CN118221779B - Sturgeon epidermis mucus recombinant antibacterial peptide ALKW, plasmid, recombinant bacterium and application thereof - Google Patents
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a sturgeon epidermis mucus recombination antibacterial peptide ALKW, a plasmid, a recombination bacterium and application thereof, belonging to the fields of genetic engineering and biotechnology, wherein the amino acid sequence of the sturgeon epidermis mucus recombination antibacterial peptide ALKW is shown as SEQ ID NO.1, the invention also provides a gene for encoding the antibacterial peptide ALKW16, a pichia pastoris recombination expression vector and the recombination bacterium comprising the gene, and application thereof in preparing a bacteriostatic agent or a bactericide, the antibacterial activity of the antibacterial peptide ALKW is stronger than that of the original antibacterial peptide AL16, and the antibacterial peptide has good thermal stability, pH stability, pepsin stability and trypsin stability.
Description
Technical Field
The invention belongs to the field of genetic engineering and biotechnology, and in particular relates to sturgeon epidermis mucus recombinant antibacterial peptide ALKW, plasmid, recombinant bacteria and application thereof.
Background
Listeria monocytogenes is a common gram-positive bacterium with great harm, mainly food-borne pollution, and foods which are easy to be polluted by the listeria monocytogenes are raw milk, cheese, meat and meat products, eggs, vegetable salad, aquatic products and the like, and the listeria monocytogenes poisoning can cause various diseases such as septicemia and the like. Listeria monocytogenes has a high capacity to survive normal heat processing and frozen storage at 4 ℃, and many countries have taken measures to control Listeria in foods and have established corresponding standards. For example, in GB 29921-2013, the limits in meat products (cooked meat products and ready-to-eat raw meat products) are specified as n=5, c=0, m=0 (/ 25 g). For thousands of years, food preservation and fresh keeping have been an important issue for humans. The antibacterial peptide has a very wide development prospect in food preservation and fresh-keeping, for example, nisin (Nisin) is used as a safe and efficient fresh-keeping antibacterial means, and is applied to the preservation and fresh-keeping of dairy products and meat products.
The antibacterial peptide has the advantages of broad-spectrum antibacterial activity, low toxicity, biocompatibility and the like, and is widely applied to the fields of food preservation, animal health protection, medical treatment and health and the like. At present, china is the country with the largest sturgeon cultivation yield in the world. In addition to caviar and fish, a major product, many by-products of sturgeon are also widely used in various fields: sturgeon skull and spine were used to develop chondroitin sulfate; viscera and the like are used to develop fish oils; the fish skin is used for tanning and extracting collagen; while a large amount of by-product mucus is not yet utilized effectively. Studies show that sturgeon skin mucus contains multiple immune related factors such as lectin, lysozyme, calmodulin, immunoglobulin, proteolytic enzyme, polysaccharide compounds, antibacterial peptide and the like, and can resist the invasion of bacteria, fungi and viruses. Therefore, the antibacterial peptide in the sturgeon skin mucus is researched, the antibacterial effect and the action mechanism of the antibacterial peptide are discussed, the high-value utilization of the sturgeon low-value material is further realized, and the rapid development of the sturgeon industry is promoted.
Disclosure of Invention
The invention aims at the antibacterial peptide AL16 derived from sturgeon epidermis mucus as a basis, obtains the antibacterial peptide mutant by the technical means of error-prone PCR, constructs the heterologous expression of a recombinant vector in Pichia pastoris GS115, and results show that the antibacterial activity of the recombinant antibacterial peptide ALKW of sturgeon epidermis mucus on escherichia coli, staphylococcus aureus and listeria monocytogenes is obviously improved compared with that of the original antibacterial peptide AL 16.
The invention is realized based on the following technical scheme:
the amino acid sequence of the sturgeon skin mucus recombinant antibacterial peptide ALKW and the amino acid sequence of the sturgeon skin mucus recombinant antibacterial peptide ALKW are shown as SEQ ID NO. 1; the method comprises the following steps: APATKKAPWLLPWWLL.
The nucleotide sequence of the amino acid sequence of the coding SEQ ID NO. 1 is shown as SEQ ID NO. 2, the SEQ ID NO. 2 is a mutant obtained by translating the coding nucleotide sequence into the coding nucleotide sequence based on the antibacterial peptide AL16 of the original amino acid sequence SEQ ID NO. 3, artificially synthesizing the nucleotide fragment and performing error-prone PCR reaction on the artificially synthesized fragment.
SEQ ID NO. 1：APATKKAPWLLPWWLL；
SEQ ID NO. 2：
GCTCCTGCTACTAAGAAGGCTCCTTGGTTGTTGCCTTGGTGGTTGTTG；
SEQ ID NO. 3：APATPAAPALLPLWLL。
The invention also provides a pichia pastoris recombinant expression vector containing the antibacterial peptide gene.
The invention relates to a recombinant Pichia pastoris GS115 strain capable of expressing the sturgeon epidermis mucus recombinant antibacterial peptide ALKW.
The invention provides a preparation method of sturgeon epidermis mucus recombination antibacterial peptide ALKW and 16, which specifically comprises the following steps: translating the amino acid sequence of the original sturgeon epidermis mucus recombination antibacterial peptide AL16 into a nucleotide sequence, artificially synthesizing the nucleotide sequence, obtaining an error-prone PCR product by adopting an error-prone PCR technical means, inserting the error-prone PCR product into a pichia pastoris recombination expression vector to construct a recombination expression vector, and sequencing and verifying the recombination expression vector; and (3) electrically converting the recombinant antimicrobial peptide into pichia pastoris for fermentation expression to obtain the sturgeon epidermis mucus recombinant antimicrobial peptide ALKW.
The invention relates to application of the recombinant antibacterial peptide ALKW, the gene, the recombinant expression vector or the recombinant bacterium in preparation of a bacteriostat or a bactericide, wherein the bacterium is at least one of escherichia coli, salmonella, staphylococcus aureus and listeria monocytogenes.
Compared with the prior art, the invention has the beneficial effects that:
The invention obtains the sturgeon epidermis mucus recombinant antibacterial peptide ALKW and the preparation method of the sturgeon epidermis mucus recombinant antibacterial peptide ALKW in pichia pastoris GS115 by an error-prone PCR method.
The invention relates to a preparation method of a sturgeon epidermis mucus recombinant antibacterial peptide ALKW and a pichia pastoris GS115 heterologous expression method.
The invention characterizes the antibacterial activity of sturgeon epidermis mucus recombination antibacterial peptide ALKW, which has stronger inhibitory activity to escherichia coli, salmonella, staphylococcus aureus and listeria monocytogenes than the original antibacterial peptide AL 16.
The sturgeon epidermis mucus recombinant antibacterial peptide ALKW and 16 provided by the invention have good thermal stability and pH stability.
The sturgeon epidermis mucus recombinant antibacterial peptide ALKW and the preparation method thereof have the advantages of good pepsin stability and trypsin stability.
The characteristics are beneficial to the application of the sturgeon epidermis mucus recombinant antibacterial peptide ALKW16 in a bacteriostatic agent or bactericide.
Drawings
FIG. 1 is a schematic diagram of PCR amplified nucleic acid electrophoresis of an error-prone PCR product recombinant expression vector;
FIG. 2 is a schematic representation of the antibacterial activity of sturgeon epidermal mucus recombinant antibacterial peptide ALKW;
FIG. 3 is a schematic illustration of the thermal stability of sturgeon skin mucus recombinant antimicrobial peptide ALKW;
FIG. 4 is a schematic illustration of pepsin stability of sturgeon epidermal mucus recombinant antibacterial peptide ALKW;
FIG. 5 is a schematic illustration of trypsin stability of sturgeon epidermal mucus recombinant antibacterial peptide ALKW;
FIG. 6 is a schematic illustration of the pH stability of sturgeon skin mucus recombinant antimicrobial peptide ALKW.
Detailed Description
The technical scheme of the present invention is further explained by examples below, but the scope of the present invention is not limited in any way by the examples.
EXAMPLE 1 error-prone PCR of sturgeon epidermal mucus recombinant antibacterial peptide ALKW Gene
The amino acid sequence of the sturgeon epidermis mucus-derived antibacterial peptide AL16 is taken as an original sequence (SEQ ID NO. 3: APATPAAPALLPLWL), translated into a coded nucleotide sequence (a complementary sequence) by using the vector NTI software, and the nucleotide fragment is artificially synthesized. Error-prone PCR reaction is carried out on the artificially synthesized fragment, the adopted PCR amplification enzyme is phanta ℃ high-fidelity amplification enzyme, and a PCR reaction system (50 mu L) is as follows: buffer 10. Mu.L; dNTP 4. Mu.L; phanta [ mu ] L; 2. Mu.L of a front primer; 2. Mu.L of the rear primer; 1 mu L of synthetic template; ddH 2 O30.5. Mu.L. The PCR reaction conditions were: 95. pre-denaturation at 3 deg.c min; 95. denaturation at 30℃ 30 sec, annealing at 56℃30 sec, elongation at 72℃1 min, 35 cycles; 72. 10℃ min total extension; 4. DEG C. After the completion of the PCR reaction, the target bands of the PCR products were detected by agarose gel electrophoresis, as shown in FIG. 1. Further purifying and recovering to obtain the error-prone PCR gene fragment of sturgeon epidermis mucus antibacterial peptide AL 16.
EXAMPLE 2 construction of error-prone PCR product recombinant expression vector for sturgeon epidermal mucus antimicrobial peptide AL16
The linearized pichia pastoris expression vector is obtained by a PCR method. The PCR reaction system (50. Mu.L) was: buffer 25. Mu.L; dNTP 1. Mu.L; phanta [ mu ] L; 2. Mu.L of a front primer; 2. Mu.L of the rear primer; 1 mu L of carrier template; ddH 2 O18.5. Mu.L. The PCR reaction conditions were: 95. pre-denaturation at 3 deg.c min; 95. denaturation at 30℃ 30 sec, annealing at 58℃30 sec, elongation at 72℃8 min, 35 cycles; 72. 10℃ min total extension; 4. DEG C. After the detection by nucleic acid electrophoresis, further purification and recovery were performed, and the error-prone PCR product of example 1 was ligated. The connection system is as follows: CE II Buffer 2. Mu.L; exnase II 0.5. Mu.L; linearization of 0.5. Mu.L of carrier; error-prone PCR gene 1. Mu.L; ddH 2 O1. Mu.L. The connection conditions are as follows: 37. 30 ℃ and min. The ligation product was then transformed into E.coli DH 5. Alpha. And further positive transformants were screened and verified by sequencing.
Example 3 electrotransformation and fermentative expression of sturgeon epidermal mucus antibacterial peptide ALKW16
The recombinant expression vector in example 2 was linearized and electrotransformed into pichia pastoris GS 115. After screening positive transformants, inoculating the positive transformants into BMGY liquid medium for fermentation expression. The fermentation process uses 1% methanol for induction, and the induction is performed once every 24 h times, and the total induction is 3 times. And collecting fermentation liquor, purifying and then carrying out subsequent antibacterial activity verification.
Example 4 determination of antibacterial Activity of sturgeon epidermal mucus recombinant antibacterial peptide ALKW16
The indicator bacteria used in this example are E.coli, salmonella, staphylococcus aureus, listeria monocytogenes;
Determination of Minimum Inhibitory Concentration (MIC), MIC was determined by microdilution: preparing bacterial suspension, respectively diluting four bacteria cultured to a growth log phase to a concentration of 10 5 CFU/mL, mixing the purified sturgeon epidermis mucus antibacterial peptide ALKW solution and the original antibacterial peptide AL16 solution with the bacterial suspension in an equivalent manner in a first column of a 96-well plate, and sequentially diluting in a concentration gradient manner. Equal amounts of diluted bacterial suspensions were added to all samples of 96-well plates, and E.coli, salmonella, staphylococcus aureus, listeria monocytogenes were incubated at 37℃for 18 h. The turbidity of each well was observed visually after incubation, wherein the minimum concentration that allowed the wells to be clarified was the MIC of sturgeon epidermis mucus antimicrobial peptide ALKW to each bacteria.
The results are shown in Table 1 and FIG. 2, and the antibacterial activity of sturgeon epidermal mucus recombinant antibacterial peptide ALKW is improved relative to AL 16. The MIC of the recombinant antimicrobial peptide ALKW to Escherichia coli is 300 mu M, the MIC of the recombinant antimicrobial peptide ALKW to Acipenser sinensis epidermis is 200 mu M; the MIC of AL16 for Salmonella is 350. Mu.M, while the MIC of sturgeon epidermis mucus recombinant antimicrobial peptide ALKW for Staphylococcus aureus is 260. Mu.M; the MIC of AL16 for listeria monocytogenes was 320 μm, while the MIC of sturgeon epidermal mucus recombinant antimicrobial peptide ALKW for staphylococcus aureus was 240 μm. The antibacterial activity results show that the antibacterial activity of the recombinant antibacterial peptide ALKW of sturgeon epidermis mucus is obviously improved compared with that of the original sequence AL 16.
TABLE 1 MIC (μM) of sturgeon epidermal mucus recombinant antibacterial peptide ALKW and the original antibacterial peptide AL16 for different bacteria tested
。
Example 5 measurement of the thermal stability of sturgeon epidermal mucus recombinant antibacterial peptide ALKW16
The indicator bacteria used in this example are listeria monocytogenes, the thermal stability results are shown in fig. 3, wherein the abscissa is the treatment time of boiling water bath, the inhibitory activities of the sturgeon epidermis mucus recombination antibacterial peptide ALKW and AL16 on each bacteria are reduced to different degrees after high-temperature treatment, the reduction amplitude of the sturgeon epidermis mucus recombination antibacterial peptide ALKW is far smaller than that of the original sequence AL16, and 90% antibacterial rate can be still maintained after 30 min treatment. The results show that the sturgeon skin mucus recombinant antibacterial peptide ALKW has better heat stability, can resist high temperature for a long time, and can be suitable for general heat processing treatment of food.
Example 6 determination of digestive enzyme stability of sturgeon epidermal mucus recombinant antibacterial peptide ALKW16
The indicator bacteria used in this example were listeria monocytogenes, and the results of the digestive enzyme stability measurement of sturgeon epidermis mucus recombinant antimicrobial peptide ALKW are shown in fig. 4 and 5, wherein fig. 4 is pepsin and fig. 5 is trypsin. The sturgeon epidermis mucus recombinant antibacterial peptide ALKW and the original sequence AL16 have better stability in pepsin and pepsin. After protease treatment is carried out on 120 min, the inhibition rate of sturgeon epidermis mucus recombination antibacterial peptide ALKW on the bacteria to be tested can still reach more than 90%, and no obvious trend is seen.
Example 7 determination of the pH stability of sturgeon epidermal mucus recombinant antibacterial peptide ALKW16
The indicator bacteria used in this example were staphylococcus aureus, and the pH stability measurement results are shown in fig. 6, wherein the abscissa indicates pH value and the ordinate indicates inhibition rate. The sturgeon epidermis mucus recombinant antibacterial peptide ALKW and the original sequence antibacterial peptide AL16 can maintain more than 95% of antibacterial activity after being treated in an alkaline environment and an acidic environment for 120: 120 min, which shows that the sturgeon epidermis mucus recombinant antibacterial peptide has good pH stability and tolerance to acid and alkali.
Claims (5)
1. The sturgeon skin mucus recombinant antibacterial peptide ALKW is characterized in that the amino acid sequence of the sturgeon skin mucus recombinant antibacterial peptide ALKW is shown as SEQ ID NO. 1.
2. A gene encoding the recombinant antibacterial peptide ALKW according to claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 2.
3. A recombinant expression vector comprising the gene of claim 2, wherein the vector is pichia pastoris.
4. Recombinant bacterium comprising the gene of claim 2, wherein the strain of recombinant bacterium is recombinant pichia pastoris GS115.
5. The use of the recombinant antimicrobial peptide ALKW of claim 1, the gene of claim 2, the recombinant expression vector of claim 3 or the recombinant bacterium of claim 4 in the preparation of a bacteriostatic or bacteriocidal agent, wherein the bacteriostatic or bacteriocidal agent is capable of inhibiting or killing at least one of escherichia coli, salmonella, staphylococcus aureus, listeria monocytogenes.
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Citations (2)
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|---|---|---|---|---|
| WO2004018706A2 (en) * | 2002-08-22 | 2004-03-04 | National Research Council Of Canada | A genomic approach to identification of novel broad-spectrum antimicrobial peptides from bony fish |
| CN102584974A (en) * | 2012-02-22 | 2012-07-18 | 华中农业大学 | Acipenser sinensis antibacterial peptide and preparation method and application thereof |
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| CN111269290B (en) * | 2020-02-28 | 2022-11-08 | 贵州翁松鲟鱼加工有限公司 | Preparation method of sturgeon anti-inflammatory peptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004018706A2 (en) * | 2002-08-22 | 2004-03-04 | National Research Council Of Canada | A genomic approach to identification of novel broad-spectrum antimicrobial peptides from bony fish |
| CN102584974A (en) * | 2012-02-22 | 2012-07-18 | 华中农业大学 | Acipenser sinensis antibacterial peptide and preparation method and application thereof |
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