CN102358753B - Trypsin inhibitor active fragment derivative, and preparation method and application thereof - Google Patents

Abstract

The invention relates to a trypsin inhibitor active fragment derivative Argc. Compared with the wild fragment in structure, 3 pairs of disulfide bonds of the trypsin inhibitor active fragment derivative Argc are converted into 2 pairs. The amino acid sequence of the trypsin inhibitor active fragment derivative Argc is SEQ ID NO:1. The preparation method comprises the following steps: chemically synthesizing gene sequences of the MBTI-Argc fragment, transforming a pGEX-4T-1-Argc expression vector to colibacillus DH5alpha to construct colibacillus engineering bacteria pGEX-4T-1-Argc/DH5alpha, inoculating the engineering bacteria into an LB (lysogeny broth) culture medium containing ampicillin, carrying out shake culture at 37 DEG C until A600nm is 0.4-0.6, adding IPTG (isopropyl-beta-D-thiogalactopyranoside) until the final concentration is 0.5 mmol/L, inducing expression for 3-5 hours, collecting the thallus, and purifying the supernatant by affinity chromatography to obtain the target protein. The Argc is applicable to cell trauma repair and wound healing.

Description

A trypsin inhibitor active fragment derivative and its preparation method and application

technical field

The invention relates to the technical field of bioengineering, in particular to the construction and expression of engineering bacteria derived from mung bean trypsin inhibitor arginine fragment derivative protein and its application in cell damage repair.

Background technique

Trypsin inhibitor (TI) refers to a polypeptide or protein with trypsin inhibitory activity, and is an important biochemical drug and biochemical reagent. Mung bean trypsin inhibitor (MBTI) belongs to the Bowman-Birk type inhibitor, which contains two active sites of lysine and arginine, located in Lys-Ser (20-21) and Arg-Ser (47-48), respectively. At present, the research on MBTI mostly focuses on the full-length MBTI fragment or the Lys active fragment, but less research on the Arg active fragment.

The Arg active fragment contains seven sulfhydryl groups, and the three pairs of disulfide bonds they contain play an important role in the activity and structural stability of the protein. This patent uses the chemical synthesis method to obtain the coding gene sequence of the Arg fragment. Compared with the natural mung bean trypsin inhibitor arginine fragment, the obtained recombinant protein derivative is obviously different: its structure changes, and the number of disulfide bonds is different. decreased, the number of free thiol groups increased. Moreover, the recombinant protein has specific biological activity on cells: the fragment basically does not have the activity of trypsin inhibitor, and can promote cell migration and proliferation.

Wound healing refers to the recovery process after the body is subjected to external force and the tissue is severed or defective, including the regeneration of various tissues, cell proliferation, migration, granulation tissue hyperplasia, collagen secretion, and tissue reconstruction. Cell migration is the first stage of wound repair. In recent years, due to the important role of oxygen free radicals in the pathogenesis of ischemic tissue damage, the cell damage mediated by it has attracted extensive attention. High concentrations of oxygen free radicals can inhibit and poison cells. The sulfhydryl group contained in protein has anti-oxidation effect, which can effectively eliminate free radicals, thereby inhibiting cell apoptosis. It can combine with active oxygen free radicals, reduce their toxic effects, and play a defensive role in cell damage caused by oxygen free radicals.

Contents of the invention

The object of the present invention is to provide a trypsin inhibitor active fragment derivative and its preparation method and application.

The gene sequence of trypsin inhibitor active fragment derivative (Argc) of the present invention is derived from an active region of mung bean trypsin inhibitor, contains 27 amino acid residues, and its amino acid sequence is SEQ ID NO: 1; Its structure and wild fragment Compared with the protein derived from 3 pairs of disulfide bonds to 2 pairs in the wild fragment, it does not have the activity of trypsin inhibitor, but promotes cell migration and proliferation.

The nucleotide sequence of Argc of the present invention is made up of 81 nucleotides, and its nucleotide sequence is SEQ ID NO:2.

A vector containing a nucleotide sequence encoding Argc is a recombinant pGEX-4T-1-Argc obtained after cloning the nucleotide fragment encoding Argc into a prokaryotic expression vector. The vector is a plasmid containing M13 promoter.

An Argc engineering bacterium, which contains the aforementioned carrier. The pGEX-4T-1-Argc recombinant is transformed into a bacterium, such as Escherichia coli DH5α, to construct an engineering bacterium.

Argc of the present invention is obtained by the following method: chemically synthesize the gene sequence of the MBTI-Argc fragment, connect the sequence to the pGEX-4T-1 vector, obtain the recombinant pGEX-4T-1-Argc, and transform the recombinant into the large intestine Bacillus DH5α, construct the Escherichia coli engineering bacterium pGEX-4T-1-Argc/DH5α, inoculate the engineering bacterium in LB medium containing ampicillin, shake and cultivate at 37°C until the A 600nm is 0.4-0.6, add IPTG to make the final concentration 0.5mmol/L, induce expression for 3-5 hours, collect the bacteria, and purify the supernatant by affinity chromatography to obtain the target protein.

MTT method and cell scratch experiment were used to study the biological activity of Argc. The experiment proved that Argc acts on cells and can effectively promote the growth and migration of various cells. Scratch test showed that Argc can promote the repair of cells after mechanical damage, and the results of disulfide bond assay showed that Argc also has anti-oxidation function. Therefore, this derivative can be applied to cell damage repair and wound healing.

Description of drawings

Figure 1: Identification of the GST-Argc recombinant plasmid (the first lane is the recombinant plasmid double digested by BamH I and Xho I)

Figure 2: GST-Argc Glutathione Sepharose 4 Fast Flow affinity chromatography (the first lane is the purified GST-Argc, and the second lane is the GST protein control with noitomorph)

Figure 3: Relative activity of trypsin inhibitors of GST-Argc

Figure 4: Effect of GST-Argc on cell proliferation

Figure 5: Effect of GST-Argc on cell migration

Detailed ways

Example 1: Synthesis of Argc fragment encoding gene and construction of recombinant expression plasmid pGEX-4T-1-Arg

According to the amino acid sequence of the MBTI-Arg fragment reported in the literature, we entrusted Dalian Bao Biology Co., Ltd. to synthesize the coding gene of the Argc fragment (with BamH I and Xho I restriction sites inserted at both ends), double digestion with BamH I and Xho I, and gel recovery of the target gene The fragment was ligated with the pGEX-4T-1 vector cut with the same BamH I and Xho I double enzymes to obtain the recombinant expression plasmid pGEX-4T-1-Argc. The recombinant plasmid was identified by double enzyme digestion and sequencing (see Figure 1).

Embodiment 2: Expression and purification of Argc fragment protein

Transfer the recombinant expression plasmid pGEX-4T-1-Argc into Escherichia coli DH5α, screen for positive clones, pick a single colony and inoculate it in 5 mL LB medium for overnight culture, then inoculate it in 1 L of medium, and culture it with shaking at 37°C until A600nm When it reaches 0.5, add IPTG with a concentration of 0.5mol/L, and continue to cultivate for 4h to collect the bacteria. The cells were resuspended in PBS buffer, ultrasonically disrupted, and the supernatant was collected. The supernatant was purified by GST affinity chromatography (see Figure 2). After the total protein is fully adsorbed, wash the affinity column with PBS (pH 7.4) buffer, and stop washing when the UV absorption value is 0. 50mmol/L Tris-HCl (pH 8.0, 10mM GSH) buffer was eluted, the eluate was collected, and the protein concentration was determined by the Coomassie brilliant blue method.

Embodiment 3: trypsin inhibitor activity assay

With BAPA (benzoyl-dl-Arg-P-nitroanilide hydrochloride) as a substrate, 100 μL of fusion proteins of different concentrations were added, and the activity of trypsin inhibitor was measured at 410 nm by ultraviolet absorption method ( See Figure 3).

Example 4: Quantitative determination of disulfide bonds

For the determination of the content of free sulfhydryl groups, the concentration of the sample to be tested was detected by the CBB method. Take 200 μL of the sample to be tested, add 4.8 g of urea to dilute to 1 mL, and incubate at 37° C. for 4 hours. Add 2mL Tris-Gly buffer, 50μLDTNB, and measure the absorbance at 412nm after 5 minutes. The content of free sulfhydryl groups in the polypeptide was judged according to the absorbance value. The number of disulfide bonds can be calculated according to the total number of sulfhydryl groups. Calculation method:

$\mathrm{<span\; class="notranslate">\; \&mu;MSH</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; g</span>}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 73.53</span>}{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 412</span>}}}{\mathrm{<span\; class="notranslate">\; D.</span>}}$

A ₄₁₂ : Absorbance value at 412nm D: Dilution factor C: Protein concentration (mg/ml)

From the above formula, μM SH/g(GST-Argc)=105.62 (the control GST protein has no absorbance value)

Free SH number = 105.62M/10 ⁶ = 3.06 (M: relative molecular weight of GST-Argc)

Number of pairs of disulfide bonds = 7 (number of total thiol groups) - 3.06 (number of free thiol groups) / 2 = 2

Embodiment 5: MTT assay detects the effect on cell proliferation

The SW480, SW620, HepG2 and MDCK cells in the exponential growth phase were introduced into 96-well culture plates at 1× ¹⁰⁴ /well, and after incubation for 24 hours at 37°C in an incubator containing 5% CO2, different concentrations of fusion proteins were added to reach the final concentration. 2.0μM, 5.0μM, 10.0μM. Four replicate wells were set up for each concentration, and the elution buffer containing GST protein was used as a control. After 24 hours of incubation, the liquid in the wells was discarded. After washing with PBS, new medium was added, and 20 μl of MTT solution (5 mg/ml) was added to each well, and the incubation was continued for 4 hours before the culture was terminated. Carefully aspirate and discard the culture supernatant in the wells, add 150 μl DMSO to each well, and shake for 10 minutes to fully melt the crystals. 490nm wavelength, the light absorption value of each well was measured on an enzyme-linked immunosorbent monitor (see Figure 4).

Embodiment 6: cell migration experiment

Seed HL7702 cells on a 24-well plate at a density of 5-10×10 ⁵ cells/well. After the cells adhere to the wall and form a single layer of cells, use a 10 μL sterile pipette tip to vertically streak, wash with PBS, and measure under a microscope Scratch diameter, and set up the experimental group and the control group respectively. The experimental group was added with purified protein Argc at a final concentration of 7.5 μM, and the control group was added with the same concentration of GST protein. After 24 hours of incubation, the distance of the cells moving outward at the scratch was measured under a microscope (see Figure 5).

Claims (4)

1. A trypsin inhibitor active fragment derivative Argc, whose structure is changed from 3 pairs of disulfide bonds to 2 pairs compared with the wild fragment, and its amino acid sequence is SEQ ID NO: 1, which can promote cell migration and proliferation effect. 2. the gene of coding Argc described in claim 1, its nucleotide sequence is: SEQ ID NO:2. 3. A vector comprising the nucleotide sequence according to claim 2. 4. An Argc engineering bacterium, containing the carrier according to claim 3.

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