CN105254753A - Protease inhibitor cystatin and immunosuppressive effect thereof - Google Patents

Abstract

The invention discloses a protease inhibitor cystatin and an immunosuppressive effect thereof. The protease inhibitor cystatin originates from Dermacentor silvarum, has obvious inhibitory activity on cathepsin L, can remarkably inhibit activation of macrophages and dendritic cells, can remarkably inhibit antigen presentation and expression of inflammatory cytokines and is an immunosuppressive molecule with application prospect.

Description

A protease inhibitor cystatin and its immunosuppressive effect

technical field

The invention relates to the application field of biomedicine, in particular to a protease inhibitor cystatin and its immunosuppressive effect.

Background technique

Cysteine cathepsin is a type of protease found in the cells of various animal tissues (especially in lysosomes), consisting of cathepsins A, B, C, D, E, L and other enzymes. In immune cells, cathepsins are involved in the degradation, processing and presentation of antigens, and are key proteases for initiating immune responses. Recent studies have also shown that cathepsins are also involved in some other important physiological and pathological processes in cells, such as apoptosis, protein processing, tumorigenesis, inflammation, and neurodegenerative diseases.

The Cystatin protein family is an important class of inhibitors of cathepsin. They regulate the function of intracellular cathepsins and play an important role in maintaining the homeostasis of intracellular proteins. In view of the important role of cathepsin family in cellular immune response and many disease processes, the role of its inhibitor Cystatin in immune regulation has also been paid more and more attention. Cystatin C, for example, is the most potent inhibitor of cysteine cathepsins B, H, L, and S, which play important regulatory roles in dendritic cells and macrophages. Extracellular CystatinC can be taken up by other cells, enter the lysosome, and inhibit the activity of cathepsin in the lysosome. CystatinC has been shown to regulate the processing and maturation of MHC class II molecules in antigen-presenting cells, thereby affecting antigen presentation. The level of intracellular CystatinC is also related to the activation degree of DC and the expression level of cytokines, suggesting that Cystatin activity affects the maturation of DC. In macrophages, Cystatin B and C were shown to affect the nitric oxide production of activated macrophages, and this function was not dependent on the protease inhibitory activity of Cystatin, suggesting a new mechanism of Cystatin regulation of inflammation. CystatinF is highly expressed in CD56dimCD16 ⁺ NK cell subsets, and regulates NK cells to participate in the immune response and cell killing of inflammatory sites.

Contents of the invention

The technical problem mainly solved by the present invention is to provide a protease inhibitor cystatin and its immunosuppressive effect, which has low preparation cost, small molecular weight, stable property, easy absorption and strong inhibitory effect.

In order to solve the above-mentioned technical problems, a technical solution adopted by the present invention is: provide a protease inhibitor cystatin, the gene sequence of the protease inhibitor cystatin is:

ATGGCTCACAGGGCAGGTATTATTGCAGTTCTCACAGCGCTGGTCGCTGTAACCCTTGCAATTCCTGGAGGCTGGTCGACGAAGGAGCCCTCATCCAGCCCCAAGTACAAAGAACTGGCCCACTTCGCTGTCGCTCAGCGCGTTGAAGGTCTGCAAAAGTACGACACAGTTCTCGAACTCACAAAGGTGGAGACTCAGGTCGTTGCTGGCGTTAACTACCGCCTAACCTTCACCATTGCCGGATCCGATTGCACAATAGGTGAAGTTGAGTACAATGCTGAACGTTGCCCGGCGAAGGATAATCAGGCAAAGGCAACCTGCACTGCGGTGGTCTACGAGAGGCCCTGGGAAAACGTGCGATCCCTCACTTCCCTCAACTGCGCTTGA。

In a preferred embodiment of the present invention, the protease inhibitor cystatin is derived from Derma sylvia.

In a preferred embodiment of the present invention, the cloning and protein expression process of the protease inhibitor cystatin is: (1) cloning the cystatin gene into the prokaryotic expression vector pGEX-6P-2; (2) expressing cystatin recombinantly in Escherichia coli; (3) Purify the recombinant cystatin protein by column chromatography.

In a preferred embodiment of the present invention, the process of cloning and protein expression of the protease inhibitor cystatin is as follows: (1) extract the RNA of leathery tick tick, prepare cDNA, design specific primers according to the homologous gene, and amplify the protein from the cDNA library. Add the protein coding region fragment of the target gene, and connect the coding region of the target gene into the Escherichia coli expression vector pGEX-6P-2 after double digestion; (2) The constructed recombinant pGEX-6P-2 target gene expression plasmid Transform into Escherichia coli BL21 strain, induce the expression of the recombinant target protein with IPTG; (3) Purify the recombinant target protein with GST medium, and remove the GST tag by enzymatic digestion with specific protease on the column.

In a preferred embodiment of the present invention, the protease inhibitor cystatin has an inhibitory effect on the activity of cathepsin L.

In a preferred embodiment of the present invention, the dose of the protease inhibitor cystatin is linearly related to the inhibitory effect on the activity of cathepsin L, and the more the dose of the protease inhibitor cystatin is used, the inhibition of the activity of cathepsin L The stronger the effect.

In a preferred embodiment of the present invention, in RAW264.7 cells, the protease inhibitor cystatin has an inhibitory effect on cathepsin in the cells.

In a preferred embodiment of the present invention, the protease inhibitor cystatin can inhibit the activation of macrophages and dendritic cells, and can inhibit the antigen presentation of immune cells

In a preferred embodiment of the present invention, the protease inhibitor cystatin can inhibit macrophage activation caused by bacterial endotoxin lipopolysaccharide LPS.

In a preferred embodiment of the present invention, the protease inhibitor cystatin can inhibit the activation of macrophages caused by pathogens.

The beneficial effects of the present invention are: the protease inhibitor cystatin and its immunosuppressive effect of the present invention, the prokaryotically expressed protein has biological activity, low preparation cost, small molecular weight, stable properties, easy absorption, poor immunogenicity, and is not easy to be absorbed by the host Neutralizing degradation, tick protease inhibitors have immunosuppressive and immunomodulatory effects on macrophages and dendritic cells.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative work, wherein:

Fig. 1 is the expression and purification figure of protease inhibitor cystatin of the present invention;

Fig. 2 is the result figure that cystatin purified protein of the present invention inhibits cathepsinL activity;

Fig. 3 is the result figure that cystatin of different dosages of the present invention is to cathepsinL activity inhibitory effect;

Fig. 4 is the result graph of the inhibitory activity of cystatin to cathepsinL in RAW264.7 cells of the present invention;

Fig. 5 is a graph showing the results of cystatin of the present invention inhibiting the expression of interleukin 1 (IL1β) in macrophages induced by LPS;

Fig. 6 is a graph showing the results of cystatin of the present invention inhibiting the production of inflammatory cytokines (mRNA and protein levels) by macrophages induced by the pathogen Borrelia (Bb).

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

In the process of biological research on insect hosts of vector-borne diseases, cystatins, a class of cathepsin inhibitors encoded in the saliva of ticks, the medical vector insects, were obtained. Such cystatins in tick saliva are important for blood-sucking and pathogen transmission of ticks.

The present invention obtains cystatin, a new protease inhibitor in forest leather ticks, and identifies its protease inhibitory activity, confirms that it has obvious inhibitory activity on cathepsin L (cathepsinL), and is effective on cathepsin L in mammalian cells. CathepsinL has a strong inhibitory activity. Cystatin can significantly inhibit the activation of macrophages and dendritic cells, and can significantly inhibit the antigen presentation of immune cells and the expression of cytokines. It is an immunosuppressive molecule with application prospects.

Embodiment one:

Protein expression of cystatin in leather tick tick

(1) Clone the cystatin gene into the prokaryotic expression vector pGEX-6P-2

Cystatin is a sequence-determined antagonist of the cysteine protease of the wood leather tick.

Extract the RNA of forest leather tick, prepare cDNA, design specific primers according to the homologous gene, amplify the protein coding region fragment of the target gene from the cDNA library, and connect the coding region of the target gene to the Escherichia coli expression vector pGEX after double digestion In -6P-2, it was confirmed as a completely new sequence by sequencing. The sequence of the protease inhibitor cystatin is:

ATGGCTCACAGGGCAGGTATTATTGCAGTTCTCACAGCGCTGGTCGCTGTAACCCTTGCAATTCCTGGAGGCTGGTCGACGAAGGAGCCCTCATCCAGCCCCAAGTACAAAGAACTGGCCCACTTCGCTGTCGCTCAGCGCGTTGAAGGTCTGCAAAAGTACGACACAGTTCTCGAACTCACAAAGGTGGAGACTCAGGTCGTTGCTGGCGTTAACTACCGCCTAACCTTCACCATTGCCGGATCCGATTGCACAATAGGTGAAGTTGAGTACAATGCTGAACGTTGCCCGGCGAAGGATAATCAGGCAAAGGCAACCTGCACTGCGGTGGTCTACGAGAGGCCCTGGGAAAACGTGCGATCCCTCACTTCCCTCAACTGCGCTTGA。

(2) Recombinant expression of cystatin in Escherichia coli

The constructed recombinant pGEX-6P-2 target gene expression plasmid was transformed into Escherichia coli BL21 strain, and IPTG was used to induce the expression of the recombinant target protein.

(3) Purification of recombinant cystatin protein by column chromatography

Use GST medium to purify the recombinant target protein, and use specific protease to digest on the column to remove the GST tag. The recombinant cystatin protein was concentrated and detoxified for activity testing.

The leather tick is a good model organism for the study of tick-borne diseases, and its whole genome sequencing has been completed. After the above steps, the cDNA of the full-length cystatin was cloned, and the expression in the protein GST prokaryotic expression system was realized. The cystatin protein obtained after purification is 12.1 kD, as shown in Figure 1 .

Embodiment two:

Inhibitory Identification of Cystatin Purified Proteins on CathepsinL Activity

1. We detected cathepsinL activity according to InnoZyme CathepsinLActivityAssayKit, FluorogenicCat.No.CBA023 instructions, and the selection of reagents is shown in the table below.

The specific operation is as follows:

(1) Add activation buffer (activation buffer), AMC, cathepsin L (cathepsin L) (100ng/well), sample buffer (sample buffer) respectively, the current volume of each group is 100ul, shake slowly at room temperature for 5min.

(2) Add GST, CYSTATIN, inhibitor (inhibitor), diluent (diluent), the current volume of each group is 150ul, shake slowly for 5min.

(3) Add the substrate (substrate), the current volume of each group is 200ul, shake the incubator at 37°C for 1 to 2 hours, measure the fluorescence at 360nm and 460nm.

The results are shown in Figure 3, and it was found that compared with the negative control GST, the inhibitory activity of cystatin was 93.87% (the inhibitory activity of the positive control was 91.9%).

2. In order to further clarify the inhibitory effect of cystatin, different doses of purified cystatin protein were used for detection, as shown in the table below.

The results are shown in Figure 3. It was found that the dose of cystatin was linearly related to the inhibitory effect, and the inhibitory effect was significantly enhanced with the increase of the dose. When the dose of cystatin was 1ng per well (concentration: 5ng/ml), the activity of cathepsinL was inhibited by 50%.

3. In RAW264.7 cells, detect the inhibition of cathepsinL activity by cystatin (200ng/well), as shown in the table below, and the specific operation is as follows.

(1) Add activationbuffer, AMC, RAW264.7 cell lysate, samplebuffer, the current volume of each group is 100ul, shake slowly at room temperature for 15min.

(2) Add GST, CYSTATIN, diluent, the current volume of each group is 150ul, shake slowly for 15min.

(3) Add substrate, the current volume of each group is 200ul, shake at 37°C for 1 to 2 hours, measure fluorescence at 360nm and 460nm.

The results are shown in Figure 4. It was found that, compared with the negative control, the inhibitory activity of cystatin on cathepsinL was 90.77%.

Therefore, it can be seen from the above results that cystatin has a significant inhibitory effect on purified cathepsinL; in RAW264.7 cells, and under the condition of the presence of cathepsinB inhibitors, cystatin has a certain inhibitory effect on cathepsin in cells (such as cathepsinL, etc.) effect.

Embodiment three:

Immunosuppressive activity of Cystatin

1. Cystatin can inhibit the activation of macrophages caused by bacterial endotoxin lipopolysaccharide (LPS), and reduce the expression level of inflammatory cytokines.

The specific operation is as follows:

(1) Add 0.25uM, 1uM, 4uM different concentrations of cystatin or the highest concentration control protein GST to the isolated mouse peritoneal macrophages, and pre-incubate for 2 hours.

(2) Add bacterial endotoxin lipopolysaccharide at a concentration of 200ng/ml to the above cells and stimulate for 4 hours.

(3) Collect the cell supernatant, extract the total cellular RNA, and reverse transcribe it into cDNA. The expression levels of inflammatory cytokines were detected by real-time quantitative PCR. The results are shown in Figure 5. The expression level of the inflammatory cytokine interleukin 1 (IL1β) was inhibited by cystatin from Derma tick, indicating that cystatin has immunosuppressive activity.

2. cystatin can inhibit the activation of macrophages caused by pathogenic bacteria Borrelia (Bb), and reduce the expression level of inflammatory cytokines.

The specific operation is as follows:

(1) Add cystatin or control protein GST at a concentration of 3 uM to the isolated mouse peritoneal macrophages, and pre-incubate for 2 hours.

(2) Add spirochete bacteria Bb at a concentration of 10 ⁴ /ml to the above cells and stimulate for 4 hours.

(3) Collect the cell supernatant, extract the total cellular RNA, and reverse transcribe it into cDNA. The expression levels of inflammatory cytokines were detected by real-time quantitative PCR.

The results are shown in Figure 6. It was found that cystatin could significantly inhibit the production of inflammatory cytokines (interleukin 1 (IL1β), tumor necrosis factor (TNFα), interleukin 6 (IL6) ability. It shows that cystatin can inhibit the activation of macrophages caused by pathogens.

The above descriptions are only examples of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the description of the present invention, or directly or indirectly used in other related technical fields, shall be The same reasoning is included in the patent protection scope of the present invention.

Claims (10)

1. a proteinase inhibitor cystatin, is characterized in that, the gene order of described proteinase inhibitor cystatin is:

ATGGCTCACAGGGCAGGTATTATTGCAGTTCTCACAGCGCTGGTCGCTGTAACCCTTGCAATTCCTGGAGGCTGGTCGACGAAGGAGCCCTCATCCAGCCCCAAGTACAAAGAACTGGCCCACTTCGCTGTCGCTCAGCGCGTTGAAGGTCTGCAAAAGTACGACACAGTTCTCGAACTCACAAAGGTGGAGACTCAGGTCGTTGCTGGCGTTAACTACCGCCTAACCTTCACCATTGCCGGATCCGATTGCACAATAGGTGAAGTTGAGTACAATGCTGAACGTTGCCCGGCGAAGGATAATCAGGCAAAGGCAACCTGCACTGCGGTGGTCTACGAGAGGCCCTGGGAAAACGTGCGATCCCTCACTTCCCTCAACTGCGCTTGA。

2. proteinase inhibitor cystatin according to claim 1, is characterized in that, described proteinase inhibitor cystatin derives from dermacentor silvarum.

3. proteinase inhibitor cystatin according to claim 1, is characterized in that, clone and the protein expression process of described proteinase inhibitor cystatin are: (1) clone cystatin gene is in prokaryotic expression carrier pGEX-6P-2; (2) Recombinant protein expression cystatin; (3) the cystatin albumen of the method purification of Recombinant of column chromatography.

4. proteinase inhibitor cystatin according to claim 3, it is characterized in that, clone and the protein expression process of described proteinase inhibitor cystatin are: (1) extracts dermacentor silvarum RNA, preparation cDNA, according to homologous gene design Auele Specific Primer, from cDNA library, amplify the protein-coding region fragment of goal gene, be connected in coli expression carrier pGEX-6P-2 by after the coding region double digestion of goal gene; (2) by the restructuring pGEX-6P-2 destination gene expression Plastid transformation that builds in e. coli strain bl21, with IPTG abduction delivering restructuring target protein; (3) with GST medium purification restructuring target protein, the method for cutting with specific protease enzyme on post, removes GST label.

5. proteinase inhibitor cystatin according to claim 1, is characterized in that, the activity of described proteinase inhibitor cystatin to kethepsin cathepsinL is inhibited.

6. proteinase inhibitor cystatin according to claim 5, it is characterized in that, the using dosage of described proteinase inhibitor cystatin is with linear to the inhibition of kethepsin cathepsinL activity, the using dosage of described proteinase inhibitor cystatin is more, and the inhibition of described cathepsinL activity is stronger.

7. proteinase inhibitor cystatin according to claim 1, is characterized in that, in RAW264.7 cell, described proteinase inhibitor cystatin is inhibited to the kethepsin in cell.

8. proteinase inhibitor cystatin according to claim 1, is characterized in that, described proteinase inhibitor cystatin can suppress the activation of scavenger cell and dendritic cell, the antigen presentation of energy Immunosuppression cell.

9. proteinase inhibitor cystatin according to claim 8, is characterized in that, the macrophage activation that described proteinase inhibitor cystatin energy anti-bacteria induced by lipopolysaccharide LPS causes.

10. proteinase inhibitor cystatin according to claim 8, is characterized in that, the macrophage activation that described proteinase inhibitor cystatin can suppress pathogenic agent to cause.