CN113845584A - Preparation method of recombinant avian epidermal growth factor - Google Patents
Preparation method of recombinant avian epidermal growth factor Download PDFInfo
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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
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
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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
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
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Abstract
The invention belongs to the field of biological pharmacy, and particularly relates to a preparation method of a recombinant avian Epidermal Growth Factor (EGF). The EGF (cEGF) gene sequence information of an avian is determined by utilizing a bioinformatics approach, the preparation method expresses in a lactobacillus expression system by virtue of modified plasmids, no chemical reagent is added for induction, the expression efficiency is high, the antibiotic resistance level is low, and the problem of antibiotic resistance is effectively prevented.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a preparation method of a recombinant avian epidermal growth factor.
Background
Epidermal Growth Factor (EGF) belongs to polypeptide, can be separated from body fluid secreted and produced by salivary gland, kidney, mammary gland, placenta and duodenum Brunner gland, and is also closely related to intestinal structure and function of animals. The intestinal tract is one of tissues with faster cell renewal speed in vivo, and EGF is a regulatory factor and a trophic factor of intestinal homeostasis, has the capacity of promoting the proliferation and differentiation of gastrointestinal epithelial cells and has an effect on intestinal cell renewal. EGF has multiple biological functions, and exogenous supply of EGF can improve the activity of digestive enzyme in intestinal tracts, inhibit the colonization of harmful bacteria in the intestinal tracts and improve the capacity of intestinal cells to absorb nutrients, thereby improving the utilization rate of feed and the digestion and absorption of the nutrients. Feeding weaned young rabbits infected with enteropathogenic escherichia coli with EGF can find a decrease in colonization numbers of pathogenic bacteria in various sections of small intestine and colon, suggesting that EGF protects the intestine by effectively alleviating colonization of pathogenic bacteria. EGF has been shown to accelerate epithelial cell recovery and improve gastrointestinal disorders such as necrotic enteritis, diarrhea and short bowel syndrome. EGF for human use is listed in the list of cosmetic additives in Japan at present, and is not widely applied in livestock and poultry though being researched.
Currently, there are two major ways for EGF production, one is isolation and extraction from tissue or body fluid components (201711285671.7), and the other is the use of genetic recombination techniques to obtain recombinant EGF protein. The latter is widely developed and applied due to mature technology, low cost, simple process and easy large-scale production. The related patents also mainly focus on human EGF and porcine EGF recombinant technology, and other animal aspects have no relevant records for a while. At present, pig EGF (pEGF) is mainly obtained in two ways, one way is separation from sow milk, the process is complex and the quantity is quite limited, the other way is obtained by expression of engineering bacteria, and the engineering bacteria which adopt biotechnology to express exogenous pEGF at present comprise escherichia coli (single parsley and the like, 202010611379.5), bacillus (Wangxiang and the like, 201910391304.8), lactic acid bacteria (Belleville and the like, 2015; Liushujie and the like, 2021), pichia pastoris and the like (Liujie and the like, 202011586279.8) and the like.
In the existing preparation method of EGF, the following two problems mainly exist: at present, EGF gene sequence information is only clear for animals such as human, pig, dog and the like, and sequence information of other animals is not recorded clearly and related research is not recorded; secondly, the existing means for recombinant expression have obvious short plates, and if the use amount of antibiotics is large, the hidden danger of drug-resistant gene diffusion exists; inducer toxicity; the complicated separation and purification operation leads to an increase in cost. Therefore, there is an urgent need to develop EGF sequence information of other animals and improve methods for preparing EGF, so as to reduce the production cost of EGF and the hidden trouble in subsequent applications.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of a recombinant poultry epidermal growth factor, which expresses in a lactobacillus expression system by means of modified plasmids, does not need to add chemical reagents for induction, has high expression efficiency and low antibiotic resistance level, effectively prevents the problem of antibiotic resistance, and solves the defects in the prior art. In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a recombinant avian epidermal growth factor (rcEGF) having a nucleotide sequence as set forth in SEQ ID No. 4.
In a second aspect of the present invention, there is provided a recombinant vector for expressing a recombinant avian epidermal growth factor, wherein the recombinant vector comprises a nucleotide sequence of a gene fragment of a signal peptide, an anchor protein, an ampicillin resistance sequence, and a cief sequence, and is a recombinant lactobacillus expression vector pLL32 a-cief.
In one embodiment, the nucleotide sequences of the gene segments of the signal peptide, the dockerin, the ampicillin resistance sequence, and the ciegf sequence are set forth in SEQ ID NO: 1-4.
In a third aspect of the present invention, there is provided a method for constructing the above recombinant vector, wherein the method comprises the following steps:
(1) carrying out double enzyme digestion on an artificially synthesized ampicillin resistance gene sequence and a pMG36e plasmid, carrying out agarose gel electrophoresis separation on enzyme digestion products, cutting gel, recovering a target band and a carrier band, connecting the target band and the carrier band by using T4 ligase, transforming the target band and the carrier band into MC1061 competent cells, and screening by using ampicillin to obtain a new plasmid pLL32 a;
(2) carrying out double enzyme digestion on an artificially synthesized gene fragment containing a signal peptide, an anchor protein and a cEGF sequence and a pLL32a plasmid, carrying out agarose gel electrophoresis separation on the enzyme digestion product, cutting gel to recover a target band and a carrier band, connecting the target band and the carrier band by using T4 ligase, transforming the target band and the carrier band into MC1061 competent cells, and screening by using ampicillin to obtain a recombinant plasmid pLL32 a-cEGF.
The fourth aspect of the invention provides a preparation method of recombinant avian epidermal growth factor, wherein the method is to express the recombinant vector constructed above in lactic acid bacteria to obtain recombinant EGF.
In one embodiment, the lactic acid bacteria are lactococcus lactis. In a preferable mode, the lactic acid bacteria is lactococcus lactis MG 1363.
In one embodiment, the method comprises the steps of:
(1) the constructed recombinant vector is electrically transformed into a lactic acid bacteria competent cell, after the electrical transformation is finished, the lactic acid galactococcus with the recombinant plasmid is transferred into a recovery culture medium for static culture at 37 ℃ for 2 hours, then an MRS plate with 0.5ug/ml ampicillin resistance is coated, and the constant temperature culture at 37 ℃;
(2) selecting a single colony growing on the plate, carrying out pure culture, extracting plasmids, and carrying out sequencing identification on the plasmids to obtain recombinant bacteria;
(3) and (3) carrying out shake culture on the recombinant bacteria in an MRS liquid culture medium containing 0.5ug/ml of ampicillin at 37 ℃ for 4h, then reducing the culture temperature to 30 ℃ for continuous culture, centrifuging the fermentation liquor after terminating the fermentation, adding a proper amount of lysozyme for cracking the bacteria after resuspending the bacteria, and obtaining the recombinant avian epidermal growth factor.
Preferably, the electrotransformation conditions are: 12kV/cm, 200 omega and 25 muF.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the invention fills up the technical blank in the research and application aspects of the poultry recombinant epidermal growth factor;
2. compared with other expression systems, the recombinant avian epidermal growth factor expressed by the modified lactobacillus system does not need to be induced by adding a chemical reagent, has high expression efficiency and low antibiotic resistance level, and effectively prevents the problem of antibiotic resistance.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1: prediction of transmembrane structure;
FIG. 2: predicting a secondary structure;
FIG. 3: predicting subcellular localization;
FIG. 4: performing double enzyme digestion identification on cEGF plasmid;
FIG. 5: carrying out double enzyme digestion identification on pLL32a plasmid;
FIG. 6: the detection result of the recombinant bacterium western blot is as follows: lane 1 is lactococcus lactis 1363-empty bacteria supernatant; lane 2 is lactococcus lactis 1363-empty bacteria precipitation; lane 3 is recombinant lactococcus lactis 1363-supernatant; lane 4 is recombinant lactococcus lactis 1363-precipitate; lane 5 is recombinant lactococcus lactis 1363-inactivated bacteria.
Detailed Description
The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.
Example 1: determination of avian EGF Gene sequences
The literature states that the mature EGF protein consists of about 53 amino acids, 159 nucleotides. At present, EGF sequences of only a few species such as human, pig, dog, mouse, horse and the like are clear. Although the related sequence of the avian EGF can be found in the GENBANK database, the sequence described is only the precursor nucleotide sequence of the avian EGF and not the nucleotide sequence corresponding to the mature EGF protein. To determine the nucleotide sequence corresponding to the avian mature EGF protein, we found that their key structural residues showed high degree of conservation compared to the mature EGF nucleotide sequences of human, porcine, murine, equine, etc., especially three glycine residues (residues 18, 36 and 39), six cysteine residues ( residues 6, 14, 20, 31, 33 and 42) and tyrosine residue (residue 37). Finally, we confirmed the nucleotide sequence and amino acid sequence of mature avian EGF (cEGF) based on the highly conserved nature of the key structural residues of the mature EGF protein, and performed transmembrane structure, secondary structure and subcellular localization prediction by TMHMM, SOPMA and PSORT online software (FIGS. 1-3) for subsequent experiments.
Example 2: construction of recombinant vectors and expression of proteins of interest
1. Carrying out double enzyme digestion (mlu I and Xho I) on an artificially synthesized ampicillin resistance gene sequence (shown as SEQ ID NO: 3) and pMG36e (vast Ling organism, MLCC1262) plasmid, carrying out agarose gel electrophoresis separation on enzyme digestion products, cutting gel, recovering a target band and a carrier band, connecting the bands by using T4 ligase, transforming the bands into MC1061 competent cells, and screening by using 100ug/ml ampicillin to obtain a new plasmid pLL32a (the sequence is shown as SEQ ID NO: 5);
2. carrying out double enzyme digestion (Xba I and Hind III) (shown in a figure 4-5) on an artificially synthesized gene fragment containing a signal peptide (shown in a figure SEQ ID NO: 1), an anchor protein (shown in a figure SEQ ID NO: 2) and a cEGF sequence (shown in a figure SEQ ID NO: 4) and a pLL32a plasmid, carrying out agarose gel electrophoresis separation on enzyme digestion products, cutting gel, recovering a target band and a carrier band, connecting the bands by using T4 ligase, transforming the bands into MC1061 competent cells, and screening by using 100ug/ml ampicillin to obtain a recombinant plasmid pLL32 a-cEGF;
3. the recombinant plasmid pLL32a-cEGF was electrotransformed into L.lactis MG1363 competent cells under the following conditions: 12kV/cm, 200 omega and 25 muF. After the electrotransformation is finished, the lactococcus lactis with the recombinant plasmid is transferred into a resuscitation medium and is statically cultured for 2 hours at 37 ℃, then an MRS plate with 0.5ug/ml ampicillin resistance is coated, and the culture is carried out at constant temperature of 37 ℃. The resuscitation medium comprises the following components: 0.5mol glucose + MRS liquid medium +2mmol calcium chloride +20mmol magnesium chloride.
4. And (3) selecting a single colony growing on the plate, carrying out pure culture, extracting plasmids, and sending the plasmids to Shanghai workers for sequencing identification to obtain the recombinant bacteria.
5. The recombinant strain is placed in MRS liquid culture medium containing 0.5ug/ml ampicillin and cultured for 4h under shaking at 37 ℃, and then the culture temperature is reduced to 30 ℃ for continuous overnight culture. After overnight culture, 2 tubes of 1ml fermentation liquor are respectively taken, after thallus precipitation is obtained by centrifugation, 100ul PBS is added into one tube to be suspended and placed in a 65 ℃ water bath kettle for inactivation treatment for 1 hour, and then a sample is prepared for standby; the other tube was used to resuspend the cells with 50ul PBS and add an appropriate amount of lysozyme to lyse the cells, after centrifugation, the supernatant and the pellet were taken separately and prepared for use. And (3) carrying out SDS-PAGE electrophoresis on the three samples and the blank lactococcus lactis, then carrying out membrane transfer, and carrying out Western blot verification by using a His tag antibody.
As a result, as shown in FIG. 6, the recombinant bacterium successfully expressed recombinant cEGF (i.e., rcEGF), and the recombinant protein was mainly concentrated on the surface portion of the cell body.
Example 3: activity verification of recombinant proteins
Recombinant bacteria expressing recombinant EGF in the example 2 are prepared into bacterial powder to be fed to experiments of feeding 817 white feather broilers in cage culture. The feeding is divided into an experimental group and a control group according to the feeding batch. In the experiment, 3 barns with the same feeding conditions were selected as an experimental group and a control group (see the following table). The experimental group is added with the EGF in the daily feed of the broiler chicken, and the control group is fed with the conventional feed under the same drinking water and other feeding conditions.
The experimental groups were as follows:
the results are tabulated below:
as can be seen from the table, it is,the average daily gain of the experimental group using EGF was 2g more than that of the control group per chicken. Go out The weight of the experimental group is 90g more than that of the control group. The market rates of the experimental groups were compared with those of the control group The marketing rate is higher than that of a control group by 2 percent, which indicates that the chicken flocks of the experimental group are uniform, regular, healthy and strong, and the defective chicken are few. Comparing with control group The average material weight ratio of the experimental group is 0.1, and EuropeComparison of the index group 152 with the control group 142 for a difference of 10, indicating EGF Has obvious functions of promoting growth, improving survival rate, reducing marketing time and other comprehensive indexes.
Experiments prove that the total-price granulated feed added with EGF in the 817 white feather broiler whole-course feeding process can effectively improve the slaughter weight of 817 white feather broiler breeding, reduce the feed-weight ratio, improve the marketing rate by 2 percent, effectively improve the European index of 817 white feather broilers of hybrid varieties, and improve the economic benefit.
Example 4: research on antibiotic resistance level of recombinant bacteria
MRS plates were prepared at ampicillin concentrations of 0.5, 1, 3, 5, 7, and 10ug/ml, and the recombinant bacteria of example 2 were plated on the prepared MRS plates and cultured overnight at 37 ℃.
The result shows that the recombinant bacteria can not grow on an MRS plate with the concentration of ampicillin more than 5ug/ml, which indicates that the recombinant bacteria prepared by the invention has extremely low antibiotic resistance.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> Jiangsu Sanjie bioengineering, Inc.; dalian Sanjie animal drugs Co Ltd
<120> preparation method of recombinant avian epidermal growth factor
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<141> 2021-11-08
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Claims (9)
1. A recombinant avian epidermal growth factor (rcEGF), wherein the nucleotide sequence of rcEGF is shown in SEQ ID No. 4.
2. A recombinant vector for expressing recombinant avian epidermal growth factor, which comprises a nucleotide sequence of a gene fragment of a signal peptide, an anchor protein, an ampicillin resistance sequence and a cEGF sequence, wherein the recombinant vector is a recombinant lactobacillus expression vector pLL32 a-cEGF.
3. The recombinant vector according to claim 2, wherein the nucleotide sequences of the gene segments of the signal peptide, the dockerin, the ampicillin resistance sequence and the cEGF sequence are set forth in SEQ ID NO: 1-4.
4. The method of constructing a recombinant vector according to claim 2 or 3, comprising the steps of:
(1) carrying out double enzyme digestion on an artificially synthesized ampicillin resistance gene sequence and a pMG36e plasmid, carrying out agarose gel electrophoresis separation on enzyme digestion products, cutting gel, recovering a target band and a carrier band, connecting the target band and the carrier band by using T4 ligase, transforming the target band and the carrier band into MC1061 competent cells, and screening by using ampicillin to obtain a new plasmid pLL32 a;
(2) carrying out double enzyme digestion on an artificially synthesized gene fragment containing a signal peptide, an anchor protein and a cEGF sequence and a pL 32a plasmid, carrying out agarose gel electrophoresis separation on the enzyme digestion product, cutting gel to recover a target band and a carrier band, connecting the target band and the carrier band by using T4 ligase, transforming the target band and the carrier band into MC1061 competent cells, and screening by using ampicillin to obtain a recombinant plasmid pLL32 a-cEGF.
5. A method for preparing recombinant avian epidermal growth factor, which comprises expressing the recombinant vector constructed in claim 4 in lactic acid bacteria to obtain recombinant EGF.
6. The method according to claim 5, wherein the lactic acid bacterium is lactococcus lactis.
7. The method according to claim 6, wherein the lactic acid bacterium is lactococcus lactis MG 1363.
8. The method of any one of claims 5 to 7, wherein the method comprises the steps of:
(1) the recombinant vector constructed in the claim 4 is electrically transformed into a lactic acid bacteria competent cell, after the electrical transformation is finished, the lactic acid galactococcus with the recombinant plasmid is transferred into a recovery culture medium for static culture for 2 hours at 37 ℃, then an MRS plate with 0.5ug/ml ampicillin resistance is coated, and the constant temperature culture is carried out at 37 ℃; the resuscitation medium comprises the following components:
(2) selecting a single colony growing on the plate, carrying out pure culture, extracting plasmids, and carrying out sequencing identification on the plasmids to obtain recombinant bacteria;
(3) and (3) carrying out shake culture on the recombinant bacteria in an MRS liquid culture medium containing 0.5ug/ml of ampicillin at 37 ℃ for 4h, then reducing the culture temperature to 30 ℃ for continuous culture, centrifuging the fermentation liquor after terminating the fermentation, adding a proper amount of lysozyme for cracking the bacteria after resuspending the bacteria, and obtaining the recombinant avian epidermal growth factor.
9. The method of claim 8, wherein the electrotransformation conditions are: 12kV/cm, 200 omega and 25 muF.
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