CN107253984B - Growth-promoting composite polypeptide and application thereof - Google Patents
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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
- C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
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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
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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Abstract
The invention belongs to the field of biological agents, and relates to a growth-promoting composite polypeptide for promoting growth and enhancing immunity and application thereof, wherein the production steps comprise: the growth promoting compound polypeptide comprises the following steps of 1) designing the growth promoting compound polypeptide, 2) constructing a recombinant expression vector, 3) constructing the expression of engineering bacteria, 4) identifying an expression product, and 5) roughly preparing a fermentation product.
Description
Technical Field
The invention belongs to the field of biological agents, and relates to a growth-promoting composite polypeptide with functions of promoting growth and enhancing immunity and application thereof.
Background
Growth Hormone (GH) is a species-specific single-chain polypeptide hormone synthesized and secreted by the animal's own pituitary, is widely present in vertebrates, and promotes the growth and development of the body by promoting protein synthesis, accelerating fat metabolism, indirectly stimulating bone growth, and the like. GH secretion is co-regulated by the interaction of other growth factors in an autocrine or paracrine fashion. Among these growth factors, GH-releasing hormone (GHRH), GH-release inhibiting hormone (GHRIH) and growth hormone releasing peptide (Ghrelin) play a major regulatory role, wherein Ghrelin and GHRH act synergistically to promote the activity of the hypothalamic-associated region and are capable of stimulating the body's release of growth hormone.
The discovery of Ghrelin is a reverse process. The first chemical synthesis reported was ghrelin-6 (GHRPs-6) synthesized by Bowers et al in 1982. In 1993, Smith et al synthesized Growth Hormone Secretagogues (GHSs) on the basis of GHRPs-6. With the development of biotechnology, Howard et al cloned GHS-R (growth hormone secretagogues) cDNA in 1996, and proved that GHS-R is highly conserved in human, pig, cow and rat. In 1999 Kojima et al obtained the highest activity of somatotropin from stomach tissue and purified a polypeptide consisting of 28 amino acid residues with the same activity, named Ghrelin (ghre is the original European language and is the meaning of growth), by examining various extracts of internal organs of rats. The research finds that the Ghrelin precursor protein of human and rat has 117 amino acid residues in total, and the front 23 peptide at the N terminal is a secretion signal peptide; the minimum active center of Ghrelin consists of the first 4 amino acid segments at the N-terminal, and the P-R (proline-arginine) structure at the C-terminal is the recognition site. The Ghrelin has an endogenous active site and is the third amino acid residue, and since the carboxyl group of the amino acid is octanoyl esterified, the initial research shows that octanoyl esterification is the essential structure of the Ghrelin related activity, but is not the only one, and other types of acyl esterification also have corresponding functions. Human and rat Ghrelin differ in amino acids 11 and 12 and have 89% homology. With the progress of research, it has been reported that the amount of non-octanoyl esterified Ghrelin in human blood is much larger than that of octanoyl esterified Ghrelin, and that non-octanoyl esterified Ghrelin also has endocrine function and can promote cell proliferation.
In the GHRPs-6 amino acid sequence, the second amino acid Trp and the fifth amino acid Phe are D-type amino acids, namely D-Trp and D-Phe, and have biological activity. The synthetic peptides similar to GHRPs-6 are also used today as hexapeptide growth hormone releasing peptide-2 (GHRPs-2), heptapeptide growth hormone releasing peptide-1 (GHRPs-1) and D-2-methyl Trp substituted HEXA, which is a derivative of D-Trp. The four GHRPs amino acid sequences are known as follows:
GHRPs-1:Ala-His-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2
GHRPs-2:D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2
GHRPs-6:His-D-Trp-Ala-Trp-D-Phe-Lys-NH2
HEXA:His-D-2-methyl Trp-Ala-Trp-D-Phe-Lys-NH2
smith et al have suggested that the aromatic amino acids such as Trp, D-Phe and terminal Ala, His in such peptides are important components of peptide activity. Walker et al, by comparing the effects on Growth Hormone (GH) release activity in monkeys, dogs and rats by parenteral administration of GHRPs-6po and intraduodenal administration, indicate that entry of GHRPs-6 into the intestinal tract facilitates GH release, i.e., GHRPs-6 is expected to be used as a drug in place of parenteral administration. Comparing these four peptides, all were found to have the structure Ala-Trp-D-Phe-Lys-NH 2. A novel polypeptide with growth promoting effect is obtained by integrating synthetic peptide consensus (AWFK) into the Ghrelin sequence and substituting amino acids from 10 th to 13 th of the Ghrelin sequence.
Saccharomyces cerevisiae (Saccharomyces cerevisiae), a unicellular eukaryotic organism, has long been used in the food industry, and the earliest applications dates back to thousands of years in the wine and bread industries, and physiological substances rich in proteins, fats, carbohydrates, vitamins and other metabolites are recognized as GRAS organisms (Generally recognized class). Because the yeast does not produce toxin in the fermentation process, the yeast expression system is safe and reliable and is the first yeast expression system to be used. The saccharomyces cerevisiae expresses the foreign protein and has the advantages that: (1) the thallus has the advantages of fast reproduction, short growth period, simple process, common culture conditions and low production cost. (2) The genetic background is clear, and the whole genome sequence sequencing was completed as early as 1996. (3) The expressed protein is modified to make the recombinant protein structure approach to the natural conformation and keep the corresponding function.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a growth-promoting composite polypeptide and application thereof.
The invention is realized by the following steps: a growth-promoting complex polypeptide, comprising: comprising Ghrelin and GHRPs polypeptide sequences.
The amino acid sequence of the growth-promoting composite polypeptide is shown as SQE ID NO. 1.
A preparation method of growth-promoting composite polypeptide is characterized in that: the method comprises the following steps:
step 1), design of growth-promoting composite polypeptide
The primary structure of Ghrelin is: GSSFLSPEHQRVQQRKESKKPPAKLQPR, GHRPs the common part of the primary structure is: AWFK; replacing the GHRPs consensus amino acid sequence with the 10 th to 13 th amino acid sequences of the Ghrelin sequence;
step 2), construction of recombinant expression vector
1) Primer design
According to the polypeptide sequence, obtaining a nucleotide sequence by referring to preferred codons of saccharomyces cerevisiae, designing a primer by using NCBI primer-Blast, and obtaining the primer sequence as follows:
f: CGCGCATCCGGTTCTTCTTTCTT GCATCC marker BamHI enzyme cutting site
R: TGCAGATCATCTTGGTTGCAACT AGATCA is marked as the restriction site XbaI
M1-F:CTTCTTTCTTGTCTCCAGAACACGCTTGGTT
M1-R:TTAGATTCCTTTCTTTGCTTGAACCAAGCGT
M2-F:GAAAGGAATCTAAGAAGCCACCAGCTAAGT
M2-R:TTGCAACTTAGCTGGTGGCTTCTTAGATTC
2) PCR reaction
According to the designed primer, carrying out PCR amplification for 4 times in an experiment to obtain a target gene; the specific reaction conditions are as follows:
m1 gene fragment amplification
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, M1-F1 muL and M1-R1 muL, and sterile water is added to reach the reaction volume of 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 65 ℃, 45s at 72 ℃, 35 cycles, 10min at 72 ℃ and 4 ℃ storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of a reaction product, and recording a target gene as M1;
m2 gene fragment amplification
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, M2-F1 muL and M2-R1 muL, and sterile water is added to reach the reaction volume of 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 66 ℃, 45s at 72 ℃, 35 cycles, 10min at 72 ℃ and 4 ℃ storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of a reaction product, and recording a target gene as M2;
g Gene fragment amplification
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, 11 muL of M, 21 muL of M, and sterile water is supplemented until the reaction volume is 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 65 ℃, 45s at 72 ℃, 10 cycles, and finally 10min at 72 ℃ and 4 ℃ for storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of the reaction product, and recording the target gene as G;
amplification of GM Gene fragment
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, 1 muL of F and 1 muL of R, and sterile water is added to reach the reaction volume of 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 65 ℃, 45s at 72 ℃, 30 cycles, and finally 10min at 72 ℃ and 4 ℃ for storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of a reaction product, and marking the target gene as GM;
connecting GM gene obtained by PCR amplification with a PMD19-T vector, transferring the GM gene into DH5 alpha competent cells for blue-white screening and monoclonal amplification; at the same time, pYES2 vector plasmid is transferred into DH5 alpha competent cells to carry out monoclonal amplification on an ampicillin culture medium;
3) construction of expression vectors
Extracting PMD19-T plasmid and pYES2 vector plasmid; simultaneously, carrying out double digestion for 5h by using BamH I and XbaI respectively at the temperature of 37 ℃; after agarose gel electrophoresis, the gel recovery kit recovers the target gene GM 'and pYES 2' carriers after enzyme digestion;
the two DNA fragments GM 'and pYES 2' after enzyme digestion are carried out for 12 hours at 37 ℃ under the action of T4 ligase; subjecting the ligation product to agarose gel electrophoresis to obtain a band of about 5.9kb, which is denoted as pYES 2/GM'; transferring pYES 2/GM' into DH5 alpha, screening the thallus successfully transferred by the expression vector on an ampicillin culture medium, and carrying out monoclonal amplification; extracting plasmids, sequencing, comparing a sequencing result with a correct base sequence, and marking the plasmids with consistent sequences as pYES 2/GM;
step 3), expression construction of engineering bacteria
Extracting pYES2/GM plasmid from the plasmid extraction kit, converting the plasmid into competent saccharomyces cerevisiae by an electrotransformation method, coating an SD solid culture medium for culturing for 72h, selecting a monoclonal colony, culturing for 72h by using a YEPD liquid culture medium, and inoculating the colony to a YEPD fresh liquid culture medium according to the proportion of 1% for amplification culture;
step 4), identification of expression product
After the fermentation of the engineering bacteria is finished, centrifugally separating thalli and supernate; diluting thalli for inducing and expressing the growth promoting composite polypeptide and original thalli induced under the same condition to the same number of thalli under the same volume, crushing under the same condition, centrifuging, and detecting by HPLC to determine the expression condition of the composite protein;
step 5) crude preparation of fermentation product
After fermentation, standing the culture medium, allowing the culture to stand for precipitation, and filtering to obtain thalli containing the compound protein; drying to obtain polypeptide bacterial powder mixed with growth promoting composite polypeptide and yeast live bacteria.
Can be added into feed or drinking water as feed additive.
The invention has the following advantages:
1. the growth-promoting peptide is recombined in yeast cells and expresses active growth-promoting composite polypeptide, and the growth-promoting composite polypeptide can increase the resistance of livestock and poultry, accelerate the growth of the livestock and poultry and improve the utilization rate of feed;
2. the growth-promoting composite polypeptide expression strain saccharomyces cerevisiae can be directly used as a feed additive, and has the effects of regulating the balance of gastrointestinal flora, supplementing mycoprotein, improving the immunity of livestock and poultry and the like;
3. the growth-promoting composite polypeptide which is purified or not purified can be directly prepared into the feed additive, the production process is simplified, the production period is shortened, and the production cost is reduced.
Drawings
FIG. 1 is a schematic representation of the attachment process of a recombinant growth-promoting complex polypeptide carrier.
Detailed Description
Example 1: a preparation method of growth-promoting composite polypeptide is characterized in that: the method comprises the following steps:
step 1), design of growth-promoting composite polypeptide
The primary structure of Ghrelin is: GSSFLSPEHQRVQQRKESKKPPAKLQPR, GHRPs the common part of the primary structure is: AWFK; replacing the GHRPs consensus amino acid sequence with the 10 th to 13 th amino acid sequences of the Ghrelin sequence; forming a composite amino acid polypeptide chain with the active sites of Ghrelin and GHRPs. The primary structure is as follows: GSSFLSPEHAWFKQRKESKKPPAKLQPR, isoelectric point 5.46, molecular weight 6815 Da;
step 2), construction of recombinant expression vector
1) Primer design
According to the polypeptide sequence, obtaining a nucleotide sequence by referring to preferred codons of saccharomyces cerevisiae, designing a primer by using NCBI primer-Blast, and obtaining the primer sequence as follows:
f: CGCGCATCCGGTTCTTCTTTCTT GCATCC marker BamHI enzyme cutting site
R: TGCAGATCATCTTGGTTGCAACT AGATCA is marked as the restriction site XbaI
M1-F:CTTCTTTCTTGTCTCCAGAACACGCTTGGTT
M1-R:TTAGATTCCTTTCTTTGCTTGAACCAAGCGT
M2-F:GAAAGGAATCTAAGAAGCCACCAGCTAAGT
M2-R:TTGCAACTTAGCTGGTGGCTTCTTAGATTC
2) PCR reaction
According to the designed primer, carrying out PCR amplification for 4 times in an experiment to obtain a target gene; the specific reaction conditions are as follows:
m1 gene fragment amplification
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, M1-F1 muL and M1-R1 muL, and sterile water is added to reach the reaction volume of 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 65 ℃, 45s at 72 ℃, 35 cycles, 10min at 72 ℃ and 4 ℃ storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of a reaction product, and recording a target gene as M1;
m2 gene fragment amplification
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, M2-F1 muL and M2-R1 muL, and sterile water is added to reach the reaction volume of 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 66 ℃, 45s at 72 ℃, 35 cycles, 10min at 72 ℃ and 4 ℃ storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of a reaction product, and recording a target gene as M2;
g Gene fragment amplification
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, 11 muL of M, 21 muL of M, and sterile water is supplemented until the reaction volume is 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 65 ℃, 45s at 72 ℃, 10 cycles, and finally 10min at 72 ℃ and 4 ℃ for storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of the reaction product, and recording the target gene as G;
amplification of GM Gene fragment
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, 1 muL of F and 1 muL of R, and sterile water is added to reach the reaction volume of 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 65 ℃, 45s at 72 ℃, 30 cycles, and finally 10min at 72 ℃ and 4 ℃ for storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of a reaction product, and marking the target gene as GM;
connecting GM gene obtained by PCR amplification with a PMD19-T vector, transferring the GM gene into DH5 alpha competent cells for blue-white screening and monoclonal amplification; at the same time, pYES2 vector plasmid is transferred into DH5 alpha competent cells to carry out monoclonal amplification on an ampicillin culture medium;
3) construction of expression vectors
Extracting PMD19-T plasmid and pYES2 vector plasmid; simultaneously, carrying out double digestion for 5h by using BamH I and XbaI respectively at the temperature of 37 ℃; after agarose gel electrophoresis, the gel recovery kit recovers the target gene GM 'and pYES 2' carriers after enzyme digestion;
the two DNA fragments GM 'and pYES 2' after enzyme digestion are carried out for 12 hours at 37 ℃ under the action of T4 ligase; subjecting the ligation product to agarose gel electrophoresis to obtain a band of about 5.9kb, which is denoted as pYES 2/GM'; transferring pYES 2/GM' into DH5 alpha, screening the thallus successfully transferred by the expression vector on an ampicillin culture medium, and carrying out monoclonal amplification; extracting plasmids, sequencing, comparing a sequencing result with a correct base sequence, and marking the plasmids with consistent sequences as pYES 2/GM;
step 3), expression construction of engineering bacteria
Extracting pYES2/GM plasmid from the plasmid extraction kit, converting the plasmid into competent saccharomyces cerevisiae by an electrotransformation method, coating an SD solid culture medium for culturing for 72h, selecting a monoclonal colony, culturing for 72h by using a YEPD liquid culture medium, and inoculating the colony to a YEPD fresh liquid culture medium according to the proportion of 1% for amplification culture;
step 4), identification of expression product
After the fermentation of the engineering bacteria is finished, centrifugally separating thalli and supernate; diluting thalli for inducing and expressing the growth promoting composite polypeptide and original thalli induced under the same condition to the same number of thalli under the same volume, crushing under the same condition, centrifuging, and detecting by HPLC to determine the expression condition of the composite protein; detecting to obtain that the maximum expression quantity of fermentation culture of the fermentation tank reaches 1.3 g/L;
step 5) crude preparation of fermentation product
After fermentation, standing the culture medium, allowing the culture to stand for precipitation, and filtering to obtain thalli containing the compound protein; drying to obtain polypeptide bacterial powder mixed with growth promoting composite polypeptide and yeast live bacteria.
Example 2: experiment of growth promoting composite polypeptide effect
1) Test for growth promoting effect of nursery pig
A healthy ternary hybrid pig with the weight of 40 heads and 30 kg is selected and randomly divided into an experimental group and a control group according to the principle of half each male pig and half each female pig. The control group was fed basal diet without any additives; the test group is fed with basic daily ration, and simultaneously, the growth-promoting composite polypeptide bacteria powder daily ration is added, and the addition amount of the growth-promoting composite polypeptide bacteria powder daily ration is 0.5 per mill of the total amount of the basic daily ration. And during the comparative feeding test, the free feeding and free drinking of the experimental pigs are ensured, and the feeding management is carried out according to the conventional method of a pig farm. The test is carried out for 7 d in a pre-test period, the adaptation condition of the test pig to the test condition is observed, and a 42d formal test period is carried out after the adaptation is determined. Feeding is stopped for 12 hours before the test is started and after the test is finished, and the test pigs are weighed on an empty stomach.
TABLE 1 basic diet formula
| Composition (I) | Content (mass fraction) |
| Corn (corn) | 60 % |
| Bean cake | 25 % |
| Fish meal | 1 % |
| Bran | 12. 5 % |
| Shell powder | 0. 5 % |
| Bone meal | 0. 7 % |
| Salt | 0. 3 % |
TABLE 2 Effect of growth promoting Complex Polypeptides on growth Performance of nursery pigs
| Group of | Daily gain (g/head) | Daily consumption material (g/head) | Meat ratio of materials |
| Control group | 424.80±21.0 | 1250±120 | 2.95±0.13 |
| Experimental group | 453.28±29.6 | 1230±110 | 2.72±0.14 |
The results show that: the growth-promoting composite polypeptide can effectively improve the growth performance of nursery pigs. In the experimental period, the average daily gain of the experimental group added with the growth-promoting composite polypeptide is obviously higher than that of the control group, and the difference of the two groups is obvious (p<0.05). The average daily material consumption of the experimental group and the control group are relatively close and have no significant difference (1)p>0.05); the feed meat is reduced by 7.8 percent compared with the experimental group and the control group, and the difference between the two groups is obvious (p<0.05). Test results show that the growth-promoting composite polypeptide feed can improve the feed conversion rate, increase the daily gain and remarkably promote the growth of nursery pigs under the condition of not changing the feed intake of the pigs.
Sequence listing
<110> Henan jin Volkswagen bioengineering Co., Ltd
<120> growth promoting complex polypeptide and application thereof
<160>1
<170>PatentIn version 3.5
<210>1
<211>28
<212>PRT
<213> Artificial sequence
<400>1
Gly Ser Ser Phe Leu Ser Pro Glu His Ala Trp Phe Lys Gln Arg Lys
1 5 10 15
Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg
20 25
Claims (3)
1. A growth-promoting complex polypeptide, comprising: the amino acid sequence of the polypeptide is shown as SQE ID NO. 1.
2. A preparation method of growth-promoting composite polypeptide is characterized in that: the method comprises the following steps:
step 1), design of growth-promoting composite polypeptide
The primary structure of Ghrelin is: GSSFLSPEHQRVQQRKESKKPPAKLQPR, GHRPs the common part of the primary structure is: AWFK; replacing the GHRPs consensus amino acid sequence with the 10 th to 13 th amino acid sequences of the Ghrelin sequence;
step 2), construction of recombinant expression vector
1) Primer design
According to the polypeptide sequence, obtaining a nucleotide sequence by referring to preferred codons of saccharomyces cerevisiae, designing a primer by using NCBI primer-Blast, and obtaining the primer sequence as follows:
f: CGCGCATCCGGTTCTTCTTTCTT GCATCC marker BamHI enzyme cutting site
R: TGCAGATCATCTTGGTTGCAACT AGATCA is marked as the restriction site XbaI
M1-F:CTTCTTTCTTGTCTCCAGAACACGCTTGGTT
M1-R:TTAGATTCCTTTCTTTGCTTGAACCAAGCGT
M2-F:GAAAGGAATCTAAGAAGCCACCAGCTAAGT
M2-R:TTGCAACTTAGCTGGTGGCTTCTTAGATTC
2) PCR reaction
According to the designed primer, carrying out PCR amplification for 4 times in an experiment to obtain a target gene; the specific reaction conditions are as follows:
m1 gene fragment amplification
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, 1 muL of M1-F and 1 muL of M1-R, and sterile water is added to reach the reaction volume of 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 65 ℃, 45s at 72 ℃, 35 cycles, 10min at 72 ℃ and 4 ℃ storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of a reaction product, and recording a target gene as M1;
m2 gene fragment amplification
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, 1 muL of M2-F and 1 muL of M2-R, and sterile water is added to reach the reaction volume of 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 66 ℃, 45s at 72 ℃, 35 cycles, 10min at 72 ℃ and 4 ℃ storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of a reaction product, and recording a target gene as M2;
g Gene fragment amplification
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, 11 muL of M, 21 muL of M, and sterile water is supplemented until the reaction volume is 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 65 ℃, 45s at 72 ℃, 10 cycles, and finally 10min at 72 ℃ and 4 ℃ for storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of the reaction product, and recording the target gene as G;
amplification of GM Gene fragment
The PCR reaction system was mixed by ice: 5 muL of 10 XPCR buffer, 4 muL of dNTP, 0.25 muL of Pfu enzyme, 1 muL of F and 1 muL of R, and sterile water is added to reach the reaction volume of 50 muL; putting the mixture into a preheated PCR amplification instrument, and carrying out amplification according to the following procedures: 5min at 94 ℃, 45s at 94 ℃, 30s at 65 ℃, 45s at 72 ℃, 30 cycles, and finally 10min at 72 ℃ and 4 ℃ for storage; carrying out agarose gel electrophoresis on the PCR product, recovering agarose gel of a reaction product, and marking the target gene as GM;
connecting GM gene obtained by PCR amplification with a PMD19-T vector, transferring the GM gene into DH5 alpha competent cells for blue-white screening and monoclonal amplification; at the same time, pYES2 vector plasmid is transferred into DH5 alpha competent cells to carry out monoclonal amplification on an ampicillin culture medium;
3) construction of expression vectors
Extracting PMD19-T plasmid and pYES2 vector plasmid; simultaneously, carrying out double digestion for 5h by using BamH I and XbaI respectively at the temperature of 37 ℃; after agarose gel electrophoresis, the gel recovery kit recovers the target gene GM 'and pYES 2' carriers after enzyme digestion;
the two DNA fragments GM 'and pYES 2' after enzyme digestion are carried out for 12 hours at 37 ℃ under the action of T4 ligase; subjecting the ligation product to agarose gel electrophoresis to obtain a band of 5.9kb, which is denoted as pYES 2/GM'; transferring pYES 2/GM' into DH5 alpha, screening the thallus successfully transferred by the expression vector on an ampicillin culture medium, and carrying out monoclonal amplification; extracting plasmids, sequencing, comparing a sequencing result with a correct base sequence, and marking the plasmids with consistent sequences as pYES 2/GM;
step 3), expression construction of engineering bacteria
Extracting pYES2/GM plasmid from the plasmid extraction kit, converting the plasmid into competent saccharomyces cerevisiae by an electrotransformation method, coating an SD solid culture medium for culturing for 72h, selecting a monoclonal colony, culturing for 72h by using a YEPD liquid culture medium, and inoculating the colony to a YEPD fresh liquid culture medium according to the proportion of 1% for amplification culture;
step 4), identification of expression product
After the fermentation of the engineering bacteria is finished, centrifugally separating thalli and supernate; diluting thalli for inducing and expressing the growth promoting composite polypeptide and original thalli induced under the same condition to the same number of thalli under the same volume, crushing under the same condition, centrifuging, and detecting by HPLC to determine the expression condition of the composite protein;
step 5) crude preparation of fermentation product
After fermentation, standing the culture medium, allowing the culture to stand for precipitation, and filtering to obtain thalli containing the compound protein; drying to obtain polypeptide bacterial powder mixed with growth promoting composite polypeptide and yeast live bacteria.
3. Use of a growth-promoting complex polypeptide according to claim 1 for the preparation of a feed supplement.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2489008A1 (en) * | 2002-07-19 | 2004-01-29 | Cytos Biotechnology Ag | Ghrelin-carrier conjugates |
| CN1634985A (en) * | 2003-12-26 | 2005-07-06 | 李宁 | Pig Ghrelin derivative, its encoding gene and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2489008A1 (en) * | 2002-07-19 | 2004-01-29 | Cytos Biotechnology Ag | Ghrelin-carrier conjugates |
| CN1634985A (en) * | 2003-12-26 | 2005-07-06 | 李宁 | Pig Ghrelin derivative, its encoding gene and application |
Non-Patent Citations (2)
| Title |
|---|
| A Nonpeptidyl Growth Hormone Secretagogue;Roy G.Smith 等;《SCIENCE》;19930611;第260卷;全文 * |
| Ghrelin基因多态性及其临床意义;项莹 等;《国际遗传学杂志》;20081215;第31卷(第6期);全文 * |
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