CN114805552A - BmSPI39 mutant and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a BmSPI39 mutant and application thereof. The BmSPI39 is composed of the 25 th to the 98 th positions in SEQ ID NO.1, and the BmSPI39 mutant is obtained by mutating alanine at the 56 th position shown in SEQ ID NO.1 of the BmSPI39 amino acid sequence 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 inhibitory activity on subtilisin and elastase, and also obtains trypsin inhibitory activity after mutation into arginine or lysine, and the mutant can be used for preparing trypsin inhibitors and has good application prospect.

Description

BmSPI39 mutant and application thereof

Technical Field

The invention is a divisional application with application number 202111080025.3, and the application date is 09/15/2021.

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a BmSPI39 mutant and application thereof.

Background

Silkworm is a spun silk insect with huge economic value, has a great amount of basic research accumulation, and becomes one of the best models of insect biochemistry, genetics and genomics. The previous researches of the inventor carry out systematic identification on immunity-related silkworm protease inhibitors, and the inventor finds that a plurality of TIL (trypsin inhibitor-like cysteine-rich domain) protease inhibitors are up-regulated and expressed after microbial feeding infection, and suggests that the TIL protease inhibitors can participate in the immune process of silkworms. Further research shows that the TIL protease inhibitor BmSPI39 of a silkworm not only can strongly inhibit activities of subtilisin, proteinase K, Beauveria bassiana body wall degradation protease CDEP-1 and aspergillus melleus protease, but also can block excessive and harmful blackening of the silkworm induced by the Beauveria bassiana body wall degradation protease CDEP-1.

The activity and function of the BmSPI39 are clear, but the action mechanism of the activity is not completely clear, and the research on potential amino acid sites which can 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

One of the objects of the present invention is to provide BmSPI39 mutant, BmSPI39 consisting of SEQ ID No.1 from position 25 to position 98, and BmSPI39 mutant obtained by mutating the BmSPI39 amino acid sequence, alanine at position 56 as shown in SEQ ID No.1, to 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 other purpose of the invention is to provide a construction method of the BmSPI39 mutant, which is to perform site-directed mutagenesis on the gene sequence of the wild type 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 site-directed mutation primers, so that the upstream primer and the downstream primer both have mutation sites;

s2, taking a BmSPI39-p28 vector as a template, using DNA polymerase, carrying out PCR amplification by using a specific mutation primer, and detecting a reaction product by agarose gel electrophoresis;

s3, carrying out enzyme digestion reaction by using DpnI to process a PCR product;

s4, transforming the PCR product treated by the enzyme digestion reaction into a Trans1-T1 competent cell, selecting positive clone for sequencing verification, and extracting mutant plasmid;

s5, transferring the mutant plasmid into a host expression strain, and inducing expression to obtain the BmSPI39 mutant.

It is a further object of the present invention to provide genes encoding the BmSPI39 mutants.

The fourth object of the present invention is to provide a plasmid carrying the gene.

The fifth purpose of the invention is to provide a host expression strain carrying the plasmid.

It is yet another object of the present invention to provide the use of the BmSPI39 mutant as a subtilisin and elastase inhibitor.

The seventh object of the present invention is to provide the use of the BmSPI39 mutant as a subtilisin, elastase and trypsin inhibitor, wherein the BmSPI39 mutant is a BmSPI39 mutant in which the BmSPI39 amino acid sequence, alanine at position 56 as shown in SEQ ID No.1, is mutated to lysine or arginine.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the amino acid sequence of BmSPI39, and alanine at position 56 as shown in SEQ ID NO.1 are subjected to site-directed mutagenesis according to the structure analysis of BmSPI39, so that the obtained mutants of BmSPI39(A56R), BmSPI39(A56K), BmSPI39(A56S), BmSPI39(A56T), BmSPI39(A56Q), BmSPI39(A56Y), BmSPI39(A56M), and BmSPI39(A56L) have enhanced subtilisin inhibition activity. BmSPI39(A56D), BmSPI39(A56E), BmSPI39(A56H), BmSPI39(A56C), BmSPI39(A56V), BmSPI39(A56N), BmSPI39(A56I), BmSPI39(A56F), BmSPI39(A56W) have reduced inhibitory activity against subtilisin. Except that the elastase inhibitory activity of BmSPI39(a56E), BmSPI39(a56S), BmSPI39(a56T), BmSPI39(a56Q) was increased, the elastase inhibitory activity of the remaining mutants was decreased. Further, BmSPI39(A56R) and BmSPI39(A56K) also acquired 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 is a partial BmSPI39 mutant PCR product (A) and a agarose gel electrophoresis image of the mutant plasmid (B).

FIG. 2 is an SDS-PAGE analysis of the BmSPI39 mutant protein, wherein "M" represents the protein molecular weight standard. "S" refers to soluble protein. "U" means insoluble protein. "Control" is a cell lysate of BL21(DE3) strain transformed with p28 empty vector. Arrows indicate that the bmsipi 39 mutant expresses protein.

FIG. 3 shows the elastase activity staining of the BmSPI39 mutant, wherein "Control" is the cell lysate of BL21(DE3) strain transformed with p28 empty vector. "EI" indicates elastase inhibitory activity and "CB" indicates Coomassie blue staining. The arrow indicating elastase inhibitory activity indicates a protease activity inhibition band. The arrow of coomassie brilliant blue staining indicates the coomassie brilliant blue staining band corresponding to the protease inhibitor.

FIG. 4 shows a comparison of the activity of BmSPI39 mutants against different proteases, with silkworm larvae hemolymph at 5 th day of age as positive control. "Control" is a cell lysate of BL21(DE3) strain transformed with p28 empty vector. "SI" refers to subtilisin inhibitory activity. "CI" means chymotrypsin inhibitory activity. "TI" means trypsin inhibitory activity. "EI" means elastase inhibitory activity. "CB" means Coomassie brilliant blue staining. The arrow for trypsin inhibitory activity indicates the protease activity inhibition band, and the arrow for coomassie brilliant blue staining indicates the coomassie brilliant blue staining band corresponding to the protease inhibitor.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

The domestic serine protease inhibitor BmSPI39-p28 recombinant expression vector is stored by the research institute of physiology and application of vitamin D of Shanxi university of science and technology.

Example 1

Construction and activity research of BmSPI39 mutant

1. Mutant primer design

The amino acid sequence of BmSPI39 consists of positions 25 to 98 of SEQ ID No.1, wherein positions 1 to 24 are signal peptide sequences, and with reference to the gene sequence of BmSPI39, as shown in SEQ ID No.2, site directed mutagenesis primers were designed for PCR amplification of BmSPI39 from 5 'to 3' of the gene. Mutant templates, desired mutations, DNA polymerase and primer sequences for bmsipi 39 are shown in table 1, respectively.

2. PCR amplification

(1) When the DNA Polymerase used for PCR amplification was FastPfu DNA Polymerase, the reaction system (25. mu.L) is shown in Table 2, and the amplification procedure is shown in Table 3. The amplification products were detected by electrophoresis on a 1% agarose gel.

TABLE 2 PCR reaction System

TABLE 3 PCR amplification procedure

(2) When the DNA Polymerase used for PCR amplification was Easypfu DNA Polymerase, the reaction system (25. mu.L) is shown in Table 4, and the amplification procedure is shown in Table 5. The PCR product was detected by electrophoresis on a 1% agarose gel.

TABLE 4 PCR reaction System

TABLE 5 PCR amplification procedure

3. The PCR amplification product is digested by Dpn I, and the template plasmid is removed.

Enzyme cutting conditions are as follows: 30min at 37 ℃. The Dpn I cleavage system is shown in Table 6.

TABLE 6 Dpn I cleavage System

4. Transferring the PCR product after the Dpn I treatment into a Trans1-T1 competent cell by the following steps:

(1) taking out competent cells (100 μ L) from an ultralow temperature refrigerator at minus 80 ℃, putting the competent cells into ice until the competent cells are in a half-melting state, separating out 50 μ L of the competent cells, putting the competent cells into another sterilized and precooled sterile centrifuge tube, adding 10 μ L of PCR products treated by Dpn I into each tube, slightly blowing and sucking the PCR products uniformly, and standing the mixture on the ice for 30 min.

(2) Heat shock at 42 deg.C for 90s, gently take out, and cool in ice for 5 min.

(3) 900. mu.L of non-resistant liquid medium was added and incubated at 37 ℃ and 220rpm for 1 h.

(4) Centrifuging at 3500rpm for 5min, discarding 800 μ L supernatant, blowing and sucking the rest bacteria liquid, mixing, adding into solid culture medium plate, uniformly coating for 5min, and performing inverted culture at 37 deg.C for about 12 h.

5. Gene sequencing verification and glycerol strain preparation

(1) Selecting single colonies which grow fully in a flat plate and have round and moist edges, and selecting the single colonies into a 1.5mL centrifuge tube, and carrying out shake culture for 3-4 h, wherein the conditions are as follows: 200. mu.L of the bacterial sample was sequenced at 37 ℃ and 220rpm by Biotechnology engineering (Shanghai) Ltd.

(2) Preparing glycerol bacteria: adding 200 μ L of 50% glycerol into 300 μ L of bacteria liquid, mixing, quick freezing with liquid nitrogen, and storing at-80 deg.C for a long time.

6. Plasmid extraction

The extraction procedure refers to the Trans plasmid extraction kit to obtain the BmSPI39 mutant plasmid.

7. Transformation into host expression strains

The bmsipi 39 mutant plasmid was transferred into BL21(DE3) competent cells and the transformation procedure was referenced to step 4.

8. Inducible expression

(1) A single colony was picked up into a 1.5mL EP tube and cultured with shaking at 37 ℃ and 220rpm for 12 hours.

(2) Adding 150 μ L of bacterial liquid into 15mL of liquid culture medium, and performing shaking culture at 37 deg.C and 220rpm to OD ₆₀₀ ＝0.6～1.0。

(3) IPTG was added to a final concentration of 0.2mM for induction expression, and the optimum induction conditions were 37 ℃ at 220rpm for 5 hours.

(4) After centrifugation at 4 ℃ and 6000rpm for 20min, the cells were collected, resuspended by pipetting with 1 Xbinding buffer (1.5mL), and all the cells were transferred to a 2.0mL EP tube.

(5) Centrifugation was carried out at 6000rpm at 4 ℃ for 10min, the supernatant was discarded, and the suspended cells were aspirated with 1 Xbinding buffer (1.0 mL).

(6) Centrifugation at 4 ℃ and 6000pm for 10min, discarding the supernatant, and resuspension by pipetting 1 Xthe binding buffer (450. mu.L).

(7) And (4) carrying out ultrasonic crushing for 15min until the bacterial liquid becomes transparent. Centrifugation at 16000g for 30min at 4 ℃ separates the supernatant from the pellet, which is resuspended by aspiration with 1 Xbinding buffer (250. mu.L).

9、SDS-PAGE

Protein samples were compressed with 5% concentrated gel (Table 7), protein was separated by 16.5% SDS-PAGE (Table 8), stained with Coomassie Brilliant blue, and the procedure was as follows:

(1) 10. mu.L of each supernatant and precipitate was collected, and 5. mu.L of 3 XSDS loading buffer was mixed well and boiled for 10 min.

(2) And (3) dropping all samples into the gel holes, performing constant current electrophoresis until bromophenol blue completely enters the electrophoresis buffer solution, and leaving no residue in the gel. Electrophoresis conditions: the gel was concentrated to 10mA and the gel was separated to 15 mA.

(3) Coomassie brilliant blue staining: the concentrated gel was removed, the gel was separated by soaking in Coomassie brilliant blue staining solution and stained with gentle shaking for 15 min.

(4) And (3) decoloring: and (4) after the dyeing solution is recovered, decoloring the dyeing solution by using a Coomassie brilliant blue decoloring solution until the background is transparent and the strips are clear.

TABLE 75% SDS-PAGE gels

TABLE 816.5% SDS-PAGE gels

10. Activity staining of BmSPI39 mutant

The 10% separation gel formulation is shown in Table 9, and the 4% concentrate gel formulation is shown in Table 10. The specific steps of the 4 Xnative-PAGE in-gel active staining are as follows

(1) Protein samples were run with 4 × Native-PAGE loading buffer at 1: and 3, uniformly mixing in proportion, completely dropping into the glue holes, and stopping electrophoresis when the constant current electrophoresis is carried out until the bromophenol blue is 2-3 mm away from the glue edge. Electrophoresis conditions: the gel was concentrated at 10mA and separated at 15mA at 4 ℃.

(2) The concentrated gel was cut off, and the gel was incubated in protease solution at 37 ℃ for 30min in the dark at 45 rpm.

(4) Recovering the protease solution from the ddH ₂ And O, slightly cleaning the surface of the gel twice, and standing for 30min in a dark place at 37 ℃.

(5) Adding a mixed solution of a staining solution and a matrix solution, and staining each gel for 15min at 37 ℃ in a dark place at 45 rpm.

(6) The staining solution was decanted off and ddH was added ₂ And O stops the reaction.

TABLE 910% 4 × Native-PAGE separation gel

TABLE 104% 4 × Native-PAGE gel concentrate

Wherein, the liquid A: tris 36.3g, 1M HC 148 mL, TEMED 0.230mL, plus 100mL ddH ₂ And (4) fixing the volume by O, and storing at 4 ℃ in a dark place.

And B, liquid B: tris 5.98g, 1M HC 148 mL, TEMED 0.46mL, plus 100mL ddH ₂ And (4) fixing the volume by O, and storing at 4 ℃ in a dark place.

And C, liquid C: acrylamide 30g, methylene bisacrylamide 0.8g, adding 100mL ddH ₂ O constant volume, filtering with 0.45 μm filter membrane, and storing at 4 deg.C in dark place.

And (3) liquid D: acrylamide 10g, methylene bisacrylamide 2.5g, adding 100mL ddH ₂ O constant volume, filtering with 0.45 μm filter membrane, and storing at 4 deg.C in dark place.

E, liquid E: riboflavin 8mg, add 200mL ddH ₂ And (4) fixing the volume by O, and storing at 4 ℃ in a dark place.

And G, liquid: ammonium persulfate 0.7g, 100mL ddH ₂ And (4) fixing the volume by O, and storing at 4 ℃ in a dark place.

The principle of active staining is as follows: protease decomposes matrix (N-acetyl-DL-phenylalanine-beta-naphthyl ester, N-acetyl-D, L-phenylalanine-beta-naphthol ester), and the generated beta-naphthol dyes the gel to be purple red through diazo coupling reaction. The endoprotease inhibitor inhibits protease activity and therefore is not stained at the site where the inhibitor is present, which appears as a white band.

11. Results and analysis

(1) The invention designs 19 pairs of site-directed mutagenesis primers, and PCR amplification is carried out by taking a BmSPI39 wild type gene sequence as a template. The PCR product was transformed into Trans1-T1 competent cells, and plasmid extraction was performed after sequencing. The result shows that the PCR amplification product of the P1 site mutant of the BmSPI39 presents a single band, the band is bright, the molecular weight is between 3000 and 5000bp (figure 1A), and the plasmid extraction is good and consistent with the expectation (figure 1B).

(2) In order to express high amount of protein, BL21(DE3) was used as the optimal host strain for BmSPI39 to induce expression, and 16.5% SDS-PAGE was selected to separate and detect BmSPI39 mutant protein. The results are shown in FIG. 2: the BmSPI39 mutant protein is expressed in a soluble form in the supernatant, the expression amount is high, the expression amount is almost not expressed in the precipitate, the apparent molecular weight is between 6.5 and 9.5kDa, and the result shows that the BmSPI39 mutant protein is normally expressed.

(3) The invention takes N-acetyl-D, L-phenylalanine-beta-naphthyl ester (N-acetyl-D, L-phenylalanine-beta-naphthyl ester) as a substrate, and 6 mu L of BmSPI39 protein supernatant is extracted. The results of active staining show that bmsipi 39 is able to strongly inhibit elastase activity. This is the first clear inhibitory activity of bombyx mori protease inhibitors on elastase (fig. 3).

(4) SDS-PAGE detection shows that the P1 site mutant protein of the BmSPI39 is expressed in high amount in the supernatant and can be used for subsequent experimental study. Based on our previous analysis of potential amino acid sites affecting the inhibitory specificity of small molecule TIL class protease inhibitors, the P1 residue may be one of the key sites affecting the inhibitory activity and inhibitory specificity of the protease inhibitor bmsipi 39.

The results show that: mutants of BmSPI39(a56R), BmSPI39(a56K), BmSPI39(a56S), BmSPI39(a56T), BmSPI39(a56Q), BmSPI39(a56Y), BmSPI39(a56M), BmSPI39(a56L) have enhanced inhibitory activity against subtilisin, compared to BmSPI39 (WT). BmSPI39(A56D), BmSPI39(A56E), BmSPI39(A56H), BmSPI39(A56C), BmSPI39(A56V), BmSPI39(A56N), BmSPI39(A56I), BmSPI39(A56F), BmSPI39(A56W) have reduced inhibitory activity against subtilisin. Except for the enhancement of elastase inhibitory activity by BmSPI39(a56E), BmSPI39(a56S), BmSPI39(a56T), BmSPI39(a56Q), the remaining mutants had reduced elastase inhibitory activity (fig. 4). Notably, BmSPI39(a56R), BmSPI39(a56K) had 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. Therefore, it is intended that the appended claims be interpreted as including 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 changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

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Claims (3)

1. Use of a bmsipi 39 mutant in the preparation of a protease inhibitor, wherein the protease is subtilisin, elastase, and trypsin; BmSPI39 consists of positions 25 to 98 of SEQ ID NO. 1;

   the BmSPI39 mutant is obtained by mutating alanine at position 56 in the amino acid sequence of BmSPI39 as shown in SEQ ID NO.1 into lysine or arginine.
2. 2. The use of claim 1 wherein the BmSPI39 mutant is obtained by site directed mutagenesis of the wild type BmSPI39 gene sequence shown in SEQ ID No.2 using a site directed mutagenesis primer.
3. 3. The use of claim 2 wherein the BmSPI39 mutant is constructed by a method comprising the steps of:

   s1, designing site-directed mutation primers, so that the upstream primer and the downstream primer both have mutation sites;

   s2, taking a BmSPI39-p28 vector as a template, using DNA polymerase, carrying out PCR amplification by using a specific mutation primer, and detecting a reaction product by agarose gel electrophoresis;

   s3, carrying out enzyme digestion reaction by using Dpn I to process a PCR product;

   s4, transforming the PCR product treated by the enzyme digestion reaction into a Trans1-T1 competent cell, selecting positive clone for sequencing verification, and extracting mutant plasmid;

   s5, transferring the mutant plasmid into a host expression strain, and inducing expression to obtain the BmSPI39 mutant.

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