Atlantic salmon growth hormone genetic engineering saccharomycetes and construction method thereof

Abstract

The invention discloses Atlantic salmon growth hormone genetic engineering saccharomycetes and a construction method thereof, and belongs to the technical field of genetic engineering. According to the present invention, the Atlantic salmon growth hormone gene in GenBank is modified, the gene is cloned to the pichia pastoris expression vector (X33), the pichia pastoris engineering bacterium for expressing the Atlantic salmon growth hormone is constructed, and the Atlantic salmon recombinant growth hormone fusion protein is obtained through the induced expression; when the pichia pastoris engineering bacteria are used as a functional additive to be mixed with fish feed to feed fishes, the feed added with the engineering bacteria can significantly improve the weight of fish fries and improve the feed conversion rate.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an Atlantic salmon growth hormone genetically engineered yeast and a construction method thereof. Background Art

[0002] In the development of modern aquaculture technology, intensive farming models are often combined with high-energy-density feeds. While this farming method can increase fish weight, it is often accompanied by negative effects such as abnormal lipid deposition, deterioration of muscle texture, and changes in body characteristics. In contrast, GH intervention technology can not only significantly shorten the breeding cycle but also address the quality issues caused by intensive farming. The use of growth hormone in aquaculture is an effective way to increase production and improve the quality of aquatic products. In the aquaculture sector, the current methods for applying growth hormone in aquaculture are mainly divided into two categories: exogenous administration and genetic engineering. Transgenic fish bred through genetic engineering grow rapidly and their quality is also improved; exogenous administration can also exert the growth-promoting effects of growth hormone.

[0003] Research has shown that adding appropriate amounts of GH to feed can stimulate fish growth. This is due to its absorption into the bloodstream by the intestinal epithelial cells of teleost fish through pinocytosis. Studies have used recombinant rainbow trout growth hormone yeast mixed with feed and fed to fish, finding that it significantly promoted fish growth. However, directly administering recombinant growth hormone (rGH) as a feed additive is not always optimal. Sire et al. demonstrated that rectal infusion of rGH is significantly more effective than oral administration. This is apparently due to the breakdown of the administered growth hormone by proteases secreted by the digestive system, rendering it ineffective. Consequently, large-scale GH administration through feeding requires the use of protective agents. When encapsulated growth hormone was fed to rainbow trout, blood absorption was approximately two-fold higher than that of unencapsulated growth hormone, demonstrating that encapsulation and other methods can effectively prevent the degradation of recombinant growth hormone by proteases in the body when fed exogenously. With the continued advancement of research on the use of bacterial protein feeds in aquaculture, the direct application of GH-expressing yeast has become more economical and convenient. Huang Xiaonan et al., by feeding fermented growth hormone-engineered yeast, increased the average weight gain of large yellow croaker fry and drumstick fry by 27.9% and 51.0%, respectively, after 45 days. However, Li Jing et al. found that adding recombinant growth hormone to feed is not necessarily better, suggesting that excessive addition of growth hormone to feed can burden the fish's digestive system and trigger negative feedback inhibition of growth hormone. Furthermore, various factors can affect the amount of growth hormone added to feed, depending on the feeding conditions and the species. To date, no such reports have been published on growth hormone in Atlantic salmon. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an Atlantic salmon growth hormone genetically engineered yeast and a construction method thereof, so as 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 an Atlantic salmon growth hormone gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0006] The invention also discloses a cloning vector containing the Atlantic salmon growth hormone gene.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the cloning vector is the recombinant plasmid pPICzαA-SGH.

[0008] The present invention also discloses a recombinant engineering bacterium, which is a yeast engineering bacterium X33-pPICzαA-SGH formed by transferring the above cloning vector into Pichia pastoris X33.

[0009] The present invention also discloses a method for constructing the above-mentioned recombinant engineering strain, comprising the following steps: S1, modification of Atlantic salmon growth hormone fusion protein gene; S2, ligating the modified gene fragment to the eukaryotic vector pPICzαA to obtain the recombinant plasmid pPICzαA-SGH; S3. The recombinant plasmid pPICzαA-SGH was transformed into Pichia pastoris X33 by electroporation to obtain the engineered yeast strain X33-pPICzαA-SGH.

[0010] The present invention has the following beneficial effects: The present invention found that the average body weight of the experimental group supplemented with the engineered yeast bacteria was significantly different from that of the control group (P<0.05). The weight gain rate of fish fed with the engineered yeast bacteria (X33-pPICzαA-SGH) was 6.7% higher than that of the control group, and the feed conversion rate was 12.4% higher. This demonstrates that the engineered yeast bacteria (X33-pPICzαA-SGH) disclosed in the present invention has a growth-promoting effect on fish and can be used as a functional feed additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the map of the pPICzαA-SGH recombinant plasmid; Figure 2 This is the linearization map of the pPICzαA-SGH recombinant plasmid; Figure 3 PCR results of GS115 Pichia pastoris colony for the construction of pPIC9K-SGH expression vector; Figure 4Electrophoresis analysis of X33-pPICzαA-SGH yeast supernatant protein; Figure 5 For X33-pPICzαA-SGH yeast precipitation induced protein electrophoresis analysis; Figure 6 Western blot analysis of the supernatant of the induced expression of the recombinant yeast strain X33-pPICzαA-SGH; Figure 7 The results of protein purification from the supernatant of the recombinant yeast strain X33-pPICzαA-SGH are shown. DETAILED DESCRIPTION

[0012] 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.

[0013] Example 1 A method for constructing a recombinant engineering strain comprises the following steps: S1, modification of Atlantic salmon growth hormone fusion protein gene; Based on the Atlantic salmon growth hormone gene (gene ID X14305.1) registered in GenBank, which is 633 bp in length, encodes 211 amino acids, has a molecular weight of approximately 23.8 kDa, and an isoelectric point of 6.93, the target gene (Atlantic salmon growth hormone gene) was fully codon-modified without changing the amino acid sequence, according to the codon preference of Pichia pastoris. An EcoRI (GAATTC) site was added to the N-terminus of the gene, and a 6×His and NotI (GCGGCCGC) restriction site were added to the C-terminus of the gene.

[0014] The nucleotide sequence of the Atlantic salmon growth hormone gene is shown below: (SEQ ID NO: 1).

[0015] S2. The modified gene fragment was digested and connected to the eukaryotic vector pPICzαA, and then the recombinant plasmid pPICzαA-SGH was obtained using the Novozymes FastPure® Plasmid Mini Kit (DC201) (see Figure 2). 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.

[0016] Plasmid linearization: Use SpeedyCut Sac I rapid digestion enzyme reagent to identify the recombinant plasmid by Sac I digestion. Prepare 20 μL of double enzyme digestion reaction system with the following system: Nuclease-free water 16 μL, 10× SpeedyOne Buffer 2 μL, recombinant plasmid DNA 1 μg, SpeedyCut Sac I 1 μL, incubate at 37°C for 30 min, and then perform 1% agarose gel electrophoresis to detect linearized and non-linearized plasmids.

[0017] The recombinant vector plasmid was digested with Sac I and then subjected to agarose gel electrophoresis. The theoretical value of the linearized fragment was 4158 bp (vector 3559 bp + target gene 599 bp), and the recombinant plasmid pPICzαA-SGH was obtained. The linearization results of the plasmid are shown in Figure 2. Figure 2 As shown, the letter "M" represents Marker, the number "1" represents the enzyme-digested linearized plasmid, and the number "2" represents the original plasmid.

[0018] S3. The recombinant plasmid pPICzαA-SGH was electroporated and introduced into Pichia pastoris X33 to obtain the yeast engineering strain X33-pPICzαA-SGH, specifically: S301. Transformation: The competent X33 yeast strain and the identified linearized recombinant plasmid pPICzαA-SGH were transformed into X33 Pichia pastoris at 1500 V. The recombinant X33 Pichia pastoris were then spread on MD solid plates containing 50 µg / mL bleomycin antibiotics and cultured at room temperature for 2-3 days.

[0019] S302, Bacterial liquid PCR: Select several single colonies and culture them overnight in YPD liquid medium. Take the bacterial liquid and perform PCR verification using universal yeast primers AOX-1 (5' AOX-1 and 3' AOX-1) and a linearized recombinant plasmid as a positive control. The primers and sequences used are as follows: 5´ AOX-1 (5′ sequencing primer): 5´-GACTGGTTCCAATTGACAAGC-3´ (SEQ ID NO: 2); 3´ AOX-1 (3' sequencing primer): 5´-GCAAATGGCATTCTGACATCC-3´ (SEQ ID NO: 3); The results are as follows Figure 3 As shown, the theoretical amplification size is about 1131 bp, which is consistent with the actual value. The 7 transformants in lanes 1-7 were selected for the next experiment.

[0020] Example 2 Induce the expression of a small amount of protein in the positive recombinant yeast strain. The specific steps are as follows: (1) Pick a single colony of the verified positive strain and inoculate it into 10 mL YPD liquid medium for overnight culture. Then take 1 mL of the bacterial solution and inoculate it into 25 mL BMGY medium. Place it in a constant temperature shaker and ferment it at 30 ° C and 250 rpm / min. When the bacterial solution OD 600 After reaching 2-3, take the bacterial solution and place it in a centrifuge, centrifuge at 4500 rpm for 5-8 minutes, and discard the supernatant; (2) Induced expression: Replace 25 mL of BMMY medium and culture in a constant temperature shaker at 25°C and 250 rpm / min. Add 0.5% methanol every 12 hours to induce expression. Harvest the bacteria after 72 hours, take the bacterial liquid and place it in a centrifuge. Centrifuge at 5000 rpm for 30 minutes. Keep the supernatant and precipitate for SDS-PAGE.

[0021] The electrophoresis results of the X33-pPICzαA-SGH recombinant yeast strain are as follows Figure 4 and Figure 5 As shown in the figure, the letter "M" represents Marker, the number "1" represents induced expression yeast, and the number "2" represents uninduced expression yeast. The theoretical size of the target protein is about 24.21kDa. The SDS-PAGE detection chart of the supernatant ( Figure 4 ) No obvious difference bands were detected at the theoretical molecule, while whole-cell detection showed a lighter band near the target protein size ( Figure 5 ), indicating that a small amount of target protein was successfully induced to express.

[0022] Example 3 TCA precipitation was performed using the trichloroacetic acid concentration kit (C510011) from Shanghai Sangon Biotechnology Service Co., Ltd. The specific steps were as follows: (1) Take 200 μL of the sample protein solution to be concentrated and add it to a 1.5 mL centrifuge tube. Then add 50 μL of precipitation solution A, vortex for 10 seconds, incubate on ice for 1 hour, and then place it in a refrigerated centrifuge and centrifuge at 4°C and 15,000 rpm for 15 minutes. Finally, remove the supernatant and retain the precipitate.

[0023] (2) Add 600 μL of washing solution B and vortex for 10 seconds, then transfer to ice and let it stand for 10 minutes. Centrifuge at 4°C and 12000 rpm for 15 minutes to obtain a precipitate; pour out the supernatant in a fume hood and air dry for 30 minutes.

[0024] (3) Add 20 μL of dissolving solution C and vortex for 10 seconds, add 1 μL of blending solution D to turn it back to blue, then boil for 5 minutes, centrifuge at room temperature and 12,000 rpm for 5 minutes, and finally take the supernatant for SDS-PAGE electrophoresis relative quantification and molecular weight analysis.

[0025] Example 4 Western blot experiments were performed using the Western Blot Kit (C600393) from Shanghai Sangon Biotechnology Service Co., Ltd. The specific steps are as follows: (1) Transfer ① Prepare PVDF membrane and filter paper of appropriate size and soak the PVDF membrane in methanol for 30 seconds to make it hydrophilic. Gently pry open the glass plate of the gel after electrophoresis, remove the concentrated gel and the surrounding unnecessary area, take out the gel containing the protein band, and then immerse the gel, membrane and filter paper in transfer buffer for 20 minutes to thoroughly soak them.

[0026] ② Arrange the membrane and protein gel in the form of a "sandwich" from the positive electrode to the negative electrode: sponge, filter paper, protein gel, PVDF membrane, filter paper, sponge.

[0027] ③ Press the transfer template tightly and place it in the transfer chamber, add transfer buffer, and make sure the membrane faces the positive electrode to transfer the membrane; after the transfer is completed, remove the PVDF membrane and wash it with deionized water.

[0028] (2) Immunoblotting ① Use 0.1% TBST to prepare 5% skim milk, rinse the PVDF membrane with 0.1% TBST, add an appropriate amount of skim milk, and block on a shaker for 60 minutes; then add an appropriate amount of blocking solution again, add mouse anti-6×His monoclonal antibody at a volume ratio of 1:1000, squeeze out the air, seal the bag, and incubate overnight at 4°C.

[0029] ② Soak the membrane in 0.1% TBST and wash on a shaker three times for 10 minutes each time. Then add blocking solution to cover the membrane. Add HRP-labeled goat anti-mouse IgG at a volume ratio of 1:50,000. Squeeze out the air and seal the bag. Incubate on a shaker at room temperature for 60 minutes. Finally, wash the membrane three times with TBST and once with TBS for 10 minutes each time.

[0030] ③ Prepare DAB substrate solution: Dissolve 1 packet of DAB in 10 mL of 1× developer solution and add 10 μL of hydrogen peroxide to develop the blotting reaction. Stop the reaction when the expected band appears. Pour off the solution and rinse repeatedly with distilled water. Dry the membrane and place it in the dark.

[0031] The results of X33-pPICzαA-SGH recombinant yeast are as follows Figure 6 As shown in the figure, specific bands were detected near the theoretical molecular weight, and the band signals were relatively diffuse. This may be due to the increase in the molecular weight of the expressed target protein caused by post-translational modification in eukaryotic cells, and the fact that the protein expression amount was small and unstable, and it was easily degraded by the environment and proteases, resulting in diffuse bands.

[0032] Example 5 Protein Purification Purify the protein using the PurKine™ Histidine Tag Protein Purification Kit (Ni-NTA resin). The specific steps are as follows: (1) Prepare buffer: prepare lysis buffer, wash buffer and elution buffer without 8 M urea.

[0033] (2) Sample preparation: Transfer the induced Pichia culture medium to a centrifuge tube, centrifuge at 5000 rpm for 10 min, and collect the supernatant.

[0034] (3) Water washing: Fix the gravity column, drain the protective liquid, add 20 mL of deionized water to the column at a flow rate of 1.5 mL / min to wash the resin and remove ethanol.

[0035] (4) Equilibration: Add 10 mL of lysis buffer to the column at a flow rate of 1.5 mL / min to equilibrate the medium so that the medium and sample are in the same buffer system. Drain the lysis buffer.

[0036] (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.

[0037] (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.

[0038] (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.

[0039] (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.

[0040] (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.

[0041] (10) Storage: Store the resin in an equal volume of PBS containing 20% ​​ethanol to prevent bacterial contamination at 4°C.

[0042] (11) SDS-PAGE protein electrophoresis: Analyze the eluate by SDS-PAGE protein electrophoresis to detect the purification effect.

[0043] The results of X33-pPICzαA-SGH recombinant yeast are as follows Figure 7As shown, the eluate sample was concentrated and then analyzed by SDS-PAGE, revealing a suspected band around 25 kDa. However, the amount obtained was very small, making subsequent experiments difficult. It is speculated that this may be due to low protein expression and protein instability and susceptibility to degradation, resulting in only a small amount of the target protein being purified.

[0044] Example 6 A feeding experiment was conducted using grass carp (Goldfish) as an example. Juvenile fish of uniform size were selected and divided into two tanks, with 30 fish per tank. A fermentation-induced yeast solution was added to the basal feed at a concentration of 1 wt% and mixed thoroughly. This served as the experimental group's feed, while the control group received the basal feed. The experiment lasted two weeks. The water temperature was maintained at 26°C, with water changes every other day. Feed was administered twice daily. Kanamycin was administered at a final concentration of 10 mg / L every seven days. After removing abnormal data from the collected experimental data, fish growth was compared and analyzed. Animal experiments were conducted to determine the growth-promoting effect of recombinant Atlantic salmon growth hormone expressed in the X33-pPICzαA-SGH expression system on grass carp juveniles. Fish weight and total feed intake were measured over a 14-day period. The results are shown in Table 1.

[0045] Table 1 Results of feeding goldfish fry with X33-pPICzαA-SGH recombinant yeast strain

[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 can be seen from Table 1, the average body weight of the experimental group was significantly different from that of the control group (P<0.05); the weight gain rate of the experimental fish was 6.56% higher than that of the control group, and the feed conversion rate was 12.38% higher, indicating that the recombinant yeast constructed in the present invention has the effect of promoting the growth of fry.

Claims

1. An Atlantic salmon growth hormone gene, characterized in that The nucleotide sequence of the Atlantic salmon growth hormone gene is shown in SEQ ID NO:

1.

2. A cloning vector, characterized in that: The cloning vector contains the Atlantic salmon growth hormone gene according to claim 1.

3. The cloning vector according to claim 2, characterized in that The cloning vector is the recombinant plasmid pPICzαA-SGH.

4. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria is a yeast engineered bacteria X33-pPICzαA-SGH formed by transferring the cloning vector according to claim 2 or 3 into Pichia pastoris X33.

5. The method for constructing the recombinant engineering strain according to claim 4, characterized in that: The following steps are involved: S1, modification of Atlantic salmon growth hormone fusion protein gene; S2, ligating the modified gene fragment to the eukaryotic vector pPICzαA to obtain the recombinant plasmid pPICzαA-SGH; S3. The recombinant plasmid pPICzαA-SGH was transformed into Pichia pastoris X33 by electroporation to obtain the engineered yeast strain X33-pPICzαA-SGH.

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