CN114805552B - BmSPI39 mutant and application thereof - Google Patents
BmSPI39 mutant and application thereof Download PDFInfo
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- CN114805552B CN114805552B CN202210609374.8A CN202210609374A CN114805552B CN 114805552 B CN114805552 B CN 114805552B CN 202210609374 A CN202210609374 A CN 202210609374A CN 114805552 B CN114805552 B CN 114805552B
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a BmSPI39 mutant and application thereof. BmSPI39 is formed by 25 th to 98 th bits in SEQ ID NO.1, and the BmSPI39 mutant is obtained by mutating alanine at 56 th bit of BmSPI39 amino acid sequence as shown in SEQ ID NO.1 into arginine, lysine, serine, threonine, glutamine, tyrosine, methionine, leucine, aspartic acid, glutamic acid, histidine, cysteine, valine, asparagine, isoleucine, phenylalanine, tryptophan, proline or glycine. The BmSPI39 mutant has inhibition activity on subtilisin and elastase, and has trypsin inhibition activity after being mutated into arginine or lysine, and the mutant can be used for preparing trypsin inhibitors, and has good application prospect.
Description
Technical Field
The invention is a divisional application with the application number of 202111080025.3, and the application date is 2021, 09 and 15.
The invention belongs to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a BmSPI39 mutant and application thereof.
Background
Silkworm is a silk insect with great economic value, has a great amount of basic research accumulation, and becomes one of the best models of insect biochemistry, genetics and genomics. Earlier studies carried out systematic identification of immune-related silkworm protease inhibitors, found that many TIL (trypsin inhibitor-like cysteine-rich) protease inhibitors were up-regulated after microbial feeding infection, suggesting that TIL protease inhibitors may be involved in the silkworm immune process. Further researches show that the TIL protease inhibitor BmSPI39 of the silkworm can not only strongly inhibit activities of subtilisin, proteinase K, beauveria bassiana body wall degradation protease CDEP-1 and aspergillus melleus protease, but also block excessive and harmful blackening of the silkworm induced by beauveria bassiana body wall degradation protease CDEP-1.
The activity and function of BmSPI39 are clear, but the action mechanism of the activity is not completely clear, and the research on potential amino acid sites which possibly influence the inhibition specificity of the TIL protease inhibitor is limited, which directly influences the genetic modification and industrial application of the inhibitor.
Disclosure of Invention
It is an object of the present invention to provide a BmSPI39 mutant, wherein BmSPI39 is composed of 25 th to 98 th bits in SEQ ID NO.1, and the BmSPI39 mutant is obtained by mutating alanine at 56 th bit of BmSPI39 amino acid sequence as shown in SEQ ID NO.1 into arginine (R), lysine (K), serine (S), threonine (T), glutamine (Q), tyrosine (Y), methionine (M), leucine (L), aspartic acid (D), glutamic acid (E), histidine (H), cysteine (C), valine (V), asparagine (N), isoleucine (I), phenylalanine (F), tryptophan (W), proline (P) or glycine (G).
The second object of the invention is to provide a construction method of the BmSPI39 mutant, which comprises the step of carrying out site-directed mutagenesis on a gene sequence of a wild BmSPI39 shown as SEQ ID NO.2 by using a site-directed mutagenesis primer to obtain the BmSPI39 mutant.
Further, the construction method of the BmSPI39 mutant is characterized by comprising the following steps:
s1, designing a site-directed mutagenesis primer, so that both an upstream primer and a downstream primer have mutation sites;
s2, using a BmSPI39-p28 carrier as a template, using DNA polymerase, carrying out PCR reaction amplification by using a specific mutation primer, and detecting a reaction product by using agarose gel electrophoresis;
s3, performing enzyme digestion reaction by using DpnI to treat a PCR product;
s4, converting the PCR product treated by the enzyme digestion reaction into a Trans1-T1 competent cell, selecting positive clones for sequencing verification, and extracting mutant plasmids;
s5, transferring the mutant plasmid into a host expression strain, and performing induced expression to obtain a BmSPI39 mutant.
It is a further object of the present invention to provide a gene encoding the BmSPI39 mutant.
It is a fourth object of the present invention to provide a plasmid carrying the gene.
It is a fifth object of the present invention to provide host expression strains harboring said plasmids.
The sixth object of the present invention is to provide the use of said BmSPI39 mutant as subtilisin and elastase inhibitor.
The invention aims at providing the application of the BmSPI39 mutant as a subtilisin, elastase and trypsin inhibitor, wherein the BmSPI39 mutant is a BmSPI39 mutant in which the 56 th alanine shown as SEQ ID NO.1 is mutated into lysine or arginine.
Compared with the prior art, the invention has the following beneficial effects:
according to BmSPI39 structural analysis, the invention carries out site-directed mutation on alanine at the 56 th site of BmSPI39 amino acid sequence as shown in SEQ ID NO.1, thus the obtained mutants BmSPI39 (A56R), bmSPI39 (A56K), bmSPI39 (A56S), bmSPI39 (A56T), bmSPI39 (A56Q), bmSPI39 (A56Y), bmSPI39 (A56M) and BmSPI39 (A56L) have enhanced inhibition activity on subtilisin. BmSPI39 (A56D), bmSPI39 (A56E), bmSPI39 (A56H), bmSPI39 (A56C), bmSPI39 (A56V), bmSPI39 (A56N), bmSPI39 (A56I), bmSPI39 (A56F) and BmSPI39 (A56W) have reduced inhibitory activity on subtilisin. Except that BmSPI39 (A56E), bmSPI39 (A56S), bmSPI39 (A56T) and BmSPI39 (A56Q) have enhanced elastase inhibitory activity, the other mutants have reduced elastase inhibitory activity. Furthermore, bmSPI39 (A56R) and BmSPI39 (A56K) also obtained trypsin inhibitory activity.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 shows gel electrophoresis detection of PCR products (A) of part of BmSPI39 mutant and mutant plasmids (B).
FIG. 2 is a SDS-PAGE analysis of BmSPI39 mutant proteins, wherein "M" represents a protein molecular weight standard. "S" means a soluble protein. "U" means insoluble protein. "Control" is the cell lysate of BL21 (DE 3) strain transformed with the p28 empty vector. Arrows indicate the BmSPI39 mutant expressed protein.
FIG. 3 shows the activity staining of elastase of BmSPI39 mutant, in which "Control" is the cell lysate of BL21 (DE 3) strain transformed with p28 empty vector. "EI" means elastase inhibitory activity and "CB" means Coomassie brilliant blue staining. Arrows of elastase inhibitory activity indicate protease activity inhibition bands. The coomassie blue stained arrow indicates the coomassie blue stained band corresponding to the protease inhibitor.
FIG. 4 shows comparison of BmSPI39 mutant activities against different proteases, with silkworm larva haemolymph at 5 th day of age 5 as positive control. "Control" is the cell lysate of BL21 (DE 3) strain transformed with the p28 empty vector. "SI" means subtilisin inhibitory activity. "CI" means chymotrypsin inhibitory activity. "TI" indicates trypsin inhibitory activity. "EI" means elastase inhibitory activity. "CB" means Coomassie brilliant blue staining. Arrows of trypsin inhibition activity indicate protease activity inhibition bands, and arrows stained with coomassie brilliant blue indicate coomassie brilliant blue staining bands corresponding to protease inhibitors.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
The recombinant expression vector of the silkworm serine protease inhibitor BmSPI39-p28 is stored by the vitamin D physiological and application research institute of Shaanxi university.
Example 1
BmSPI39 mutant construction and activity research
1. Mutant primer design
The amino acid sequence of BmSPI39 is composed of 25 th to 98 th positions in SEQ ID NO.1, wherein the 1 st to 24 th positions are signal peptide sequences, and the BmSPI39 gene sequence is referred to, as shown in SEQ ID NO.2, and site-directed mutagenesis primers are designed for the BmSPI39 for the PCR amplification of 5 'to 3' of the gene. The mutant template, desired mutation, DNA polymerase and primer sequences of BmSPI39 are shown in table 1, respectively.
2. PCR amplification
(1) When FastPfu DNA Polymerase was used as a DNA polymerase for PCR amplification, the reaction system (25. Mu.L) was shown in Table 2, and the amplification procedure was shown in Table 3. The amplified product was detected by 1% agarose gel electrophoresis.
TABLE 2 PCR reaction System
TABLE 3 PCR amplification procedure
(2) When EasyPfu DNA Polymerase was used as a DNA polymerase for PCR amplification, the reaction system (25. Mu.L) was shown in Table 4, and the amplification procedure was shown in Table 5. The PCR products were detected by 1% agarose gel electrophoresis.
TABLE 4 PCR reaction System
TABLE 5 PCR amplification procedure
3. The PCR amplification product was digested with Dpn I and the template plasmid was removed.
Enzyme cutting conditions: 37℃for 30min. The Dpn I cleavage system is shown in Table 6.
TABLE 6 Dpn I cleavage System
4. Transferring the PCR product treated by Dpn I into a Trans1-T1 competent cell, wherein the method comprises the following steps:
(1) Taking out competent cells (100 mu L) from a refrigerator at-80 ℃ and ultra-low temperature, putting the competent cells into ice until 50 mu L of competent cells are separated out in a semi-molten state, putting the competent cells into another sterile centrifuge tube subjected to sterilization and precooling, adding 10 mu L of PCR products treated by Dpn I into each tube, slightly blowing and sucking the PCR products, uniformly mixing the PCR products, and standing the PCR products on ice for 30min.
(2) Heat shock at 42 ℃ for 90s, gently take out and cool in ice for 5min.
(3) 900. Mu.L of non-resistant liquid medium was added and incubated at 37℃for 1h at 220 rpm.
(4) Centrifuging at 3500rpm for 5min, discarding 800 μl supernatant, blowing and sucking the rest bacterial liquid, mixing, adding into solid culture medium plate, spreading for 5min, and culturing at 37deg.C for 12 hr.
5. Gene sequencing verification and glycerinum preparation
(1) Selecting single colonies with full growth and round edges in a flat plate, picking the single colonies into a 1.5mL centrifuge tube, and carrying out shake culture for 3-4 hours under the conditions that: 200. Mu.L of the bacterial sample was taken at 37℃and 220rpm and sent to the engineering (Shanghai) Co., ltd for sequencing.
(2) Preparing glycerol bacteria: 200 mu L of 50% glycerol is taken, 300 mu L of bacterial liquid is added, and after uniform mixing and quick freezing by liquid nitrogen, the mixture is placed at the temperature of minus 80 ℃ for long-term storage.
6. Plasmid extraction
The extraction step refers to a Trans plasmid extraction kit to obtain the BmSPI39 mutant plasmid.
7. Transformation into host expression strains
The BmSPI39 mutant plasmid was transferred into BL21 (DE 3) competent cells, the transformation step was referred to in step 4.
8. Induction of expression
(1) Single colonies were picked into 1.5mL EP tubes and cultured with shaking at 220rpm at 37℃for 12h.
(2) 150 mu L of bacterial liquid is added into 15mL of liquid culture medium, and the culture is oscillated at 37 ℃ and 220rpm until the OD is reached 600 ＝0.6～1.0。
(3) IPTG was added to a final concentration of 0.2mM for induction expression, with optimal induction conditions of 37℃and 220rpm for 5h.
(4) The cells were collected by centrifugation at 6000rpm at 4℃for 20min, resuspended by pipetting with 1 Xthe binding buffer (1.5 mL), and all the cells were transferred to a 2.0mL EP tube.
(5) Centrifugation was performed at 6000rpm at 4℃for 10min, the supernatant was discarded, and the resuspended cells were aspirated by blowing 1 Xthe binding buffer (1.0 mL).
(6) Centrifugation was performed at 6000pm at 4℃for 10min, the supernatant was discarded, and the 1 Xbinding buffer (450. Mu.L) was subjected to a pipetting resuspension.
(7) And (5) ultrasonic crushing for 15min until the bacterial liquid becomes transparent. The supernatant was separated from the pellet by centrifugation at 16000g for 30min at 4℃and the pellet was resuspended by pipetting with 1 Xof binding buffer (250. Mu.L).
9、SDS-PAGE
Protein samples (Table 7) were compressed with 5% gel concentrate, and proteins were separated by 16.5% SDS-PAGE (Table 8), stained with Coomassie Brilliant blue, as follows:
(1) 10. Mu.L of each of the supernatant and the pellet was taken, 5. Mu.L of a 3 XSDS loading buffer was uniformly mixed, and the mixture was boiled for 10 minutes.
(2) And (3) all samples are spotted into gel holes, constant-current electrophoresis is carried out until bromophenol blue completely enters an electrophoresis buffer solution, and no residue exists in the gel. Electrophoresis conditions: the gel was concentrated to 10mA and the gel was separated to 15mA.
(3) Coomassie brilliant blue staining: the concentrated gel was excised, the separation gel was soaked with coomassie blue staining solution and stained with gentle shaking for 15min.
(4) Decoloring: after the dyeing liquid is recovered, the coomassie brilliant blue decolorization liquid is used for decolorization until the background is transparent, and the band is clear.
TABLE 7 5% SDS-PAGE concentrate gel
TABLE 8 16.5% SDS-PAGE separating gel
10. BmSPI39 mutant reactive staining
The 10% split gum formulation is shown in Table 9 and the 4% concentrated gum formulation is shown in Table 10. The specific steps of the active staining in the 4 Xnative-PAGE gel are as follows
(1) Protein samples were loaded onto 4 XNative-PAGE buffer at 1:3, uniformly mixing the materials in proportion, and uniformly dispensing all the materials into a glue hole, and stopping electrophoresis when the constant current electrophoresis is carried out until the distance between bromophenol blue and the edge of the glue is 2-3 mm. Electrophoresis conditions: concentrated gel at 4deg.C, concentrated gel at 10mA, and separated gel at 15mA.
(2) The concentrated gel was excised and the isolated gel was incubated in protease solution at 37℃and 45rpm for 30min in the absence of light.
(4) Recovering protease solution, and using ddH 2 O lightly wash the glue surface twice and keep it stand for 30min at 37 deg.C in dark.
(5) Adding the mixed solution of the dyeing solution and the matrix solution, and dyeing 40mL of each piece of glue for 15min at 37 ℃ and 45 rpm.
(6) Pouring out the staining solution and adding ddH 2 O stops the reaction.
TABLE 9 10%4 Xnative-PAGE separation gel
TABLE 10 4%4 XNative-PAGE concentrated gel
Wherein, solution A: tris 36.3g,1M HC1 48mL,TEMED 0.230mL, 100mL ddH 2 O constant volume, and storing at 4deg.C in dark place.
And (2) liquid B: tris 5.98g,1M HC1 48mL,TEMED 0.46mL, 100mL ddH 2 O constant volume, and storing at 4deg.C in dark place.
And C, liquid: 30g of acrylamide, 0.8g of methylene bisacrylamide and 100mL of ddH are added 2 O constant volume, 0.45 μm filter membrane suction filtration, 4 ℃ light-shielding preservation.
And D, liquid: 10g of acrylamide, 2.5g of methylene bisacrylamide and 100mL of ddH are added 2 O constant volume, 0.45 μm filter membrane suction filtration, 4 ℃ light-shielding preservation.
E, liquid: riboflavin 8mg, 200mL ddH was added 2 O constant volume, and storing at 4deg.C in dark place.
G, liquid: ammonium persulfate 0.7g, 100mL ddH 2 O constant volume, and storing at 4deg.C in dark place.
The principle of reactive staining is as follows: protease breaks down the matrix (N-acetyl-DL-phenylalanine-beta-naphthalate, N-acetyl-D, L-phenylalanine-beta-naphthol ester) and the resulting beta-naphthol dyes the gel to a purplish red color by diazo coupling. The inhibitors of the proteases within the gel inhibit the protease activity and therefore the site is not stained and the site of the inhibitor appears as a white band.
11. Results and analysis
(1) The invention designs 19 pairs of site-directed mutagenesis primers, and uses BmSPI39 wild type gene sequence as a template for PCR amplification. The PCR products were transformed into Trans1-T1 competent cells, sequenced and plasmid extracted. The results show that the PCR amplified product of the P1 site mutant of BmSPI39 appears as a single band, the band is brighter, the molecular weight is between 3000 and 5000bp (FIG. 1A), and the plasmid extraction is well consistent with expectations (FIG. 1B).
(2) In order to express high amounts of protein, BL21 (DE 3) is used as an optimal host strain of BmSPI39 for induction expression, and 16.5% SDS-PAGE is selected for separation detection of BmSPI39 mutant protein. The results are shown in FIG. 2: bmSPI39 mutant protein is expressed in a soluble form in supernatant, the expression level is high, the BmSPI39 mutant protein is hardly expressed in precipitation, the apparent molecular weight is between 6.5 and 9.5kDa, and the result shows that the BmSPI39 mutant protein is expressed normally.
(3) The invention takes N-acetyl-D, L-phenylalanine-beta-naphthalene ester (N-acetyl-D, L-phenylalanine-beta-phenylalanyl ester) as a substrate, and the BmSPI39 protein supernatant is taken to 6 mu L. The results of the activity staining showed that BmSPI39 can strongly inhibit elastase activity. This is the first time that it was clear that the inhibitory activity of the silkworm protease inhibitor against elastase (FIG. 3).
(4) The SDS-PAGE detection shows that the P1 site mutant protein of BmSPI39 is expressed in high amount in supernatant, and can be used for subsequent experimental study. Based on previous analysis of potential amino acid sites affecting the inhibition specificity of small molecule TIL type protease inhibitors, the P1 residue may be one of key sites affecting the inhibition activity and inhibition specificity of the protease inhibitor BmSPI 39.
The results show that: compared with BmSPI39 (WT), the mutants BmSPI39 (A56R), bmSPI39 (A56K), bmSPI39 (A56S), bmSPI39 (A56T), bmSPI39 (A56Q), bmSPI39 (A56Y), bmSPI39 (A56M) and BmSPI39 (A56L) have enhanced inhibitory activity on subtilisin. BmSPI39 (A56D), bmSPI39 (A56E), bmSPI39 (A56H), bmSPI39 (A56C), bmSPI39 (A56V), bmSPI39 (A56N), bmSPI39 (A56I), bmSPI39 (A56F) and BmSPI39 (A56W) have reduced inhibitory activity on subtilisin. Except that BmSPI39 (A56E), bmSPI39 (A56S), bmSPI39 (A56T), and BmSPI39 (A56Q) had enhanced elastase inhibitory activity, the remaining mutants had reduced elastase inhibitory activity (FIG. 4). Notably, bmSPI39 (A56R), bmSPI39 (A56K) has not only subtilisin and elastase inhibitory activity, but also trypsin inhibitory activity (FIG. 4).
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Sequence listing
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<400> 36
gggagcaatg cgcctacgca gctggtg 27
<210> 37
<211> 27
<212> DNA
<213> artificial sequence
<400> 37
caccagctgc gtaccggcat tgctccc 27
<210> 38
<211> 27
<212> DNA
<213> artificial sequence
<400> 38
gggagcaatg ccggtacgca gctggtg 27
<210> 39
<211> 30
<212> DNA
<213> artificial sequence
<400> 39
caccagctgc gtagtggcat tgctcccaac 30
<210> 40
<211> 29
<212> DNA
<213> artificial sequence
<400> 40
ttgggagcaa tgccactacg cagctggtg 29
Claims (3)
- The application of BmSPI39 mutant in preparing protease inhibitor, which is characterized in that the protease is subtilisin, elastase and trypsin; bmSPI39 is composed of 25 th to 98 th positions in SEQ ID NO. 1;the BmSPI39 mutant is characterized in that alanine at the 56 th position shown in SEQ ID NO.1 in a BmSPI39 amino acid sequence is mutated into lysine or arginine.
- 2. The use according to claim 1, characterized in that the BmSPI39 mutant is obtained by site-directed mutagenesis of the gene sequence of the wild BmSPI39 as shown in SEQ ID NO.2 with a site-directed mutagenesis primer.
- 3. The use according to claim 2, wherein the method for constructing the BmSPI39 mutant comprises the steps of:s1, designing a site-directed mutagenesis primer, so that both an upstream primer and a downstream primer have mutation sites;s2, using a BmSPI39-p28 carrier as a template, using DNA polymerase, carrying out PCR reaction amplification by using a specific mutation primer, and detecting a reaction product by using agarose gel electrophoresis;s3, performing enzyme digestion reaction by using Dpn I to treat a PCR product;s4, converting the PCR product treated by the enzyme digestion reaction into a Trans1-T1 competent cell, selecting positive clones for sequencing verification, and extracting mutant plasmids;s5, transferring the mutant plasmid into a host expression strain, and performing induced expression to obtain a BmSPI39 mutant.
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| CN102584987A (en) * | 2012-03-12 | 2012-07-18 | 西南大学 | Protease inhibitor BmSPI39 and preparation method and application thereof |
| CN105175534A (en) * | 2015-10-23 | 2015-12-23 | 西南大学 | Application of domestic silkworm cocoon antifungal protease inhibitor in fungus prevention and preparation method of domestic silkworm cocoon antifungal protease inhibitor |
| CN113151200A (en) * | 2021-05-10 | 2021-07-23 | 安徽省农业科学院水稻研究所 | Plant ACCase mutant protein and gene sequence and application thereof |
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| TW472085B (en) * | 1997-09-04 | 2002-01-11 | Nat Science Council | Mutant type subtilisin YaB and its application |
| CN112940095B (en) * | 2021-02-07 | 2022-04-15 | 西南大学 | Bombyx mori BmSPI51 mutant protein BmSPI51M and preparation method and application thereof |
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| CN102584987A (en) * | 2012-03-12 | 2012-07-18 | 西南大学 | Protease inhibitor BmSPI39 and preparation method and application thereof |
| CN105175534A (en) * | 2015-10-23 | 2015-12-23 | 西南大学 | Application of domestic silkworm cocoon antifungal protease inhibitor in fungus prevention and preparation method of domestic silkworm cocoon antifungal protease inhibitor |
| CN113151200A (en) * | 2021-05-10 | 2021-07-23 | 安徽省农业科学院水稻研究所 | Plant ACCase mutant protein and gene sequence and application thereof |
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