阅读说明：本技术 可抑制灿烂弧菌胞外产物毒性的CgTIMP重组蛋白及制备方法 (CgTIMP recombinant protein capable of inhibiting toxicity of extracellular products of vibrio splendidus and preparation method thereof ) 是由 王玲玲 王伟林 刘兆群 孔宁 宋林生 于 2019-12-02 设计创作，主要内容包括：本发明公开一种可抑制灿烂弧菌胞外产物毒性的<I>Cg</I>TIMP重组蛋白,蛋白氨基酸序列如SEQ ID NO.1所示。该重组蛋白具有抑制灿烂弧菌胞外产物酶活性的作用,在制备灿烂弧菌胞外产物毒性抑制药物等方面具有应用价值。(The invention discloses a method for inhibiting toxicity of extracellular products of vibrio splendidus Cg The protein amino acid sequence of the TIMP recombinant protein is shown as SEQ ID NO. 1. The recombinant protein has the function of inhibiting the enzymatic activity of extracellular products of vibrio splendidus, and has application value in the aspects of preparing toxicity inhibiting medicines of the extracellular products of vibrio splendidus and the like.)

1. A method for inhibiting toxicity of extracellular product of vibrio splendidusCgA TIMP recombinant protein characterized in that: the protein amino acid sequence is shown as SEQ ID NO. 1.

2. The method for inhibiting toxicity of extracellular products of Vibrio splendidus according to claim 1CgThe preparation method of the TIMP recombinant protein is characterized by comprising the following steps in sequence:

a. carrying out PCR amplification on the coding region fragment of the oyster TIMP628 gene by using specific primers P1 and P2, wherein the DNA sequence of the primer P1 is shown as SEQ ID NO.2, and the DNA sequence of the primer P2 is shown as SEQ ID NO. 3;

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min, then the following cycle was entered: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, 35 cycles in total, and final extension at 72 ℃ for 10 minutes;

b. purifying and recovering the amplified fragment, connecting the amplified fragment with a pMD19-T vector, screening positive clones after transformation, and extracting plasmids; use ofNdeI andBamh I enzyme cutting plasmid, purifying and recovering the target fragment generated by the enzyme cutting plasmid;

c. combining the recovered target fragment with the proteinNdeI andBamh I restriction enzyme expression vector pET-30a connection and transformation into Escherichia coliTransetta(DE 3);

d. a single clone was picked, inoculated into 200 mL of LB liquid medium at 220 rpm, and cultured at 37 ℃ to OD₆₀₀= 0.4-0.8, 1 mmol L of final concentration is added^-1Culturing for 4 hours after the inducer IPTG is added, centrifuging for 10 minutes at 4 ℃ and 5000 rpm, and collecting thalli;

e. purifying and renaturing the obtained thallus to obtain the product capable of inhibiting the toxicity of the extracellular product of the vibrio splendidusCgTIMP recombinant proteins.

3. The composition according to claim 2, wherein the toxicity of extracellular products of Vibrio splendidus is inhibitedCgThe preparation method of the TIMP recombinant protein is characterized in that the step e sequentially comprises the following steps:

(1) packing the nickel sepharose FF into a column with the volume of 1.6 multiplied by 20 cm and the volume of a column bed being 10 mL;

(2) using buffer solution I to balance 2-5 bed volumes, the flow rate is 2 mL min^-1(ii) a The buffer I had the following composition: 50 mmol L^-1Tris-HCl buffer, pH 7.4; 50 mmol L^-1NaCl；8 mol L^-1Urea;

(3) d, resuspending the thallus collected in the step d with a buffer solution I, carrying out 150W ultrasonic disruption for 30 minutes, carrying out 12000 rmp, centrifuging for 30 minutes at 4 ℃, taking the supernatant, filtering the supernatant with a 0.45-micron filter membrane, and passing through a column at the flow rate of 1 mL min^-1；

(4) Washing with buffer solution I for 2-5 bed volumes at a flow rate of 2 mL min^-1；

(5) Washing with buffer solution II for 2-5 bed volumes at a flow rate of 2 mLmin^-1(ii) a The buffer solution II is prepared by adding 50 mmol L of buffer solution I^-1Imidazole;

(6) eluting with buffer IIICollecting the protein of (4); the buffer solution III is prepared by adding 400 mmol L of buffer solution I^-1Imidazole;

(7) the collected proteins were renatured by dialysis.

4. The method for inhibiting toxicity of extracellular products of Vibrio splendidus according to claim 1CgThe TIMP recombinant protein is characterized by being applied to preparing a medicament for inhibiting the toxicity of extracellular products of vibrio splendidus.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a method for inhibiting toxicity of extracellular products of vibrio splendidusCgTIMP recombinant protein and a preparation method thereof.

Background

Oyster (Chang mu Li)Crassostrea gigas，Cg) Is an internationally important seaOne of the economic shellfish is cultured in water. In recent years, diseases caused by bacteria and viruses cause the large-scale death of crassostrea gigas, and huge economic losses are brought. Researches show that vibrio splendidus is one of the main pathogenic bacteria, and Extracellular products (ECP) and metalloproteases in the vibrio splendidus have strong toxicity to oyster juvenile mollusks and can obviously inhibit adhesion and phagocytic activity of oyster immune cells. In mammals, tissue metalloproteinase inhibitor (TIMP) proteins can inhibit the activity of body tissue metalloproteinases and thus inhibit cell migration, and play a key role in inhibiting tumor migration. So far, no relevant report that the Ostrea gigas TIMP can inhibit the toxicity of extracellular products of Vibrio splendidus is available.

Disclosure of Invention

The present invention is to solve the above problems of the prior art and to provide a method for inhibiting the toxicity of extracellular products of Vibrio splendidusCgTIMP recombinant proteins.

A method for inhibiting toxicity of extracellular product of vibrio splendidusCgA TIMP recombinant protein characterized in that: the protein amino acid sequence is shown as SEQ ID NO. 1.

The above can inhibit toxicity of extracellular product of Vibrio splendidusCgThe preparation method of the TIMP recombinant protein is characterized by comprising the following steps in sequence:

a. carrying out PCR amplification on the coding region fragment of the oyster TIMP628 gene by using specific primers P1 and P2, wherein the DNA sequence of the primer P1 is shown as SEQ ID NO.2, and the DNA sequence of the primer P2 is shown as SEQ ID NO. 3;

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min, then the following cycle was entered: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, 35 cycles in total, and final extension at 72 ℃ for 10 minutes;

b. purifying and recovering the amplified fragment, connecting the amplified fragment with a pMD19-T vector, screening positive clones after transformation, and extracting plasmids; use ofNdeI andBamh I enzyme cutting plasmid, purifying and recovering the target fragment generated by the enzyme cutting plasmid;

c. combining the recovered target fragment with the proteinNdeI andBamh I restriction enzyme expression vector pET-30a connection and transformation into Escherichia coliTransetta(DE 3);

d. a single clone was picked, inoculated into 200 mL of LB liquid medium at 220 rpm, and cultured at 37 ℃ to OD₆₀₀= 0.4-0.8, 1 mmol L of final concentration is added^-1Culturing for 4 hours after the inducer IPTG is added, centrifuging for 10 minutes at 4 ℃ and 5000 rpm, and collecting thalli;

e. purifying and renaturing the obtained thallus to obtain the product capable of inhibiting the toxicity of the extracellular product of the vibrio splendidusCgTIMP recombinant proteins.

The step e is as follows in sequence:

(1) packing the nickel sepharose FF into a column with the volume of 1.6 multiplied by 20 cm and the volume of a column bed being 10 mL;

(2) using buffer solution I to balance 2-5 bed volumes, the flow rate is 2 mL min^-1(ii) a The buffer I had the following composition: 50 mmol L^-1Tris-HCl buffer, pH 7.4; 50 mmol L^-1NaCl；8 mol L^-1Urea;

(3) d, resuspending the thallus collected in the step d with a buffer solution I, carrying out 150W ultrasonic disruption for 30 minutes, carrying out 12000 rmp, centrifuging for 30 minutes at 4 ℃, taking the supernatant, filtering the supernatant with a 0.45-micron filter membrane, and passing through a column at the flow rate of 1 mL min^-1；

(4) Washing with buffer solution I for 2-5 bed volumes at a flow rate of 2 mL min^-1；

(5) Washing with buffer solution II for 2-5 bed volumes at a flow rate of 2 mLmin^-1(ii) a The buffer solution II is prepared by adding 50 mmol L of buffer solution I^-1Imidazole;

(6) eluting the target protein by using a buffer solution III and collecting; the buffer solution III is prepared by adding 400 mmol L of buffer solution I^-1Imidazole;

(7) the collected proteins were renatured by dialysis.

The above can inhibit toxicity of extracellular product of Vibrio splendidusCgThe TIMP recombinant protein is applied to the preparation of the drugs for inhibiting the toxicity of extracellular products of vibrio splendidus.

The invention analyzes the immune fine of crassostrea gigas under the stress of pathogenic bacteriaScreening out a matrix metalloproteinase inhibitor capable of inhibiting the toxicity of the extracellular product of pathogenic vibrio splendidus on the basis of the cell transcriptome (CgTIMP) to obtain the recombinant protein (r) of the matrix metalloproteinase inhibitor geneCgTIMP 628) and the recombinant protein has the function of inhibiting the enzymatic activity of extracellular products of vibrio splendidus, and has application value in the aspects of preparing toxicity inhibiting medicaments of the extracellular products of the vibrio splendidus and the like.

Drawings

FIG. 1 shows a recombinant protein r according to an embodiment of the present inventionCgTIMP628 induction and purification effect chart.

FIG. 2 shows a real-time example of recombinant protein r of the present inventionCgThe toxicity inhibiting effect of TIMP628 on extracellular product ECP of vibrio splendidus is shown.

Detailed Description

The invention can inhibit the toxicity of extracellular products of vibrio splendidusCgThe protein amino acid sequence of the TIMP recombinant protein is shown as SEQ ID NO. 1.

Inhibiting toxicity of extracellular products of vibrio splendidusCgThe preparation method of the TIMP recombinant protein is characterized by comprising the following steps in sequence:

a. carrying out PCR amplification on the coding region fragment of the oyster TIMP628 gene by using specific primers P1 and P2, wherein the DNA sequence of the primer P1 is shown as SEQ ID NO.2, and the DNA sequence of the primer P2 is shown as SEQ ID NO. 3;

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min, then the following cycle was entered: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, 35 cycles in total, and final extension at 72 ℃ for 10 minutes;

b. purifying and recovering the amplified fragment, connecting the amplified fragment with a pMD19-T vector, screening positive clones after transformation, and extracting plasmids; use ofNdeI andBamh I enzyme digestion plasmid, and purifying and recovering the target fragment generated by the enzyme digestion plasmid by using a glue recovery and purification kit (Dalibao bioengineering Co., Ltd.);

c. combining the recovered target fragment with the proteinNdeI andBamh I restriction enzyme expression vector pET-30a connection and transformation into Escherichia coliTransetta(DE 3);

d. a single clone was picked, inoculated into 200 mL of LB liquid medium at 220 rpm, and cultured at 37 ℃ to OD₆₀₀=0.6, add final concentration 1 mmol L^-1Culturing for 4 hours after the inducer IPTG is added, centrifuging for 10 minutes at 4 ℃ and 5000 rpm, and collecting thalli;

e. purifying and renaturing the obtained thallus to obtain the product capable of inhibiting the toxicity of the extracellular product of the vibrio splendidusCgThe TIMP recombinant protein comprises the following specific steps in sequence:

(1) packing the nickel sepharose FF into a column with the volume of 1.6 multiplied by 20 cm and the volume of a column bed being 10 mL;

(2) using buffer solution I to balance 2-5 bed volumes, the flow rate is 2 mL min^-1(ii) a The buffer I had the following composition: 50 mmol L^-1Tris-HCl buffer, pH 7.4; 50 mmol L^-1NaCl；8 mol L^-1Urea;

(3) d, resuspending the thallus collected in the step d with a buffer solution I, carrying out 150W ultrasonic disruption for 30 minutes, carrying out 12000 rmp, centrifuging for 30 minutes at 4 ℃, taking the supernatant, filtering the supernatant with a 0.45-micron filter membrane, and passing through a column at the flow rate of 1 mL min^-1；

(4) Washing with buffer solution I for 2-5 bed volumes at a flow rate of 2 mL min^-1；

(5) Washing with buffer solution II for 2-5 bed volumes at a flow rate of 2 mLmin^-1(ii) a The buffer solution II is prepared by adding 50 mmol L of buffer solution I^-1Imidazole;

(6) eluting the target protein by using a buffer solution III and collecting; the buffer solution III is prepared by adding 400 mmol L of buffer solution I^-1Imidazole;

(7) dialyzing the collected protein for renaturation: the collected denatured and purified product was renatured by dialysis with 2 mM reduced glutathione, 0.4mM oxidized glutathione, 1 mM EDTA, 50 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 1% glycine and a gradient of decreasing urea concentration, gradually changing from the initial 6M to 4M, 3M, 2M, 1M, 0M. Glycerol was not added when the dialysate was subjected to the last dialysis (urea 0M), and was dialyzed at 4 ℃ for 12 hours each time. To obtain a compound which can inhibit the toxicity of extracellular products of vibrio splendidusCgTIMP recombinant protein rCgTIMP628，The amino acid sequence is shown in SEQ ID NO.1 through determination.

Length: 208 amino acids

Type (2): amino acids

Chain type: single strand

The characteristics are as follows: the molecular weight is 23.95 kDa, the isoelectric point is 6.18, and the peptide has a conserved TIMP structural domain.

The invention can inhibit the toxicity of extracellular products of vibrio splendidusCgTIMP recombinant protein (r)CgTIMP 628) before and after induction and purification effects are shown in fig. 1. M in FIG. 1: marker; 1: inducing a pre-mycoprotein; 2: induced mycoprotein; 3: after purification rCgTIMP 628. As can be seen, the monoclonal cultures of the examples of the present invention were induced to obtain clear bands.

Experiment: the invention can inhibit the toxicity of extracellular products of vibrio splendidusCgTIMP recombinant protein (r)CgTIMP 628) for inhibiting toxicity of extracellular products of vibrio splendidus

The method comprises the following specific steps:

1. culture of vibrio splendidus and preparation of extracellular product ECP thereof

Vibrio splendidus (Vibrio splendidusChina general microbiological culture Collection center (CGMCC) number: CGMCC 1.6380), cultured with 2216E liquid medium at 16 deg.C and 220 rpm to logarithmic growth phase. Then, the cells were diluted to about 1X 10 with 0.85% NaCl physiological saline⁷CFU, 100. mu.L of the bacterial solution was spread evenly on 2216E solid medium plate with sterilized cellophane spread thereon, and cultured at 16 ℃ for 48 hours. Removing cellophane full of bacteria, scraping off thallus, and thoroughly resuspending with normal saline solution at 12,000gAfter centrifugation for 10 minutes, the supernatant is taken to be the extracellular product ECP of the vibrio splendidus. Adjusting the concentration to 0.1 mgmL with physiological saline^-1And (5) standby.

2. Recombinant protein r of the present exampleCgDetermination of toxicity inhibition of extracellular product ECP of vibrio splendidus by TIMP628

150. mu.L of ECP solution from 4 tubes was added to 100. mu.L of PBS solution, EDTA solution (50 mM), and purified Trx-tagged protein (0.1 mg mL)^-1Recombinant protein control protein) or recombinant protein r of the embodiments of the inventionCgTIMP628（0.1 mgmL^-1) Incubate at 16 ℃ for 1 hour. Then 250. mu.L of azocasein substrate (5 mg mL) was added separately^-1Dissolved in 50 mM Tris-HCl buffer, pH 8.0), reacted at 25 ℃ for 20 minutes, then precipitated the undegraded substrate by adding 1750. mu.L of 5% (v/w) trichloroacetic acid TCA at 4 ℃ and 3000gThe supernatant was centrifuged for 5 minutes. 100 mu L of supernatant was added with 0.5M NaOH to terminate the reaction, and OD was measured with a microplate reader₄₄₀And calculating the enzyme activity according to the light absorption value, wherein the enzyme activity is stronger when the light absorption value is larger. Three replicates were set for each sample, the mean values were plotted based on the 3 measurements, and the study data were statistically analyzed using SPSS6.0 software for significant differences (P<0.05) with a mark. The results are shown in FIG. 2.

In fig. 2: PBS: a buffer solution; EDTA: a metal ion chelating agent; trx: purification of Trx tag protein rCgTIMP 628: the recombinant proteins of the embodiments of the present invention.

The experimental result shows that the crassostrea gigas tissue metalloproteinase inhibitor gene recombinant protein r of the inventionCgTIMP628 can inhibit the protease activity (toxicity) of extracellular product ECP of vibrio splendidus, and can be used as a medicine for inhibiting the toxicity of the extracellular product of vibrio splendidus.

Sequence listing

<110> university of Dalian ocean

<120> CgTIMP recombinant protein capable of inhibiting toxicity of extracellular products of vibrio splendidus

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