阅读说明：本技术 BmSPI38突变体及其应用 (BmSPI38 mutant and application thereof ) 是由 李游山 张�杰 于 2021-09-15 设计创作，主要内容包括：本发明属于基因工程和酶工程技术领域,具体涉及BmSPI38突变体及其应用。BmSPI38是由SEQ ID NO.1中第23位至第80位组成,所述BmSPI38突变体是将BmSPI38氨基酸序列中,如SEQ ID NO.1所示第54位的甘氨酸突变为突变为精氨酸、赖氨酸、丝氨酸、苏氨酸、丙氨酸、谷氨酰胺、天冬氨酸、组氨酸、半胱氨酸、脯氨酸、缬氨酸、天冬酰胺、酪氨酸、甲硫氨酸、亮氨酸、苯丙氨酸、异亮氨酸、色氨酸或谷氨酸获得的。本发明的BmSPI38突变体均对枯草杆菌蛋白酶和弹性蛋白酶具有抑制活性,且当突变为精氨酸或赖氨酸后,还获得了胰蛋白酶抑制活性,此类突变体可用于制备胰蛋白酶抑制剂,应用前景良好。(The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a BmSPI38 mutant and application thereof. The BmSPI38 is composed of 23 rd to 80 th bits of SEQ ID NO.1, and the BmSPI38 mutant is obtained by mutating glycine at 54 th bit shown in SEQ ID NO.1 in the amino acid sequence of BmSPI38 into arginine, lysine, serine, threonine, alanine, glutamine, aspartic acid, histidine, cysteine, proline, valine, asparagine, tyrosine, methionine, leucine, phenylalanine, isoleucine, tryptophan or glutamic acid. The BmSPI38 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.)

A bmsipi 38 mutant, wherein bmsipi 38 consists of positions 23 through 80 of SEQ ID No.1, and the bmsipi 38 mutant is obtained by mutating glycine at position 54 of the bmsipi 38 amino acid sequence, as shown in SEQ ID No.1, to arginine, lysine, serine, threonine, alanine, glutamine, aspartic acid, histidine, cysteine, proline, valine, asparagine, tyrosine, methionine, leucine, phenylalanine, isoleucine, tryptophan, or glutamic acid.

2. The method for constructing the bmsipi 38 mutant according to claim 1, wherein the bmsipi 38 mutant is obtained by site-directed mutagenesis of the gene sequence of wild-type bmsipi 38 as shown in SEQ ID No.2 using a site-directed mutagenesis primer.

3. The method of constructing the bmsipi 38 mutant of claim 2, 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 BmSPI38-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 then extracting mutant plasmid;

s5, transferring the mutant plasmid into a host expression strain, and inducing expression to obtain the BmSPI38 mutant.

4. A gene encoding the bmsipi 38 mutant of claim 1.

5. A plasmid carrying the gene of claim 4.

6. A host expression strain carrying the plasmid of claim 5.

7. The use of the bmsipi 38 mutant of claim 1 as a subtilisin and elastase inhibitor.

8. The use of the BmSPI38 mutant of claim 1 as a subtilisin, elastase and trypsin inhibitor wherein the BmSPI38 mutant is a BmSPI38 mutant wherein the glycine at position 54 in the BmSPI38 amino acid sequence is mutated to lysine or arginine as set forth in SEQ ID No. 1.

Technical Field

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a BmSPI38 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 BmSPI38 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 BmSPI38 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 BmSPI38 mutant, BmSPI38 consisting of positions 23 to 80 in SEQ ID No.1, and BmSPI38 mutant obtained by mutating the amino acid sequence of BmSPI38, glycine at position 54 as shown in SEQ ID No.1, to arginine (R), lysine (K), serine (S), threonine (T), alanine (a), glutamine (Q), aspartic acid (D), histidine (H), cysteine (C), proline (P), valine (V), asparagine (N), tyrosine (Y), methionine (M), leucine (L), phenylalanine (F), isoleucine (I), tryptophan (W), or glutamic acid (E).

The other purpose of the invention is to provide a construction method of the BmSPI38 mutant, which is to perform site-directed mutagenesis on the gene sequence of the wild type BmSPI38 shown as SEQ ID NO.2 by using a site-directed mutagenesis primer to obtain the BmSPI38 mutant.

Further, the construction method of the BmSPI38 mutant comprises the following steps:

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

s2, taking BmSPI38-p28 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 BmSPI38 mutant.

It is a further object of the present invention to provide genes encoding the BmSPI38 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 BmSPI38 mutant as a subtilisin and elastase inhibitor.

The seventh object of the present invention is to provide the use of the BmSPI38 mutant as a subtilisin, elastase and trypsin inhibitor, wherein the BmSPI38 mutant is a BmSPI38 mutant in which the 54 th glycine in the BmSPI38 amino acid sequence, 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:

the invention carries out site-directed mutagenesis on BmSPI38 amino acid sequence and glycine at position 54 as shown in SEQ ID NO.1 according to the BmSPI38 structural analysis, thereby obtaining mutants with enhanced activity of inhibiting subtilisin and elastase, wherein the mutants comprise BmSPI38(G54R), BmSPI38(G54K), BmSPI38(G54S), BmSPI38(G54T), BmSPI38(G54A) and BmSPI38 (G54Q); mutants with reduced subtilisin and elastase inhibitory activity include BmSPI38(G54D), BmSPI38(G54H), BmSPI38(G54C), BmSPI38(G54P), BmSPI38(G54V), BmSPI38(G54N), BmSPI38(G54Y), BmSPI38(G54M), BmSPI38(G54L), BmSPI38 (G54F); mutants with very weak inhibitory activity against subtilisin and elastase compared to the wild type include BmSPI38(G54I), BmSPI38(G54W), and mutants with inhibitory activity against subtilisin, elastase and trypsin, including BmSPI38(G54R), BmSPI38 (G54K).

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 BmSPI38 mutant PCR product (A) and a agarose gel electrophoresis image of the mutant plasmid (B).

FIG. 2 is an SDS-PAGE analysis of the BmSPI38 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 Origami 2(DE3) strain transformed with p28 empty vector. Arrows indicate that the bmsipi 38 mutant expresses protein.

FIG. 3 shows the elastase activity staining of the BmSPI38 mutant, wherein "Control" represents the Control induced by transformation of Origami 2(DE3) with BmSPI38-p 28. "EI" indicates elastase inhibitory activity and "CB" indicates Coomassie blue staining. The arrow for elastase 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.

FIG. 4 shows a comparison of the activity of BmSPI38 mutants against different proteases, with silkworm larvae hemolymph at 5 th day of age as positive control. "Control" is a cell lysate of Origami 2(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. Arrows of subtilisin inhibitory activity and elastase inhibitory activity indicate protease activity inhibition bands, and arrows of coomassie brilliant blue staining indicate coomassie brilliant blue staining bands corresponding to protease inhibitors.

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 BmSPI38-p28 recombinant expression vector is stored by the institute of physiology and application of vitamin D of Shanxi university of science and technology.

Example 1

Construction and activity research of BmSPI38 mutant

1. Mutant primer design

The amino acid sequence of BmSPI38 consists of positions 23 to 80 of SEQ ID No.1, wherein positions 1 to 22 are signal peptide sequences, and with reference to the gene sequence of BmSPI38, as shown in SEQ ID No.2, site directed mutagenesis primers were designed for 5 'to 3' PCR amplification of BmSPI 38. Mutant templates, desired mutations, DNA polymerase and primer sequences for bmsipi 38 are shown in table 1, respectively.

P1 site mutation primer of BmSPI38 in Table 1

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 DpnI cleavage System

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

(1) and (3) taking out the competent cells (100 mu 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 mu L of the competent cells, putting the competent cells into another precooled sterile centrifuge tube, adding 10 mu L of PCR products treated by Dpn I into each tube, slightly blowing, sucking and uniformly mixing the products, 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 transferred to Biotechnology engineering (Shanghai) Ltd for sequencing at 37 ℃ and 220 rpm.

(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 step refers to a Trans plasmid extraction kit to obtain the mutant plasmid.

7. Transformation into host expression strains

The BmSPI38 mutant plasmid was transferred into Origami 2(DE3) competent cells and the transformation procedure was referred 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 OD₆₀₀＝0.6～1.0。

(3) IPTG was added to a final concentration of 0.2mM for induction of expression at 16 ℃ at 220rpm for 20 h.

(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. BmSPI38 mutant Activity staining

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 ddH₂And 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 added₂And 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 ddH₂And (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 ddH₂And (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 ddH₂O 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 ddH₂O 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 ddH₂And (4) fixing the volume by O, and storing at 4 ℃ in a dark place.

And G, liquid: ammonium persulfate 0.7g, 100mL ddH₂And (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-D, L-phenylalanine-beta-naphthyl ester, N-acetyl-D, L-phenylalanine-beta-naphthyl ester), and the generated beta-naphthol dyes the gel into 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 BmSPI38 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 BmSPI38 presents a single band, the band is bright, the molecular weight is between 3000-5000 bp (FIG. 1A), and the plasmid extraction is good and consistent with the expectation (FIG. 1B).

(2) In order to express high amount of protein, Origimi 2(DE3) is used as the optimal host strain of BmSPI38 for induced expression, and 16.5% SDS-PAGE is selected for separating and detecting BmSPI38 mutant protein. The results are shown in FIG. 2: the BmSPI38 mutant protein is in a soluble form in the supernatant and is expressed in a high amount, but is hardly expressed in the precipitate, the apparent molecular weight of the BmSPI38 mutant protein is between 6.5 and 9.5kDa, and partial mutants such as BmSPI38(G54V), BmSPI38(G54Y), BmSPI38(G54M) and BmSPI38(G54I) are not only expressed in the supernatant in a high amount, but also expressed in a small amount in the precipitate, and the result shows that the BmSPI38 mutant protein is normally expressed.

(3) The invention takes N-acetyl-D, L-phenylalanine-beta-naphthyl ester as a substrate, and 6 mu L of BmSPI38 protein supernatant is extracted. The results of active staining showed that bmsipi 38 was able to strongly inhibit elastase activity (fig. 3). This is the first clear inhibitory activity of bombyx mori protease inhibitors on elastase.

(4) SDS-PAGE detection shows that the P1 site mutant protein of the BmSPI38 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 38.

The results show that: the bmsipi 38 mutants had substantially identical inhibitory activity against subtilisin and elastase. BmSPI38(G54R), BmSPI38(G54K), BmSPI38(G54S), BmSPI38(G54T), BmSPI38(G54A), BmSPI38(G54Q) have enhanced inhibitory activity against subtilisin and elastase, while BmSPI38(G54D), BmSPI38(G54H), BmSPI38(G54C), BmSPI38(G54P), BmSPI38(G54V), BmSPI38(G54N), BmSPI38(G54Y), BmSPI38(G54M), BmSPI38(G54L), BmSPI38(G54F) have reduced inhibitory activity against subtilisin and elastase. BmSPI38(G54I), BmSPI38(G54W) had very weak inhibitory activity against subtilisin and elastase (fig. 4). Notably, BmSPI38(G54R), BmSPI38(G54K) possess simultaneous subtilisin, elastase and trypsin inhibitory activity.

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.

Sequence listing

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cgcttgcgtc ccggccctca ttcaaac 27

<210> 38

<211> 25

<212> DNA

<213> Artificial sequence

<400> 38

aatgagggcc gggacgcaag cggtg 25

<210> 39

<211> 29

<212> DNA

<213> Artificial sequence

<400> 39

accgcttgcg tcgtggccct cattcaaac 29

<210> 40

<211> 27

<212> DNA

<213> Artificial sequence

<400> 40

tgaatgaggg ccacgacgca agcggtg 27