Method for preparing Atlantic salmon growth hormone recombinant gene protein

Abstract

The invention discloses a method for recombinant gene protein of Atlantic salmon growth hormone, and belongs to the technical field of gene engineering. Escherichia coli expression whole gene modification is carried out on an Atlantic salmon growth hormone gene, then a modified connecting pET-28a (+) plasmid is introduced into an Escherichia coli BL21 (DE3) strain, a recombinant Escherichia coli (BL21 (DE3) / pET-28a) expression line is constructed, and an Atlantic salmon growth hormone (rSGH) fusion protein is expressed. After the Atlantic salmon growth stimulating fusion protein is purified, auxin capable of promoting the growth of the Atlantic salmon is prepared, then the pure Atlantic salmon growth stimulating fusion protein is fed to fishes, the weight gain rate of the fishes is 16.8% higher than that of a control group, and the feed conversion rate is 21.7% higher. It is proved that the purified Atlantic salmon growth hormone (rSGH) fusion protein has a growth promoting effect and shows a certain commercial application value.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for recombining Atlantic salmon growth hormone gene protein. Background Art

[0002] In recent years, my country has made significant progress in transgenic breeding of important marine and freshwater aquaculture fish. For example, carp transgenic for rainbow trout growth hormone (GH) contain more protein, less fat, and less muscle water than ordinary carp. Using microinjection, Wang Yaping et al. developed a "whole-fish" GH gene transgenic carp strain. The average weight of the F1 generation was significantly higher than that of control fish; the average weight of the F2 generation was 1.8 to 2.5 times that of control fish. The transgenic fish also had a higher feed utilization rate. Exogenous administration of GH can also exert the growth-promoting effects of fish growth hormone. Studies have shown that adding appropriate amounts of GH to feed can stimulate fish growth. This is because the small intestinal epithelial cells of bony fish absorb large molecules through pinocytosis and transfer them intact to the blood. This property has led to the mixing of recombinant GH into feed for administration, but direct administration is not very efficient. Sire et al. administered recombinant growth hormone to eels via oral and rectal infusion, finding that rectal infusion was significantly more effective than oral administration. Furthermore, when rainbow trout (average 100 g) were fed 200 μg of encapsulated growth hormone mixed in their feed, blood absorption was approximately two times higher than that of unencapsulated growth hormone. Therefore, recombinant growth hormone has broad application prospects as a feed additive.

[0003] To date, there have been no reports or patents related to the prokaryotic expression, purification, and application of modified Atlantic salmon growth hormone genes and fusion proteins. This is primarily due to the small scale of Atlantic salmon farming in my country, which requires stringent conditions and relies on imported fertilized eggs for hatching and raising into commercial fish. This has led to a lag in growth hormone-related research and patents. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for recombining Atlantic salmon growth hormone gene protein to solve the technical problem of slow growth rate of fish.

[0005] To achieve the above object, the technical solution adopted by the present invention is: to provide a method for producing recombinant gene protein of Atlantic salmon growth hormone, comprising the following steps: S1, modification of Atlantic salmon growth hormone fusion protein gene; S2, ligating the modified gene fragment to the vector to obtain the recombinant plasmid pET28a-SGH; S3. The recombinant plasmid pET28a-SGH was transformed into Escherichia coli BL21, and then induced at 37°C and an IPTG concentration of 1.5 mM for 6 hours. Finally, the recombinant Atlantic salmon growth hormone protein rSGH was obtained by purification.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the nucleotide sequence of the Atlantic salmon growth hormone gene after S1 modification is shown in SEQ ID NO: 1.

[0007] Furthermore, the expression level of recombinant Atlantic salmon growth hormone protein rSGH was 0.35-0.5 mg / mL.

[0008] The present invention has the following beneficial effects: A GenBank-listed Atlantic salmon growth hormone gene was fully modified for expression in Escherichia coli and a 6Xhis purification tag was added to its 3-terminus. The modified pET-28a(+) plasmid was then introduced into the E. coli BL21(DE3) strain to construct a recombinant E. coli (BL21(DE3) / pET-28a) expression system, expressing an Atlantic salmon growth hormone (rSGH) fusion protein. The Atlantic salmon growth hormone fusion protein was purified by ultrasonic disruption and nickel ion affinity chromatography to produce a growth hormone that promotes fish growth. Feeding the purified Atlantic salmon growth hormone fusion protein to fish resulted in a 16.8% higher weight gain and a 21.7% higher feed conversion rate than the control group. This confirms the growth-promoting effects of the purified Atlantic salmon growth hormone (rSGH) fusion protein, reduces the risks associated with long aquaculture operations, and demonstrates promising commercial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is the map of the pET28a-SGH recombinant plasmid; Figure 2 This is the enzyme digestion identification map of the pET28a-SGH recombinant plasmid vector; Figure 3 SDS-PAGE protein electrophoresis analysis of rSGH protein induced expression; Figure 4 The expression of rSGH protein was induced by different IPTG concentrations and analyzed by SDS-PAGE protein electrophoresis; Figure 5 SDS-PAGE protein electrophoresis analysis of rSGH protein expression induced by different temperature gradients; Figure 6 The rSGH protein was expressed by gradient induction at different time points and analyzed by SDS-PAGE protein electrophoresis. Figure 7SDS-PAGE protein electrophoresis analysis of the supernatant of bacteria with large-scale induced expression of rSGH protein; Figure 8 This is the SDS-PAGE protein electrophoresis analysis of the purified rSGH protein. DETAILED DESCRIPTION

[0010] 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.

[0011] Example 1 A method for recombinant gene protein of Atlantic salmon growth hormone comprises the following steps: S1. Modify the Atlantic salmon growth hormone gene. Based on the Atlantic salmon growth hormone gene accession number X14305.1 in GenBank, the gene is 633 bp long, encodes 211 amino acids, has a molecular weight of 23.8 kDa, and an isoelectric point of 6.93. Based on the codon preference of E. coli, without changing the amino acid sequence, the target gene was codon-optimized. An Nde I (CATATG) site was added to the N-terminus of the gene, along with a native ATG promoter. A 6×His and TAATAG double terminator were added to the C-terminus of the gene, along with an Xho I (CTCGAG) restriction site.

[0012] The nucleotide sequence of the modified Atlantic salmon growth hormone gene is shown below: (SEQ ID NO: 1).

[0013] S2. The modified gene fragment was digested and connected to the vector pET28a(+) containing Kan resistance. Then, the recombinant plasmid pET28a-SGH 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.

[0014] S3. Transform the recombinant plasmid pET28a-SGH into Escherichia coli BL21 to construct a prokaryotic expression engineered bacterium. Use SpeedyCut XhoI and NdeI enzyme reagents to perform double enzyme digestion of the plasmid with XhoI and NdeI. Prepare a 20μL double enzyme digestion reaction system with the following system: 15μL Nuclease-free water, 2μL 10× SpeedyOne Buffer, 1μL recombinant plasmid DNA, 1μL SpeedyCut XhoI, 1μL SpeedyCut NdeⅠ, incubate at 37°C for 30min, and confirm the enzyme digestion results by agarose gel electrophoresis. The band results are as follows: Figure 2 As shown in the figure, the letter "M" represents the marker, the number "1" represents the double-enzyme digestion product of the pET28a-SGH recombinant plasmid vector, and the number "2" represents the original plasmid. After enzyme digestion, the recombinant plasmid showed two bands of approximately 590 bp and 5.3 kbp, respectively. As expected, the two bands represent the inserted recombinant SGH 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).

[0015] Then, the protein was induced at 37° C. and 1.5 mM IPTG for 6 h, and finally purified to obtain the recombinant Atlantic salmon growth hormone protein rSGH.

[0016] Example 2 The constructed recombinant E. coli strain was subjected to prokaryotic small-scale expression. The specific steps were as follows: (1) Preparation of culture medium: Prepare LB (Luria-Bertani) solid culture medium and LB liquid culture medium. After autoclaving and cooling, add kanamycin solution stock solution to the solid and liquid culture media in a clean bench to make the final kanamycin concentration of 30 μg / mL.

[0017] (2) Streak the prokaryotic expression engineered bacteria constructed in Example 1 on a Kan LB plate, screen the successfully recombinant transformed strains using Kan resistance, and culture them in a 37°C incubator overnight.

[0018] (3) Pick a single colony with good growth status from the recombinant transformed Escherichia coli BL21 (DE3), inoculate 1 mL into 5 mL of Kan LB liquid medium, and culture the bacteria at 37°C and 200 rpm / min for 6 hours until the logarithmic growth phase (OD 600 Around 0.5).

[0019] (4) Add IPTG solution to the bacterial solution to a final concentration of 1 mM to induce expression. No IPTG solution was added to the control group. The culture was continued at 37°C and 200 rpm / min for 6 h, and the bacteria were harvested for later use.

[0020] The results are as follows Figure 3 As shown in the figure, the letter "M" represents the protein marker, the number "1" indicates the induced recombinant expression bacteria, and the number "2" indicates the uninduced recombinant expression bacteria. A clear band is observed at approximately 23 kDa, while 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 rSGH protein, confirming that this band is the induced expression target protein band.

[0021] 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), IPTG concentration (0.5mM, 1mM and 1.5mM) and induction time (4h, 6h, 8h and 10h) were optimized respectively.

[0022] Effect of IPTG concentration on induction results Figure 4 As shown in the figure, the letter "M" represents a protein marker, the number "1" indicates the recombinant expression strain induced with 0.5mM IPTG, the number "2" indicates the recombinant expression strain induced with 1mM IPTG, the number "3" indicates the recombinant expression strain induced with 1.5mM IPTG, and the number "4" indicates the uninduced control. Compared with the uninduced control, the rSGH E. coli expression system showed a differential band near 23kDa at all three IPTG concentrations, demonstrating that the target protein was successfully induced. Furthermore, the color of the 23kDa band deepened with increasing IPTG concentration, indicating an increase in the concentration of the expressed target protein. The rSGH E. coli expression system showed better expression at 1.5mM IPTG than at 0.5mM and 1mM IPTG induction concentrations.

[0023] The effect of culture temperature on induction results Figure 5 As shown, the letter "M" represents a protein marker, the number "1" represents a recombinant expression bacterium induced at 25°C, the number "2" represents a recombinant expression bacterium induced at 30°C, the number "3" represents a recombinant expression bacterium induced at 37°C, and the number "4" represents a recombinant expression bacterium induced at 44°C. The rSGH Escherichia coli expression system constructed by the present invention shows obvious bands near 23kDa at culture temperatures of 30°C, 37°C and 44°C, proving that the target proteins are successfully induced to express. The protein expression level at 37°C is higher than that at 30°C, and when the expression level continues to rise to 44°C, there is no significant difference in protein expression from that at 37°C, which indicates that temperature affects the induced expression of the target protein, and 37°C is the optimal induced expression culture temperature for this expression system.

[0024] The effect of induction time on induction results Figure 6As shown in the figure, wherein the letter "M" represents protein Marker, the number "1" represents 4h induction recombinant expression bacteria, the number "2" represents 6h induction recombinant expression bacteria, the number "3" represents 8h induction recombinant expression bacteria, and the number "4" represents 10h induction recombinant expression bacteria. The rSGH Escherichia coli expression system constructed by the present invention all shows obvious bands near 23kDa under 4 induction culture times, proving that the target protein is successfully induced to express. Protein electrophoresis result shows that the Escherichia coli expression system has target protein expression when 4h, and protein expression level improves when 6h. When 8h, target protein expression level is similar to 6h, and subsequent extended culture time protein expression level decreases instead. Therefore, the 6h induction time is shorter and the expression level is higher, which is a more suitable induction expression culture time.

[0025] Example 4 The expression pattern of the recombinant protein was explored by ultrasonically disrupting the induced expression bacterial solution. 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.

[0026] (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).

[0027] (3) 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.

[0028] (4) Centrifuge the bacterial solution (7000 rpm for 15 min), discard the supernatant, and collect the bacteria.

[0029] (5) Add 7 mL of lysis buffer without 8 M urea to a 15 mL centrifuge tube, oscillate to suspend, and ultrasonically disrupt in an ice bath (ultrasonication time: 30 min, power: 250 W).

[0030] (6) Centrifuge the broken liquid (at 4°C, 10,000 rpm for 30 min) and collect the supernatant.

[0031] (7) Repeat steps (5) and (6), and store the collected supernatant and precipitate in a refrigerator at 4°C for later use.

[0032] The results after lysis are as follows Figure 7As shown in the figure, the letter "M" represents the protein marker, the number "1" indicates induced recombinant expression, and the number "2" indicates uninduced recombinant expression. No differential bands were observed in the supernatant. This indicates that the induced expression of the target protein failed to achieve extracellular secretion and existed as insoluble inclusion bodies.

[0033] Example 5 Purify the protein using the PurKine™ His-Tagged Protein Purification Kit (Ni-NTA resin). The specific steps are as follows: (1) Prepare buffers: prepare lysis buffer without 8 M urea, lysis buffer containing 8 M urea, wash buffer, and elution buffer.

[0034] (2) Sample preparation: Take protein inclusion bodies, add lysis buffer containing 8 M urea in a ratio of 1 mg of cells to lysis buffer in 10 mL, and oscillate to suspend; filter with a 0.22 μm syringe filter and place on ice until use.

[0035] (3) Water washing: Fix the gravity column and drain the protective solution. Add 20 mL of deionized water to the column at a flow rate of 2 mL / min to wash the resin and remove the ethanol.

[0036] (4) Equilibration: Add 10 mL of lysis buffer to the column at a flow rate of 2 mL / min to equilibrate the medium so that the medium and sample are in the same buffer system. Drain the lysis buffer.

[0037] (5) Sample loading: Add the protein extract sample to the resin at a flow rate of 1 mL / min to ensure that the target protein is incubated with Ni 2+ Full contact.

[0038] (6) Secondary equilibration: Add 20 mL of lysis buffer to the column at a flow rate of 1 mL / min to equilibrate the medium and ensure stable binding of the sample and the medium.

[0039] (7) Washing: Add 20 mL of washing buffer to the column at a flow rate of 1 mL / min to remove non-specifically adsorbed impurities.

[0040] (8) Elution: Add 20 mL of elution buffer to the column at a flow rate of 1 mL / min and collect the eluate. The eluate is the target protein solution.

[0041] (9) Water washing: Add 6 mL of lysis buffer and 10 mL of deionized water to the column at a flow rate of 1 mL / min to balance the Ni-NTA resin and wash the resin medium.

[0042] (10) SDS-PAGE protein electrophoresis: Analyze the eluate by SDS-PAGE protein electrophoresis to detect the purification effect.

[0043] The bacterial suspension expressing a large amount of protein was crushed and then purified. The flow-through, washing solution and eluate collected during the rSGH protein purification were detected by SDS-PAGE protein electrophoresis. The results were as follows. Figure 8 As shown in the figure, the letter "M" represents the protein marker, the number "1" represents the flowthrough, the number "2" represents the wash buffer, and the number "3" represents the eluate. The flowthrough contains multiple protein bands, including a large protein at 23 kDa, which is consistent with the target protein size. The wash buffer also contains multiple, lighter protein bands, also at 23 kDa, containing a large amount of the target protein. The eluate only has a clear band at approximately 23 kDa, which is identified as the target protein rSGH band.

[0044] Example 6 A feeding experiment was conducted using grass carp (Goldfish) as an example. Juveniles of uniform size were selected and divided into two tanks, with 30 fish in each tank. Refolded and purified recombinant Atlantic salmon 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°C, and the water was changed every other day. Feed was administered twice daily. Kanamycin was administered at a final concentration of 10 mg / L every seven days. After removing outliers from the collected experimental data, the growth status of the fish was compared and analyzed. Animal experiments were conducted to determine the effect of recombinant Atlantic salmon growth hormone on the growth of grass carp juveniles. Fish weight and total feed intake were measured over a 14-day comparative experiment. The results are shown in Table 1.

[0045] Table 1 Results of feeding goldfish fry with E. coli rSGH recombinant protein

[0046] Note: “*” in the table indicates that there is a significant difference between the experimental group and the control group (P<0.05).

[0047] G = (w1-w2) / w2×100%; W = (w1-w2) / D×100%; H=h / (h1-h2); Among them, G is weight gain rate, %; w1 is the average final weight, g; w2 is the average initial weight, g; W is daily weight gain, g / tail; D is the number of feeding days, days; H is feed conversion rate, %; h is feed consumption, g; h1 is the final total weight, g; h2 is the initial total weight, g.

[0048] As shown in Table 1, the average body weights of the experimental groups were significantly different from those of the control group (P < 0.05). The weight gain rate of the experimental fish was 16.8% higher than that of the control group, and the feed conversion rate was 21.7% higher. This demonstrates that the recombinant protein expressed by the prokaryotic expression system for Atlantic salmon growth hormone constructed in this invention is biologically active after purification and can significantly promote the growth of juvenile fish and improve feed utilization.

Claims

1. A method for producing recombinant gene protein of Atlantic salmon growth hormone, characterized in that: The following steps are involved: S1, modification of the Atlantic salmon growth hormone gene; S2, ligating the modified gene fragment to the vector to obtain the recombinant plasmid pET28a-SGH; S3. The recombinant plasmid pET28a-SGH was transformed into Escherichia coli BL21, and then induced at 37°C and an IPTG concentration of 1.5 mM for 6 hours. Finally, the recombinant Atlantic salmon growth hormone protein rSGH was obtained by purification.

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

1.

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

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