Method for recombination of sebastes schlegeli growth hormone gene protein

Abstract

The invention discloses a method for recombinant gene protein of sebastes schlegeli growth hormone, and belongs to the technical field of gene engineering. The sebastes schlegeli growth hormone gene is subjected to escherichia coli expression whole gene modification, and a label for 6Xhis purification is added; then, a recombinant escherichia coli (BL21 (DE3) / pET-28a) prokaryotic expression system is constructed, and sebastes schlegeli growth hormone fusion protein is expressed. The purified sebastes schlegeli growth hormone fusion protein is fed to fishes, and the average weight gain rate of the fishes is remarkably higher than that of a control group (Plt; 0.001), the average feed conversion ratio (FCR) is significantly reduced to 1.28 (Plt; the sebastes schlegeli growth hormone fusion protein prepared by the method provided by the invention has the advantages that the growth factor of the sebastes schlegeli growth hormone fusion protein is greatly improved, the feed utilization efficiency is obviously improved, the obtained sebastes schlegeli growth hormone fusion protein has a growth promoting effect, various risks caused by long breeding time are reduced, and the sebastes schlegeli growth hormone fusion protein shows a certain commercial application value.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a method for recombining gene protein of growth hormone of flatfish mullet. Background Art

[0002] The physiological processes of fish growth and development are precisely regulated by growth hormone (GH). As a core factor in the growth axis of vertebrates, the analysis of the molecular mechanism of GH has dual value for aquaculture technology innovation and growth regulation network research.

[0003] In recent years, breakthroughs in non-invasive drug delivery technologies have significantly improved the safety of recombinant growth hormone (rGH). In a study of yellow catfish (Pelteobagrus fulvidraco), Jiang Lijuan et al. constructed the pET-PfGH prokaryotic expression system, producing highly purified rGH (>95%). Using a water immersion method, they found that the specific growth rate (SGR) of experimental juveniles significantly increased by 29.67% compared with the control group. Similarly, feeding transgenic GH algae (Chlamydomonas reinhardtii) to flounder (Paralichthys olivaceus) increased their growth rate by 28%, with no genotoxicity markers detected in muscle tissue. Feng Hao et al. successfully expressed black carp (Mylopharyngodon piceus) GH using the pET-28a vector, obtaining a recombinant protein with a purity of 92%. Western blot analysis confirmed that the protein had the same immunogenicity as native GH. Wang Wei et al. achieved secretory expression of grass carp growth hormone at a concentration of 50 mg / L in Pichia pastoris. Li Yinghua et al., using an intracellular expression strategy, increased carp growth hormone production to 200 mg / L. Lü Qingji et al., using Pichia pastoris fermentation technology, successfully obtained a recombinant protein of grouper growth hormone. Xie Dizhi et al., by optimizing induction conditions, confirmed that this recombinant protein significantly promoted tilapia growth. Long Shaojun, through artificial synthesis, obtained a Pichia pastoris strain that efficiently secreted fish growth hormone, ultimately achieving an expression level of 80 mg / L, a higher level. Therefore, recombinant growth hormone has broad application prospects as a feed additive.

[0004] To date, there have been no reports or patents related to the prokaryotic expression, purification, and application of the modified or fusion protein of the S. philippinarum growth hormone gene. This is primarily due to the small scale of S. philippinarum aquaculture in my country, which requires stringent conditions and relies on imported fertilized eggs and hatching to produce commercial fish. This has led to a lag in research and patents related to growth hormone. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for recombining gene protein of growth hormone of Scorpionfish philoprogenitor to solve the technical problem of slow growth rate of fish.

[0006] To achieve the above object, the technical solution adopted by the present invention is to provide a method for recombinant gene protein of growth hormone of Scorpionfish philoprolifera, comprising the following steps: S1. Modify the growth hormone gene of Scorpionfish xu's; S2, ligating the modified gene fragment to the vector to construct the recombinant plasmid XGH-pET-28a; S3. The recombinant plasmid XGH-pET-28a was transformed into Escherichia coli BL21, and then induced at 37°C and 1 mM IPTG concentration for 8 h. Finally, the recombinant scorpionfish growth hormone protein was obtained by purification.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the nucleotide sequence of the S1-modified Scorpionfish growth hormone gene is shown in SEQ ID NO: 1.

[0008] Furthermore, the expression level of the recombinant Scorpionfish growth hormone protein was 0.35-0.5 mg / mL.

[0009] The present invention has the following beneficial effects: the scorpionfish growth hormone gene, registered in GenBank, was fully expressed in Escherichia coli and a 6Xhis purification tag was added. Subsequently, a recombinant Escherichia coli (BL21(DE3) / pET-28a) prokaryotic expression system was constructed, and the scorpionfish growth hormone fusion protein was expressed. The scorpionfish growth hormone fusion protein was purified by ultrasonic disruption and affinity chromatography. Finally, the purified scorpionfish growth hormone fusion protein was fed to fish. The average weight gain rate and average daily weight gain of the fish were significantly higher than those of the control group (P<0.001) (P<0.05), and the average feed conversion ratio (FCR) was significantly reduced to 1.28 (P<0.05). Feed utilization efficiency was significantly improved, demonstrating that the scorpionfish growth hormone fusion protein has a growth-promoting effect and reduces various risks associated with long-term culture. The scorpionfish growth hormone fusion protein demonstrates considerable commercial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the map of the XGH-pET-28a prokaryotic recombinant plasmid; Figure 2 This is the enzyme digestion identification map of XGH-pET-28a(+) prokaryotic recombinant plasmid vector; Figure 3 This is the SDS-PAGE detection image of protein induced expression; Figure 4 The SDS-PAGE detection diagram of induction at different temperatures; Figure 5 The SDS-PAGE detection diagrams were obtained under different induction time periods; Figure 6 This is the freeze-fracture SDS-PAGE detection image of induced expression; Figure 7 This is the SDS-PAGE detection diagram of the second protein purification result. DETAILED DESCRIPTION

[0011] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.

[0012] Example 1 A method for recombining gene protein of growth hormone of Scorpionfish xu's, comprising the following steps: S1. Modification of the Sebastes schlegelii growth hormone gene. Using the GenBank accession number U89917.1, multiple sequence alignment analysis was performed using the Genetyx bioinformatics platform. This analysis identified highly conserved regions within the Sebastes schlegelii growth hormone (XGH) gene. Based on sequence homology and functional domain integrity, a 615-bp fragment was extracted. This region encodes a mature peptide containing 205 amino acid residues. The theoretical molecular weight of the recombinant protein is 21.7 kDa.

[0013] The nucleotide sequence of the modified Scorpionfish growth hormone gene is shown below: (SEQ ID NO: 1).

[0014] S2. The modified gene fragment was digested and connected to the vector pET28a(+) containing Kan resistance. Then, the recombinant plasmid XGH-pET-28a was obtained using the Novozymes FastPure® Plasmid Mini Kit (DC201) (see the map). Figure 1 The plasmid was constructed and purified by Shanghai Sangon Biotechnology Service Co., Ltd. The specific steps are as follows: (1) Take 3 mL of overnight culture solution for 14 h, add it to a centrifuge tube, centrifuge at 10,000 rpm (11,500 × g) for 1 min, discard the culture medium, and invert the tube onto absorbent paper to absorb the residual liquid; (2) Add 250 μL of Buffer P1 to the centrifuge tube containing the bacterial pellet and mix thoroughly using a pipette or vortex. (3) Add 250 μL of Buffer P2 to the tube prepared in step 2 and mix gently by inverting the tube 8-10 times to fully lyse the cells. (4) Add 350 μL of Buffer P3 to the solution prepared in step 3. Immediately and gently invert the solution 8-10 times to allow the solution to completely neutralize Buffer P2. A white flocculent precipitate should appear. Centrifuge at 12,000 rpm (13,400 × g) for 10 min. (5) Place the FastPure DNA Mini Columns adsorption column in a 2 mL Collection Tube. Carefully transfer the supernatant from step 4 to the adsorption column using a pipette, taking care not to aspirate the precipitate. Centrifuge at 12,000 rpm (13,400 × g) for 30–60 seconds, then discard the waste liquid in the collection tube and place the adsorption column back into the collection tube. (6) Add 500 μL of Buffer PW1 to the adsorption column, centrifuge at 12,000 rpm (13,400 × g) for 30–60 sec, discard the waste liquid, and return the adsorption column to the collection tube; (7) Add 600 μL of Buffer PW2 to the adsorption column, centrifuge at 12000 rpm (13400 × g) for 30-60 seconds, discard the waste liquid, and return the adsorption column to the collection tube; (8) Repeat step 7; (9) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm (13,400 × g) for 1 min to dry the adsorption column and completely remove the remaining rinse solution in the adsorption column; (10) Place the adsorption column in a new sterilized 1.5 mL centrifuge tube, add 30-100 μL of Elution Buffer to the center of the column membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12000 rpm (13400 × g) for 1 minute to elute the DNA; (11) Discard the adsorption column and store the DNA product at -20°C to prevent DNA degradation.

[0015] S3. Transform the recombinant plasmid XGH-pET-28a into Escherichia coli BL21 (ED3) to construct a prokaryotic expression engineered bacterium.

[0016] SpeedyCut XhoI and NdeI enzyme reagents were used to perform double enzyme digestion of the plasmid with XhoI and NdeI. A 20 μL double enzyme digestion reaction system was prepared with the following contents: 15 μL Nuclease-free water, 2 μL 10× SpeedyOne Buffer, 1 μL recombinant plasmid DNA, 1 μL SpeedyCut XhoI, and 1 μL SpeedyCut NdeⅠ. The mixture was incubated at 37°C for 30 min. The digestion results were confirmed by agarose gel electrophoresis. The band results were as shown in Figure 2. Figure 2 As shown in the figure, the letter "M" represents the marker, the number "1" represents the original plasmid, and the number "2" represents the double-enzyme digestion product of the XGH-pET-28a recombinant plasmid vector. After enzyme digestion, two bands of approximately 576 bp and 5.3 kbp appeared in the recombinant plasmid, respectively. As expected, the two bands represent the inserted recombinant XGH target gene and the vector sequence, respectively, indicating that the target gene was successfully inserted into the pET-28a(+) vector and E. coli BL21 (ED3).

[0017] Then the cell was induced at 37°C and 1 mM IPTG for 8 hours, and finally purified to obtain the recombinant Scorpionfish growth hormone protein.

[0018] Example 2 The constructed recombinant E. coli strain was subjected to prokaryotic small-scale expression. The specific steps were as follows: (1) Inoculation: Escherichia coli BL21 (DE3) containing the prokaryotic expression engineered bacteria (containing the pET-XGH recombinant plasmid) constructed in Example 1 was streaked onto LB solid medium. A glycerol solution was taken with an inoculation loop and streaked onto four zones on an LB agar plate containing kanamycin (Kan, final concentration 50 μg / mL). The plate was placed upside down in a 37°C constant temperature incubator and incubated for 12-16 hours until single colonies were clearly visible. The plate should be preheated to room temperature in advance to reduce interference from condensed water. The diameter of the recombinant colonies usually has regular edges.

[0019] (2) Activation of recombinant strains: Use a sterile inoculation loop to select single clones with regular morphology and transfer them to a 15 mL sterile centrifuge tube containing 5 mL of Kan-LB liquid medium.

[0020] (3) Culture: Place the above centrifuge tube in a constant temperature shaking incubator (37°C, 200 rpm) and continue to culture for 6-8 hours. Monitor the OD 600 The value (sampled every 1 hour) confirms that the bacterial solution has entered the OD 600 =0.4~0.6 of the logarithmic growth period.

[0021] (4) IPTG induction: The bacterial solution in the logarithmic growth phase (OD 600≈0.6) Add 0.22 μm filter-sterilized IPTG stock solution (50 mg / mL) to a final concentration of 1 mM, mix gently, and return to the shaking incubator (37°C, 200 rpm). Set up a parallel culture without IPTG as an uninduced control group and continue culturing for 6-7 h until the stationary phase. Take 1 mL of the culture sample for subsequent SDS-PAGE analysis.

[0022] (5) Bacterial solution treatment: In a clean bench, take 1 mL of bacterial solution and centrifuge (13000 rpm, 3 min), discard the supernatant, add 30 μL of 1× PBS buffer to the precipitate, oscillate and suspend, then add 7 μL of 5× protein loading buffer, mix well, boil for 5 min, centrifuge (13000 rpm, 3 min), and take the supernatant as the loading sample.

[0023] The results are as follows Figure 3 As shown in the figure, the letter "M" represents the protein marker, the number "1" represents the uninduced recombinant expression bacteria, and the number "2" represents the induced recombinant expression bacteria. A distinct band is observed at approximately 15-25 kDa of the marker, whereas the uninduced positive clones lack a band at the corresponding position. The molecular weight of this band is consistent with the theoretical molecular weight of the XCH protein (21.7 kDa), confirming that this band is the target protein band for induced expression.

[0024] Example 3 In order to explore the effects of different induction conditions on the expression of target proteins by recombinant bacteria, the culture temperature (25°C, 30°C, 37°C and 44°C) and induction time (4h, 6h, 8h and 10h) were optimized respectively.

[0025] The effect of culture temperature on induction results Figure 4 As shown in the figure, the letter "M" represents the protein marker, the number "1" represents the precipitate from the recombinant expression bacteria induced at 25°C, the number "2" represents the precipitate from the recombinant expression bacteria induced at 30°C, the number "3" represents the precipitate from the recombinant expression bacteria induced at 37°C, and the number "4" represents the precipitate from the recombinant expression bacteria induced at 44°C. The precipitates all show the target band located between 15 and 25 kDa of the marker, with a molecular weight of approximately 21.7 kDa. The target band is brightest at 37°C (lane 4), while at 44°C (lane 5), the protein degrades. Therefore, the recombinant protein is expressed most strongly at 37°C, followed by 25 and 30°C, and least at 44°C.

[0026] The effect of induction time on induction results Figure 5As shown in the figure, the letter "M" indicates a protein marker, the number "1" represents the precipitate from the recombinant expression bacteria after 2 hours of induction, the number "2" represents the precipitate from the recombinant expression bacteria after 4 hours of induction, the number "3" represents the precipitate from the recombinant expression bacteria after 6 hours of induction, the number "4" represents the precipitate from the recombinant expression bacteria after 8 hours of induction, and the number "5" represents the precipitate from the recombinant expression bacteria after 10 hours of induction. The target band appears in all precipitates (lanes 2-5), with a value of 21.7 kDa, ranging from 15 to 25 kDa. The band is most prominent after 8 and 10 hours of IPTG induction. Considering expression efficiency and other factors, 8 hours was selected as the optimal induction time.

[0027] Example 4 The supernatant of the bacterial solution was detected by ultrasonic disruption to explore the expression of the bacterial solution and improve the expression of the recombinant protein. The specific steps are as follows: (1) Streak the prokaryotic expression system engineered bacteria on a Kan LB plate, screen the successfully recombinant transformed strains using Kan resistance, and culture them in a 37°C incubator overnight.

[0028] (2) Pick a single colony with good growth status and inoculate it into 250mL KanLB liquid culture medium in a large conical flask. Incubate the bacteria at 37℃ for 6 hours until the logarithmic growth phase (OD 600 Around 0.5).

[0029] (3) When the bacterial solution OD 600 =0.5, add IPTG solution to the bacterial solution to a final concentration of 1 mM to induce expression, and continue to culture the bacteria at 37°C and 200 rpm for 6 h.

[0030] (4) Centrifuge the bacterial solution (7000 rpm for 15 min), discard the supernatant, and collect the bacteria.

[0031] (5) Transfer 200 mL of bacterial suspension to a centrifuge tube and centrifuge at 7000 rpm for 15 min. Discard the supernatant. Weigh the pellet and add lysis buffer (without urea) at a ratio of 1:10 (w / v) to resuspend the cells. Disrupt the cells by ultrasonication on ice (2 s, 4 s intervals, 30 min, 250 W).

[0032] (6) Centrifuge the fragmentation solution at 10,000 rpm for 30 minutes and discard the supernatant. Repeat the ultrasonic fragmentation in an ice bath. The white, dense mass at the bottom of the centrifuge tube is the inclusion body. Weigh the precipitate and add lysis buffer (containing urea) at a ratio of 1:10 (w / v) to resuspend the cells.

[0033] After the bacterium is broken, the results are as follows Figure 6As shown in the figure, numbers "1" and "4" represent markers, number "2" represents the IPTG-induced precipitate, number "3" represents the precipitate without IPTG, number "5" represents the supernatant from the first IPTG-induced precipitate, number "6" represents the supernatant from the first IPTG-induced precipitate without IPTG, number "7" represents the supernatant from the second IPTG-induced precipitate, and number "8" represents the supernatant from the second IPTG-induced precipitate. No distinct bands were observed in the supernatant. This indicates that the induced protein of interest failed to achieve extracellular secretion and was present as insoluble inclusion bodies.

[0034] Example 5 PurKine™ Histidine Tag Protein Purification Kit (Ni-NTA resin) was used for protein purification, purchased from Yacoin Biotechnology Co., Ltd. The specific steps are as follows: (1) Column pretreatment: After fixing the gravity column vertically, remove the upper and lower end plugs in turn to allow the protective solution to drain naturally. Then slowly add 5 mL of lysis buffer to rinse the column to remove residual impurities and activate the column bed. After fixing the gravity column vertically, remove the upper and lower end plugs, allow the protective solution to drain naturally, and slowly add 5 mL of lysis buffer to rinse.

[0035] (2) Resin equilibration: Inject 5 mL of equilibration buffer into the column and allow the buffer to drain naturally due to gravity. This process is intended to allow the resin immobilized with metal ions to fully contact the equilibration buffer to ensure that the resin is in the optimal binding state.

[0036] (3) Protein loading and binding: The protein extract was slowly introduced into the column and incubated at room temperature for 13 min to promote the full binding of the target protein with the metal ions on the resin, while the flow-through was collected.

[0037] (4) Elution of impurities: Add 13 mL of wash buffer in sequence and collect the eluate step by step to effectively remove non-specifically adsorbed impurity proteins and improve purification specificity.

[0038] (5) Elution of target protein: Add 5 mL of elution buffer to elute the target protein, collect the eluate and analyze it by SDS-PAGE.

[0039] (6) Column regeneration: Use 3 mL of regeneration buffer to thoroughly elute the residual protein, then rinse with 5 mL of deionized water to restore the column to its original state and prepare for subsequent purification experiments. The results were analyzed by SDS-PAGE at a concentration of 12%. Figure 7The letter "M" represents the marker, the number "1" represents the eluate, the number "2" represents the flow-through, and the number "3" represents the wash buffer. The target band (21.7 kDa) appears near the marker 15-25 kDa in the eluate (lane 1), wash buffer (lane 2), and flow-through (lane 3).

[0040] Example 6 A feeding experiment was conducted using grass carp as an example. Juvenile fish of uniform size were selected and divided into two tanks, with 30 fish kept in each tank. Purified prokaryotic growth hormone protein was added to the basal feed at a dosage of 15 μg / g and mixed thoroughly. This was used as the feed for the experimental group, while the control group was fed the basal feed. The experiment lasted for two weeks. The water temperature was maintained at 26 ± 1°C, and 30% of the water was changed every three days to maintain stable water quality. Feed was administered twice daily. Throughout the breeding process, the fish in both the experimental group (treated with prokaryotic recombinant growth hormone) and the control group remained in good spirits and displayed normal behavior.

[0041] After the experiment, the individual weight and total weight of each group of fish were weighed (accurate to 0.01g), and the growth and nutrient utilization indicators were calculated, including weight gain (WG), weight gain rate (WGR), and feed conversion ratio (FCR) of the control group and the experimental group. The calculation formula is as follows: WG=W t -W0; WGR=(W t -W0) / W0×100%; FCR=W1 / W2; Where W t is the weight of the fish at the end of the experiment, g; W0 is the initial weight of the fish at the beginning of the experiment, g; W1 is the total feed consumption of each group of fish, g; W2 is the total weight of each group of fish, g.

[0042] The statistical results of growth performance are shown in Table 1.

[0043] Table 1 Results of feeding goldfish fry with E. coli recombinant protein

[0044] Note: “*” in the table indicates that there is a significant difference between the experimental group and the control group (P<0.05).

[0045] As shown in Table 1, the average weight gain of fish in the experimental group was significantly higher than that in the control group (P < 0.05). The average tail weight of the control group was 5.24 g at the beginning of the trial and increased to 6.45 g at the end, with an average weight gain of 1.21 g, an average weight gain rate of 23.12%, and an average daily weight gain of 0.086 g per fish. The average tail weight of the experimental group was 4.26 g at the beginning of the trial and increased to 5.74 g at the end of the trial, with an average weight gain of 1.48 g, a significant increase compared to the control group (P < 0.05). The average weight gain rate of fish in the experimental group reached 34.70%, which was extremely significantly higher than that in the control group (P < 0.001). The average daily weight gain reached 0.11 g per fish, a 27.9% increase compared to the experimental group. This indicates that the addition of prokaryotic recombinant growth hormone to feed has a significant growth-promoting effect on fish (P < 0.05). The increase in weight gain and weight gain rate in the experimental group may be related to the fact that prokaryotic expression of recombinant growth hormone accelerates protein synthesis in fish and improves metabolic efficiency, especially in the group with lower initial weight, suggesting that the treatment has a compensatory effect on individuals with delayed growth.

[0046] The total feed intake per fish in the control group was 1.80g, and the average feed conversion ratio (FCR) was calculated to be 1.54, meaning that the control fish consumed 1.54g of feed for every gram of weight gain. The experimental group, supplemented with prokaryotically expressed recombinant growth hormone, maintained the same feed input as the control group, but the FCR was significantly reduced to 1.28 (P<0.05), significantly improving feed utilization efficiency. This means that this supplementation method saved 0.26g of feed for every gram of weight gain. This indicates that recombinant growth hormone can significantly improve feed utilization efficiency and, in practical applications, can reduce feed waste in aquaculture. This has important economic value in high-density aquaculture scenarios, reducing feed costs and minimizing water pollution.

Claims

1. A method for recombining gene protein of growth hormone of Scorpionfish xu, characterized in that: The following steps are involved: S1. Modify the growth hormone gene of Scorpionfish xu's; S2, ligating the modified gene fragment to the vector to construct the recombinant plasmid XGH-pET-28a; S3. The recombinant plasmid XGH-pET-28a was transformed into Escherichia coli BL21, and then induced at 37°C and 1 mM IPTG concentration for 8 h. Finally, the recombinant scorpionfish growth hormone protein was obtained by purification.

2. The method for producing recombinant gene protein of growth hormone of Scorpio humilis according to claim 1, characterized in that: The nucleotide sequence of the S1-modified Scorpionfish growth hormone gene is shown in SEQ ID NO:

1.

3. The method for producing recombinant gene protein of growth hormone of Scorpio humilis according to claim 1, characterized in that: The expression level of the recombinant Scorpionfish growth hormone protein is 0.35-0.5 mg / mL.

Images