Preparation method and application of oral recombinant fusion fish growth hormone and PEGylation thereof

Abstract

The invention discloses a preparation method and application of an oral recombinant fusion fish growth hormone and a PEGylated fish growth hormone, and relates to the fields of gene recombination technology and pharmaceuticals. The amino acid sequence of the fusion fish growth hormone in the invention comprises a transduction protein domain sequence, a linker protein sequence and a universal fish growth hormone amino acid sequence. The invention adopts a Piscinenodavirus (PNV) transduction domain sequence, a Grass Carp Reovirus (GCRV) transduction domain sequence, and a Protein Kinase Activated Receptor 2 (Par2) TMhelix1 as a cell-penetrating peptide to guide the growth hormone into cells, thereby improving the protein transduction rate. Meanwhile, mPEG-NH2 is used to PEGylate the fusion protein, thereby improving the half-life of the fusion protein and making it suitable for use in juvenile fish.

Description

Technical Field

[0001] The present invention relates to the fields of gene recombination technology and pharmaceuticals, and in particular to an oral recombinant fusion fish growth hormone and a PEGylated recombinant fusion fish growth hormone, as well as a preparation method of the recombinant fusion fish growth hormone and the PEGylated recombinant fusion fish growth hormone and their application in promoting fish growth. Background Art

[0002] Growth hormone (GH) is widely present in various vertebrates. It is a growth regulator with a wide range of physiological functions, affecting nearly all tissue types and cells. It promotes bone growth, nitrogen retention, accelerates protein synthesis, and influences glucose and lipid metabolism. Because of these functions, GH has been mass-produced using genetic engineering techniques. This exogenous GH can be injected or fed as an additive to animals to promote growth. This holds great promise in the fish farming industry, with significant economic and social benefits.

[0003] Prokaryotic expression is a type of protein recombinant method that involves using gene cloning techniques to construct an expression vector and introduce it into a prokaryotic expression strain for expression within a specific prokaryotic organism or cell. The advantages of prokaryotic expression are the rapid production of gene products at a relatively low cost. The method is also relatively simple, capable of expressing a wide range of proteins, making it suitable for large-scale production. Intracellular expression is the primary method for prokaryotic expression of exogenous proteins. However, the reducing nature of the prokaryotic cytoplasm hinders the formation and stabilization of disulfide bonds, leading to inaccurate protein folding and the formation of insoluble inclusion bodies. Protein kinetic modeling studies have shown that the yield of active protein also depends on the protein's synthesis, folding, and aggregation rates. When a foreign protein is highly expressed in prokaryotic cells, inclusion bodies can form if the aggregation rate of nascent peptide chains exceeds the folding rate.

[0004] Currently, Patent ZL201410650626 discloses a recombinant tilapia growth hormone; Patent ZL00116393 discloses a silver carp growth hormone; Patent ZL201210189101 discloses a recombinant oral growth hormone for tilapia; Patent ZL202110052253 discloses a liposome-encapsulated recombinant veterinary growth-promoting protein and its preparation method and application; Among them, Patent ZL201410650626 and other similar patents are all developed based on a single fish species. The growth hormones of fish vary greatly, and the applicability design of multiple fish species has not been carried out. In addition, the high osmotic pressure of marine aquaculture is not conducive to the development of new products. Characteristics will have a great impact on the activity of the protein, so this solution is applicable to fewer fish species; Patents ZL201410650626, ZL00116393, etc. have not been optimized for oral administration, and the absorption rate of growth hormone is low; the oral growth hormone developed by Patent ZL201210189101 uses HIV type I transduction protein, and it has been verified that the transduction rate of growth hormone is low when used on gastric fish; the method disclosed in Patent ZL202110052253 uses lipids to form an oil film on the water surface. Although the transduction rate is high, it is easy to cause water pollution and fish hypoxia, and is therefore not suitable for aquaculture. Summary of the Invention

[0005] The main purpose of the present invention is to provide a preparation method and application of oral recombinant fusion fish growth hormone and its PEGylation, which can effectively solve the problems in the background technology.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides an oral recombinant fusion fish growth hormone. The amino acid sequence of the fusion fish growth hormone consists of a transduction protein domain sequence, a linker protein sequence and a universal fish growth hormone amino acid sequence. The transduction protein domain sequence includes a PNV transduction protein domain, a GCRV transduction protein domain and a Par2 transduction protein domain.

[0008] A further improvement is that the amino acid sequence of the PNV transduction protein domain is shown as SEQ ID NO.7, the amino acid sequence of the GCRV transduction protein domain is shown as SEQ ID NO.11, the amino acid sequence of the Par2 transduction protein domain is shown as SEQ ID NO.14, the linker protein sequence is shown as SEQ ID NO.9, and the universal fish growth hormone amino acid sequence is shown as SEQ ID NO.5.

[0009] The present invention also provides a method for preparing the above-mentioned oral recombinant fusion fish growth hormone, comprising the following steps:

[0010] (1) Obtaining the fusion fish growth hormone gene sequence;

[0011] (2) Construction of engineered bacteria;

[0012] (3) After fermentation and purification, fused fish growth hormone is obtained.

[0013] A further improvement is that obtaining the fused fish growth hormone gene sequence specifically comprises the following steps:

[0014] (1) Obtaining the transduction protein domain sequence;

[0015] (2) obtaining the amino acid sequence of universal fish growth hormone;

[0016] (3) using a flexible linker protein sequence to connect the transduction protein domain sequence and the universal fish growth hormone amino acid sequence to obtain a fusion fish growth hormone amino acid sequence;

[0017] (4) Using DNAMAN software and BL21 codon preference optimization, reverse translation was performed and the restriction site NCOⅠ was added upstream and the restriction site XhoⅠ was added downstream to obtain the fusion fish growth hormone gene sequence.

[0018] A further improvement is that the universal fish growth hormone amino acid sequence is obtained by removing the signal peptide sequence, the intramembrane sequence and the transmembrane sequence to obtain the carp growth hormone sequence, the sea bass growth hormone effective sequence, the salmon growth hormone effective sequence and the catfish growth hormone effective sequence respectively, and then the amino acid sequences are consistent and spliced.

[0019] A further improvement is that the engineered bacteria are constructed by double-digesting the fusion protein gene sequence and the pET32a plasmid with endonucleases, connecting the double-digested products, picking positive clones, culturing at constant temperature, and sequencing.

[0020] A further improvement is that the specific steps of the fermentation purification are:

[0021] (1) Taking the qualified engineered bacteria cultured after sequencing, adding IPTG, inducing harvest, centrifuging, collecting the bacteria, cyclically crushing, collecting the crushed bacterial liquid, centrifuging to obtain the precipitate and dissolving it in washing buffer, stirring, centrifuging, washing, crushing, and fully dissolving it in dissolving buffer, and then centrifuging to obtain the supernatant, which is the inclusion body lysate;

[0022] (2) The inclusion body lysate was slowly added to 4 times the volume of refolding buffer, allowed to stand overnight, and then dialyzed against 50 times the volume of dialysis fluid overnight. The supernatant was collected by centrifugation.

[0023] The present invention also provides a method for preparing the oral recombinant fusion fish growth hormone through PEGylation, comprising the following steps:

[0024] (1) Take 0.5 g / mL fusion fish growth hormone, dilute the fusion fish growth hormone to 0.01 mM / mL with MES dialyzate, stir and add EDC solid powder, add NHS after complete dissolution, and mix thoroughly;

[0025] (2) Add β-mercaptoethanol, stir, inactivate EDC, adjust pH, add PBS containing 0.05 g / mL 5K molecular weight mPEG-NH2 and mix thoroughly, stir for 2 hours, and dialyze overnight to obtain PEGylated fusion fish growth hormone.

[0026] The present invention also provides an application of the oral recombinant fusion fish growth hormone in a medicament for promoting fish growth.

[0027] The present invention also provides an application of the PEGylated recombinant fusion growth hormone obtained by the preparation method in a medicine for promoting fish growth.

[0028] Cell penetrating peptides (CPPs) are a class of small polypeptides that can mediate the passage of exogenous substances across the cell membrane. Many CPPs are derived from the protein transduction domains of viral structural proteins that directly interact with host cells. The most widely used cell penetrating peptide is derived from the transactivating transcriptional activator (Tat) of human immunodeficiency virus type 1 (HIV-1). Whether TAT is a single cell penetrating peptide or carries other macromolecules such as proteins, oligonucleotides, or liposomes, it can exhibit high cell penetrating activity when penetrating the cell membrane. The cell penetrating efficiency is related to the number of arginines, with peptides containing 6 or more arginines having higher cell penetrating efficiency than those containing fewer than 5 arginines.

[0029] PEGylation, or polyethylene glycol modification, is the process of coupling activated PEG to a protein or peptide molecule. PEG modification technology is used for the chemical modification of a variety of proteins and peptides. PEG modification has the effects of extending half-life, reducing or eliminating immunogenicity, reducing toxic side effects, and enhancing physical, chemical, and biological stability. Polyethylene glycol (PEG) has irreplaceable advantages in protein modification due to its low toxicity, non-antigenicity, good amphiphilicity, and FDA-approved biocompatibility. Methoxy polyethylene glycol amino (mPEG-NH2) is a reactive PEG derivative that can be used to modify proteins, peptides, and other materials through carboxyl (-COOH) or other amine-reactive chemical groups.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention is based on the consistency design of the growth hormones of four common economic fish species, salmon, sea bass, carp and catfish, covering freshwater fish and marine fish, fish with stomach and fish without stomach, and designs an artificial fish growth hormone protein that can be applied to common freshwater and marine economic fish.

[0032] 2. The present invention uses the transduction domain sequence of Piscine nodavirus (PNV), the transduction domain sequence of grass carp reovirus (GCRV), and the protein kinase receptor 2 (Par2)TM helix 1 as cell-penetrating peptides to guide auxin into cells. The PNV transduction domain and the GCRV transduction domain are used for the preparation of fusion proteins for the first time in the field. The PNV transduction domain sequence is preferred, and its protein transduction rate in fish without stomach is 83% higher than that of the existing HIV type I transduction domain sequence (TAT) and in fish with stomach. The protein transduction rate is 228% higher.

[0033] 3. The present invention uses mPEG-NH2 to PEGylate the fusion protein, which can specifically bind to the C-terminus of the fusion protein, avoid the inactivation of the N-terminal transduction protein, and increase the half-life of the fusion protein, making it suitable for use in the juvenile fish stage. Compared with non-PEGylated auxin, it has a 5.4% higher weight gain effect on juvenile fish and a 13.3% higher growth effect on juvenile fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the alignment result of effective auxin sequences of carp, sea bass, salmon and catfish;

[0035] Figure 2 The following are the electrophoresis images of the purified recombinant fish growth hormones; wherein: M: protein marker 26610, lane 1: empty bacteria control, lane 2: purified recombinant fGH sample, lane 3: purified recombinant PNV-fGH sample, lane 4: purified recombinant GCRV-fGH sample, lane 5: purified recombinant Par2-fGH sample, lane 6: purified recombinant TAT-fGH sample;

[0036] Figure 3 This is an electron micrograph of the prepared recombinant universal fish growth protein liposomes; the diameter of the liposomes is about 100 nm;

[0037] Figure 4The following is a Page electrophoresis diagram of the PEGylated recombinant fish auxins; M: Protein Marker 26610, Lane 1: PEGylated sample fGH-mPEG, Lane 2: PEGylated sample PNV-fGH-mPEG, Lane 3: PEGylated sample GCRV-fGH-mPEG, Lane 4: PEGylated sample Par2-fGH-mPEG, Lane 5: PEGylated sample TAT-fGH-mPEG. The PEGylated proteins have an artificially high molecular weight due to cross-linking between PEG and SDS. In this case, the PEGylated auxins are approximately 43 kD in size, but the Page electrophoresis diagram shows approximately 80 kD.

[0038] Figure 5 This is an electron micrograph of the prepared PEGylated recombinant universal fish growth protein liposomes; the diameter of the liposomes is about 100 nm;

[0039] Figure 6 The graph shows the changes in the concentration of each recombinant growth hormone in rat serum over time. DETAILED DESCRIPTION

[0040] The present application is further described in detail below in conjunction with the accompanying drawings (tables). It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0041] The present invention will be further described in detail below with reference to the embodiments.

[0042] Example 1: Preparation of universal fish growth hormone

[0043] (1) Obtaining the auxin gene sequence

[0044] With reference to Genbank accession number AAA49208.1, the signal peptide sequence, the intramembrane sequence and the transmembrane sequence were removed to obtain the effective amino acid sequence of carp growth hormone as shown in SEQ ID NO.1;

[0045] With reference to Genbank accession number AAG09621.1, the signal peptide sequence, the intramembrane sequence and the transmembrane sequence were removed to obtain the effective amino acid sequence of sea bass growth hormone as shown in SEQ ID NO.2;

[0046] With reference to Genbank accession number NP_001117148.1, the signal peptide sequence, the intramembrane sequence and the transmembrane sequence were removed to obtain the effective amino acid sequence of salmon growth hormone as shown in SEQ ID NO.3;

[0047] With reference to Genbank accession number AAN84786.1, the signal peptide sequence, the intramembrane sequence and the transmembrane sequence were removed to obtain the effective amino acid sequence of catfish growth hormone as shown in SEQ ID NO.4;

[0048] DNAMAN was used to compare the four amino acid sequences, and the consistency was 71.81%. Figure 1 The sequences were aligned and spliced to obtain the artificial amino acid sequence shown in SEQ ID NO.5.

[0049] DNAMAN software was used, combined with BL21 codon preference optimization, for reverse translation. The restriction site NCOⅠ was added upstream, and the restriction site XhoⅠ was added downstream to obtain the artificial gene sequence shown in SEQ ID NO.6. The gene sequence was sent to BGI for synthesis, and the target gene sequence was obtained after qualified sequencing.

[0050] (2) Construction of engineered bacteria

[0051] The synthesized gene and pET32a plasmid were double-digested using NCOⅠ and XhoⅠ endonucleases. The double-digested products were ligated using ligase and introduced into Bl21 (DE3) competent cells. The cells were spread on ampicillin LB plates and cultured at 37°C. Single colonies grown on the ampicillin LB plates were picked and sent to BGI for sequencing. The sequencing results were consistent with those of the target gene, indicating that the expression bacteria were successfully constructed and recorded as fGH engineered bacteria.

[0052] (3) Fermentation and purification

[0053] Sequencing-qualified fGH-engineered bacteria were cultured on a shaker at 37°C, 220 rpm, until an OD600 of approximately 2.0 was reached. 1‰ IPTG was added, and the shaker temperature was adjusted to 32°C. After induction for approximately 5 hours, the cells were harvested. The cells were centrifuged at 7000 rpm for 15 minutes at 4°C, and then disrupted five times using a high-pressure homogenizer at a pressure of 600-700 bar. The resulting suspension was collected and centrifuged at 7000 rpm for 15 minutes at 4°C. The supernatant was discarded, and the pellet was collected. Each 10g of the pellet was dissolved in 100 mL of wash buffer (pH 8.0): 2 M urea, 50 mM Tris-HCl, 1 mM EDTA, and 0.5% Triton X-100. The cells were washed on a magnetic stirrer for 2 hours, centrifuged at 12000 rpm for 15 minutes, and the supernatant was discarded. The pellet was then washed twice. The washed precipitate was gradually crushed and fully dissolved with 45mL dissolution buffer (pH 8.0): 100mM NaCL, 50mM Tris-HCL, 1mM EDTA, 7M guanidine hydrochloride, 0.1% β-mercaptoethanol. Stir overnight on a magnetic stirrer, then centrifuge at 4°C and 12000r / min for 15min, and the supernatant was the inclusion body solubilization product. The inclusion body solubilization product was slowly added to 4 times the volume of refolding buffer (pH 8.0): 2.5M urea, 50mMTris-HCL, 100mM NaCL, 0.1% β-mercaptoethanol. Let it stand at 4°C overnight, and then place it in 50 times the volume of dialysis solution (pH 7.0): 50mM Tris-HCL, 100mM NaCL. Dialyze at 4°C for more than 6h, centrifuge at 12000r / min for 15min, and collect the supernatant, which is the target product, marked as fGH, as shown. Figure 2 Lane 2.

[0054] Example 2: Preparation of PNV transduction domain fused with fish growth hormone

[0055] (1) Obtaining the fusion protein gene sequence

[0056] With reference to Genbank accession number ALL27039.1, the amino acid sequence of PNV was obtained to obtain the transduction domain sequence shown in SEQ ID NO.7.

[0057] The artificial amino acid sequence obtained according to the method in Example 1 is shown in SEQ ID NO.5.

[0058] The PNV transduction domain sequence and the artificial amino acid sequence were connected using a flexible linker protein to obtain the target amino acid sequence as shown in SEQ ID NO.8; the flexible linker protein amino acid sequence is shown in SEQ ID NO.9.

[0059] DNAMAN software was used, combined with BL21 codon preference optimization, for reverse translation. The restriction site NCOⅠ was added upstream, and the restriction site XhoⅠ was added downstream to obtain the artificial gene sequence shown in SEQ ID NO.10. The gene sequence was sent to BGI for synthesis, and the target gene sequence was obtained after qualified sequencing.

[0060] (2) Construction of engineered bacteria

[0061] The target gene synthesized above was used to construct an engineered bacterium according to the engineering bacterium construction method in Example 1 to obtain an engineered bacterium named PNV-fGH.

[0062] (3) Fermentation and purification

[0063] The qualified PNV-fGH engineered bacteria were obtained by sequencing, and the target product was obtained according to the fermentation and purification steps in Example 1, which was labeled as PNV-fGH. Figure 2 Lane 3.

[0064] Example 3: Preparation of GCRV transduction domain fused with fish growth hormone

[0065] (1) Obtaining the fusion protein gene sequence

[0066] With reference to Genbank accession number AFP55032.1, the amino acid sequence of GCRV was obtained to obtain the transduction domain sequence shown in SEQ ID NO.11.

[0067] The artificial amino acid sequence obtained according to the method in Example 1 is shown in SEQ ID NO.5.

[0068] The GCRV transduction domain sequence and the artificial amino acid sequence were connected using a flexible linker protein to obtain the target amino acid sequence as shown in SEQ ID NO.12.

[0069] DNAMAN software was used, combined with BL21 codon preference optimization, for reverse translation. The restriction site NCOⅠ was added upstream, and the restriction site XhoⅠ was added downstream to obtain the artificial gene sequence shown in SEQ ID NO.13. The gene sequence was sent to BGI for synthesis, and the target gene sequence was obtained after qualified sequencing.

[0070] (2) Construction of engineered bacteria

[0071] The target gene synthesized above was used to construct an engineered bacterium according to the engineering bacterium construction method in Example 1, and an engineered bacterium named GCRV-fGH was obtained.

[0072] (3) Fermentation and purification

[0073] The qualified GCRV-fGH engineered bacteria were obtained by sequencing, and the target product was obtained according to the fermentation and purification steps in Example 1, which was labeled as GCRV-fGH. Figure 2 Lane 4.

[0074] Example 4: Preparation of Par2™ helix 1 fusion fish growth hormone

[0075] (1) Obtaining the fusion protein gene sequence

[0076] With reference to Genbank accession number NM_001140517.1, the amino acid sequence of Par2 was obtained to obtain the TM helix 1 sequence shown in SEQ ID NO.14.

[0077] The artificial amino acid sequence obtained according to the method in Example 1 is shown in SEQ ID NO.5.

[0078] The Par2™ helix1 sequence and the artificial amino acid sequence were connected using a flexible linker protein to obtain the target amino acid sequence as shown in SEQ ID NO.15.

[0079] DNAMAN software was used, combined with BL21 codon preference optimization, for reverse translation. The restriction site NCOⅠ was added upstream, and the restriction site XhoⅠ was added downstream to obtain the artificial gene sequence shown in SEQ ID NO.16. The gene sequence was sent to BGI for synthesis, and the target gene sequence was obtained after qualified sequencing.

[0080] (2) Construction of engineered bacteria

[0081] The target gene synthesized above was used to construct an engineered bacterium according to the engineering bacterium construction method in Example 1 to obtain an engineered bacterium named Par2-fGH.

[0082] (3) Fermentation and purification

[0083] The Par2-fGH engineered bacteria that passed the sequencing were fermented and purified according to the steps in Example 1 to obtain the target product, which was labeled as Par2-fGH. Figure 2 Lane 5.

[0084] Example 5: Preparation of HIV type 1 TAT transduction domain fused to fish growth hormone

[0085] (1) Obtaining the fusion protein gene sequence

[0086] With reference to Genbank accession number 5SVZ_B, the sequence of HIV type I transduction domain TAT was obtained as shown in SEQ ID NO.17.

[0087] The artificial amino acid sequence obtained according to the method in Example 1 is shown in SEQ ID NO.5.

[0088] The TAT transduction domain sequence and the artificial amino acid sequence were connected using a flexible linker protein to obtain the target amino acid sequence as shown in SEQ ID NO.18.

[0089] DNAMAN software was used, combined with BL21 codon preference optimization, for reverse translation. The restriction site NCOⅠ was added upstream, and the restriction site XhoⅠ was added downstream to obtain the artificial gene sequence shown in SEQ ID NO.19. The gene sequence was sent to BGI for synthesis, and the target gene sequence was obtained after qualified sequencing.

[0090] (2) Construction of engineered bacteria

[0091] The target gene synthesized above was constructed by engineering bacteria according to the engineering bacteria construction method in Example 1 to obtain an engineered bacterium named TAT-fGH.

[0092] (3) Fermentation and purification

[0093] The qualified TAT-fGH engineered bacteria were obtained by sequencing, and the target product was obtained according to the fermentation and purification steps in Example 1, which was labeled as TAT-fGH. Figure 2 Lane 6.

[0094] Example 6 Preparation of liposome-encapsulated fish growth hormone

[0095] Take 500mL of anhydrous ethanol on a magnetic stirrer, dissolve 100g of soybean lecithin with a purity of ≥98% and 60g of cholesterol in anhydrous ethanol, seal with plastic wrap, and stir for 30 minutes. Ultrasonicate in an ice bath in an ultrasonic disruptor for 20 minutes, power 300W, ultrasonicate for 1S and rest for 1S, then place in a 42°C water bath for use; take 1g of freeze-dried fGH and dissolve it in 100mL of PBS with pH 7.0, place in a 37°C water bath for use. Measure with a thermometer, mix and stir the two for 10 minutes after the temperature of the two is reached, and remove the ethanol by rotary evaporation. Filter using a 0.45μm oil-based filter membrane to obtain liposome-encapsulated fGH, which has a double-layer membrane structure and a particle size of about 100nm. Figure 3 . Denoted as L-fGH.

[0096] Example 7 PEGylation of fGH

[0097] 0.5 g / mL fGH was dialyzed overnight at 4°C using MES dialysate (0.1 M MES, 0.5 M NaCl, pH 6.0) using a 5Kd dialysis bag. The fGH was diluted to 0.367 g / mL (0.01 mM / mL) with MES dialysate. 100 mL of the solution was placed on a magnetic stirrer at 150 rpm. 1.553 g of EDC solid powder (final concentration 0.1 mM) was added. Once dissolved, 2.878 g of NHS (final concentration 0.25 mM) was added, mixed thoroughly, and allowed to stand at room temperature for 15 minutes. EDC was inactivated by adding 7 μL of β-mercaptoethanol (final concentration 1 mM) and stirring at 30 rpm for 3 minutes. The pH was adjusted to 7.2. 100 mL of PBS (pH 7.2) containing 0.05 g / mL 5K molecular weight mPEG-NH2 was added, mixed thoroughly, and stirred at 90 rpm for 2 hours at room temperature. Use 7kD dialysis bag, dialyze overnight with 50 volumes of PBS (pH 7.2) to obtain PEGylated fGH, which is recorded as fGH-mPEG. Figure 4 Lane 1.

[0098] Example 8 Preparation of Liposome-Encapsulated PEGylated Fish Growth Hormone

[0099] Take 1 g of freeze-dried fGH-mPEG in Example 7 and follow the method for preparing liposome-encapsulated fish growth hormone in Example 6 to obtain liposome-encapsulated fGH-mPEG, which has a double-layer membrane structure and a particle size of about 100 nm. Figure 5 . It is denoted as L-fGH-mPEG.

[0100] Example 9 PEGylation of PNV-fGH

[0101] 0.5 g / mL PNV-fGH obtained in Example 2 was taken and PEGylated according to the fGH PEGylation method in Example 7 to obtain PEGylated PNV-fGH, which was designated as PNV-fGH-mPEG. Figure 4 Lane 2.

[0102] Example 10 PEGylation of GCRV-fGH

[0103] 0.5 g / mL GCRV-fGH obtained in Example 3 was taken and PEGylated according to the fGH PEGylation method in Example 7 to obtain PEGylated GCRV-fGH, which was recorded as GCRV-fGH-mPEG. Figure 4 Lane 3.

[0104] Example 11 PEGylation of Par2-fGH

[0105] 0.5 g / mL Par2-fGH obtained in Example 4 was taken and PEGylated according to the fGH PEGylation method in Example 7 to obtain PEGylated Par2-fGH, which was designated as Par2-fGH-mPEG. Figure 4 Lane 4.

[0106] Example 12 PEGylation of TAT-fGH

[0107] 0.5 g / mL TAT-fGH obtained in Example 5 was taken and PEGylated according to the fGH PEGylation method in Example 7 to obtain PEGylated TAT-fGH, which was designated as TAT-fGH-mPEG. Figure 4 Lane 5.

[0108] Example 13 Verification of the Effect of Solution Osmotic Pressure on the Activity of Each Auxin

[0109] Tilapia growth hormone, designated ZL-GH, was prepared with reference to Patent ZL201410650626. Eleven samples, including those from Examples 1 to 5, 7, and 9 to 12, were dialyzed overnight at 4°C using 100-fold volumes of 0.9% NaCL solution (hypotonic solution) or 3.5% NaCL solution (hypertonic solution), diluted to 1 mg / mL, and allowed to stand at 4°C for 24 hours. Aseptically, the samples were filtered through a 0.22 μm sterile filter and diluted to 0.1 mg / mL using sterile 0.9% NaCL solution or 3.5% NaCL solution, respectively, for later use.

[0110] Digest 3T3 cells and adjust the concentration to 1.5 × 10 4 / mL, add 100 μL to each well of a 96-well cell culture plate, culture overnight at 37°C in a 5% CO2 environment, wash twice with PBS, and aspirate the PBS. Add 100 μL each of the above hypotonic solutions of ZL-GH, fGH, PNV-fGH, GCRV-fGH, Par2-fGH, TAT-fGH, fGH-mPEG, PNV-fGH-mPEG, GCRV-fGH-mPEG, Par2-fGH-mPEG, and TAT-fGH-mPEG to the culture plate. Prepare eight replicate wells for each auxin, and set up eight wells as a 0.9% NaCl control. To another culture plate, 100 μL of each hypertonic solution of ZL-GH, fGH, PNV-fGH, GCRV-fGH, Par2-fGH, TAT-fGH, fGH-mPEG, PNV-fGH-mPEG, GCRV-fGH-mPEG, Par2-fGH-mPEG, and TAT-fGH-mPEG was added dropwise. Eight replicate wells were prepared for each auxin, and a 3.5% NaCl control was set up in eight wells in 5% CO2. The plates were incubated at 37°C for 48 h. The liquid was discarded, and 25 μL of MTT solution (5 mg / mL, pH 7.2 in PBS) was added to each well and incubated for another 5 h. 120 μL of DMSO solution was added to each well, and the plates were shaken at low speed for 15 min to fully dissolve the crystals. The absorbance of each well was measured at 570 nm using a microplate reader. The average value of each group was calculated, and the background control was subtracted. As shown in the table below, compared with ZL-GH, the auxins described in this case (not encapsulated in liposomes) have better tolerance to high osmotic pressure, and the differences in activity are all within 10%.

[0111] Table 1 Activity differences of various auxins under different osmotic pressures

[0112]

[0113] Example 14 Detection of serum concentration of each growth hormone after oral administration

[0114] (1) Materials

[0115] Sixty-five healthy male SD rats (purchased from Anhui Provincial Experimental Animal Center) weighing approximately 200 g were selected and divided into 13 groups.

[0116] (2) Method

[0117] After 12 hours of fasting (except for water), SD rats were given ZL-GH and each of the growth hormones prepared in Examples 1 to 12. The mixture was diluted to 1 mg / mL with PBS and administered 1 mL to each rat via gavage. Blood was collected from the tail of the rats (200 μL) before the start of the experiment (0 h) and at 1, 3, 5, 7, 12, 18, 24, and 48 h after gavage. The blood was stored at 4°C for 1 h, centrifuged at 5000 rpm for 15 min, and immediately frozen at -70°C. After the final blood draw, the serum was also frozen at -70°C for at least 2 h to ensure that all serum was frozen and thawed once during ELISA testing. The fish growth hormone ELISA assay kit (orb1146928) from Biorbyt was used according to the manufacturer's instructions to determine the levels of each fish growth hormone in rat serum. This indicates the levels of each growth hormone in rat serum.

[0118] (3) Results

[0119] ZL-GH, fGH, PNV-fGH, GCRV-fGH, Par2-fGH, TAT-fGH, and L-fGH reached peak concentrations in rat serum after about 5 hours. Among them, the fusion proteins PNV-fGH, GCRV-fGH, Par2-fGH, and TAT-fGH had significantly higher levels in serum than ZL-GH and fGH. After reaching their peak concentrations in serum, they began to slowly decline, with a half-life of about 16 hours. fGH-mPEG, GCRV-fGH-mPEG, Par2-fGH-mPEG, and TAT-fGH-mPEG reached their peak concentrations in rat serum after about 7 hours, and began to slowly decline after reaching their peak, with a half-life of about 30 hours. PNV-fGH-mPEG and L-fGH-mPEG reached their peak concentrations in rat serum after about 5 hours, and began to slowly decline after reaching their peak, with a half-life of about 30 hours. However, the peak concentrations of all PEGylated growth hormones in rat serum were lower than those of non-PEGylated ones. As shown in Table 2 and Figure 6 shown.

[0120] The results showed that the fusion proteins of L-GH and transducin auxin, PNV-fGH, GCRV-fGH, Par2-fGH, and TAT-fGH, are more suitable for applications with higher concentration requirements and more frequent use. Among the PEGylated auxins, PNV-fGH-mPEG and L-fGH-mPEG are more suitable for applications with lower concentration requirements and less frequent use.

[0121] Table 2 Changes of growth hormone concentrations in rat serum over time

[0122]

[0123]

[0124] Example 15 Transduction rate detection of each growth hormone after oral administration

[0125] (1) Materials

[0126] Fish species selection: Grass carp was used as freshwater stomachless fish: 225±25g / tail, 40 tails, randomly divided into 8 groups, 5 tails in each group; mandarin fish with stomach was used as freshwater fish: 135±15g / tail, 40 tails, randomly divided into 8 groups, 5 tails in each group; large yellow croaker was used as marine stomach fish: 90±10g / tail, 40 tails, randomly divided into 8 groups, 5 tails in each group; purchased from Anhui Provincial Experimental Animal Center.

[0127] (2) Method

[0128] After a 12-hour fast, PNV-fGH, GCRV-fGH, Par2-fGH, TAT-fGH, L-fGH, PNV-fGH-mPEG, and L-fGH-mPEG were diluted with PBS to 1 mg / mL and gavage administered to each fish. A control group was gavage administered with 1 mL of PBS. Five hours later, small intestinal segments were removed from the fish, rinsed with PBS, and 0.4 g of ileum was excised from each fish. A total of 2 g per group was pooled and thoroughly homogenized using a homogenizer with 2 mL of PBS. The supernatant was centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. The content of each fish growth hormone in the intestinal supernatant was determined using a Biorbyt Fish Growth Hormone ELISA Kit (orb1146928) according to the manufacturer's instructions. This assay indicates the level of each growth hormone transduced into intestinal cells. Transduction efficiency = (intestinal supernatant content of each fish - control content) / gavage dose.

[0129] (3) Results

[0130] In terms of whether farmed fish have stomachs, L-fGH was the highest in both grass carp and mandarin fish, followed by PNV-fGH. TAT-fGH was 42% lower in fish with stomachs than in fish without stomachs, while PNV-fGH was 8% lower in fish with stomachs than in fish without stomachs. In terms of whether farmed fish are freshwater or marine, L-fGH was the highest in both mandarin fish and large yellow croaker, followed by PNV-fGH. TAT-fGH was 91% lower in freshwater fish than in marine fish, while PNV-fGH was 17% lower in freshwater fish than in marine fish. This suggests that PNV-fGH has better universality than TAT-fGH. Of the PEGylated products, PNV-fGH-mPEG and L-fGH-mPEG, L-fGH-mPEG had a higher transduction rate, as shown in Table 3.

[0131] Table 3 Growth hormone small intestinal transduction rate

[0132]

[0133] Example 16 Verification of Growth-Promoting Performance of Growth Hormone in Fish

[0134] (1) Verification of adult fish growth performance

[0135] Fish species: Grass carp (225 ± 25 g / fish, 70 randomly divided into 7 groups of 10) were used as freshwater stomach-less fish. Mandarin fish (135 ± 15 g / fish, 70 randomly divided into 7 groups of 10) were used as freshwater stomach-forming fish. Yellow croaker (90 ± 10 g / fish, 70 randomly divided into 7 groups of 10) were used as marine stomach-forming fish. All fish were purchased from the Anhui Provincial Experimental Animal Center. New Hope Liuhe Company's Yuyuhuan brand general-purpose growout feed was used. The feeds were ground into powder. 10 g of auxin (10 mg / mL) was added to 10 kg of each feed, including ZL-GH, TAT-fGH, PNV-fGH, L-fGH, PNV-fGH-mPEG, and L-PNV-fGH-mPEG. The mixture was thoroughly mixed and re-formed. The water temperature was controlled at 25°C and the fish were fed for 30 days. Based on the initial weight of the fish, 5% of the feed was added for the first 10 days, 7.5% for the middle 10 days, and 10% for the last 10 days. Feed was added once in the morning and evening. The experiment was terminated after 30 days and the fish were weighed.

[0136] Because L-fGH and L-PNV-fGH-mPEG contain more lipids, the water becomes turbid and the water surface becomes oily during the feeding period, and the fish mortality rate exceeds 30%, so they are not counted. No deaths or morbidity occurred in other groups. As shown in Table 4 below, the group with PNV-fGH added had the best growth-promoting effect on freshwater fish, marine fish, fish with stomachs, and fish without stomachs compared with other groups, followed by PNV-fGH-mPEG. ZL-GH had the worst growth-promoting effect among all groups. TAT-fGH performed better on fish without stomachs and freshwater fish, and performed worse on fish with stomachs and marine fish. This shows that PNV-fGH-mPEG and PNV-fGH in the present invention have better growth-promoting effects than the single growth hormone and TAT fusion scheme in other patents, and PNV-fGH is better than PNV-fGH-mPEG.

[0137] Table 4 Statistics of adult fish growth performance

[0138]

[0139]

[0140] (2) Verification of juvenile fish growth performance

[0141] 150 2±0.5cm grass carp fry (purchased from Anhui Provincial Experimental Animal Center) were selected and divided into 3 groups of 50 each. The feeding feed was selected from the Yuyuhuan brand grass carp fry feed of New Hope Liuhe Company. Take PNV-fGH and PNV-fGH-mPEG and add 10g of 10mg / mL growth hormone to 10kg feed, mix thoroughly and dry. Control the water temperature to 28°C, feed for 15 days, feed once in the morning and evening, and feed 10g each time. The experiment was terminated after 15 days to measure body length and weigh. Dead fish were found in the process and fished out in time. As shown in Table 5 below, the group with the addition of PNV-fGH-mPEG was better than the group with the addition of PNV-fGH in terms of survival rate, weight gain and growth, and both were better than the control group in terms of survival rate, weight gain and growth. This shows that PNV-fGH-mPEG in the present invention has a better growth-promoting effect on fry than PNV-fGH.

[0142] Table 5 Statistics of fry growth performance

[0143]

[0144] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. An oral recombinant fusion fish growth hormone, characterized in that: The amino acid sequence of the fused fish growth hormone is shown in any one of the following: PNV-fGH: as shown in SEQ ID NO.8; GCRV-fGH: as shown in SEQ ID NO. 12; Par2-fGH: as shown in SEQ ID NO.

15.

2. A method for preparing the oral recombinant fusion fish growth hormone according to claim 1, characterized in that: The following steps are involved: (1) Obtaining the fusion fish growth hormone gene sequence; (2) Construction of engineered bacteria; (3) After fermentation and purification, fused fish growth hormone is obtained.

3. The method for preparing an oral recombinant fusion fish growth hormone according to claim 2, characterized in that: The acquisition of the fused fish growth hormone gene sequence specifically comprises the following steps: (1) Obtaining the transduction protein domain sequence; (2) Obtaining the amino acid sequence of universal fish growth hormone; (3) Using a flexible linker protein sequence, the transduction protein domain sequence and the universal fish growth hormone amino acid sequence are connected to obtain the amino acid sequence of the fused fish growth hormone; (4) Using DNAMAN software and BL21 codon preference optimization, reverse translation was performed and the restriction site NCO Ⅰ was added upstream and the restriction site Xho Ⅰ was added downstream to obtain the fusion fish growth hormone gene sequence.

4. The method for preparing an oral recombinant fusion fish growth hormone according to claim 3, characterized in that: The universal fish growth hormone amino acid sequence is obtained by removing the signal peptide sequence, the intramembrane sequence and the transmembrane sequence to obtain the carp growth hormone sequence, the sea bass growth hormone effective sequence, the salmon growth hormone effective sequence and the catfish growth hormone effective sequence respectively, and then the amino acid sequences are consistent and spliced.

5. The method for preparing an oral recombinant fusion fish growth hormone according to claim 2, characterized in that: The engineering bacteria are constructed by double-digesting the fusion protein gene sequence and the pET32a plasmid with endonucleases, connecting the double-digested products, picking positive clones, culturing at constant temperature, and sequencing.

6. The method for preparing an oral recombinant fusion fish growth hormone according to claim 2, characterized in that: The specific steps of the fermentation purification are: (1) Take the qualified engineered bacteria cultured after sequencing, add IPTG, induce harvest, centrifuge, collect the bacteria, cyclically crush, collect the broken bacterial liquid, centrifuge to obtain the precipitate and dissolve it in washing buffer, stir, centrifuge, wash the precipitate, crush it and fully dissolve it in dissolution buffer, then centrifuge to obtain the supernatant which is the inclusion body lysate; (2) Slowly add the inclusion body dissolved product to 4 times the volume of refolding buffer, let it stand overnight, then dialyze it against 50 times the volume of dialysate overnight, and centrifuge to collect the supernatant.

7. A method for preparing oral recombinant fusion fish growth hormone by PEGylation according to claim 1, characterized in that: The following steps are involved: (1) Take 0.5 g / mL fusion fish growth hormone, dilute the fusion fish growth hormone to 0.01 mM / mL with MES dialyzate, stir and add EDC solid powder, add NHS after complete dissolution, and mix thoroughly; (2) Add β-mercaptoethanol, stir, inactivate EDC, adjust pH, add PBS containing 0.05 g / mL 5K molecular weight mPEG-NH2 and mix thoroughly, stir for 2 hours, and dialyze overnight to obtain PEGylated fusion fish growth hormone.

8. Use of the oral recombinant fusion fish growth hormone as claimed in claim 1 in the preparation of a medicine for promoting fish growth.

9. Use of the PEGylated recombinant fusion auxin obtained by the preparation method of claim 7 in the preparation of a fish growth promoting agent.

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