CN112940095B - Bombyx mori BmSPI51 mutant protein BmSPI51M and preparation method and application thereof - Google Patents
Bombyx mori BmSPI51 mutant protein BmSPI51M and preparation method and application thereof Download PDFInfo
- Publication number
- CN112940095B CN112940095B CN202110174143.4A CN202110174143A CN112940095B CN 112940095 B CN112940095 B CN 112940095B CN 202110174143 A CN202110174143 A CN 202110174143A CN 112940095 B CN112940095 B CN 112940095B
- Authority
- CN
- China
- Prior art keywords
- bmspi51m
- bmspi51
- mutant protein
- protein
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The invention discloses a bombyx mori BmSPI51 mutant protein BmSPI51M and a preparation method and application thereof, wherein the amino acid sequence of the mutant protein BmSPI51M is shown in SEQ ID NO. 4; the nucleotide sequence is shown as SEQ ID NO.2, the mutant protein is mutated from 'KGSFP' to 'RGGFR' from the active site of silkworm BmSPI51, the inhibition effect on fungi is improved under the condition of not reducing the inhibition activity and self-stability to pancreatin after prokaryotic recombinant expression, and the mutant protein can be used for inhibiting drug-resistant fungi.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a bombyx mori BmSPI51 mutant protein BmSPI51M, and further relates to a preparation method and application of the mutant protein BmSPI 51M.
Background
The silkworm cocoon is composed of silk fiber and is a protective layer of pupa. It has a multilayer structure to protect pupae; it also has excellent mechanical properties. Various protease inhibitors have also been found in silkworm cocoons, which are effective in inhibiting the activity of proteases in fungi and bacteria. 80 serine protease inhibitors were found in silkworm and named BmSPI 1-80. These protease inhibitors are currently recognized as 11 classes, anti-stasin, TIL, serpin, Kunitz, Kazal, BPTI, amfpi, Bowman-Birk, WAP, Pacifastin, alpha-macrobulin, and the like. Among them, TIL type, Kunitz type and Kazal type are found in cocoons. Wherein the BmSPI51 protein is a protease inhibitor with the highest content in silkworm cocoons and belongs to Kunitz type. In our previous studies, the bmsipi 51 protein was shown to be effective in inhibiting the growth of fungi such as saccharomyces cerevisiae (saccharomyces cerevisiae), Candida albicans (Candida albicans), and beauveria bassiana (beauveria bassiana). Due to the long-term abuse of antibiotic drugs, the drug resistance of microorganisms is stronger and stronger, so that the drug resistance becomes a great obstacle to people.
Therefore, the activity of the BmSPI51 protein for inhibiting the growth of fungi is further improved, and the BmSPI51 protein has strong stability, so that the problem of the drug resistance of the fungi at present can be effectively solved.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a bombyx mori BmSPI51 mutein BmSPI 51M; the second purpose of the invention is to provide a recombinant expression vector containing the bombyx mori BmSPI51 mutant protein BmSPI 51M; the third object of the present invention is to provide a transformant containing the recombinant expression vector; the fourth purpose of the invention is to provide a preparation method of the mutant protein BmSPI 51M; the invention also aims to provide an application of the mutant protein BmSPI51M in preparation of a fungal inhibitor.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the bombyx mori BmSPI51 mutein BmSPI51M is shown in SEQ ID NO.4, and the amino acid sequence of the mutein BmSPI51M is shown in SEQ ID NO. 4.
Preferably, the nucleotide sequence of the mutant protein BmSPI51M is shown as SEQ ID NO. 2.
2. The recombinant expression vector contains the bombyx mori BmSPI51 mutant protein BmSPI 51M.
Preferably, the expression vector is obtained by connecting a sequence shown in SEQ ID NO.2 into a PET-sumo expression vector through BamHI and NotI enzyme cutting sites.
3. A transformant containing the recombinant expression vector; preferably, the transformant is the strain BL21(DE 3).
4. The preparation method of the mutant protein BmSPI51M is characterized in that a recombinant expression vector containing a sequence shown in SEQ ID NO.2 is expressed in a host.
5. The application of the mutant protein BmSPI51M in preparing a fungal inhibitor.
In the present invention, the fungus may be other fungi; preferably, the fungus is a yeast.
The invention has the beneficial effects that: the invention discloses a bombyx mori BmSPI51 mutant protein BmSPI51M, wherein an active site is mutated from KGSFP to RGGFR, the inhibition effect on fungi can be increased after prokaryotic recombinant expression, the inhibition activity and stability on pancreatin are not reduced, and the bombyx mori mutant protein BmSPI can be used for inhibiting drug-resistant fungi and effectively solving the problem of drug resistance of the current fungi.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows mutations in the BmSPI51 protein sequence (A. nucleotide sequence; B. amino acid sequence);
FIG. 2 shows proteins purified from BmSPI51 and BmSPI 51M;
FIG. 3 shows that BmSPI51 and BmSPI51M have higher stability (A: inhibitory activity of BmSPI51 and BmSPI51M on the enzyme inhibitor under different pH conditions; B: inhibitory activity of BmSPI51 and BmSPI51M on the enzyme inhibitor under different temperature conditions).
FIG. 4 shows the difference between the bacteriostatic activity of BmSPI51 and BmSPI51M (A: Saccharomyces cerevisiae growth curve; B: BmSPI51 and BmSPI51M inhibition of Saccharomyces cerevisiae growth).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 BmSPI51 protein mutant sequences
Downloading a base sequence of the BmSPI51 protein, and mutating a codon corresponding to an active site, namely mutating 'AAG GGC AGT TTT CCA' to 'AGG GGC GGT TTC CGA', namely mutating the active site from 'KGSFP' to 'RGGFR', wherein the sequences before and after mutation are shown in figure 1, the nucleotide sequence before mutation (BmSPI51) is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No. 2; the nucleotide sequence of the mutant (BmSPI51M) is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
If mutation is required, the mutant nucleotide sequence is synthesized and ligated by pUC57 vector.
Example 2 expression vector construction
By utilizing a prokaryotic expression vector PET-sumo, target fragments BmSPI51 and BmSPI51M are respectively connected to the PET-sumo expression vector through BamHI and NotI enzyme cutting sites, and the prokaryotic expression vectors PET-sumo-BmSPI51 and PET-sumo-BmSPI51M are successfully constructed and then used for prokaryotic expression of protein.
1. The target fragment is linked to an expression vector
And connecting the recovered target fragment with a PET-sumo vector, wherein the connection system is as follows (the total system is 10 mu L):
the ligation was carried out at 16 ℃ for 1.5-2 h.
2. Transformation of
(1) Taking the Trans1-T1 competent cells out of a refrigerator at the temperature of-80 ℃, and putting the competent cells on ice for thawing;
(2) adding 10 μ L of the ligation product into the milky competent cells in an ultraclean bench, and standing on ice for 20-25 min;
(3) placing the sample on a metal bath at 42 ℃ for heat shock for 90s, and standing for 2-3min on ice;
(4) adding 500 μ L LB non-resistant liquid culture medium (operating in a clean bench) into 1.5mL centrifuge tube, culturing at 37 deg.C and 220rpm for 45-60min to activate bacteria;
(5) uniformly coating 100 mu L of bacterial liquid on an LB solid culture medium plate containing kanamycin antibiotic in an ultra-clean workbench;
(6) upright placing in 37 deg.C constant temperature incubator for about 3-10min, and then inversely culturing in 37 deg.C incubator overnight;
3. screening and sequencing of Positive clones
In an ultra-clean workbench, picking monoclonal bacterial plaques into LB liquid culture medium containing 500 mu L of kanamycin resistance, performing shake culture at 37 ℃ and 220rpm, and taking out bacterial liquid for PCR verification after the bacterial liquid becomes turbid.
4. Extraction of plasmids
(1) Carrying out amplification culture on the positive strains preliminarily screened by the PCR in a 10mL centrifuge tube to 3mL kana culture medium, culturing for 12h at 37 ℃ and 220rpm, centrifuging the cultured bacterial liquid for 3min at room temperature and 12000g, and discarding the supernatant;
(2) adding 250 mu L of resuspension buffer RB (added with RNaseA), and resuspending the thalli until no caking thalli exist;
(3) adding 250 μ L lysis buffer LB, slowly turning over for 4-6 times, and fully lysing until blue bright solution is formed, wherein the process is not more than 4 min;
(4) adding 350 μ L yellow neutralization buffer NB, slowly turning over for 4-6 times, standing at room temperature for 5min, and centrifuging at 12000g at room temperature for 12 min;
(5) transferring the supernatant obtained in the step (4) to a DNA binding column, centrifuging at 12000g for 40s at room temperature, and discarding the filtrate. Taking care not to suck the sediment in the tube (4);
(6) the DNA binding column is put back into a centrifuge tube, 700 mu L of washing buffer WB is added, centrifugation is carried out for 1min at 12000g and room temperature, and the filtrate is discarded;
(7) repeating the step (6) once;
(8) putting the DNA binding column back into a 2mL centrifuge tube, and emptying for 2min at 12000g at room temperature to thoroughly throw off the redundant liquid on the binding column;
(9) the DNA binding column was transferred to a new centrifuge tube and left for 2min to allow ethanol to evaporate sufficiently. Dropping 30-50 μ L of ddH at 65 deg.C into DNA binding column2O, standing for 1min, centrifuging at 12000g at room temperature for 2min, washing the DNA, and measuring the concentration. The plasmid was stored in a freezer at-30 ℃ for use.
5. Double enzyme digestion verification (10. mu.L system)
Detecting the product after double enzyme digestion by 1.2% agarose gel, taking 150 microliter of strains which are detected to be positive, sending the strains to a company for sequencing detection, and screening positive clones to extract plasmids for later use.
Example 3 purification of prokaryotic expression proteins
The correct recombinant plasmids PET-sumo-BmSPI51 and PET-sumo-BmSPI51M were transferred into BL21(DE3) expressing strains, respectively, and then expanded for culture, specifically, 350. mu.L of kanamycin-resistant LB liquid medium was added to a 1.5mL centrifuge tube, a single colony was picked up and placed therein, and the cells were cultured to turbidity at 37 ℃ at 220 rpm. The activated cells were cultured in an expanded state until the OD value was about 0.4 to 0.6. The group without IPTG addition was also set as a blank control group. Then culturing for 4h at 37 ℃ with 220rpm of a shaking table, and culturing for 20h at 16 ℃ with 220rpm of a shaking table; after the culture, the bacterial liquid is placed at 13000g and centrifuged for 7min at 4 ℃, 4mL of bacterial liquid thalli after induction expression are collected, and a supernatant culture medium is discarded. The cells were resuspended thoroughly in 750. mu.L of precooled protein buffer, and the pellet was carefully transferred to a 1.5mL centrifuge tube. Then, ultrasonication was carried out:
(1) fixing 1.5mL of a centrifugal tube containing the bacterial liquid by using a buoy, and suspending the centrifugal tube on an ice-water mixture;
(2) setting the program of the crusher: the power is 21%, the crushing is carried out for 1s, the stopping is carried out for 3s, and the crushing time is set to be 3min and 30 s;
(3) centrifuging the crushed solution at 13000g for 12min at 4 deg.C, collecting supernatant and precipitate, and detecting.
(4) The pellet was resuspended in 30. mu.L of 8M urea.
And (3) detecting the small-amount induced expression of the protein:
(1) preparation of a sample:
10 μ L of protein sample
5×SDS-PAGE Loading Buffer 4μL
(2) Electrophoresis: and (3) dropping the prepared protein samples into the glue holes in a certain sequence, and setting a 15mA constant current electrophoresis.
(3) Dyeing: the running albumin glue was placed in fresh staining solution (Coomassie brilliant blue) and stained on a horizontal shaker for 25 min.
(4) And (3) decoloring: and (3) decoloring the protein glue by using the prepared fresh decoloring solution.
(5) Scanning: scanning the destained albumin glue, and storing the picture (for later use).
And (3) purifying the recombinant protein:
(1) bacterial liquid enlargement induction
And selecting a bacterium solution under the optimal expression condition for activation, and carrying out a large amount of induced expression.
(2) Centrifugal bacteria collection
The cells were centrifuged at 8000g and 25 ℃ for 25min to collect the cells from the bottom of the flask.
(3) Ultrasonic crushing:
diluting thallus collected by each 1L of bacterial liquid to about 100mL by using a precooled binding buffer, and performing high-pressure crushing by using a high-pressure crushing instrument, wherein the bacterial liquid is circularly crushed for 3-4 times.
(4) Centrifuging and taking a supernatant:
and (3) centrifuging the high-pressure crushed thalli for 30min at 4 ℃ under 13000g, collecting supernatant protein after centrifugation, and placing the supernatant protein on ice to prevent protein degradation.
(5) Nickel column affinity chromatography
a) Adding a Binding Buffer balance nickel column with 5 times of column volume;
b) filtering the protein sample by using a filter membrane of 0.45 mu m, enabling the sample to slowly flow through the nickel column filler at the low temperature of 4 ℃, fully combining the protein sample with the filler in the column, and collecting flow-through liquid;
c) eluting the protein with a gradient concentration of imidazole (20mM, 50mM, 100mM, 250mM, 500mM imidazole);
d) collecting eluents with various imidazole concentrations, and detecting the purity of a sample;
e) excising the sumo tag from the collected sample using a commercially available enzyme;
f) performing nickel column affinity chromatography again, collecting unbound protein, and detecting sample purity;
the results of the detection are shown in FIG. 2. The results show that the purified protein is a single band.
Example 4 enzyme inhibition Activity assay
After the two proteins BmSPI51 and BmSPI51M are incubated overnight at different pH and different temperature, the pancreatin activity is detected, and the reaction system is as follows:
Trypsin(0.01M) 1μL
protein sample (0.1M) 1. mu.L
20mM Tris-HCl pH 8.0 make up 100. mu.L
After reacting for 30min at room temperature, adding 100 μ L of fluorogenic substrate, processing in dark for 60min, and reading data under the conditions of excitation wavelength of 490nm and emission wavelength of 535 nm. The results are shown in FIG. 3 and show that BmSPI51M has strong stability at different pH and different temperature.
Example 5 measurement of inhibitory Rate of fungal Activity
(1) Diluting Saccharomyces cerevisiae to concentration (1 × 10) for bacteriostasis experiment5one/mL);
(2) and (3) a bacteriostatic activity detection system: PDB medium, broth, protein samples were added to a sterile 96-well plate to make a total volume of 210. mu.L (70. mu.L broth, 70. mu.L medium, remaining 70. mu.L BmSPI51 protein and PBS buffer) to make a working concentration of 0.005M (about 0.03mg/mL) for BmSPI51 or BmSPI51M protein. EDTA solution at a concentration of 100mM was used as a positive control, and PBS (pH 7.5) was used as a negative control;
(3) and (3) fungus culture: culturing the prepared bacteriostatic system at 40rpm and 29 ℃;
(4) and (3) detecting the dynamics of the growth of the fungi: the system was subjected to OD once every 12h600Measuring the value, detecting the growth condition of the fungus, and recording the data;
(5) after the experiment is finished, a bacteriostatic growth kinetic curve graph is drawn according to the data, and the result is shown in fig. 4.
Through the verification of antibacterial activity, the growth curve of yeast is monitored by measuring the change of OD value in the growth process of fungi, and the inhibitory activity of two proteins on saccharomyces cerevisiae is observed, so that the inhibitory activity of BmSPI51M is obviously stronger than that of BmSPI 51.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of southwest
<120> bombyx mori BmSPI51 mutant protein BmSPI51M and preparation method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 168
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aatcctgatt gtttgctgcc aataaagacg ggcccttgca agggcagttt tccaagatat 60
gcctacgatt catcggagga caaatgcgtt gaattcatct acggaggttg ccaagcgaat 120
gctaataatt ttgagactat agaagagtgc gaagcagcct gcctgtaa 168
<210> 2
<211> 55
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Asn Pro Asp Cys Leu Leu Pro Ile Lys Thr Gly Pro Cys Lys Gly Ser
1 5 10 15
Phe Pro Arg Tyr Ala Tyr Asp Ser Ser Glu Asp Lys Cys Val Glu Phe
20 25 30
Ile Tyr Gly Gly Cys Gln Ala Asn Ala Asn Asn Phe Glu Thr Ile Glu
35 40 45
Glu Cys Glu Ala Ala Cys Leu
50 55
<210> 3
<211> 168
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aatcctgatt gtttgctgcc aataaagacg ggcccttgca ggggcggttt ccgaagatat 60
gcctacgatt catcggagga caaatgcgtt gaattcatct acggaggttg ccaagcgaat 120
gctaataatt ttgagactat agaagagtgc gaagcagcct gcctgtaa 168
<210> 4
<211> 55
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Asn Pro Asp Cys Leu Leu Pro Ile Lys Thr Gly Pro Cys Arg Gly Gly
1 5 10 15
Phe Arg Arg Tyr Ala Tyr Asp Ser Ser Glu Asp Lys Cys Val Glu Phe
20 25 30
Ile Tyr Gly Gly Cys Gln Ala Asn Ala Asn Asn Phe Glu Thr Ile Glu
35 40 45
Glu Cys Glu Ala Ala Cys Leu
50 55
Claims (9)
1. Bombyx mori BmSPI51 mutein BmSPI51M characterized in that: the amino acid sequence of the mutant protein BmSPI51M is shown in SEQ ID NO. 4.
2. A gene encoding a bombyx mori BmSPI51 mutein BmSPI51M characterized in that: the nucleotide sequence of the mutant protein BmSPI51M is shown as SEQ ID NO. 3.
3. A recombinant expression vector comprising the gene of claim 2.
4. The recombinant expression vector of claim 3, wherein: the expression vector is obtained by connecting a sequence shown by SEQ ID NO.3 into a PET-sumo expression vector through BamHI and NotI enzyme cutting sites.
5. A transformant containing the recombinant expression vector according to claim 3 or 4.
6. The transformant according to claim 5, characterized in that: the transformant is BL21(DE3) strain.
7. Method for the preparation of the mutein bmsipi 51M according to claim 1 or 2, characterized in that: the recombinant expression vector containing the sequence shown in SEQ ID NO.3 is expressed in a host.
8. Use of the mutein bmsipi 51M according to claim 1 or 2 for the preparation of fungal inhibitors.
9. Use according to claim 8, characterized in that: the fungus is yeast.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110174143.4A CN112940095B (en) | 2021-02-07 | 2021-02-07 | Bombyx mori BmSPI51 mutant protein BmSPI51M and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110174143.4A CN112940095B (en) | 2021-02-07 | 2021-02-07 | Bombyx mori BmSPI51 mutant protein BmSPI51M and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112940095A CN112940095A (en) | 2021-06-11 |
| CN112940095B true CN112940095B (en) | 2022-04-15 |
Family
ID=76244300
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110174143.4A Active CN112940095B (en) | 2021-02-07 | 2021-02-07 | Bombyx mori BmSPI51 mutant protein BmSPI51M and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112940095B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114805552B (en) * | 2021-09-15 | 2023-05-12 | 陕西理工大学 | BmSPI39 mutant and application thereof |
| CN113621056B (en) * | 2021-09-15 | 2022-06-24 | 陕西理工大学 | BmSPI38 mutant and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5663143A (en) * | 1988-09-02 | 1997-09-02 | Dyax Corp. | Engineered human-derived kunitz domains that inhibit human neutrophil elastase |
| CN102584987A (en) * | 2012-03-12 | 2012-07-18 | 西南大学 | Protease inhibitor BmSPI39 and preparation method and application thereof |
| CN105175534B (en) * | 2015-10-23 | 2019-04-02 | 西南大学 | Application and preparation method thereof of the silkworm silk cocoon antifungal protein enzyme inhibitor in antimycotic |
-
2021
- 2021-02-07 CN CN202110174143.4A patent/CN112940095B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112940095A (en) | 2021-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Noda et al. | The Botrytis cinerea xylanase Xyn11A contributes to virulence with its necrotizing activity, not with its catalytic activity | |
| CN112940095B (en) | Bombyx mori BmSPI51 mutant protein BmSPI51M and preparation method and application thereof | |
| EP2319927B1 (en) | Secretion expression of antibiotic peptide cad in bacillus subtilis and expression system of recombination bacillus subtilis | |
| von Bargen et al. | Interactions between the tomato spotted wilt virus movement protein and plant proteins showing homologies to myosin, kinesin and DnaJ-like chaperones | |
| CN113980112A (en) | Expression vector and expression product of king cobra antimicrobial peptide OH-CATH30 and construction and preparation method thereof | |
| Outram et al. | The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and provides insight into Snn3 recognition and pro‐domain protease processing of fungal effectors | |
| Day et al. | Chloroplast outer membrane β-barrel proteins use components of the general import apparatus | |
| CN111349177B (en) | Preparation method and application of fusion antibacterial peptide CAT | |
| CN110922457A (en) | Plant immune induced resistance protein FgPII1 secreted by fusarium graminearum and application thereof | |
| Weinthal et al. | Characterization of nuclear localization signals in the type III effectors HsvG and HsvB of the gall-forming bacterium Pantoea agglomerans | |
| CN110776569B (en) | Diblock fusion protein with adhesion-freeze resistance dual functions and synthesis method and application thereof | |
| CN108003243B (en) | Method for purifying protein | |
| CN109134662B (en) | Visual antibacterial peptide fusion protein, and preparation method and application thereof | |
| CN107058363B (en) | Method for realizing efficient secretory expression of small molecule peptides based on amyloid protein and application thereof | |
| CN109305996B (en) | Fusarium graminearum secretory protein elicitor FgHrip1 and application thereof | |
| WO2017156457A1 (en) | Multimeric defensin proteins and related methods | |
| CN112457413B (en) | Method for fusion expression of antibacterial peptide by using SUMO | |
| CN109055416A (en) | A kind of preparation method of soluble recombinant protein | |
| US8357509B2 (en) | Microbial expression of tobacco osmotin for biocidal and medical applications | |
| JPH06501396A (en) | Isolation and characterization of a novel protease from Streptomyces lividans | |
| Liu et al. | Heterologous expression and purification of Cry1Ac22 toxin from Bacillus thuringiensis W015-1 | |
| CN110616230B (en) | Method for promoting secretory expression of zearalenone degrading enzyme ZHD protein and application | |
| CN108841846B (en) | Recombinant fluorescent protein with large Stokes shift and preparation method thereof | |
| CN111850022A (en) | Method for quickly adding or replacing protein tag of target gene | |
| CN105349536A (en) | Specific PCR primer for pathogenesis-related protein PR10 gene of tobacco and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |