CN112094833A - Bacteriostatic protein, coding gene, application and strain method thereof - Google Patents
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Abstract
The invention discloses a bacteriostatic protein, a coding gene, application and a strain method thereof. According to the invention, chicken lysozyme is taken as a starting template, an amino acid sequence of the scorpion-derived antimicrobial peptide IsCT is superimposed on the N end of chicken lysozyme protein through rational design, and simultaneously, an antioxidant peptide and an amino acid linker are directionally introduced to obtain a mutant Lyic; then according to the preference of pichia pastoris codon, obtaining the gene lys-Is corresponding to the mutant Lyic by adopting a whole-gene synthesis technology, wherein the nucleotide sequence of the lys-Is shown as SEQ ID NO. 1; thirdly, connecting the lysozyme mutant gene lys-Is into an expression vector pPICZ alpha A, transferring into pichia pastoris X33, fermenting and culturing recombinant bacteria, and separating and purifying the lysozyme mutant Lyic; and finally, the bacteriostatic effect of the lysozyme mutant Lyic and the original lysozyme on common pathogenic bacteria is analyzed in a comparative way. The lysozyme mutant Lyic provided by the invention has an effectively improved antibacterial effect on common pathogenic bacteria (escherichia coli, salmonella and staphylococcus aureus), and lays a foundation for the wide application of chicken lysozyme.
Description
Technical Field
The invention belongs to the technical field of biotechnology, and particularly relates to a bacteriostatic protein, and a coding gene, application and a strain thereof.
Background
Many problems have been raised in recent years with the abuse of antibiotics such as: the emergence of super drug-resistant bacteria; antibiotic residues, and the like. From the sustainable development point of view, the usage and dosage of antibiotics must be effectively controlled by human beings. From the 90 s, many researchers at home and abroad begin to search for an antibiotic substitute, and through many researches, lysozyme as a natural protein can effectively kill many microorganisms, so that the lysozyme is expected to become the antibiotic substitute and is applied to many fields. Lysozyme as a glycoside hydrolase can decompose the cell wall of microorganisms, so that bacteria lose the protection of the cell wall and are broken and killed under the action of intracellular high osmotic pressure, thereby killing target microorganisms.
The chicken lysozyme is the lysozyme which is most researched at present, and is mainly present in egg white of fresh eggs, and the chicken lysozyme is commercially produced at present. With the intensive research and application, the chicken lysozyme has poor bacteriostatic effect on part of gram-positive bacteria (such as staphylococcus aureus) and many gram-negative bacteria (such as escherichia coli), and the application of the chicken lysozyme in many fields is limited. Therefore, the antibacterial effect of chicken lysozyme on different pathogenic microorganisms is improved, and the method has important significance. In our previous experiments, the scorpion-derived antimicrobial peptide IsCT has a good bacteriostatic effect (patent application No. CN104263749A), and can effectively inhibit pathogenic bacteria such as escherichia coli, salmonella, staphylococcus aureus and the like. On the basis, the invention adds the amino acid sequence of the scorpion source antibacterial peptide IsCT to the N end of the chicken lysozyme protein through rational design to obtain the lysozyme mutant Lyic, thereby effectively improving the bacteriostatic property of the chicken lysozyme to common pathogenic bacteria (escherichia coli, salmonella and staphylococcus aureus) and laying a foundation for the wide application of the chicken lysozyme.
Disclosure of Invention
In order to solve the problems, the invention discloses a multi-tag text classification method based on tag relevance, compared with original lysozyme, the lysozyme mutant Lyic disclosed by the invention has the advantages that the bacteriostatic effect on common pathogenic bacteria (escherichia coli, salmonella and staphylococcus aureus) is effectively improved, and a foundation is laid for the wide application of chicken lysozyme.
A bacteriostatic protein, the amino acid sequence of which is shown in SEQ ID NO. 2.
A coding gene of bacteriostatic protein, which comprises a nucleotide sequence for coding an amino acid sequence shown as SEQ ID NO. 2.
In a further improvement, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.
A strain comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2.
In a further improvement, the strain is pichia pastoris X33.
The application of the bacteriostatic protein is shown in SEQ ID No.2, and the bacteriostatic protein is used for inhibiting staphylococcus aureus, escherichia coli and salmonella.
In a further improvement, the staphylococcus aureus is staphylococcus aureus ATCC25923, the escherichia coli is escherichia coli ATCC25922, and the salmonella is salmonella ATCC 13076.
In a further improvement, the using concentration range of the bacteriostatic protein is 0.125mg/mL to 1 mg/mL.
In a further improvement, the using concentration of the bacteriostatic protein is 1 mg/mL.
Compared with the prior art, the invention has the following advantages and effects:
compared with the original chicken lysozyme, the mutant Lyic has an effectively improved bacteriostatic effect on common pathogenic bacteria (escherichia coli, staphylococcus aureus and salmonella).
Drawings
FIG. 1 shows the electrophoresis of the Lyic SDS-PAGE protein gel of the lysozyme mutant.
FIG. 2 shows the bacteriostatic effect of the lysozyme mutant Lyic recombinant bacteria cultured in a 24-well plate. In fig. 1-24 are the first through twenty-fourth plate holes, respectively.
FIG. 3 is a graph showing the bacteriostatic effect of lysozyme mutant Lyic and original chicken lysozyme on Staphylococcus aureus.
FIG. 4 is a diagram showing the bacteriostatic effect of lysozyme mutant Lyic and original chicken lysozyme on Salmonella.
FIG. 5 shows the bacteriostatic effect of lysozyme mutant Lyic and original chicken lysozyme on Escherichia coli.
Detailed Description
The present invention will be described in more detail with reference to the accompanying drawings and examples.
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The molecular biology experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions; the reagents and biomaterials, if not specifically indicated, are commercially available. Experimental materials and reagents involved in the present invention:
according to the invention, chicken lysozyme is taken as a starting template, an amino acid sequence of the scorpion-derived antimicrobial peptide IsCT is superimposed on the N end of chicken lysozyme protein through rational design, and simultaneously, an antioxidant peptide and an amino acid linker are directionally introduced to obtain a mutant Lyic; then according to the preference of pichia pastoris codon, obtaining the gene lys-Is corresponding to the mutant Lyic by adopting a whole-gene synthesis technology, wherein the nucleotide sequence of the lys-Is shown as SEQ ID NO. 1; thirdly, connecting the lysozyme mutant gene lys-Is into an expression vector pPICZ alpha A, transferring into pichia pastoris X33, fermenting and culturing recombinant bacteria, and separating and purifying the lysozyme mutant Lyic; and finally, the bacteriostatic effect of the lysozyme mutant Lyic and the original lysozyme on common pathogenic bacteria is analyzed in a comparative way, and the effectiveness of the mutant Lyic is verified. The method comprises the following specific steps:
1. bacterial strains and vectors
Coli strain Top10, Pichia pastoris X33, expression vector pPICZ alpha A were all purchased from commercial sources.
2. Enzyme and kit
Q5 high fidelity Taq enzyme MIX was purchased from NEB; plasmid extraction, gel purification kit purchased from Tiangen Biotechnology (Beijing) Ltd; restriction enzymes were purchased from daisies technologies (beijing) ltd; zeocin was purchased from Invitrogen, and the protein assay kit was purchased from Shanghai Producer, Inc.
3. Culture medium
The E.coli medium was LB (1% (w/v) peptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, pH 7.0). LBZ is LB medium plus 25. mu.g/mL Zeocin (bleomycin).
The yeast medium was YPD (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose). The yeast selection medium was YPDZ (YPD +100mg/L zeocin).
Yeast Induction Medium BMGY (1% (w/V) yeast extract, 2% (w/V) peptone, 1.34% (w/V) YNB, 0.00004% (w/V) Biotin, 1% glycerol (V/V)) and BMMY medium (the remainder was identical to BMGY except that 0.5% (V/V) methanol was used instead of glycerol).
Note: YNB is Yeast Nitrogen source Base (Yeast Nitrogen Base); biotin is Biotin.
Example 1 lysozyme mutant design
The research shows that the antibacterial effect of the lysozyme can be improved by overlapping the antibacterial peptide on the basis of the lysozyme amino acid, scorpion source antibacterial peptide IsCT (CN104263749A) with good antibacterial effect is obtained through experiments in the previous research, and the patent hopes that the antibacterial property of the chicken lysozyme is improved by overlapping the amino acid sequence of the antibacterial peptide IsCT to the chicken lysozyme protein. The chicken lysozyme mutant Lyic containing the antimicrobial peptide IsCT is designed by the following process: (1) in order to prevent the amino acid sequence of the antibacterial peptide IsCT from being wrapped in the mutant Lyic protein to influence the antibacterial activity of the mutant Lyic protein, the amino acid sequence of the antibacterial peptide IsCT is superposed on the N end of the chicken lysozyme protein; (2) because the antibacterial property of the antibacterial peptide IsCT is strong, in order to reduce the toxicity of the antibacterial peptide IsCT to an expression host, a section of antioxidant peptide (DEDTQAMP) is added in front of the antibacterial peptide IsCT to balance the toxicity of the antibacterial peptide IsCT to the host; (3) in addition, an amino acid linker (GGGSSSSGGGG) is added between the antimicrobial peptide IsCT and the chicken lysozyme, which is helpful to improve the folding processing of the mutant in an expression host. The chicken lysozyme mutant Lyic is finally obtained through the directional rational design, and the amino acid sequence of the mutant is shown in SEQ ID NO. 2.
Example 2 Synthesis of Chicken Lyic enzyme mutant Gene and construction of expression vector
According to the codon preference of pichia pastoris, the nucleotide sequence of the mutant Lyic coding Is optimized through online codon optimization software, so that the gene lys-Is obtained, and the nucleotide sequence Is shown as SEQ ID No. 1. The gene lys-Is has a full length of 486bp (without signal peptide), encodes 161 amino acids, and comprises antioxidant peptide (DEDTQAMP), antibacterial peptide IsCT (ILGKILKGIKKLF), amino acid linker (GGGSSSSGGGG) and chicken lysozyme without signal peptide (129 amino acids). The optimized gene lys-Is was obtained by whole-gene synthesis by general biosystems (Anhui) Ltd. Designing a pair of primers (the primer sequences are fw: 5'-agtc gaattcGATGAAGACACTCAAGCTATG-3' and rev: 5'-ttctctaga TTACAATCTACAAC CTCTGAT-3' respectively) according to the sequence of lys-Is for amplifying the gene lys-Is, detecting the PCR amplification result by agarose electrophoresis, purifying and recovering the PCR product, performing a ligation reaction on the amplified fragment and an expression vector pPICZ alpha A, transferring the fragment into escherichia coli Top10, verifying a recombinant transformant through bacteria liquid PCR, performing plasmid extraction on the transformant which Is verified to be correct, and performing sequencing to determine to obtain the expression vector pPICZ alpha A-lys-Is.
Example 3 recombinant Pichia pastoris construction and screening
The expression vector pPICZ alpha A-lys-Is linearized with SacI and transferred into Pichia pastoris X33, and the recombinant transformants are plated on Zeocin resistance plates (100. mu.g/ml-500. mu.g/ml) of different concentrations. The yeast recombinant transformants grown on the plates were picked up one by one with a toothpick into 24-well plates containing 2mL of BMGY medium per well, cultured at 30 ℃ for about 36h at 220rpm, and then subjected to induction culture by adding 1% (v/v) of methanol, respectively. After culturing at 30 ℃ and 220rpm for 48 hours, centrifuging and taking the supernatant to carry out bacteriostatic test determination. The specific steps of the antibacterial experiment determination are as follows: (1) micrococcus luteus (CICC10680) is cultured to the logarithmic growth phase (OD600 is about 1), and the cultured bacterial liquid is added into a sterile culture medium according to the concentration of 1 mu L/mL to prepare a plate. (2) Punching a solid plate by using a puncher with the radius of 3mm, adding different supernatant culture solutions into the holes (the adding volume is 75 mu L), and standing at a low temperature (4-8 ℃) for 2 hours; (3) the plate was taken out from the refrigerator, after 24 hours of stationary culture at 37 ℃, the zone of inhibition was determined, and the expression level of each bacterium was preliminarily determined according to the size of the zone of inhibition, as can be seen from fig. 1, among the 24 transformants selected, the zone of inhibition was largest for the 21 bacterium, and then for the 6 bacterium. Since bacterium 21 has the best bacteriostatic effect, it was used as the starting strain for the next experiment.
Example 4 Shake flask culture and Lyic purification of the Lyic mutant
The No. 21 strain is subjected to shake flask culture, and the shake flask culture process is approximately as follows: (1) firstly, inoculating No. 21 bacteria into a 50mL shake flask containing 10mL BMGY medium; (2) after overnight culture at 30 ℃ and 200rpm, obtaining the thalli by centrifugation, and resuspending the thalli by using a BMMY culture medium; (3) inoculating the resuspended thallus into a 250mL triangular flask containing 50mL BMMY culture medium, and adjusting the initial OD600 to 1.0; (4) every 24 hours, 1% methanol was added for induction and samples were taken for bacteriostatic determination.
The lyoic purification procedure is roughly as follows: the supernatant was obtained by centrifugation of the fermentation broth after 96 hours of shake flask culture, and the supernatant enzyme solution was first concentrated by ultrafiltration using a 10kDa ultrafiltration tube, followed by purification with reference to a Ni-IDA protein purification kit (Shanghai Producer Co., Ltd.). The purity of the purified Lyic was checked by SDS-PAGE protein gel electrophoresis.
Example 5 comparison of the bacteriostatic Properties of the Lyic mutant Lyic with the original hen Lysozyme
The purified lysozyme mutant Lyic is compared with the original lysozyme (obtained by purification and preparation in the early stage of a laboratory) in bacteriostatic property, and the protein concentrations of the purified lysozyme mutant Lyic and the original lysozyme are respectively determined by using a modified Bradford kit, wherein the determination method comprises the following steps: and (3) taking 100 mu L of sample diluent (adding 100 mu L of distilled water into the control group), adding 1mL of Bradford reagent, quickly mixing uniformly, standing at room temperature for 15 minutes, measuring the light absorption value at 595nm, and substituting the OD obtained by subtracting the control group from the experimental group into a standard curve to calculate the protein concentration of the target sample. The protein concentration of the sample was adjusted to 1mg/mL, 0.5mg/mL, 0.25mg/mL, and 0.125mg/mL, respectively. Three common pathogenic bacteria are selected as indicator bacteria of bacteriostatic experiments, and the three bacteria are staphylococcus aureus ATCC25923, escherichia coli ATCC25922 and salmonella ATCC13076 respectively. The procedure of the bacteriostatic test was substantially as described in example 3 except that the indicator bacteria were replaced with Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922 and Salmonella ATCC13076, respectively, and the amount of the added sample was 75. mu.L.
The bacteriostatic effect of the lysozyme mutant Lyic and the original chicken lysozyme on Staphylococcus aureus ATCC25923 is shown in FIG. 3. As can be seen from FIG. 3, the original chicken lysozyme had no inhibitory effect on Staphylococcus aureus ATCC25923 at the selected 4 concentration gradients (1mg/mL, 0.5mg/mL, 0.25mg/mL and 0.125 mg/mL). The mutant Lyic gradually weakens the bacteriostatic effect along with the reduction of the protein concentration, and compared with the original chicken lysozyme, the mutant Lyic obviously improves the bacteriostatic effect on staphylococcus aureus ATCC 25923.
The bacteriostatic effects of the lysozyme mutant Lyic and the original chicken lysozyme on Salmonella ATCC13076 are shown in FIG. 4. As can be seen in FIG. 4, the original chicken lysozyme had no inhibitory effect on Salmonella ATCC13076 at all the 4 concentration gradients selected. The mutant Lyic has improved bacteriostatic effect on salmonella ATCC13076, and the effect is best when the concentration is 1 mg/ml. The bacteriostatic performance is gradually weakened along with the reduction of the concentration, and when the concentration is 0.125mg/ml, the bacteriostatic effect is not achieved.
The bacteriostatic effects of the lysozyme mutant Lyic and the original chicken lysozyme on Escherichia coli ATCC25922 are shown in FIG. 5. As can be seen from FIG. 5, the original chicken lysozyme had no inhibitory effect on E.coli ATCC25922 at all the 4 concentration gradients selected. The bacteriostatic effect of the mutant Lyic on Escherichia coli ATCC25922 is improved, but the promotion amplitude is not as obvious as that of Staphylococcus aureus ATCC25923 and Salmonella ATCC 13076. Of the 4 concentrations tested, only 1mg/ml showed bacteriostatic effects, and none of the other concentrations inhibited E.coli ATCC 25922.
The above description is only one specific guiding embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modification of the present invention using this concept shall fall within the scope of the invention.
Sequence listing
<110> Shenzhen kang blessing biotechnology Limited company
<120> bacteriostatic protein and coding gene, application and strain method thereof
<130> 2020.3.18
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 486
<212> DNA
<213> Artificial synthesis
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gatgaagaca ctcaagctat gccaattttg ggtaagattt tgaagggaat taagaagttg 60
tttggaggtg gatcttcctc ttccggaggt ggaggtaagg ttttcggtag atgtgaattg 120
gctgctgcta tgaagagaca cggtttggac aactacagag gttactcttt gggtaactgg 180
gtttgtgttg ctaagttcga atctaacttc aacactcaag ctactaacag aaacactgac 240
ggttctactg actacggtat cttgcaaatc aactctagat ggtggtgtaa cgacggtaga 300
actccaggtt ctagaaactt gtgtaacatc ccatgttctg ctttgttgtc ttctgacatc 360
actgcttctg ttaactgtgc taagaagatc gtttctgacg gtaacggtat gtctgcttgg 420
gttgcttgga gaaacagatg taagggtact gacgttcaag cttggatcag aggttgtaga 480
ttgtaa 486
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<213> Artificial synthesis
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Asp Glu Asp Thr Gln Ala Met Pro Ile Leu Gly Lys Ile Leu Lys Gly
1 5 10 15
Ile Lys Lys Leu Phe Gly Gly Gly Ser Ser Ser Ser Gly Gly Gly Gly
20 25 30
Lys Val Phe Gly Arg Cys Glu Leu Ala Ala Ala Met Lys Arg His Gly
35 40 45
Leu Asp Asn Tyr Arg Gly Tyr Ser Leu Gly Asn Trp Val Cys Val Ala
50 55 60
Lys Phe Glu Ser Asn Phe Asn Thr Gln Ala Thr Asn Arg Asn Thr Asp
65 70 75 80
Gly Ser Thr Asp Tyr Gly Ile Leu Gln Ile Asn Ser Arg Trp Trp Cys
85 90 95
Asn Asp Gly Arg Thr Pro Gly Ser Arg Asn Leu Cys Asn Ile Pro Cys
100 105 110
Ser Ala Leu Leu Ser Ser Asp Ile Thr Ala Ser Val Asn Cys Ala Lys
115 120 125
Lys Ile Val Ser Asp Gly Asn Gly Met Ser Ala Trp Val Ala Trp Arg
130 135 140
Asn Arg Cys Lys Gly Thr Asp Val Gln Ala Trp Ile Arg Gly Cys Arg
145 150 155 160
Leu
Claims (9)
1. A bacteriostatic protein is characterized in that the amino acid sequence of the protein is shown in SEQ ID NO. 2.
2. A coding gene of a bacteriostatic protein is characterized by comprising a nucleotide sequence for coding an amino acid sequence shown as SEQ ID NO. 2.
3. The encoding gene of the bacteriostatic protein according to claim 2, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No. 1.
4. A strain comprising a nucleotide sequence encoding an amino acid sequence as set forth in SEQ ID No. 2.
5. The strain of claim 4, wherein the strain is Pichia pastoris X33.
6. The application of the bacteriostatic protein is characterized in that the amino acid sequence of the protein is shown in SEQ ID No.2, and the bacteriostatic protein is used for inhibiting staphylococcus aureus, escherichia coli and salmonella.
7. The use of a bacteriostatic protein according to claim 6 wherein the Staphylococcus aureus is Staphylococcus aureus ATCC25923, Escherichia coli is Escherichia coli ATCC25922 and Salmonella is Salmonella ATCC 13076.
8. The use of a bacteriostatic protein according to claim 6, wherein the bacteriostatic protein is used at a concentration ranging from 0.125mg/mL to 1 mg/mL.
9. The use of a bacteriostatic protein according to claim 8, wherein the bacteriostatic protein is used at a concentration of 1 mg/mL.
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| CN115975992A (en) * | 2022-10-28 | 2023-04-18 | 山东龙昌动物保健品有限公司 | Lysozyme mutant with improved enzyme activity, bile acid enzyme preparation and application |
| CN115975048A (en) * | 2022-10-28 | 2023-04-18 | 山东龙昌动物保健品有限公司 | Antibacterial compound preparation containing eucommia ulmoides leaf extract and application of antibacterial compound preparation in food preservation and fresh keeping |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115322914A (en) * | 2022-10-19 | 2022-11-11 | 天津市农业科学院 | Construction method and application of Ec-cLYZ and MEL gene co-expression recombinant pichia pastoris |
| CN115322914B (en) * | 2022-10-19 | 2024-03-22 | 天津市农业科学院 | Construction method and application of Ec-cLYZ and MEL gene co-expression recombinant Pichia pastoris |
| CN115975992A (en) * | 2022-10-28 | 2023-04-18 | 山东龙昌动物保健品有限公司 | Lysozyme mutant with improved enzyme activity, bile acid enzyme preparation and application |
| CN115975048A (en) * | 2022-10-28 | 2023-04-18 | 山东龙昌动物保健品有限公司 | Antibacterial compound preparation containing eucommia ulmoides leaf extract and application of antibacterial compound preparation in food preservation and fresh keeping |
| CN115975992B (en) * | 2022-10-28 | 2024-04-09 | 山东龙昌动物保健品股份有限公司 | Lysozyme mutant with improved enzyme activity, bile acid enzyme preparation and application |
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