCGGCACTCAACAAGGAGAGCTTCCAGAAGCCCTCACACGCCAAGTACT

[0074] CCAAGTACGACGTGTGGCAGAAGAAGAGCTCCCAGCGGCTGCAGCGGGAGGTGCCCGGCATCCTGC

[0075] GCGCCCGCCGGTACCGGTGGCAGGCCGAGGGGCTGCAAGCGGCTGAAGAAGCCAGGGCGCTGCATC

[0076] GCCCCCTCATCTCCTTGCCCAGCCAGCGGCCCCCAGCGCCGCGAGCATCCCCCGAAGCCACCGGCCC

[0077] CCAGGAATGAACCATGACTGGCCGGCTCCATTTGTGATCTCCCGGGGAGAGACTGGCGAGACCCGG

[0078] CCCCCCCCGAGCCCCTCCGTCCCCGAGCCGAGGAGCGGGGCGGCGGGGCGCCATCACGCCGCTCCG

[0079] GCCCCAACCAAACAACTGACACAAGCAAGGAGGGGATGGTGGCGGCGGCGGTGGCCGCGCGCGGC

[0080] GTCCTCCTTTTGGGGTGGGGGGGAGGGTTGTTTCGTTTCCCAGGCCTGTCTCGTTTCCCCCGCGCACC

[0081] CAGTATTTTTCAAGCCTTGTACTTGCAAAGGGACAGTTGGGCACTTGAACTTTTTTTGCTGCCGGGCA

[0082] CTGCGTGACATCCCCGGTGCCAACACCAGAAGACAAAAGAAAGAGCAAGAGGGAAAAAGGATATG

[0083] AAGTTAACTTTTTTTTTATTTTTTATTTTTTCTCAAGAAGAGGAGTCCATTCCGTGTCTTTCTTGACAA

[0084] TAAAGTGAAAAACAAAAACAAATGGGGAGAAATGTTGAGGGATATAACTTTTTTGCCTCATTCCCA

[0085] CCCCACTTTTTTTTTCCTTTTATTTTGATTTTGTTGGTAACTTTTTGTAATTTTTATAACTTTCTTTTGCA

[0086] CCTTTAAAAAGAAAACGTAACTGAGACGAAGTTCCGAGGAGACCCATCTGCATTTTTTGTGTGTGTG

[0087] CTTAATTACAAGATTCCAAATCGACTTGCACCTTTTTGAAATCATCGCTGACGGGAGAAAGAGAGGA

[0088] AGAGGAGAGAGAGAGAGAGAGAGAGAAAGAGAGAGAAGAAAAAGGAAAGGGGAGAAAAAAAAA

[0089] AGAAAAAAGTGAGCTGATGCTCACTTTAAATGGAGGAGGCGTCTGCATTGCACATTTGCCACGGATT

[0090] TAGTTGATTTATATATATAATATATTATATAACTTTTTTGGCAAAAAAGCACTATTTTTATGGGACAT

[0091] TTTGTGTAGTATCTGAAGAGGGATTGTAATGTTTAAAATGTGTTATGGTGCTAAAGTAAGTTGGAAG

[0092] GGAAAAAACCCAGAAATAGCAAGGGAGGGTGGGGAGGGAAGGACAAGATAGCGAATACTGAACTT

[0093] GAAATTTTAGCTTTTCGTTTTAGGAAAAGTGTGATAAATGTGTTTCTTTTCTCTTCTGAAATATAGCTT

[0094] GGTCATGACCATATGAGCTAGTTGGCTGTCACTCATGTACACAGATGCACGCACACACAGAAATACA

[0095] TCTACACGCATCCTTTATTTTTTTTTTTGTAGAAAACAAAACAAAACACAACACAACGATTTAGTCTA

[0096] AAAGGGAACTGTTCTAGAGACTAACTGCTTTGAATGGCGAAGGTATTGCCAGGTTACAACTCCAGGC

[0097] CCTGCTTCCGTGCGGAGCTCGGCTCCTCCATCCTCCTGACCCTGGTGGCTCGCTCGCGCGAGCCGAG

[0098] GCCCTGCCAAAGCACCAGCCCCACCATCTCCTTTTCCTCCTCTTGCCCTTCGTGTCCTTCTTGCAGAA

[0099] CCCCCCTGCCCTCCTCCCCTTGCCCCAACGAGCTGGGGCCATGGAGGATGGCAGCCGGGACTGACGG

[0100] GGACCACCCCAGTGCAGGGCAGGCGTCCCCCCCGGTGCGGTGACCTCCACCTGCCGGGTGGCTGGG

[0101] ATGGGTTGACGGGAGGGAGGAGAGGCAACTCTCTGGCTTTGAGGCTCAGGATTCGATGAGCAGATG

[0102] GGTTGGAGAGGTGGGCAGAGGGCTGTAAAATAGGTTGGTTTTTAAAATAATATATATTATCTGTCTA

[0103] GGAACCGTAAAGAGGGGGGAAACAATTACATCTCCCTGTGATATACTCTCACTTGCTAATAAAATGG

[0104] GGACACTCTGTTAACCAGTTCTGTACTTGCTTTGTCTTATATATCCATACATTTAAAACATAAAAAAA

[0105] AAGACTACTTGAATTGTATTCTATCTCGTGGTTTTGAGGGGAGGGCTAGGGGGAGTCTCGAGAGGGG

[0106] GAGGATTTTAATGTATAATCCTTTTGTTTGTTATTACCTTGCAATGTTGAGCCATGAAGCCTTTGTTTA

[0107] AAAAAATGGCACTGCAATACACGACAAAGAAAATCCTTTATTTATTTTATTAAATATATATGGAAAAGAA(SEQ ID NO:1)

[0108] 1.1.1.2 PCR amplification reaction

[0109] Using duck cDNA as template, PCR amplification was performed using primer sequences as described in Table 3. The PCR amplification results are shown in Table 3. Figure 1 As shown, the PCR amplification product was then excised and recovered using QIAqμick PCR&Gel Cleanμp Kit (refer to the kit instructions for details), and then the following fusion reaction was performed. Among them, the PCR amplification reaction system and conditions are shown in Tables 4 and 5:

[0110] Table 3 Primer names and sequences

[0111]

[0112] Table 4 PCR amplification reaction system

[0113]

[0114] Table 5 PCR amplification reaction conditions

[0115]

[0116] 1.1.2 In Fusion Recombination of Vector and Target Fragment

[0117] use Seamless Cloning and Assembly Enzyme Mix was used to construct the recombinant plasmid to obtain E. coli BL21 (DE3) carrying pET32-IGF2; wherein, the reaction system and conditions for the fusion reaction of duck IGF2 and pET32 are shown in Table 6:

[0118] Table 6 Duck IGF2 and pET32 fusion reaction system and conditions

[0119]

[0120] 1.1.3 Sequencing identification

[0121] 3-5 grown colonies were selected and sent to Shanghai Biotech Co., Ltd. for sequencing. The sequencing results were consistent with the duck IGF2 sequence through MegAlign sequence alignment, and the bacterial solution and plasmid were used for subsequent prokaryotic expression induction experiments.

[0122] 1.2 Prokaryotic expression and condition optimization of duck pET32-IGF2 gene

[0123] 1.2.1 Inducible expression of pET32-IGF2 protein

[0124] 1.2.1.1 E. coli BL21 (DE3) carrying pET32-IGF2 after sequencing analysis was inoculated into 5 ml of LB liquid medium (containing 50 μg / mL Ampicillin at a final concentration) and cultured overnight at 37°C and 200 rpm;

[0125] 1.2.1.2 Transfer to new SOC liquid medium (containing final concentration of 50 μg / mL Ampicillin) at 1:100, 37°C, 200 rpm, and culture for 2-3 hours. OD 600 When the concentration of β-glucose was 0.5-0.6, IPTG was added to a final concentration of 0.5 mM and induced at 37°C for 3 hours. The bacterial solution was collected, and the pET32 empty vector IPTG-induced group and the pET32-IGF2 IPTG-uninduced group were used as controls.

[0126] 1.2.1.3 Collect the bacterial precipitate by centrifugation (12,000×g, 1 min), add 80 μl 1× protein loading buffer, boil for 5 min, and cool to room temperature; then centrifuge (12,000×g, 5 min), and take 5 μl of the supernatant for SDS-PAGE electrophoresis analysis.

[0127] 1.2.1.4 Conclusion: See Figure 2 In lane 4, a bright band (about 27 kDa) appeared near the target molecular weight, which was basically consistent with the predicted result; while the empty vector and pET32-IGF2 recombinant plasmid uninduced groups did not show the target band, indicating that IGF2 was successfully expressed in E. coli.

[0128] 1.2.2 Identification of IGF2 prokaryotic expression products by Western Blotting

[0129] 1.2.2.1 Take 2 μl of the product before IPTG induction and after IPTG induction, and perform SDS-PAGE electrophoresis;

[0130] 1.2.2.2 Transfer: Equilibrate the SDS-PAGE gel in Tris-Glycine transfer buffer for 30 min; transfer the gel at a constant voltage of 100 V for 2 hr under cooling conditions;

[0131] 1.2.2.3 Blocking and rinsing: Block with T-TBS (containing 5% skim milk powder) at room temperature for 1 hr, then rinse with T-TBS, 5 min × 3;

[0132] 1.2.2.4 Primary antibody incubation: Add Anti-6X His at a ratio of 1:5,000 Antibody (HRP) was added to T-TBS (containing 3% skim milk powder) and incubated at room temperature for 1 hr; then rinsed with T-TBS, 5 min × 5;

[0133] 1.2.2.5 Development: Add ECLWestern Blotting Sμbstrate (mix equal volumes of solution A and solution B), incubate at room temperature for 2-3 minutes, then remove excess ECL reagent, seal with plastic wrap, place X-ray film in a dark box and expose for 3-5 minutes before development and fixing.

[0134] 1.2.2.6 The results are as follows Figure 3 As shown: Western Blot detection was performed using rabbit anti-6×His polyclonal antibody. In lane 1, no target band appeared, while in lane 2, an obvious specific hybridization band (27 kDa) appeared near 27 kDa, further confirming that IGF2 was successfully expressed in E. coli.

[0135] 1.2.3 Optimization of IGF2 gene induction expression time

[0136] 1.2.3.1 The pET32-IGF2 bacterial solution was induced with IPTG (final concentration: 0.5 mM) for 1, 2, 3, 4, and 5 hr. The pET32 empty plasmid bacterial solution was used as the control group. The bacterial solution was collected and the samples were processed and subjected to SDS-PAGE electrophoresis according to the previous method.

[0137] 1.2.3.2 Results: After the pET32-IGF2 recombinant bacteria were induced by IPTG for 1, 2, 3, 4 and 5 hours, the cells were collected for SDS-PAGE detection. Figure 4 Gel grayscale scanning analysis showed that the expression level of the recombinant protein increased with the increase of induction time, reaching the highest value at 5 hours of induction, and the proportion of the recombinant protein in the total bacterial protein was about 20%.

[0138] 1.2.4 Detection of the existence form of pET32-IGF2 gene expression product

[0139] To determine whether the pET32-IGF2 expression product exists in the cytoplasm of E. coli BL21 (DE3) as a soluble substance or in the form of inclusion bodies, the following experiment was performed:

[0140] 1.2.4.1 Take 50 ml of bacterial solution and induce with IPTG for 3 hours, collect the bacteria by centrifugation (12,000×g, 1 min), and then add 5 ml of PBS (containing 1% TritonX-100) to thoroughly resuspend the bacterial pellet;

[0141] 1.2.4.2 Ultrasonic treatment: 300W, 10S, 10S rest, total 10min;

[0142] 1.2.4.3 After ultrasound, centrifuge (12,000g, 10min). The supernatant is the soluble expressed protein and the precipitate is the inclusion body. Then add 2×SDS-PAGE loading buffer, resuspend by oscillation, boil for 10min, cool to room temperature, and then centrifuge (12,000rpm, 10min). Take the supernatant for SDS-PAGE electrophoresis.

[0143] 1.2.4.4 Results: After IPTG induction, the pET32-IGF2 bacterial solution was treated with ultrasound, and the supernatant and precipitate were obtained for SDS-PAGE electrophoresis analysis. Figure 5 ,The results showed that the proportion of the target protein band in the supernatant was low, while the precipitate contained a large amount of the target protein, indicating that the recombinantly expressed IGF2 protein mainly existed in the form of inclusion bodies.

[0144] 1.2.5 Purification of pET32-IGF2 expression product

[0145] Using HisPur TM The expression product was purified by Ni-NTA Magnetic Beads (Thermo Fisher). The specific scheme is as follows:

[0146] 1.2.5.1 Collection of bacterial culture: 100 ml of bacterial culture induced by 0.5 mM IPTG was centrifuged and resuspended in 10 ml PBS (containing 1% Tritox-100), ultrasonically disrupted and centrifuged (12,000 g, 20 min), and the supernatant was discarded and the precipitate was retained;

[0147] 1.2.5.2 Resuspension and dissolution of inclusion bodies: Add 5 ml of Eqμlibration Buffer to resuspend the inclusion body precipitate and rotate at 4°C for 1 hour to dissolve the precipitate;

[0148] 1.2.5.3 Magnetic bead pretreatment: Take 2 ml of HisPurNi-NTA Magnetic Beads, mix well, perform magnetic separation, and discard the supernatant; then add Eqμlibration Buffer to resuspend, discard the supernatant, and repeat this step twice;

[0149] 1.2.5.4 Binding of magnetic beads and proteins: Add the protein in 5.2 to the magnetic beads pretreated in 5.3, bind at room temperature for 30 minutes, then perform magnetic separation, discard the supernatant, and retain the magnetic beads;

[0150] 1.2.5.5 Rinse: Add 5 ml of Washing Buffer to the magnetic beads to resuspend, then perform magnetic separation, discard the supernatant, retain the magnetic beads, and repeat this step 2 more times;

[0151] 1.2.5.6 Elution: Add 250 μl Elution Buffer (PBS / 8M Urea, pH 8.0, with final imidazole concentrations of 100 mM / 250 mM / 350 mM / 500 mM) in sequence, then perform magnetic separation and pipette the supernatant into a new 1.5 ml centrifuge tube;

[0152] 1.2.5.7 Electrophoresis: Take 5 μl of each eluted product, add an equal volume of 2×SDS-PAGE loading buffer, boil and load all of it for electrophoresis analysis.

[0153] 1.2.5.8 Results: The inclusion body protein of recombinant bacteria was purified by HisPur TM After purification by Ni-NTA magnetic beads, the target protein was eluted with imidazole elution buffers of different concentration gradients, and the elution effect was detected by SDS-PAGE. Figure 6 The results showed that when 250mM imidazole was used as the elution solution, the elution effect was better. There was a 27kDa target band in the SDS-PAGE gel, and almost no other protein bands were observed. This showed that the duck IGF2 recombinant protein was successfully purified and isolated, providing an important basis for antibody preparation.

[0154] The amino acid sequence of the duck-derived IGF2 recombinant protein is: 7.59 kilodaltons, AYGTAETLCGGELVDTLQFVCGDRGFYFSRPVGRNNRRINRGIVEECCFRSCDLALLETYCAKSVKSE (SEQ ID NO: 2)

[0155] 1.3 Renaturation of duck pET32-IGF2 gene prokaryotic expression protein

[0156] 1.3.1 Cut the dialysis bag into corresponding lengths according to the volume of eluted protein; rinse it with deionized water first, and then soak it in deionized water at room temperature for 30 minutes;

[0157] 1.3.2 Add 200 volumes of 4 M urea buffer to the beaker and stir magnetically at 4°C overnight (replace the 4 M urea buffer once during this period);

[0158] 1.3.3 Then dialyze in refolding buffer at 4°C for 24 h (replace refolding buffer once during this period);

[0159] 1.3.4 Add 2 M urea buffer and dialyze at 4°C for 24 h;

[0160] 1.3.5 Finally, add 200 times PBS and stir magnetically at 4℃ overnight;

[0161] 1.3.6 Use ultrafiltration tube to concentrate to a certain volume.

[0162] Example 2: Application of duck-derived IGF2 recombinant protein

[0163] 2.1 Primary isolation and culture of duck granulosa cells

[0164] Laying ducks in the laying period were selected and killed by bleeding from the jugular vein. The entire ovarian tissue was taken out and placed in a sterile culture dish filled with pre-cooled PBS and quickly brought back to the laboratory, where it was washed several times with PBS containing double antibodies. The rinsed follicles were transferred to a plate containing pre-cooled PBS buffer, and the outer membrane, connective tissue and vascular network of the follicles were peeled off. The yolk was released after the follicles were cut, and the rinsed follicle membranes were cut into pieces as much as possible and placed in a 15mL centrifuge tube; 4mL of culture medium was added and repeatedly blown for 1min, and the supernatant was discarded after centrifugation at 4℃1000r / min; 4mL of 0.2% type II collagenase was added, the precipitate was resuspended, and the precipitate was placed in a 37℃ constant temperature shaker at 80r / min for digestion for 30min; 4mL of M199 complete culture medium (containing 10% serum) was added to terminate the digestion; Filtered with a 200-mesh stainless steel sieve, the filtrate was collected, and the cells were collected after centrifugation at 4℃1000r / min for 10min, and M199 complete culture medium (containing 10% FBS and 1% double antibody) was added for resuspending, and the cell density was determined and then inoculated into a 6-well culture dish, which was placed in a 37℃, 5% CO2 incubator for static culture. After 24h of culture, the subsequent related experiments can be used.

[0165] Afterwards, HE staining and immunofluorescence identification were performed on the isolated and cultured duck follicle granulosa cells, indicating that more than 90% of the isolated cells were duck ovarian granulosa cells, and subsequent experiments could be performed.

[0166] 2.2IGF2 promotes the proliferation of duck follicle granulosa cells

[0167] 2.2.1 Recovery and passage of duck granulosa cells

[0168] After cell recovery, the cells were cultured with M199 complete medium (containing 10% FBS and double antibodies) at 37°C until the confluence reached about 80%. The cells were trypsinized and passaged in 24-well plates.

[0169] 2.2.2 Transfection and EdU staining of duck granulosa cells

[0170] Granulosa cells were treated with different concentrations of IGF2 (i.e., duck-derived IGF2 recombinant protein) and divided into four groups: Control, IGF2 (50 ng / ml), IGF2 (200 ng / ml), and IGF2 (500 ng / ml). After 48 hours, the culture medium was discarded, and complete culture medium containing 50 μM EdU working solution was added and incubated for another 2 hours. Then, the cells were rinsed with PBS for 5 min × 2. 2.2.3 Cell fixation and permeabilization

[0171] 2.2.3.1 Add 200 μl 4% paraformaldehyde to each well and fix at room temperature for 30 min, then discard the fixative;

[0172] 2.2.3.2 Rinse the cells with PBS, incubate on a shaker for 5 min, and then discard the solution;

[0173] 2.2.3.3 Add 400 μl PBS containing 0.3% Triton X-100, incubate on a decolorizing shaker for 10 min, and then discard the solution;

[0174] 2.2.3.4 Rinse the cells with PBS, incubate on a shaker for 5 min, and then discard the solution;

[0175] 2.2.4 EdU detection

[0176] 2.2.4.1 Prepare ClickAdditive Solution reaction solution as required, add 300 μl of reaction solution to each well, incubate in a decolorizing shaker for 30 min at room temperature in a dark place, and discard the reaction solution;

[0177] 2.2.4.2 Rinse the cells with PBS, incubate on a decolorizing shaker for 5 min, and then discard the solution.

[0178] 2.2.5 Nuclear staining

[0179] 2.2.5.1 Add 1×Hoechst33342 reaction solution and incubate at room temperature in the dark for 10 min, then discard the reaction solution;

[0180] 2.2.5.2 Add PBS to each well for rinsing for 2 min × 3, take a photo immediately (×100), and calculate the fluorescence intensity.

[0181] 2.2.6 Experimental results

[0182] Granulosa cells were treated with different concentrations of IGF2 (i.e., duck-derived IGF2 recombinant protein) and divided into four groups: Control, IGF2 (50 ng / ml), IGF2 (200 ng / ml), and IGF2 (500 ng / ml). EdU staining was used to analyze the changes in cell proliferation ability. Figure 7The results showed that compared with the Control group, the number of EdU-positive cells in the IGF2 (200 ng / ml) group increased (P<0.05), while the number of EdU-positive cells in the IGF2 (500 ng / ml) group increased significantly (P<0.01), indicating that IGF2-induced treatment can promote the proliferation of duck ovarian granulosa cells.

[0183] 2.3IGF2 activates the PI3K-AKT pathway in duck granulosa cells

[0184] Granulosa cells were treated with 500 ng / ml IGF2 (i.e., duck IGF2 recombinant protein) and PI3K inhibitor LY294002 and divided into four groups: Control, IGF2 (500 ng / ml), LY294002 (20 μM), and IGF2 (500 ng / ml) + LY294002 (20 μM), and the expression changes of key node proteins in the PI3K-AKT signaling pathway were observed.

[0185] Western Blot: Total protein was extracted using a total protein extraction kit (containing Protease Inhibitor Cocktail) (ThermoFisher, USA), and then total protein was quantified using a BCA quantification kit (Beyotime, Shanghai, China). 8-12% separation gel and 5% concentrated gel were prepared, and electrophoresis was performed at 60V for the concentrated gel and 80V for the separation gel for about 2 hours. The membrane was transferred to a PVDF membrane at constant pressure for 2 hours. After the transfer, it was placed in T-TBS (containing 5% BSA), blocked at room temperature for 1 hour, and then rinsed with T-TBS. The primary antibody was incubated overnight; after rinsing with T-TBS, the secondary antibody was added at room temperature for 1 hour; rinsed with T-TBS. West Dura Extended Duration Substrate, 1ml ECL working solution, incubate the transfer membrane at room temperature for 1min, then remove the excess ECL reagent, put X-ray film in the dark box and expose for 5-10min before developing and fixing. Image J software was used to analyze the optical density of the bands. Each band was repeated 3 times. The relative expression of the target protein = {target protein (optical density value) / internal reference (optical density value)}×10 n The results were expressed as mean ± standard deviation.

[0186] The results show that Figure 8), after GCs were treated with IGF2, the protein expression of p-PI3K, p-AKT and IGF1R increased, and after GCs were treated with PI3K inhibitor LY294002, the protein expression of p-AKT and p-PI3K were downregulated. When IGF2 and PI3K inhibitor were added at the same time, the expression levels of p-AKT and p-PI3K, the marker gene proteins related to the PI3K-AKT signaling pathway, were higher than those in the LY294002 treatment group, but lower than those in the IGF2 addition group. This indicates that IGF2 can break the inhibited state of p-AKT and p-PI3K, activate the state of p-AKT, p-PI3K and IGF1R, and IGF2 can affect the proliferation of granulosa cells through the PI3K-AKT pathway.

[0187] 2.4 Transcriptome sequencing

[0188] The sequencing samples were divided into a control group and an IGF2-added group (i.e., duck-derived IGF2 recombinant protein). RNA was extracted from ovarian granulosa cells using a standard extraction method, and then the RNA samples were strictly quality-controlled using Agilent 2100bioanalyzer software to accurately detect RNA integrity. After the library inspection was qualified, Illumina sequencing and differential gene analysis were performed.

[0189] The sequencing results showed that 327 differentially expressed genes were screened, of which 120 were up-regulated and 207 were down-regulated. Fig. 9 A). Through heat map ( Fig. 9 B) It can be seen that the clustering analysis grouping is reasonable, proving that the sequencing grouping results are accurate. KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in ECM-receptor interaction, focal adhesion, stereoid hormone biosynthesis, and neuroactive ligand-receptor interaction ( Fig. 9 C). This suggests that IGF2 may directly or indirectly affect hormone-related signaling pathways such as stereoidhormone biosynthesis.

[0190] The experimental results of the present invention show that IGF2 can regulate the PI3K-AKT signaling pathway and IGF1R of Jinding duck, affect the proliferation level of ovarian granulosa cells, promote the growth of granulosa cells and promote follicle development.

[0191] 2.5 Statistical analysis

[0192] The present invention uses the mean ± standard error (SEM) of at least three independent experiments as quantitative data. The t test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups. p < 0.05 was set as significant, and p < 0.01 was set as extremely significant. GraphPad Prism 9.5.1 software (La Jolla, CA, USA) was used to draw statistical data.

Claims

1. A duck-derived IGF2 recombinant protein, characterized in that: The amino acid sequence of the duck-derived IGF2 recombinant protein is shown in SEQ ID NO:

2.

2. A method for preparing the duck-derived IGF2 recombinant protein as claimed in claim 1, characterized in that: It includes the following steps: (1) PCR amplification of duck IGF2 gene coding region: using duck cDNA as template, using primers IGF2-F: GACAAGGCCATGGCT GATATC GCGTACGGCACGGCGGAGA and primer IGF2-R: ACGGAGCTCGAATTC GGATCC CTACTCAGACTTGACGGACTTGG, perform PCR amplification on it, and then perform gel excision and recovery on the PCR amplification product for subsequent fusion reaction; (2) In Fusion for recombination of vector and target fragment: Seamless Cloning and Assembly Enzyme Mix was used to construct the recombinant plasmid; the reaction system for the duck IGF2 amplification product and pET32 fusion reaction was as follows: 10 μL of the system contained the following components: 2.0 μL sterile deionized water, 2.0 μL duck IGF2 amplification product, 1.0 μL pET32 (EcoRV / BamHI) (50 ng), 5.0 μL 2X Enzyme Mix; reaction conditions: place at room temperature for 20 minutes; place on ice for 2-3 minutes, and then transform all competent cells BL21 (DE3) to obtain E. coli BL21 (DE3) carrying pET32-IGF2; (3) Sequencing identification: 3-5 grown colonies were selected for sequencing; (4) Induced expression of pET32-IGF2 protein: E. coli BL21 (DE3) carrying pET32-IGF2 after sequencing analysis was cultured, and then induced with IPTG. The bacterial precipitate was collected by centrifugation, protein loading buffer was added, boiled, and cooled to room temperature; then centrifuged, and the supernatant was collected for SDS-PAGE electrophoresis analysis; (5) Identification of expression products by Western Blotting; (6) Purification of pET32-IGF2 expression product: Using HisPur TM The expressed product was purified by Ni-NTA Magnetic Beads (Thermo Fisher) to obtain duck IGF2 recombinant protein.

3. The preparation method according to claim 2, characterized in that: The reaction system for PCR amplification in step (1) is as follows: the reaction system is 50 μL, including 1.0 μL of sterile deionized water, 1.0 μL of primer IGF2-F with a concentration of 10 μM, 1.0 μL of primer IGF2-R with a concentration of 10 μM, 2.0 μL of duck cDNA, PCR SμperMix, High Fidelity 45μL; the reaction conditions for PCR amplification were: pre-denaturation at 94°C for 2 min; 94°C for 30 sec, 60°C for 30 sec, 68°C for 30 sec, 35 cycles.

4. The preparation method according to claim 2, characterized in that: The specific method of step (4) is: S1 E. coli BL21 (DE3) carrying pET32-IGF2 after sequencing analysis was inoculated into 5 mL of LB liquid medium containing 50 μg / mL Ampicillin at a final concentration of 50 μg / mL and cultured overnight at 37°C and 200 rpm; S2 was transferred to a new SOC liquid medium containing 50 μg / mL Ampicillin at a dilution of 1:100 and cultured at 37°C, 200 rpm for 2-3 hr. OD 600 When the concentration of pET32 protein was 0.5-0.6, IPTG was added to a final concentration of 0.5 mM and induced at 37°C for 1-5 hours. The bacterial culture was collected, and the pET32 empty vector IPTG-induced group and the pET32-IGF2 IPTG-uninduced group were used as controls. S3 was centrifuged at 12000×g for 1 min, the bacterial precipitate was collected, 80 μL of 1× protein loading buffer was added, boiled for 5 min, and cooled to room temperature; then centrifuged at 12000×g for 5 min, and 5 μL of supernatant was taken for SDS-PAGE electrophoresis analysis.

5. The preparation method according to claim 2, characterized in that: The specific method of step (6) is: Using HisPur TM The expression product was purified by Ni-NTA Magnetic Beads (Thermo Fisher). The specific scheme is as follows: S1 bacterial liquid collection: 100 ml of bacterial liquid induced by 0.5 mM IPTG was centrifuged and resuspended in 10 ml PBS containing 1% Tritox-100. After ultrasonic disruption, centrifuged at 12,000 g for 20 min, and the supernatant was discarded and the precipitate was retained; S2 Inclusion body resuspension and dissolution: Add 5 ml Eqμlibration Buffer to resuspend the inclusion body precipitate, rotate at 4℃ for 1 hour to dissolve the precipitate; S3 magnetic bead pretreatment: Take 2 ml of HisPurNi-NTA Magnetic Beads, mix well, perform magnetic separation, and discard the supernatant; then add Eqμlibration Buffer to resuspend, discard the supernatant, and repeat this step twice; S4 Binding of magnetic beads and proteins: Add the protein in step 5.2 to the magnetic beads pretreated in step 5.3, bind at room temperature for 30 minutes, then perform magnetic separation, discard the supernatant, and retain the magnetic beads; S5 washing: add 5 ml of Washing Buffer to the magnetic beads to resuspend, then perform magnetic separation, discard the supernatant, retain the magnetic beads, and repeat this step 2 more times; S6 elution: Add 250 μl of imidazole elution buffer with different concentration gradients to elute the target protein, then perform magnetic separation and pipette the supernatant into a new 1.5 ml centrifuge tube; S7 electrophoresis: Take 5 μl of each eluted product, add an equal volume of 2×SDS-PAGE loading buffer, boil and load all of it for electrophoresis analysis.

6. The preparation method according to claim 5, characterized in that: In step S6, the imidazole eluent is: a PBS buffer solution with a pH of 8.0, and the PBS buffer solution contains 8M urea and 100-500mM imidazole.

7. The preparation method according to claim 6, characterized in that: The imidazole eluent is a PBS buffer solution with a pH of 8.0, and the PBS buffer solution contains 8M urea and 250mM imidazole.

8. Use of the duck-derived IGF2 recombinant protein according to claim 1 in the preparation of a promoter for promoting the proliferation of duck ovarian granulosa cells.

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