KR101374757B1 - Serine protease inhibitor derived from apis cerana having antielastolytic function and composition for inhibiting inflammation comprising the same - Google Patents

Abstract

The present invention relates to a serine protease inhibitor derived from Apis cerana having an elastase inhibitory function, and a composition for inhibiting inflammation comprising the same as an effective component. Serine protease inhibitory protein derived from Apis cerana can be mass produced using polypeptide forming the serine protease inhibitory protein derived from Apis cerana and polynucleotide encoding thereof. The serine protease inhibitory protein derived from Apis cerana has excellent elastase inhibitory activity, thereby being practically used as an inflammation inhibitor.

Description

Serine protease inhibitor derived from native bee having an elastase inhibitory activity and an inhibitory composition comprising the same as an active ingredient {Serine protease inhibitor derived from Apis cerana having antielastolytic function and composition for inhibiting inflammation comprising the same}

The present invention relates to a serine protease inhibitory protein derived from native bees having an elastase inhibitory activity and a composition for inhibiting inflammation comprising the same as an active ingredient.

Honeybees are economically important insects. It provides not only pollen mediating but also products such as honey, royal jelly, propolis, pollens, wax, and venom. These bee products have been used as the main material in oriental medicine [Cherniack EP, Alternat. Med. Rev., 15: 124-135 (2010)]. Recently, many studies have been conducted on various pharmacological effects of bee venom components [Son DJ et al., Pharmacol. Ther., 115: 246-270 (2007). Bumblebee ( Bombus) , also known as a pollinated insect ignitus ) fibrinolytic function of serine protease has been reported [Choo YM et al. PLoS ONE 5: e10393 (2010)], the antifibrinolytic function of the serine protease inhibitor has been reported [Choo YM et al. PLoS ONE 7 : e10393 (2012)], serine protease inhibitory proteins found in various organisms are involved in various physiological processes, such as blood coagulation, fibrinolysis and inflammation removal. This is of great interest because of the potential for new drug development [Masci PP et al., Blood Coagul. Fibrinolysis 11: 385-393 (2000); Corral-Rodriguez MA et al., Insect Biochem. Mol. Biol., 39: 579-595 (2009); Flight SM et al., British J. Hematol., 145: 207-211 (2009)]. However, there are relatively few studies on serine protease inhibitory proteins in bees. Western Bee ( Apis) Serine protease inhibitory proteins with chymotrypsin and cathepsin G inhibitory effects have been reported in mellifera (Bania DR et al., Eur. J. Biochem., 262: 680-687 (1999); Cierpicki T et al., Protein Sci., 9: 976-984 (2000)], reports on the antifibrinolytic function of serine protease inhibitory proteins in bumblebees ( Bombus ignitus ) [Choo YM et al. PLoS ONE 7 : e10393 (2012)], the genes and functions of bee-derived serine protease inhibitor proteins that inhibit human neurophil elastase activity, which plays a major role in inflammation, have not been reported. .

Therefore, the inventors of the present invention, the native bee ( Apis) The human neutrophil elastase activity of humans in which a protein produced by cloning the gene of a serana protease inhibitor (AcCI) derived from a cerana and transforming it into an insect cell line plays a major role in inflammation. By confirming the inhibition, the present invention was completed.

An object of the present invention is to provide a serine protease inhibitory protein derived from native bees having inhibitory activity of chymotrypsin and elastase and a polynucleotide encoding the same.

Another object of the present invention to provide a recombinant expression vector comprising a polynucleotide sequence encoding a serine protease inhibitory protein derived from native bees.

Another object of the present invention to provide a pharmaceutical composition for inhibiting inflammation comprising a serine protease inhibitory protein derived from native bees as an active ingredient.

In order to achieve the above object, the present invention provides a serine protease inhibitory protein derived from native bees having inhibitory activity of chymotrypsin and elastase, and a polynucleotide encoding the same.

The present invention also provides a recombinant expression vector comprising a polynucleotide sequence encoding a serine protease inhibitory protein derived from native bee.

In addition, the present invention provides a pharmaceutical composition for inhibiting inflammation comprising a serine protease inhibitory protein derived from native bee as an active ingredient.

Using a polypeptide constituting a native serine protease protein derived from the native bee of the present invention and a polynucleotide encoding the same, a native serine protease inhibitory protein can be produced in a large quantity, and the native serine protease inhibitory protein is an elastase inhibitory activity. This is excellent and can be usefully used as an inflammation inhibitor.

1 shows homology with native bee-derived serine protease inhibitory protein and other bee-derived serine protease inhibitory protein [arrow: cleavage site of signal sequence; Asterisk: P1 region; Solid circle: a cysteine residue that forms a conserved disulfide bond].
Figure 2 is a diagram showing the results of electrophoresis of serine protease inhibitory recombinant protein derived from native bees of the present invention.
Figure 3 is a diagram stained sugar chains of serine protease inhibitory recombinant protein derived from native bee of the present invention.
4 is a diagram showing the inhibitory activity against alpha chymotrypsin of native serine protease inhibitory recombinant protein of the present invention.
5 is a diagram showing the inhibitory activity of AcCI against human neutrophil elastase and porcine pancreatic elastase of native serine protease inhibitory recombinant protein of the present invention.

The present invention provides a serine protease inhibitory protein derived from Apis cerana having inhibitory activity of chymotrypsin and elastase, and a polynucleotide encoding the same.

Hereinafter, the present invention will be described in detail.

More specifically, the amino acid sequence of the native serine protease inhibitor of the present invention and the polynucleotide encoding the serine protease inhibitor were separated as follows.

Apis received from the Department of Agricultural Biology, National Academy of Agricultural Science, RDA cerana ) cDNA library was prepared using poly (A) and mRNA extracted from worker bees and gene expression tags (ESTs) were analyzed. The native bee ESTs analysis cloned the cDNA of the gene encoding the native serine protease inhibitory protein, which is shown in SEQ ID NO: 1. In addition, the result of database analysis of the amino acid sequence translated from the cDNA encoding the serine protease inhibitory protein derived from native bees is shown in SEQ ID NO: 2.

AcCI gene derived from native bee was first isolated from native bee, and its characteristics have not been reported so far. The sequence of the gene encoding AcCI derived from native bees was revealed, and the AcCI gene derived from native bees was registered in the NCBI GenBank database with accession number JX899417 (cDNA) on October 5, 2012.

The cDNA of the native bee-derived AcCI gene obtained by analyzing the nucleotide sequence of the native bee-derived bee derived from the present invention has a nucleotide sequence of SEQ ID NO: 1 having a length of 258 bp to the stop codon, and the poly of SEQ ID NO: 1 The protein encoded by the nucleotide sequence consists of a signal sequence of 20 amino acids and a mature peptide of 65 amino acids, and has a molecular weight of 7244 Dalton and a molecular weight of 16 kDa on electrophoresis. .

The native bee ( Apis cerana) the amino acid sequence of the serine protease inhibitor gene derived from the coding is registered in the NCBI GenBank database Apis mellifera and Bombus terrestris It has high homology with the amino acid sequence of a serine protease inhibitory protein, etc.

In addition, the present invention is the native bee ( Apis A recombinant expression vector comprising a polynucleotide encoding a serine protease inhibitor protein derived from cerana ) is provided.

As used herein, the term "vector" is a DNA molecule that is a means of delivery used to transfer foreign genetic material to another cell. Typically, plasmid vectors, bacteriophage vectors, cosmid vectors, vectors using artificial chromosomes (e.g., YAC, BAC) and the like exist. Since the gene is used to express a desired genetic material in a large amount, there is a common element necessary to function as a vector, and a replication origin, a multicloning site, and a selective marker must exist. shall. The vector itself contains the transgene and vector backbone to be transplanted into the DNA sequence.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. It is preferable that the polynucleotide of the present invention is derived from eukaryotic cells and that eukaryotic cells are used as a host in consideration of the convenience of culture. The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (such as a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL 貫 promoter, , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation and a transcription / translation termination sequence. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) and the left promoter of phage l Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)] can be used as a regulatory region.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, pEGFP, pCR, etc.), phages (eg, λgt4λB, λ) which are often used in the art. -Can be produced by manipulating viruses, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell (for example, a metallothionein promoter) or a mammalian virus Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

On the other hand, the vector of the present invention includes, as a selection marker, an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamicin, carbinicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The present invention provides a serine protease recombinant protein expressed from an insect baculo virus comprising a gene encoding a serine protease inhibitory protein.

The term " recombinant protein " in the present invention is a protein produced from recombinant DNA prepared by joining two or more DNAs with recombinant DNA technology.

The recombinant DNA technology can be used to synthesize a medical protein or an industrial enzyme that can only be obtained in a trace amount from the natural state, using bacteria such as E. coli and animal cells. It can be synthesized through fermentation and cultivation of yeast and Mammalian cells including bacteria (eg E. coli ) and purification of pharmaceutical raw materials. Examples of recombinant proteins include human growth hormone (hGH), insulin (INS), and interleukin.

In addition, the present invention provides a pharmaceutical composition for inhibiting inflammation comprising a serine protease inhibitory protein derived from native bee as an active ingredient.

Serine protease inhibitory protein derived from native bees of the present invention has excellent inhibitory activity against elastase and can be usefully used for inhibiting inflammation.

The composition of the present invention may contain one or more known active ingredients having an anti-inflammatory effect together with serine protease inhibitory proteins derived from native bees.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dose of the native bee-derived serine protease inhibitory protein is about 0.1-100 mg / kg, preferably administered once to several times a day.

The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifiers for inflammation suppression.

Below. The present invention will be described in detail by examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

[ Example  1] encoding serine protease inhibitory protein derived from native bee Gene cloning.

Apis received from the Department of Agricultural Biology, National Academy of Agricultural Science, RDA cerana ) cDNA library was prepared by using Uni-ZAP XR vector and Gigapack III Gold Packing Extract (Stratagene, USA), a commercial kit using poly (A) and mRNA extracted from worker bees and expressing gene expression. sequence tags, ESTs). DNA extraction was performed using a Wizard mini-preparation kit (Promega, USA) as a commercial kit. The DNA sequence was sequenced with an automated DNA sequencer (Applied Biosystems, USA). The nucleotide sequences were compared by the BLAST program of NCBI (http://www.ncbi.nlm.nih.gov/BLAST).

As a result, the cDNA of the native bee-derived serine protease inhibitory protein gene encoding 85 amino acids by native bee ESTs analysis was cloned, and this is shown in SEQ ID NO: 1. In addition, the result of database analysis of the amino acid sequence translated from native serine protease inhibitory protein cDNA is shown in SEQ ID NO: 2.

The results of the analysis by the BLAST program of the above sequence are shown in Fig.

As shown in Figure 1, Western species Bee Apis mellifera (GenBank accession no.XP_001121077) and Western duper Bombus terrestris It showed relatively high homology with (GenBank accession no. NP_001037044), and the cysteine residues forming five disulfide bonds were well preserved.

Example 2 Preparation of a vector expressing a serine protease inhibitory protein ( AcCI ) derived from recombinant native bees .

In order to express AcCI derived from native bees, AcCI cDNAs derived from native bees are isolated from insect baculovirus ( Autographa). californica nucleopolyhedrovirus) transfection vector pBAC8 (Clontech, USA), followed by transfection vector (500 ng) and non-eukaryotic ( bAc GOZA) viral DNA (100 ng) [Je et al ., Biotechnol. Lett., 23: 575-582 (2001)] were mixed with an insect cell line Sf9 ( Spodoptera frugiperda ) using lipofectin (Clonetech) 9) by co-transfection. After 5 days, the culture was collected to prepare a recombinant baculo virus expressing AcCI from native bees. Recombinant baculovirus was propagated in Sf9 cell line and recombinant AcCI was isolated using MagneHis ^™ protein purification system (Promega, USA). Recombinant AcCI protein electrophoresis from native bees is shown in Figure 2, and the sugar chain staining results are shown in Figure 3.

As shown in Figures 2 and 3, the recombinant AcCI derived from native bees can be confirmed by the glycoprotein staining results showing a molecular weight of 7244 Da, and a molecular weight of 16 kDa on electrophoresis by adding a sugar chain.

Example 3 Validation of Serine Protease Inhibitory Recombinant Protein

1. Trypsin or Chymotrypsin  Confirm inhibitory activity

In order to verify the characterization of serine proteases as proteins that inhibit serine proteases, the representative trypsin and chymotrypsin are inhibited to determine 100 nM trypsin (Sigma, USA) and 100 nM alpha chymotrypsin (Sigma). , USA) serine protease inhibitory recombinant protein was treated in 100 mM Tris-HCl (pH 8.0) buffer containing 20 mM CaCl ₂ and 0.05% Triton X-100 at a concentration of 0 ~ 200 nM. After reaction with the substrate for 30 minutes at 37 ° C, trypsin substrate 0.5 mM BApNA (Sigma, USA) and chymotrypsin substrate 0.5 mM Suc-AAPF-pNA (Sigma, USA) were added and reacted at 37 ° C for 30 minutes . The result of measuring the reaction at OD 405 nm is shown in FIG.

As shown in Figure 4, AcCI showed an inhibitory activity specifically for alpha chymotrypsin.

2. Elastase  Confirm inhibitory activity

To confirm the inhibitory activity against elastase, a serine protease associated with inflammation, 50 mM of serine protease inhibitory recombinant proteins in human (neutrophil elastase) and porcine (porcine pancreatic elastase) elastase were administered at concentrations of 0 to 200 nM. The reaction was performed in Tris-HCl (pH 7.4) buffer for 30 minutes at 37 ° C. without substrate, and 0.5 mM S-4760 substrate (Sigma, USA) was added followed by reaction at 37 ° C. for 1 hour.

The results of measuring the reaction at O.D 405 nm are shown in FIG. 5.

As shown in Figure 5, native bee-derived serine protease inhibitory protein showed a strong inhibitory ability to human elastase, IC ₅₀ of 50% inhibitory concentration was 38.50 nM. In addition, a relatively weak inhibitory ability (IC ₅₀ 70.21 nM) was confirmed in porcine elastase.

As a result, it was confirmed that native bee-derived AcCI has excellent elastase activity, and that it can inhibit inflammation.

Formulation example  One  : Preparation of Pharmaceutical Formulations

1. Manufacturing of powder

200 ml of serine protease inhibitory protein derived from native bee lactose 100 ml

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

200 ml of serine protease inhibitory protein derived from native bee

100 ml of corn starch

Lactose 100 ml

2 ml of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

200ml Serine Protease Inhibitory Protein from Native Bees

Corn starch 100ml

Lactose 100ml

Magnesium stearate 2ml

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Preparation of injections

200ml Serine Protease Inhibitory Protein from Native Bees

       Mannitol 100ml

_{Na 2 HPO 4 · 2H 2 O} 2ml

Sterile sterilized water for injection

Injection was prepared by mixing the above components per ampoule (2) according to the usual injection preparation method.

Attach an electronic file to a sequence list

Claims (8)

Apis consisting of the amino acid sequence of SEQ ID NO: 2 having inhibitory activity of chymotrypsin and elastase cerana ) derived serine protease inhibitory protein. Claim 1 tojongbeol (Apis A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 encoding a serine protease inhibitory protein derived from cerana ). A recombinant expression vector comprising the polynucleotide of claim 2. 4. The recombinant expression vector according to claim 3, wherein the recombinant expression vector is a recombinant baculovirus. The recombinant expression vector according to claim 4, wherein the recombinant baculo virus expresses a serine protease inhibitory recombinant protein (AcCI) derived from Apis cerana . Apis consisting of the amino acid sequence of SEQ ID NO: 2 A pharmaceutical composition for inhibiting inflammation comprising a serine protease inhibitory protein derived from cerana ) as an active ingredient. The pharmaceutical composition for inhibiting inflammation according to claim 6, wherein the serine protease inhibitory protein is encoded by the nucleotide sequence of SEQ ID NO: 1. The pharmaceutical composition for inhibiting inflammation according to claim 6, wherein the serine protease inhibiting protein inhibits the activity of chymotrypsin and elastase.

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