CN112175964B - Recombinant pig growth hormone and preparation method and application thereof - Google Patents
Recombinant pig growth hormone and preparation method and application thereof Download PDFInfo
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- CN112175964B CN112175964B CN202011145343.9A CN202011145343A CN112175964B CN 112175964 B CN112175964 B CN 112175964B CN 202011145343 A CN202011145343 A CN 202011145343A CN 112175964 B CN112175964 B CN 112175964B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormones [GH] (Somatotropin)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/184—Hormones
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
Abstract
The invention discloses a recombinant pig growth hormone and a preparation method and application thereof, wherein the preparation steps are as follows: firstly, inserting a polynucleotide sequence containing a plurality of pig growth hormone full-length sequences into a pichia pastoris expression vector to obtain a recombinant plasmid; introducing the recombinant plasmid into a pichia pastoris competent cell to construct and obtain recombinant engineering bacteria; and carrying out fermentation expression and protein purification on the recombinant engineering bacteria to obtain a target protein, namely the recombinant pig growth hormone, wherein the recombinant pig growth hormone can be used as a pig feed additive. The invention has the advantages that the molecular weight of the protein is increased by adopting a multi-linkage mode, so that the problem that the hormone is digested in the stomach to lose functions is solved, and a plurality of repeated fragments exist, so that the problem of short half-life period can be better solved; in addition, the invention is produced by a recombination mode, is simpler than an extraction method and has low cost, and can be widely applied to feed additives.
Description
Technical Field
The invention relates to the technical field of gene recombination, in particular to a recombinant porcine growth hormone, a preparation method and application thereof.
Background
Recombinant proteins are proteins obtained by techniques using recombinant DNA or recombinant RNA. The production of in vitro recombinant protein is mainly divided into four major systems of prokaryotic expression and eukaryotic expression: prokaryotic expression system is prokaryotic type; eukaryotes are further classified into: yeast expression systems, insect cell expression systems, and mammalian cell expression systems.
Yeast expression system: has the advantages of both prokaryotic system and higher eukaryotic system. The yeast is a unicellular lower eukaryote, has common culture conditions and rapid growth and reproduction speed, can tolerate higher hydrostatic pressure, can be produced in large scale when used for expressing genetic engineering products, and effectively reduces the production cost. The yeast expression exogenous gene has certain post-translational processing capacity, the harvested exogenous protein has certain folding processing and glycosylation modification, and the property is more stable than that of prokaryotic expression protein, so that the yeast expression exogenous gene is particularly suitable for expressing eukaryotic genes and preparing functional expression protein. Some yeast expression systems have an exocrine signal sequence that can secrete the expressed foreign protein outside the cell and thus can be easily purified. The use of yeast expression systems to produce protein products of foreign genes also has disadvantages such as heterogeneity of the product protein, incomplete processing of signal peptides, internal degradation, multimer formation, etc., which results in structural inconsistencies of the expressed protein. In addition, excessive glycosylation of the expressed product is also frequently encountered.
The human beings 'understanding of animal growth hormone begins in the 30's of the 20 th century, and at present, the growth hormone of nearly hundreds of animals is separated and purified successively, and the research on the physiological and biochemical mechanisms is more and more intensive. Porcine Growth Hormone (PGH), a single-chain multi-skin hormone containing a single amino acid secreted by the anterior pituitary, stimulates the growth of almost all tissues. Its physiological and biochemical mechanism for promoting growth is to regulate the protein, fat and sugar metabolism of animals. A large number of research works show that the exogenous pig growth hormone can improve the growth speed of animals and the feed conversion rate, the reduction of the fat in the body can be achieved, the daily weight increase and the feed conversion rate can be improved, and the method has obvious economic benefit and social benefit.
At present, a fusion protein of pig growth hormone is developed by related mechanisms, the problem of gastrointestinal tract absorption is solved, and the problem of short half-life is not solved.
Disclosure of Invention
The invention aims to provide a recombinant porcine growth hormone, a preparation method and an application thereof, which solve the problem of short half-life by adopting a multi-connection mode.
The invention realizes the purpose through the following technical scheme:
a polynucleotide sequence comprises a guide sequence and a plurality of pig growth hormone coding gene full-length sequences which are connected in sequence, wherein the pig growth hormone coding gene full-length sequences are connected through a flexible linker sequence.
The further improvement is that the full-length sequence of the pig growth hormone coding gene is shown as SEQ ID NO. 1.
In a further improvement, the guide sequence is shown as SEQ ID NO. 2.
The further improvement is that the flexible linker sequence is shown as SEQ ID NO. 3.
In a further improvement, the polynucleotide sequence comprises at least three sets of full-length sequences of genes encoding porcine growth hormone.
A preparation method of recombinant porcine growth hormone is characterized by comprising the following steps:
(1) inserting the polynucleotide sequence into a pichia pastoris expression vector to obtain a recombinant plasmid;
(2) introducing the recombinant plasmid into a pichia pastoris competent cell to construct and obtain recombinant engineering bacteria;
(3) and carrying out fermentation expression and protein purification on the recombinant engineering bacteria to obtain a target protein, namely the recombinant pig growth hormone.
A recombinant porcine growth hormone prepared by the method.
An application of the recombinant pig growth hormone in serving as a pig feed additive.
The further improvement is that the addition amount of the recombinant pig growth hormone in the pig feed is 0.33-3.3mg/100 g.
The invention has the beneficial effects that:
(1) the invention adopts a multi-linkage mode to increase the molecular weight of protein so as to overcome the problem that the hormone is digested in the stomach and loses function;
(2) the invention adopts a multi-connection mode, has a plurality of repeated segments and can better solve the problem of short half-life period;
(3) the invention is produced by a recombination mode, is simpler than an extraction method and has low cost, and can be widely applied to feed additives.
Drawings
FIG. 1 is a graph showing the verification result of recombinant triple pig growth hormone wb, wherein M represents protein Marker 26616, lane 1 represents recombinant triple pig growth hormone, and lane 2 represents naturally extracted pig growth hormone
FIG. 2 is a graph showing the time-dependent change of the serum PGH concentration in rats.
Detailed Description
The present application will now be described in further detail with reference to the drawings, it should be noted that the following detailed description is given for illustrative purposes only and is not to be construed as limiting the scope of the present application, as those skilled in the art will be able to make numerous insubstantial modifications and adaptations to the present application based on the above disclosure.
Example 1: polynucleotide sequences
A polynucleotide sequence comprises a guide sequence and three repeated pig growth hormone coding gene full-length sequences which are sequentially connected, wherein the pig growth hormone coding gene full-length sequences are connected through a flexible linker sequence.
Wherein, the full-length sequence of the pig growth hormone coding gene refers to GENBANK accession number X53325.1, the specific sequence is shown as SEQ ID NO.1, the guide sequence is shown as SEQ ID NO.2, and the flexible linker sequence is shown as SEQ ID NO. 3.
Example 2: preparation of recombinant porcine growth hormone
(1) Obtaining a gene sequence of the recombinant porcine growth hormone:
referring to GENBANK accession number X53325.1, taking a full-length sequence of the porcine growth hormone, making three repeats, adding an enzyme cutting site EcoR I and a guide sequence at the upstream, using a flexible linker between the three repeats to protect the space structure of a target fragment, and adding an enzyme cutting site Not I and a protective base at the downstream to obtain the nucleotide sequence shown in SEQ ID No. 4.
Synthesizing the target gene sequence with the Huahua large gene, and obtaining the target gene sequence after the sequencing is qualified.
(2) Construction of engineering bacteria:
the polynucleotide sequence in the embodiment 1 is inserted into a pichia pastoris expression vector to obtain pPICZ alpha A plasmid, two endonucleases of EcoR I and Not I are used for carrying out double enzyme digestion on the synthesized gene and the pPICZ alpha A plasmid, double enzyme digestion products are connected by using ligase and are electrically transferred into X33 pichia pastoris competent cells, the pichia pastoris competent cells are coated on a YPD plate with the Zeocin of 300mg/L, the culture is carried out at the constant temperature of 30 ℃ for about 36h, colonies growing on a resistant plate are sent to a Huada gene to be sequenced and identified as positive, the construction success of the expression vector is shown, and the engineering bacterium is marked as X33/R-TPGH.
(3) Expression by fermentation
Carrying out shake culture (30 ℃, 250R/min till OD600 is 2-6) on a BMGY culture medium by using sequencing X33/R-TPGH engineering bacteria, centrifugally collecting the bacteria, carrying out heavy suspension on the bacteria by using a BMMY culture medium until OD600 is approximately equal to 1, continuing shake fermentation, and adding absolute methanol into the culture medium every 24 hours till the final concentration is 1%; and (5) stopping culturing for 48h, centrifuging at 7000rpm/min for 30min, and taking the supernatant to obtain the crude product of the target protein R-TPGH.
Filtering the crude protein, passing through Ni affinity chromatography column, eluting with Elution buffer (50mM Tris-Cl50mM reduced glutathione pH7.0) gradient, and collecting R-TPGH peak; passing the purified R-TPGH of the previous step through a desalting column, replacing the R-TPGH with a buffer solution (50mM Tris-Cl pH10.0), balancing the column with a Loading buffer (50mM Tris-Cl pH7.0), performing gradient Elution with an elusion buffer (50mM Tris-Cl 1M NaCl pH7.0) after Loading, and collecting an R-TPGH peak; the collected R-TPGH by ion exchange chromatography was passed through a molecular sieve column, and the peak of R-TPGH was collected by an elusion buffer (50mM Na2HPO 40.1M NaCl pH 7.0).
When using the BIORByt PGH antibody (orb24331) as an antibody and goat anti-mouse IgG (HRP-labeled) as a secondary antibody for WB, the sample showed a positive band at 76kD (as shown in FIG. 1), indicating that the expressed R-TPGH was PGH.
Example 3: serum concentration detection of recombinant triple pig growth hormone after oral administration
Healthy male SD rats (purchased from the center of laboratory animals, Anhui province) were selected at 3 animals, and the weight was about 200 g. After fasting and water prohibition for 12 hours, the recombinant porcine growth hormone PBS prepared in example 2 is diluted to 1mg/ml, and 1mg of the recombinant triple porcine growth hormone is administrated to each rat by gavage. 200 mu L of blood is taken from the orbit 1, 2, 4, 8, 12, 18 and 24 hours before (0 hour) and after the test, the orbit is placed for 1 hour at 4 ℃, serum is centrifugally separated at 7000rpm, the blood is frozen at-70 ℃ immediately, and the blood is frozen at-70 ℃ for more than 2 hours after the last blood sampling, so that all the serum is frozen and thawed for 1 time during ELISA detection. The porcine growth hormone content in rat serum was determined as described in Biorbyt (orb 439756). SD rats can be detected in serum after oral administration of the recombinant triple pig growth hormone for 1 hour, the peak concentration (120ng/mL) is reached after 4 hours, the peak concentration starts to slowly decrease after the peak is reached, and the half-life period is about 24 hours, as shown in table 1 and figure 2, the half-life period is far longer than that of the pig growth hormone prepared or extracted through recombination by the existing methodology, and the feeding period can be 2-3 days and 1 time.
Table 1: time-dependent table of rat serum PGH concentration
| Time (h) | 0 | 1 | 2 | 4 | 8 | 12 | 18 | 24 |
| Concentration (ng/mL) | 2 | 31 | 87 | 120 | 116 | 103 | 89 | 62 |
Example 4: growth hormone growth promotion performance verification of recombinant triple pigs
The effective protein content of the X33/R-TPGH engineering bacteria after fermentation is about 30 percent, the thalli are lyophilized after fermentation expression, and 3.3g of lyophilized yeast powder is added into 100kg of feed to be mixed and stirred uniformly. The feed is universal feed for pigs produced by Anhui Xinkang feed factory. The feed added with the freeze-dried yeast powder contains about 1mg/100g of recombinant triple pig auxin and is marked as feed A, and the common feed without the freeze-dried yeast powder is marked as feed B.
Selecting 20 healthy ternary pigs with age of 30 days and weight of 7.5-8.5 kg, dividing the pigs into 2 groups of 10 pigs each with half of the pigs in each group. Selecting 20 healthy ternary hybrid piglets with age of 60 days and weight close to 19.5-20.5 kg, dividing male piglets and female piglets into 2 groups randomly, and 10 piglets in each group. Obtaining a group A: the method comprises the following steps of (1) marking an ear tag A on 10 30-day-old pigs and 10 60-day-old pigs, and feeding the pigs with a feed A; group B: and (3) marking the ear tag B on 10 pigs of 30 days old and 10 pigs of 60 days old, and feeding the feed B. Separately culturing in two piggeries under the same conditions for 30 days.
The death and morbidity do not occur in the feeding period, the average body weight of the group A fed with the recombinant triple pig auxin feed is increased by 1.02 kg/day/pig, and the average body weight of the group B fed with the common feed is increased by 0.75 kg/day/pig. The daily average gain of group A was increased by 36% over group B as shown in Table 2. The recombined triple pig growth hormone has better growth promoting effect on pigs of different ages in days.
TABLE 2 growth Performance statistics Table (kg)
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Sequence listing
<110> Onhui Zhongzhi Biotech Co Ltd
<120> recombinant porcine growth hormone, preparation method and application thereof
<141> 2020-10-23
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tacatcccgg agggacagag gtactccatc cagaacgccc aggctgcctt ctgcttctcg 240
gagaccatcc cagcccccac gggcaaggac gaggcccagc agagatcgga cgtggagctg 300
ctgcgcttct cgctgctgct catccagtcg tggctcgggc ccgtgcagtt cctcagcagg 360
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ggcaaggacg aggcccagca gagatcggac gtggagctgc tgcgcttctc gctgctgctc 360
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cgcttcgtgg agagcagctg tgccttcggt ggtggtggag gaggaggtgg tggtggagga 720
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ctccgggccc agcacctgca ccaactggct gccgacacct acaaggagtt tgagcgcgcc 900
tacatcccgg agggacagag gtactccatc cagaacgccc aggctgcctt ctgcttctcg 960
gagaccatcc cagcccccac gggcaaggac gaggcccagc agagatcgga cgtggagctg 1020
ctgcgcttct cgctgctgct catccagtcg tggctcgggc ccgtgcagtt cctcagcagg 1080
gtcttcacca acagcctggt gtttggcacc tcagaccgcg tctacgagaa gctgaaggac 1140
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cagatcctca agcaaaccta cgacaaattt gacacaaact tgcgcagtga tgacgcgctg 1260
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tacaaggagt ttgagcgcgc ctacatcccg gagggacaga ggtactccat ccagaacgcc 1620
caggctgcct tctgcttctc ggagaccatc ccagccccca cgggcaagga cgaggcccag 1680
cagagatcgg acgtggagct gctgcgcttc tcgctgctgc tcatccagtc gtggctcggg 1740
cccgtgcagt tcctcagcag ggtcttcacc aacagcctgg tgtttggcac ctcagaccgc 1800
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gatggcagcc cccgggcagg acagatcctc aagcaaacct acgacaaatt tgacacaaac 1920
ttgcgcagtg atgacgcgct gcttaagaac tacgggctgc tctcctgctt caagaaggac 1980
ctgcacaagg ctgagacata cctgcgggtc atgaagtgtc gccgcttcgt ggagagcagc 2040
tgtgccttct agccggccgc 2060
Claims (6)
1. A polynucleotide, wherein: the pig growth hormone coding gene sequence comprises a guide sequence and three groups of pig growth hormone coding gene full-length sequences which are sequentially connected, wherein the three groups of pig growth hormone coding gene full-length sequences are connected through a flexible linker sequence, the pig growth hormone coding gene full-length sequence is shown as SEQ ID No.1, the guide sequence is shown as SEQ ID No.2, and the flexible linker sequence is shown as SEQ ID No. 3.
2. A polynucleotide according to claim 1, wherein: the sequence of the polynucleotide is shown in SEQ ID NO. 4.
3. A preparation method of recombinant porcine growth hormone is characterized by comprising the following steps:
(1) inserting the polynucleotide of claim 1 or 2 into a pichia pastoris expression vector to obtain a recombinant plasmid;
(2) introducing the recombinant plasmid into a pichia pastoris competent cell to construct and obtain recombinant engineering bacteria;
(3) and carrying out fermentation expression and protein purification on the recombinant engineering bacteria to obtain a target protein, namely the recombinant pig growth hormone.
4. A recombinant porcine growth hormone produced by the method of claim 3.
5. Use of the recombinant porcine growth hormone of claim 4 as a feed additive for pigs.
6. Use according to claim 5, characterized in that: the addition amount of the recombinant pig growth hormone in the pig feed is 0.33-3.3mg/100 g.
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