Preparation of recombinant expression pigeon prolactin and construction method of fat formation inducing model of recombinant expression pigeon prolactin

Abstract

The invention relates to the technical field of bioengineering, and discloses a method for preparing recombinant expression pigeon prolactin and constructing a fat formation inducing model of the recombinant expression pigeon prolactin, which comprises the following steps of: constructing genetically engineered bacteria for efficiently expressing pigeon-derived prolactin, establishing a two-step purification process, and performing low-temperature concentration by using PEG8000 and an ultrafiltration two-step method, so as to obtain the fat formation inducing pigeon prolactin. The high-purity and high-activity recombinant pigeon prolactin protein is obtained, and meanwhile, an in-vitro organ culture technology is utilized to construct an in-vitro model for inducing the formation of craw tissue fat by the recombinant pigeon prolactin. The invention provides a brand-new platform for researching a molecular mechanism of bird prolactin for regulating fat metabolism.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for preparing recombinantly expressed pigeon prolactin and constructing a model thereof for inducing fat formation. Background Art

[0002] Prolactin (PRL) is a pleiotropic hormone that is widely involved in various physiological processes in mammals, including reproduction, metabolism, and immunity. Recent studies have revealed that avian prolactin plays a crucial role in regulating fat metabolism, particularly its mechanism of inducing adipogenesis in crop tissue, providing new insights into fat metabolism.

[0003] However, recombinant mammalian prolactin is often used in avian physiology research or artificial breeding, but its effectiveness is suboptimal due to species differences. Avian prolactin products reported in the literature suffer from insufficient variety, low purity, and a lack of simple methods for subsequent activity verification. Furthermore, previous models of fat metabolism have often relied on drugs to induce abnormal metabolic fat accumulation in cells. These methods are complex, time-consuming, and costly, and precise control of experimental conditions is difficult. The lack of effective models for studying the mechanisms of normal fat formation in birds under normal physiological conditions has limited further exploration in this field. Summary of the Invention

[0004] Purpose of the Invention: To address the problems existing in the prior art, the present invention provides a method for preparing recombinantly expressed pigeon prolactin and constructing a model for its adipogenesis induction. By constructing a genetically engineered bacterium that efficiently expresses pigeon prolactin and establishing a two-step purification process, followed by cryogenic concentration using PEG8000 and ultrafiltration, a highly pure and active recombinant pigeon prolactin protein is obtained. Furthermore, using in vitro organ culture technology, an in vitro model of recombinant pigeon prolactin-induced crop adipogenesis is constructed. This invention provides a novel platform for studying the molecular mechanisms by which avian prolactin regulates fat metabolism.

[0005] Technical solution: On the one hand, the present invention provides a method for preparing recombinantly expressed pigeon prolactin, comprising the following steps:

[0006] Total RNA was extracted from various tissues of pigeons, and the prolactin target gene was amplified by RT-PCR; the target gene was then cloned into the pET-29 prokaryotic expression plasmid to obtain a recombinant expression plasmid containing the prolactin target gene; the recombinant expression plasmid containing the prolactin target gene was then transformed into BL21 expression bacteria and induced for expression with IPTG; finally, the bacterial culture after induced expression was separated and purified to obtain recombinantly expressed pigeon prolactin;

[0007] The nucleotide sequence of the prolactin target gene is shown in SEQ ID NO: 1.

[0008] (SEQ ID NO: 1).

[0009] Furthermore, the primers for the RT-PCR amplification are as follows:

[0010] F1:5'-CATATGTTGCCAGTCTGCCCCAAT-3';

[0011] R1: 5'-CTCGAGACAATTGCTATCGTGGATTAGG-3';

[0012] The reaction conditions for RT-PCR amplification were as follows: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles of 72°C for 7 min.

[0013] Preferably, the concentration of IPTG is 0.5 mM, and the induction expression time is 2 h.

[0014] Furthermore, the specific steps of separation and purification are:

[0015] (1) Inclusion body dissolution: The bacterial culture after induced expression is ultrasonically disrupted, washed, and centrifuged to obtain inclusion bodies; the inclusion bodies are dissolved in a dissolution buffer, shaken at 4°C for a period of time, and then centrifuged to obtain the supernatant;

[0016] (2) Nickel column purification: the supernatant was loaded onto a Ni column and the protein effluent was collected;

[0017] (3) Inclusion body refolding: The protein effluent was mixed with refolding buffer, allowed to stand for a period of time, and then placed in a dialysis bag for refolding by dialysis at 4°C.

[0018] (4) Two-step concentration: The renatured inclusion bodies were concentrated at low temperature in PGE8000, and then ultrafiltration was performed at 4°C. The supernatant was collected to obtain the recombinant expressed pigeon prolactin.

[0019] Furthermore, after step (2), the following steps are further included:

[0020] Take part of the protein eluate and elute it with different imidazole concentration gradients; take a little of the protein eluate from each gradient and mix it with SDS-loading, and then perform SDS-PAGE analysis after placing it in a metal bath at 100℃ for 5 minutes.

[0021] Furthermore, if there are foreign proteins in the eluate, the eluate is subjected to Sephadex G-50 column chromatography.

[0022] On the other hand, the present invention provides a method for constructing a model for inducing adipogenesis, wherein the model is constructed using recombinantly expressed pigeon prolactin prepared by any of the methods described above.

[0023] Furthermore, the specific construction method is as follows:

[0024] S1. Tissue Culture: Pigeon crop tissue was harvested, cut into small pieces, and inoculated into culture flasks. Complete culture medium was added for culturing.

[0025] S2.PRL induction: Add different concentrations of recombinant pigeon prolactin to the S1 culture medium and continue culturing for a period of time;

[0026] S3. Oil Red O staining: The effect of recombinantly expressed pigeon prolactin on the density and morphology of crop lipid droplets was detected by Oil Red O staining, and the distribution and number of lipid droplets were observed.

[0027] Furthermore, in S2, the concentration of the recombinantly expressed pigeon prolactin is 50-100 ng / mL.

[0028] Beneficial effects: The present invention provides a technical solution for recombinantly expressing a high-purity product of avian prolactin. Based on this solution, a prolactin-induced adipogenesis model for avian tissues and organs was established by selecting the crop as a special organ. This provides important technical support for in-depth research on the regulatory mechanism of fat metabolism in birds under normal physiological conditions. It has important scientific significance and application value. Compared with the existing technology, its specific beneficial effects are as follows:

[0029] (1) Through genetic engineering technology, the pigeon prolactin gene was successfully cloned and an efficient prokaryotic expression system was constructed, achieving efficient expression of recombinant pigeon prolactin;

[0030] (2) A complete protein purification process was established, including a two-step purification process using PEG8000 and ultrafiltration for low-temperature concentration, to obtain high-purity and high-activity recombinant pigeon prolactin protein;

[0031] (3) Innovatively utilizing in vitro organ culture technology, a model of crop adipogenesis induced by recombinant pigeon prolactin was constructed, providing a new platform for studying the molecular mechanism of pigeon prolactin regulating fat metabolism;

[0032] (4) By constructing a model of crop adipogenesis induced by recombinant pigeon prolactin, we systematically studied the role of recombinant pigeon prolactin in adipogenesis for the first time, providing important evidence for the study of the regulatory mechanism of fat metabolism in birds;

[0033] (5) The model of crop tissue fat formation induced by recombinant pigeon prolactin constructed in the present invention has the advantages of simple operation, good repeatability, and ability to simulate the in vivo environment. It can be widely used in the fields of research on the regulation mechanism of bird fat metabolism and drug screening, and has significant practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Electrophoresis of the prolactin gene expressed in various tissue samples of pigeons; M: DL1000 DNA Marker; 1, 2, 5, 6, 7: Pigeon muscle and other tissues do not express the prolactin gene; 3, 4, 7, 8: Prolactin genes (687 bp) expressed in the crop, brain, pituitary gland, and liver tissues, respectively;

[0035] Figure 2 : Electrophoresis of PCR amplification products of prolactin gene expressed in pigeon pituitary tissue samples; wherein, M: DL5000 DNA Marker; 1-2: PCR amplification products of target gene expressed in pituitary tissue (687 bp);

[0036] Figure 3: Electrophoresis of recombinant clone plasmid double enzyme digestion; M: DL5000 DNA Marker; 1-2: pMD18-T (2692 bp); 3: target gene linked to pMD18-T enzyme; 4: recombinant clone plasmid double enzyme digestion; 5: target gene (687 bp);

[0037] Figure 4 : Electrophoresis of colony PCR identification of recombinant clone strains; M: DL5000 DNA Marker; 1-3: colony PCR products (687 bp);

[0038] Figure 5 : Electrophoresis of colony PCR identification of recombinant expression strain; M: DL5000 DNA Marker; 1-6: colony PCR product (697bp);

[0039] Figure 6 : Optimization of expression of recombinant expression bacteria induced by different IPTG concentrations; M: protein molecular weight standard; 1-7: final concentrations of 0mM, 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1.0mM, and 2.0mM IPTG, respectively;

[0040] Figure 7 : Optimization of expression of recombinant expression bacteria at different induction times; M: protein standard molecular weight band; 1: uninduced recombinant expression bacteria; 2-7: expression products of recombinant expression bacteria at "0.5h", "1h", "1.5h", "2h", "2.5h", and "3h" after adding the optimal inducer, respectively;

[0041] Figure 8 : Identification of recombinant protein solubility; where M is the protein molecular weight standard; 1 is the inclusion body product; 2 is the soluble product;

[0042] Figure 9 : Target protein was eluted by gradient elution of different imidazole concentrations; wherein, M is a protein marker, and lanes 1-14 represent uninduced control, IPTG-induced control, ultrasonic precipitate, ultrasonic supernatant, flow-through, 10 mM imidazole, 20 mM imidazole, 30 mM imidazole, 40 mM imidazole, 50 mM imidazole, 60 mM imidazole, 70 mM imidazole, 80 mM imidazole, and 250 mM imidazole, respectively;

[0043] Figure 10 : Separate and purify the target protein by dextran gel;

[0044] Figure 11 : Target protein after two-step concentration;

[0045] Figure 12: Effects of PRL on lipid droplet distribution and morphology of pigeon crop tissue cultured in vitro; AC: pigeon crop tissue cultured in tissue culture medium containing 50 ng / ml PRL for 0 h (A), 36 h (B), and 48 h (C); DF: pigeon crop tissue cultured in tissue culture medium containing 100 ng / ml PRL for 0 h (D), 36 h (E), and 48 h (F);

[0046] Figure 13 : The process flow chart of the preparation of the recombinantly expressed pigeon prolactin and the model construction method of inducing adipogenesis of the present invention. DETAILED DESCRIPTION

[0047] The present invention is described in detail below with reference to the embodiments.

[0048] Embodiment 1: Prokaryotic expression of pigeon-derived prolactin

[0049] 1. Materials

[0050] 1.1 Sample

[0051] Total RNA in various tissues including pigeon pituitary tissue, crop tissue, brain tissue, heart tissue and liver tissue.

[0052] 1.2 Plasmids and strains

[0053] pMD18-T cloning plasmid, pET-29 prokaryotic expression plasmid, DH5α competent cells.

[0054] 1.3 Enzymes and reagents

[0055] Enzymes: Ex Taq DNA polymerase, enzymes NdeⅠ and XhoⅠ, T4 DNA Ligase.

[0056] Reagents: Trizol kit, plasmid extraction kit, DNA gel recovery kit, kanamycin (Kan), ampicillin (AMP), IPTG, etc.

[0057] 2 Methods

[0058] 2.1 Primer design and synthesis

[0059] A pair of primers (synthesized by Shanghai Yingjun Company) were designed based on the cDNA and genomic sequences of pigeon prolactin gene and the prokaryotic expression vector pET-29.

[0060] pigeon-PRL-F1:5'- CATATG TTGCCAGTCTGCCCCAAT-3'

[0061] pigeon-PRL-R1:5'-CTCGAGACAATTGCTATCGTGGATTAGG-3'

[0062] Among them, the underlined parts are the restriction sites of enzymes NdeⅠ and XhoⅠ, respectively.

[0063] 2.2 Amplification of the pigeon-derived prolactin target gene

[0064] Total RNA was extracted from various pigeon tissues, including the pituitary, crop, brain, liver, and heart, using the Trizol method. RT-PCR amplification yielded a large number of target gene fragments. The PCR products were then run on an electrophoretic gel to examine target gene expression. The PCR reaction was performed in a total volume of 25 μL (Table 1). After observation, the expected gene bands were excised and recovered.

[0065] The nucleotide sequence of the prolactin target gene is shown in SEQ ID NO: 1.

[0066] (SEQ ID NO: 1).

[0067] The specific conditions of the PCR reaction were as follows: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles, and 72°C for 7 min.

[0068] Table 1 PCR amplification reaction system (25 μL)

[0069]

[0070] 2.3 Enzyme ligation of target gene and pMD18-T

[0071] The recovered target gene was enzymatically ligated with pMD18-T using a 20 μL ligation system (Table 2). After reacting at 16°C for 1-5 hours, the recombinant cloning plasmid was generated and transformed into DH5α for cloning. Procedure: Add 10 μL of the ligation product, 10 μL of 5× KCM solution, and 30 μL of ddH2O to a 1.5 mL centrifuge tube (for low-temperature operation). Remove the competent DH5α from a -80°C freezer and immediately thaw. Using a pipette, pipette 50 μL of the solution and mix thoroughly. Place the mixture in an ice-water mixture for 20 minutes. Allow to stand at room temperature for 10 minutes. Then, pipette 500 μL of LB medium and incubate at 37°C with shaking at 150 rpm for 50-60 minutes. Finally, inoculate the plate onto LB medium using the spreader method and incubate overnight in an incubator. Colonies were selected for colony PCR verification using a 25 μL PCR system (Table 3).

[0072] Colony PCR verification: Select two single colonies from each of three culture dishes. After sterilizing the inoculation loop, pick a single colony and place it in a PCR tube containing 10 μL of sterile water. 5 μL of the solution is used for PCR amplification, and the results are observed by gel electrophoresis. Another 5 μL of the bacterial solution containing the target gene band is used for liquid tube culture and shaken at 37°C, 200 rpm overnight.

[0073] Table 2 Enzyme-linked reaction system (20 μL)

[0074]

[0075] Table 3 Colony PCR reaction system (25 μL)

[0076]

[0077] 2.4 Construction and identification of recombinant expression plasmids

[0078] Plasmids were extracted from overnight bacterial cultures and double-digested with NdeI and XhoI. The first digestion was performed with 10 μL of the enzyme system (Table 4) in a 37°C water bath for 1 hour. The digestion products were electrophoresed and visualized on a gel imager. The digestion was then further expanded (100 μL) (Table 4) to obtain a large amount of the target gene.

[0079] Simultaneously, the pET-29 prokaryotic expression plasmid was double-digested and then ligated to the target gene using a ligation reaction system (20 μL) (Table 2). The reaction was allowed to react at 16°C for 1-5 hours to obtain the recombinant expression plasmid, which was then transformed into DH5α. Colony PCR was performed to identify the colonies, and those that met the requirements were cultured with shaking overnight.

[0080] Table 4 Double enzyme digestion system

[0081]

[0082]

[0083] 2.5 Inducible expression of recombinant expression plasmid

[0084] After the recombinant expression plasmid is extracted, it is transformed into the BL21 expression host bacteria and cultured. Colonies are then picked for PCR and electrophoresis verification, and the bacteria containing the target gene are cultured overnight.

[0085] Optimize the expression of different IPTG concentrations: inoculate the above cultured bacteria into 7 50 mL Erlenmeyer flasks, each containing 25 mL LB medium, numbered 1 to 7, add 1.5 mL of recombinant expression BL21 bacteria solution to each flask and incubate at 37 ° C, 150 rpm on a shaker for 2-3 h until the OD 600 If the pH value is 0.6-0.8, IPTG with final concentrations of 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1.0mM and 2.0mM can be added to the above 2-7 Erlenmeyer flasks in sequence, that is, 2.5μL, 12.5μL, 25μL, 125μL, 250μL and 500μL of IPTG can be added to each flask in sequence. No IPTG is added to Erlenmeyer flask No. 1. The 7 flasks are marked in sequence according to the IPTG concentration added. Continue to culture for another 3 hours, aspirate 1 mL of each bottle of bacterial liquid and place it in a 1.5 mL centrifuge tube, centrifuge at 12000 r / min for 4 minutes, discard the supernatant, add 100 μL 3× protein loading buffer to the precipitate, mix well, boil for 5 minutes, mix again and centrifuge at 12000 rpm for 1 minute, store in a -20°C refrigerator, and perform SDS-polyacrylamide gel electrophoresis identification to determine the optimal IPTG induction concentration required for the maximum expression of prolactin protein.

[0086] Induction of expression at different times after IPTG addition: Place 25 mL of LB medium in each of seven 50 mL Erlenmeyer flasks numbered 1-7. Add 1.5 mL of overnight cultured bacteria and incubate at 37°C with shaking at 150 rpm for 2-3 hours until the OD600 reaches 0.6-0.8. Add equal amounts of IPTG at the optimal concentration to each of these 2-7 Erlenmeyer flasks. Flask 1 is left without IPTG. At this point, timing and sampling can be performed. Label flask 1 "0 h" and sample from it. Flasks 2-7 are labeled "0.5 h," "1 h," "1.5 h," "2 h," "2.5 h," and "3 h," respectively. Following IPTG addition, culture for the indicated times and immediately sample the cells. Repeat the above procedure and verify by SDS-PAGE electrophoresis to determine the optimal induction time required for maximum prolactin protein expression after IPTG induction.

[0087] 2.6 SDS-polyacrylamide gel electrophoresis identification

[0088] Glue filling and sample loading: Prepare 15% separation gel and stacking gel according to the gel preparation recipe (Table 5), and load the sample after gel filling;

[0089] Electrophoresis: After loading the sample, close the top cover and connect the electrophoresis instrument. Run the sample at low voltage (40V) for about 0.5h. After the bromophenol blue has electrophoresed into the separating gel, increase the voltage to 120V and run the electrophoresis for about 1.5h.

[0090] Staining and Destaining: After electrophoresis, remove the film and place it in the staining solution for 0.5 hours. Remove the staining solution, rinse several times with pure water, and then add the eluent to destain. During the destaining process, the eluent should be changed frequently until the bands are clear. If clear bands are urgently needed, after adding the eluent, place the large culture dish in an induction cooker with boiling water and heat it for about 5 minutes, changing the eluent. Repeat 3 to 4 times to see clear protein bands.

[0091] Table 5 Glue making formula

[0092]

[0093] 3 Results and Analysis

[0094] 3.1 Obtaining the pigeon-derived prolactin target gene

[0095] After electrophoresis of the PCR products of each tissue sample, the gel imaging instrument showed that the crop, brain, pituitary gland, and liver all expressed the prolactin gene, and the gene size was 687 bp ( Figure 1 ), which is consistent with the expected relative molecular mass of the prolactin protein gene, and the prolactin gene expression in the pituitary tissue is the highest, while other tissues such as muscle tissue do not express it. Re-verification of the gene expressed in the pituitary tissue ( Figure 2 ).

[0096] 3.2 Enzyme ligation analysis of target gene and pMD18-T

[0097] The prolactin target gene (687 bp) and pMD18-T (2692 bp) were ligated by enzyme to obtain a recombinant cloning plasmid of approximately 3379 bp ( Figure 3 ) and positive recombinant clone plasmids were identified by colony PCR. In the medium containing ampicillin, only when the target gene and the cloned plasmid were successfully linked and transformed into the DH5α competent cell could colonies grow on the plate. The empty plasmid alone and the target gene transformation could not make DH5α grow in the medium containing ampicillin. After colony PCR amplification and electrophoresis using the colonies grown on the plate as templates, the target gene was visible on the gel imager. The gene size was 687bp ( Figure 4), indicating that the target gene was successfully ligated with pMD18-T enzyme.

[0098] 3.3 Colony PCR identification of recombinant expression plasmid

[0099] pET-29 carries kanamycin resistance. In a culture medium containing kanamycin, only the recombinant expression plasmid with the prolactin target gene successfully linked to the pET-29 prokaryotic expression vector can grow into colonies when transformed into DH5α. Other strains that have not been successfully linked or transformed cannot generate colonies. Using the colonies on the plate as templates for colony PCR and running the gel, a prolactin gene band can be seen on the gel imager. The gene size is 687bp ( Figure 5 ), indicating that the recombinant expression plasmid was successfully constructed.

[0100] 3.4 SDS-PAGE analysis of recombinant expression products

[0101] After inducing the expression of BL21 strain with different final concentrations of IPTG, the results showed that the recombinant protein expression was strongest when the final concentration of IPTG was 0.5mM ( Figure 6 Based on the above conditions, the optimal time for induction expression is 2h ( Figure 7 ), and the optimal expression conditions were determined to be 0.5 mM IPTG for 2 h. SDS-PAGE analysis revealed a protein band with a molecular mass of approximately 22 kD, consistent with the predicted molecular weight of the prolactin protein.

[0102] 3.5 Protein solubility analysis

[0103] After SDS-PAGE electrophoresis of the protein samples, it was found that the recombinant protein existed in both the supernatant and the precipitate samples, indicating that the recombinant protein existed in two forms, namely soluble and inclusion bodies, but most of them appeared in the form of inclusion bodies, with a molecular weight of about 22KD ( Figure 8 ).

[0104] Implementation 2: Isolation, purification, renaturation and concentration of pigeon-derived prolactin protein

[0105] 1. Test materials

[0106] 1.1 Main Materials

[0107] Sephadex G-50, PEG8000, 6× SDS-loading buffer, TEMED, ammonium persulfate, Tris, hydrochloric acid, 30% gelatinizing solution, 6× his nickel column, dialysis bag, ultrafiltration tube, magnetic stirrer, ultrasonic disruptor, centrifuge 1.2 Main reagent configuration

[0108] Ultrasonication buffer: 50 mmol / L Tris-HCl (pH 8.0), 0.1 mol / L NaCl, 5 mmol / L EDTA. To prepare 500 ml of ultrasonication buffer, you need 3.0285 g of Tris, 2.922 g of NaCl, and 0.7306 g of EDTA.

[0109] Wash buffer: 50 mmol / L Tris-HCl (pH 8.0), 0.1 mol / L NaCl, 5 mmol / L EDTA, 2 mol / L urea, 2% Triton X-100. To prepare 500 ml of wash buffer, you need 3.0285 g of Tris, 2.922 g of NaCl, 0.7306 g of EDTA, 60.06 g of urea, and 10 ml of Triton X-100 (add more when the volume is almost 500 ml).

[0110] Dissolution buffer: 8 mol / L urea. To prepare 100 ml of dissolution buffer, 48.048 g of urea is required.

[0111] Refolding buffer: 50 mmol / L Tris-HCl (pH 8.0), 0.1 mol / L NaCl, 4 mmol / L reduced glutathione (GSH), 0.4 mmol / L oxidized glutathione (GSSG), 1% glycerol. To prepare 500 ml of refolding buffer, you need 3.0285 g of Tris, 2.922 g of NaCl, 0.61464 g of reduced glutathione, 0.122526 g of oxidized glutathione, and 5 ml of glycerol (Note: GSH: GSSG = 10:1, GSH concentration range is 2-5 mM).

[0112] PBS buffer (pH 8.0, 1 L): 8 g NaCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, 0.2 g KCl, add 1% glycerol, and sterilize by high pressure.

[0113] Equilibration buffer (500 ml, pH adjusted to 8.0 with NaOH): 10 mmol / L imidazole (0.34 g), 500 mmol / L NaCl (14.61 g), 50 mmol / L NaH2PO4 (3.45 g)

[0114] Elution Buffer 1 (500 ml, adjust pH to 8.0 with NaOH): 20 mmol / L imidazole (0.68 g), 500 mmol / L NaCl (14.61 g), 50 mmol / L NaH2PO4 (3.45 g)

[0115] Elution Buffer 2 (500 ml, adjust pH to 8.0 with NaOH): 30 mmol / L imidazole (1.02 g), 500 mmol / L NaCl (14.61 g), 50 mmol / L NaH2PO4 (3.45 g)

[0116] Elution Buffer 3 (500 ml, adjust pH to 8.0 with NaOH): 40 mmol / L imidazole (1.36 g), 500 mmol / L NaCl (14.61 g), 50 mmol / L NaH2PO4 (3.45 g)

[0117] Elution Buffer 4 (500 ml, adjust pH to 8.0 with NaOH): 50 mmol / L imidazole (1.7 g), 500 mmol / L NaCl (14.61 g), 50 mmol / L NaH2PO4 (3.45 g)

[0118] Elution Buffer 5 (500 ml, adjust pH to 8.0 with NaOH): 60 mmol / L imidazole (2.04 g), 500 mmol / L NaCl (14.61 g), 50 mmol / L NaH2PO4 (3.45 g)

[0119] Elution Buffer 6 (500 ml, adjust pH to 8.0 with NaOH): 70 mmol / L imidazole (2.38 g), 500 mmol / L NaCl (14.61 g), 50 mmol / L NaH2PO4 (3.45 g)

[0120] Elution Buffer 7 (500 ml, adjust pH to 8.0 with NaOH): 80 mmol / L imidazole (2.72 g), 500 mmol / L NaCl (14.61 g), 50 mmol / L NaH2PO4 (3.45 g)

[0121] Elution Buffer 8 (500 ml, adjust pH to 8.0 with NaOH): 250 mmol / L imidazole (8.5 g), 500 mmol / L NaCl (14.61 g), 50 mmol / L NaH2PO4 (3.45 g)

[0122] Coomassie Brilliant Blue R250 staining solution: 400 ml of methanol, 100 ml of glacial acetic acid, 1 g of Coomassie Brilliant Blue R250, and pure water to 1000 ml. Then filter the solution with filter paper and collect the filtered staining solution.

[0123] Decolorizing solution: 300 ml of methanol, 75 ml of glacial acetic acid, and dilute to 1 L with pure water.

[0124] 2 Test methods

[0125] 2.1 Isolation and purification of inclusion body proteins

[0126] 2.1.1 Inclusion body washing

[0127] Wash the precipitate after ultrasonic disruption with 20 ml of washing buffer, then centrifuge at 10,000 rpm for 5 min at 4°C to retain the precipitate. Rinse the precipitate once with sterile ultrapure water, centrifuge at 10,000 rpm for 5 min at 4°C to retain the precipitate.

[0128] 2.1.2 Inclusion body dissolution

[0129] Dissolve the precipitate in 10 ml of dissolution buffer (add 10 ul of β-mercaptoethanol), incubate at 4 degrees, 100 rpm, for 4 hours (in a shaker), then centrifuge at 4 degrees, 10,000 rpm for 10 minutes, take the supernatant, filter it through a 0.22 μm filter membrane, and set aside.

[0130] 2.1.3 Nickel column purification (6x his)

[0131] (1) Take an appropriate amount of nickel column slurry (choose the company's product by yourself and fill the amount of slurry according to the protein loading capacity of the slurry) and load it into the affinity chromatography column. Open the cap at the bottom to drain the liquid and cover the cap. Then add equilibration buffer (five times the column bed volume) to the column to balance the column (turn it upside down to mix the slurry and equilibration buffer evenly). Open the cap at the bottom to drain the equilibration buffer.

[0132] (2) Cover the cap of the lower chromatography column, pour the supernatant dissolved and filtered in 1.2.6.2 into the affinity chromatography column, turn it upside down to mix the mud sediment and protein solution, and incubate it in a shaker at 4 degrees and 200 rpm for 1 hour (turn it upside down every 20 minutes to mix the mud and protein solution).

[0133] (3) Open the cap under the chromatography column and use a clean, sterilized 15 ml centrifuge tube to collect the outflowing liquid. The collected liquid is the flow-through liquid. Take out a little and use it for SDS-PAGE analysis. Then, use different imidazole concentration gradients (10mM-250mM) with 5 times the column volume to elute the target protein. Take a little of the protein eluate from each gradient and mix it with SDS-loading, and heat it in a metal bath at 100 degrees for 5 minutes. The prepared sample is used for subsequent SDS-PAGE analysis.

[0134] (4) If there are foreign proteins in the eluate (there are foreign protein bands on the SDS-PAGE gel), the protein eluate is subjected to Sephadex G-50 column chromatography; if there are no foreign proteins, this step is omitted.

[0135] 2.2 Inclusion body renaturation

[0136] Take 5 ml of the target protein solution purified by the nickel column, add 5 ml of the refolding buffer at a ratio of 1:1, mix it by inversion, let it stand at 4 degrees for 4 hours, then transfer it to the dialysis bag and place it in a 1L beaker, and fill the beaker with 800 ml of dialysate (the dialysate is PBS buffer with 1% glycerol added). Add the rotor of the magnetic stirrer to the beaker and place it on the magnetic stirrer. Turn on the magnetic stirrer so that the dialysis bag in the beaker can rotate in the dialysate to ensure low speed and 4 degrees of dialysis refolding.

[0137] 2.3 Two-step concentration

[0138] The protein refolding liquid in the dialysis bag is buried in PGE8000 and concentrated at low temperature. It is updated in time according to the moisture absorption of PEG8000. Then the concentrate is transferred to an ultrafiltration tube and centrifuged at 4 degrees and 3000g for 10 minutes for ultrafiltration concentration. After that, the concentrated supernatant is transferred to a clean EP tube with a gun, which is the target protein solution, and the protein precipitate is discarded.

[0139] 3. Results and Analysis

[0140] 3.1 Gradient elution of target protein using different imidazole concentrations

[0141] like Figure 9 As shown, the target protein began to be eluted at a concentration of 40 mM imidazole (lane 9), but impurity protein bands (shown by blue connectors) appeared faintly above the target protein in lanes 13 and 14. The proteins in lanes 13 and 14 were passed through a dextran gel column to remove impurity proteins.

[0142] 3.2 Sephadex column chromatography

[0143] like Figure 10 As shown in the figure, after separation by dextran gel, some impurity proteins were removed and the target protein was separated and purified.

[0144] 3.3 Protein purity analysis after concentration

[0145] like Figure 11 As shown in the figure, after the two-step concentration by PEG8000 and ultrafiltration, the impurities were further removed and the protein purity was further improved.

[0146] Implementation 3: Construction of an in vitro adipogenesis induction model of pigeon crop tissue

[0147] 1. Main materials and instruments

[0148] Pigeon recombinant expressed prolactin (PRL), fetal bovine serum, DMEM / F-12, penicillin-streptomycin, bovine insulin, cortisol and epidermal growth factor, PBS buffer, modified Oil Red O staining kit, etc.;

[0149] Galaxy series carbon dioxide incubator (RS Biotech, UK), SW-CJ-1FD clean bench (Suzhou Antai Air Technology Co., Ltd.), high pressure sterilizer (ZDX-35BI, pedestal automatic electric pressure steam sterilizer), disposable culture bottles (Costar, USA), frozen slicer, etc.

[0150] 2. Tissue Culture Process and Oil Red Staining Process

[0151] A 60-week-old female American king pigeon on the 10th day of incubation (I10) was selected, and the crop tissue was collected for separate culture. The crop tissue was rinsed with PBS and then placed in tissue cleaning solution and brought into a sterile operating room at low temperature. The crop sample was rinsed with tissue cleaning solution three times in a clean bench, then soaked in tissue soaking solution for 20 minutes. After soaking, it was transferred to tissue cleaning solution and rinsed three times. The fat, connective tissue and epidermal tissue were peeled off, and the remaining tissue was cut into 2-3mm 3 Cells of uniform size were inoculated into culture flasks, 5 mL of complete culture medium (DMEM / F12 + 5% fetal bovine serum + 100 U / mL penicillin-streptomycin + 0.5 μg / mL cortisol + 10 ng / mL epidermal growth factor + 5 μg / mL bovine insulin) was added, and cultured in a cell culture incubator at 37°C and 5% CO2, with the culture medium replaced every 24 h.

[0152] 3. Experimental Design and Sample Collection

[0153] After pre-incubation of pigeon crop tissue with complete medium for 24 hours, two concentrations of PRL (50 ng / mL and 100 ng / mL) were added and cultured for 36 and 48 hours. Crops were then collected. Each crop sample was divided into two aliquots. The samples were quickly frozen in liquid nitrogen and stored at -80°C for frozen section preparation. At each time point, the PRL-induced effect was compared with a control group containing 0 ng / mL PRL.

[0154] 4. Staining of Frozen Sections with Oil Red O

[0155] The crop tissue was removed, and an appropriate volume of tissue was cut and placed on a freezing stage, embedded with embedding agent, and then placed on a freezing stage for freezing so that the embedding agent completely fixed the tissue.

[0156] The slices were sliced ​​using a freezing microtome with a thickness of about 12 μm and were picked up with adhesive slides.

[0157] The tissue-stained slide was rinsed twice in distilled water for 1 min each time, and then in 60% isopropyl alcohol for 20-30 s.

[0158] The sections were then placed in modified Oil Red O staining solution (covered) and stained for 10 min.

[0159] After staining, the sections were placed in 60% isopropanol for color separation and then briefly rinsed in distilled water.

[0160] Use filter paper to remove moisture from the surface of the slide, and then observe and take pictures under a microscope.

[0161] 5. Results

[0162] In this study, oil red staining was used to qualitatively detect the effects of PRL on lipid droplet density and morphology of pigeon crop tissue cultured in vitro. Figure 12 As shown in the figure, at 0 h, the crop tissue had almost no lipid droplets, or lipid droplets were scattered at the bottom of the epithelial nutrient layer. In the groups treated with PRL (50 ng / mL and 100 ng / mL PRL), the number of lipid droplets in the epithelial layer increased, and the number of lipid droplets continued to increase with the extension of culture time.

[0163] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A method for preparing recombinantly expressed pigeon prolactin, characterized in that: The following steps are involved: Total RNA was extracted from various tissues of pigeons, and the target gene of prolactin was amplified by RT-PCR; The product was then cloned into the pET-29 prokaryotic expression plasmid to obtain a recombinant expression plasmid containing the prolactin target gene; the recombinant expression plasmid containing the prolactin target gene was then transformed into BL21 expression bacteria and induced for expression with IPTG; finally, the bacterial culture after induced expression was separated and purified to obtain recombinant pigeon prolactin; The nucleotide sequence of the prolactin target gene is shown in SEQ ID NO:

1.

2. The preparation method of recombinantly expressed pigeon prolactin according to claim 1, wherein: The primers for the RT-PCR amplification are as follows: F1: 5'-CATATGTTGCCAGTCTGCCCCAAT-3'; R1: 5'-CTCGAGACAATTGCTATCGTGGATTAGG-3'; The reaction conditions for RT-PCR amplification were as follows: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles, and 72°C for 7 min.

3. The preparation method of recombinantly expressed pigeon prolactin according to claim 1, wherein: The concentration of IPTG was 0.5 mM, and the induction expression time was 2 h.

4. The preparation method of recombinantly expressed pigeon prolactin according to claim 1, wherein: The specific steps of separation and purification are: Inclusion body dissolution: The induced expression bacterial culture was ultrasonically disrupted, washed, and centrifuged to obtain inclusion bodies; the inclusion bodies were dissolved in a dissolution buffer, shaken at 4°C for a period of time, and then centrifuged to collect the supernatant; Nickel column purification: the supernatant was loaded onto a Ni column and the protein effluent was collected; Inclusion body refolding: the protein effluent was mixed with refolding buffer, allowed to stand for a period of time, and then placed in a dialysis bag for renaturation by dialyzing at 4°C; Two-step concentration: The renatured inclusion bodies were concentrated at low temperature in PGE8000, and then ultrafiltration was concentrated at 4°C. The supernatant was collected to obtain recombinant expressed pigeon prolactin.

5. The preparation method of recombinantly expressed pigeon prolactin according to claim 4, wherein: After step (2), the following steps are also included: Take part of the protein eluate and elute it with different imidazole concentration gradients; take a little of the protein eluate from each gradient and mix it with SDS-loading, and then perform SDS-PAGE analysis after placing it in a metal bath at 100℃ for 5 minutes.

6. The preparation method of recombinantly expressed pigeon prolactin according to claim 5, wherein: If there are foreign proteins in the eluate, the eluate is subjected to Sephadex G-50 column chromatography.

7. A method for constructing a model for inducing adipogenesis, characterized by: The model is constructed using recombinantly expressed pigeon prolactin prepared by the method according to any one of claims 1 to 6.

8. The method for constructing a model for inducing adipogenesis according to claim 7, wherein: The specific construction method is as follows: S1. Tissue Culture: Cut pigeon crop tissue into small pieces, inoculate into culture flasks, and culture in complete culture medium. S2. PRL induction: Add different concentrations of recombinant pigeon prolactin to the S1 culture medium and continue culturing for a period of time; S3. Oil Red O staining: The effects of recombinantly expressed pigeon prolactin on the density and morphology of crop lipid droplets were examined by Oil Red O staining, and the changes in the distribution and number of lipid droplets were observed.

9. The method for constructing a model for inducing adipogenesis according to claim 8, wherein: In S2, the concentration of the recombinantly expressed pigeon prolactin is 50-100 ng / mL.

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