KR20150122088A

KR20150122088A - Cell permeable mx1 recombinant protein

Info

Publication number: KR20150122088A
Application number: KR1020150056137A
Authority: KR
Inventors: 이흥규; 정희은
Original assignee: 한국과학기술원
Priority date: 2014-04-22
Filing date: 2015-04-21
Publication date: 2015-10-30
Also published as: KR101650100B1

Abstract

The present invention relates to an Mx1 recombinant protein with increased cytopermeability and a use thereof. According to the present invention, Mx1 recombinant protein has increased cytopermeability and thus directly exhibits protective effects against various types of virus in a host cell. Therefore, it is anticipated to be used for effective antiviral medicine against various viruses by using the same.

Description

Cell permeable Mx1 recombinant protein {CELL PERMEABLE MX1 RECOMBINANT PROTEIN}

The present invention relates to an Mx1 recombinant protein having increased cell permeability which has a protective effect against influenza virus and its use.

In 1962, after Lindenmann discovered that A2G mice were resistant to Influenza virus infection, a variety of follow-up studies were conducted to determine the cause of resistance. Mx (Myxovirus (influenza virus) resistance) protein, a type of GTPases (Dynamin-like large guanosine triphosphatases) that is induced by a variety of RNA viruses plays an important role in promoting the antiviral mechanism against infection of various RNA viruses (Genome Res 12, 2002, 527-530).

Mx1, a GTPase of mouse, is a nucleotide protein composed of 631 amino acids. It contains a GTPase domain, a Dynamin GTPase effector domain, a Leucine zipper element, And a nuclear localization signal. Mutation analysis for each of the domains revealed that GTP binding elements and nuclear locus signals are essential for the anti-influenza virus effect of the Mx1 protein, and that the Mx1 protein is a functional virus It has been shown that the function of anti-influenza virus is inhibited by the binding of PB2 and NP, the proteins constituting the influenza virus, in the process of functional viral ribonucleoprotein complex assembly (Journal of Virology 86 (24), 2012, 13445-13455).

This Mx1 protein was expected to be used as an antiviral agent by inhibiting the virus activation and inhibiting the virus activation by participating in the innate immune response to various virus infections. However, since the Mx1 protein can not be smoothly transferred into the cell, It is a fact that it does not become.

On the other hand, the eukaryotic cells are surrounded by the cell membrane of the phospholipid bilayer. Due to the hydrophobicity inside the bilayer, it is almost impossible for the hydrophilic substances outside the cell to pass through the cell membrane and move into the cell. Helping. Among them, the protein transduction domain (PTD) is a peptide composed of about 10 basic amino acids, and it is well known that it can bind to various substances such as proteins, peptides, DNA, and move substances into cells without special receptors . Protein transfer by PTD was first detected in the Tat protein of HIV-1 (human immunodeficiency virus-1). The amino acid sequence of the PTD of HIV-1 Tat was found to be the 49-57th RKKRRQRRR, which is the smallest unit that causes free movement of the cell membrane. Recently, various sequence PTDs have been actively developed through subsequent studies.

The present invention aims at using Mx1 protein as an effective antiviral agent, and it is an object of the present invention to provide Mx1 recombinant protein having increased cell permeability and its use.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions, and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, particular features, forms, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

In the present specification, a protein transduction domain (PTD) is a peptide consisting of 3 to 30 amino acids and binds to various substances such as proteins, peptides, and DNA, thereby allowing a peptide capable of transferring substances into cells without special receptors It says. (SEQ ID NO: 4), a trans-activator of transcription (Tat), SEQ ID NO: 5 (YGRKKRRORRR), ANTP (SEQ ID NO: 7: RQIKIWFQNRRMKWKK), a sequence having 5 to 10 arginine 1 (SEQ ID NO: 10: KETWWETWWTEWSQPKKKRKV), Pep-2 (SEQ ID NO: 6: KETWFETWFTEWSQPKKKRKV), but can bind to the Mx1 protein to form a cell membrane There is no restriction as long as it is a domain through which the Mx1 protein can be transferred into the cell.

In this specification, the term "antivirus" refers to a prophylactic or therapeutic action against viral infection that is parasitic on living cells such as animals, plants, bacteria, The virus to be inhibited is not limited, but preferably refers to a virus belonging to orthomyxoviridae and an influenza virus.

The present invention provides a cell permeable Mx1 recombinant protein in which a protein transduction domain (PTD) is bound to one side of a Myxovirus resistance 1 (Mx1) protein.

In one embodiment of the present invention, the protein transduction domain may be an arginine binding sequence, Tat, ANTP, Vp22, MTS, Pep-1, Pep-2 or the like. However, there is no restriction as long as it is a domain capable of binding a protein bound to the Mx1 protein through the cell membrane and transferring it to the interior of the cell.

In another embodiment of the present invention, the recombinant protein may be labeled on one side of the protein for ease of separation. The label may be a histidine-tag, an immunoglobulin Fc region, or the like, but is not limited thereto.

In another embodiment of the present invention, the immunoglobulin Fc gene may be combined with a tobacco etch virus nuclear inclusion endopeptase (TEV protease) sequence so that it can be removed from the cells.

The present invention also provides a polynucleotide encoding the cell permeable Mx1 recombinant protein.

The present invention also provides a recombinant expression vector comprising the polynucleotide. The expression vector may be used for eukaryotic cells or bacteria, but is not limited thereto.

The present invention also provides a pharmaceutical composition for antiviral therapy comprising a cell permeable Mx1 recombinant protein.

In the present invention, the pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages. The pharmaceutical composition may be a human.

The pharmaceutical composition of the present invention may be formulated in the form of oral preparations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterilized injection solutions according to a conventional method, have. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like in the case of oral administration. A solubilizing agent, an isotonic agent, a stabilizer and the like may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, Elixir, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other, solutions, suspensions, tablets, capsules, sustained-release preparations and the like.

Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The pharmaceutical composition of the present invention varies depending on various factors including the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease to be prevented or treated. And the dose of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the type of drug, the route of administration and the period of time, and may be 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

The Mxl recombinant protein according to the present invention is expected to be used as an effective antiviral agent against various kinds of viruses by increasing cell permeability.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic representation of a recombinant vector for bacteria according to an embodiment of the present invention. FIG.
2 is a result of confirming intracellular introduction of the recombinant Mx1 protein according to an embodiment of the present invention.
FIG. 3 is a graph showing in vitro inhibitory effect of recombinant Mx1 protein on influenza virus and showing the effect of inhibiting virus multiplication by recombinant Mx1 protein. FIG.
FIG. 4 is a graph showing in vitro inhibitory effect of recombinant Mx1 protein on influenza virus, showing inhibitory effect of recombinant Mx1 protein on viral gene expression. FIG.
FIG. 5 is a graph showing the inhibitory effect of recombinant Mx1 protein on influenza virus in vitro, showing inhibition of virus proliferation upon treatment of cells with various concentrations of recombinant Mx1 protein.
FIG. 6 is a graph showing that the survival rate of influenza virus infection is improved in a mouse to which recombinant Mx1 protein is administered, as a result of in vivo examination of the protective effect of recombinant Mx1 protein against influenza virus.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Production of recombinant Mx1 expression vector

The present inventors have found that a recombinant expression vector comprising a sequence encoding a cell permeable protein, to which a protein transduction domain sequence is bound to one side of a whole sequence of a mouse-derived Myxovirus resistance 1 (Mx1) .

Specifically, according to a preferred embodiment of the present invention, 9 arginine (PTD sequence (SEQ ID NO: 4)) is added to the 3 'terminal region of the mouse-derived myxvirus resistance gene sequence (SEQ ID NO: 1) (NheI and NotI) cleavage sites, the PCR is carried out using the novel primer sequences SEQ ID NOS: 2 and 3 as forward and backward primers. The primer sequence can be hybridized at the end of the template Mx1 sequence to form a double-stranded structure.

A variety of DNA polymerases can be used in the PCR process, and the polymerase used is a thermostable DNA polymerase from T7 polymerase from various bacteriophages or from a variety of bacteria, including Thermus aquaticus, Taq), Thermus thermophilus (Tth), and Pyrococus furious (pfu).

When carrying out the polymerization reaction, buffer solutions are used which contain co-factors such as Mg ² ⁺ and dNTPs including dATP, dTTP, dCTP and dGTP sufficiently and the polymerase reaction can take place under optimal conditions.

After the polymerase chain reaction, restriction enzymes and DNA joining enzymes were used to insert the amplified sequences into the vector. Specifically, the present inventors used E. coli overexpressing vector pET28a. The pET28a vector itself contains a His tag at the multiple restriction site so that a His tag that facilitates protein extraction can be coupled to the recombinant protein. The PCR products and vectors were treated with Nhe I and Not I restriction enzymes and the PCR products were inserted into the vector using T4 DNA ligase. Finally, recombinant proteins having the same structure as in FIG. 1 were expressed in this vector . The vector expressing the recombinant Mx1 protein prepared through such a process was named pET28a-His-Mx1-9R expression vector.

Example 2: Expression of pET28a-His-Mx1-9R in E. coli

The pET28a-His-Mx1-9R vector prepared in Example 1 was transformed into Escherichia coli BL21 (DE3) (Enzynomics) and cultured in LB solid medium containing kanamycin for 12 hours at 37 ° C. A single colony formed at this time was inoculated into LB liquid medium containing kanamycin and incubated at 37 ° C. for 3 hours. The culture was cooled to 18 ° C. and then IPTG (Isopropyl-β-D-thiogalactopyranoside) 0.1 mM concentration, and further cultured for 18 hours to express Mx1 recombinant protein added with PTD sequence.

The culture was centrifuged at high speed and the precipitated cells were suspended in a lysis buffer and dissolved using an ultrasonic generator. The lysis buffer used contained Tris-HCl, NaCl, and imidazole, and PMSF (Phenylmethanesulfonylfluoride), a protease inhibitor, was added to prevent protein degradation. The lysed cells were again centrifuged at high speed and the supernatant was added to Ni-IDA (agarose resin column) to adsorb the protein on the resin. Subsequently, the eluted buffer solution containing excess imidazole was added to separate and extract the recombinant protein adsorbed on the resin, and the filtered recombinant protein was confirmed on an 8% SDS-PAGE gel.

The recombinant Mx1-9R expressed in the above procedure formed a large amount of insoluble inclusion bodies at the time of expression, but the yield of the water-soluble proteins could be increased by lowering the expression temperature to 18 ° C.

Example 3: Confirmation of intracellular introduction of recombinant Mx1 protein Mx1-9R

This recombinant protein was inoculated into MDCK (Madin-darby canine kidney) cells at various concentrations and time to confirm the ability of the recombinant Mx1 protein Mx1-9R having the PTD sequence extracted in Example 2 to intracellularly. Since the recombinant protein Mx1-9R expresses His, it was confirmed by flow cytometry how much Mx1-9R was introduced into the inoculated cells. Specifically, Mx1-9R was inoculated into MDCK cells, cultured at 37 ° C., and cells inoculated 12 hours or 24 hours later. The cells were washed with PBS, and the extra Mx1-9R not introduced was removed. Cell fixation and permeabilization kit was used to immobilize the cells and to impart permeability, and a His-specific antibody And the Mx1-9R protein present in the cells was confirmed using a flow cytometer. As a result, it was observed that the recombinant Mx1 protein Mx1-9R was introduced into the cells at a concentration of 50 μg / ml and 100 μg / ml after 12 hours (FIG. 2).

Example 4: Confirmation of virus infection resistance of recombinant Mx1 protein

After confirming that the recombinant Mx1 protein Mx1-9R was introduced into cells through Example 3, the effect of the Mx1 protein introduced into the cells on the viral infection-defying ability of the cells was confirmed. Specifically, Mx1-9R at a concentration of 50 μg / ml was treated with MDCK cells 12 hours before the infection and cultured at 37 ° C., and infected with influenza virus PR8 at 0.01 or 0.1 MOI (multiplicity of infection). After incubation for 48 hours at 37 ° C, the virus concentration in the culture medium was measured using a plaque assay method. The mRNA of the cells was extracted and the cDNA was synthesized using reverse transcriptase. The expression of the viral RNA was analyzed by qPCR Respectively. As a result, it was confirmed that when the recombinant Mx1 protein Mx1-9R was treated, virus proliferation was inhibited by 7 times at 0.1 MOI infection and 30 times at 0.01 MOI infection as compared with the control group as shown in FIG. 3 (p value < 0.001). It was observed that the expression of NP (nucleoprotein), a viral gene, decreased from 8 times at 0.1 MOI infection to 54 times at 0.01 MOI infection (Fig. 4).

In order to examine the effect of MDCK cells treated with various concentrations of Mx1-9R on influenza virus infection, the degree of inhibition of virus proliferation was examined. Twenty-two hours before infection, 50-125 μg / ml recombinant Mx1-9R The cells were treated and infected with 0.01 MOI of influenza virus. After 48 hours, the amount of virus in the culture solution was measured. As a result, a 6 to 9-fold inhibition of virus proliferation was observed for Mx1 protein (p value <0.05) (Fig. 5).

Example 5

It was confirmed that the recombinant Mx1 protein introduced into the cell via Mx1-9R enhances the ability to protect against influenza virus infection through Example 4, and it is verified whether the same effect is also exerted in vivo using the mouse. Specifically, 1 μg of recombinant Mx1 protein per mouse was administered into the nasal cavity, and after 24 hours, 25 PFU of influenza virus PR8 was infected intranasally and the survival rate of the mice was measured. At this time, mice in which PBS was intranasally administered and infected with 25 PFU of influenza virus PR8 in the nasal cavity after 24 hours were used as a control for the evaluation of the survival rate. Euthanasia was defined as the end of the experiment when the survival rate was measured. When the body weight of the mice decreased by 25% or more compared with the day of virus infection, euthanasia was performed. As a result, it was confirmed that the recombinant protein Mx1-9R increased the ability to protect against influenza virus infection in vivo as well (P value <0.05) ).

<110> Korea Advanced Institute of Science And Technology (Kaist) <120> CELL PERMEABLE MX1 RECOMBINANT PROTEIN <130> DPB140013.k01 <150> KR KR 14/048110 <151> 2014-04-22 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1896 <212> DNA <213> Mouse_Mx1 gene <400> 1 atggattctg tgaataatct gtgcaggcac tatgaggaga aggtgcggcc ctgtattgac 60 ctcatcgaca ccctgagggc tctgggtgtg gagcaggacc tggccctgcc tgccatcgct 120 gtcattgggg accagagttc agggaagagc tctgttctgg aagcactgtc tggagtggcc 180 ctccccagag gcagtggtat tgtcaccaga tgccctctgg tgctgaaatt gaggaagctg 240 aaagaaggag aggagtggag aggcaaagtc tcctatgatg acatcgaagt ggagctctct 300 gatccttcag aggtggaaga ggccattaac aagggtcaga acttcattgc tggggtaggc 360 ctggggatca gtgataagct cattagcctg gatgtcagct ccccaaatgt cccagacctg 420 actctcattg acctgcctgg aattaccagg gtggctgtag gcaaccagcc tgcagacata 480 ggacgccaga tcaagagact tatcaagaca tacatccaaa aacaagagac catcaacctg 540 gtggtagtcc ccagcaatgt ggacattgct accacagagg ctctgagcat ggctcaggag 600 gtggaccctg aaggggatag gaccataggg gtcttgacca agcctgatct ggtggacaga 660 ggtgctgaag gcaaggtctt ggatgtgatg cggaacctgg tgtatcctct gaagaagggc 720 tacatgatag tcaagtgcag aggtcagcag gacatccaag agcagctgag cctgactgag 780 gcttttcaga aagagcaagt cttcttcaag gatcactcat acttcagcat tcttctggaa 840 gatgggaagg ccacagtgcc ctgcctggca gagagactga ctgaggagct cacctcccac 900 atctgtaaat cactgccact attggaagat caaataaata gcagtcatca gagtgcaagc 960 gaggagctgc agaagtacgg tgcagacata ccagaagatg acagaacgag gatgtccttt 1020 ctggtgaata aaatcagtgc cttcaatagg aatatcatga atttgataca agcacaggaa 1080 accgtatcag agggagacag ccggttgttt accaaactgc gaaatgagtt tcttgcttgg 1140 gatgatcata ttgaggaata ttttaaaaaa gattctcctg aggtacaaag caagatgaaa 1200 gaatttgaaa atcagtatcg tggccgggag ctgccagggt ttgtggacta caaggcattt 1260 gagagcatca tcaaaaagcg agtcaaagcc ctggaagagt ctgctgtgaa catgctgcgc 1320 agggtcacta agatggtcca aactgccttc gtaaagattt tatcaaatga ttttggtgat 1380 tttttaaacc tctgctgtac tgctaagtcc aaaattaaag aaataagatt aaaccaagaa 1440 aaggaagctg aaaacctgat ccgacttcac ttccagatgg aacagattgt ctactgccag 1500 gaccaggttt acaaggaaac cttgaagacg atcagagaga aggaagctga gaaagagaag 1560 accaaggcat taataaaccc tgctaccttt caaaataact ctcagtttcc tcaaaagggg 1620 ttgactacca ctgagatgac ccagcacctg aaagcctact accaggagtg cagacggaat 1680 attgggagac agatccctct gattatccag tacttcatcc tgaaaacatt tggggaggaa 1740 atagagaaaa tgatgcttca gcttttacag gacaccagta agtgcagctg gttcctggag 1800 gagcagagtg acaccagaga gaagaagaag ttcctgaaaa ggcggctttt aaggctggat 1860 gaggctcggc agaagcttgc caaattctcc gattaa 1896 <210> 2 <211> 34 <212> PRT <213> Nhe1_mutant_primer 5 ' <400> 2 Cys Thr Ala Gly Cys Thr Ala Gly Cys Ala Thr Gly Gly Ala Thr Thr 1 5 10 15 Cys Thr Gly Thr Gly Ala Ala Thr Ala Ala Thr Cys Thr Gly Thr Gly 20 25 30 Cys Ala <210> 3 <211> 61 <212> PRT <213> Not1_mutant_primer 3 ' <400> 3 Ala Thr Ala Ala Gly Ala Ala Thr Gly Cys Gly Gly Cys Cys Gly Cys 1 5 10 15 Cys Thr Ala Gly Cys Gly Gly Cys Gly Thr Cys Thr Gly Cys Gly Thr 20 25 30 Cys Thr Gly Cys Gly Gly Cys Gly Thr Cys Thr Gly Cys Gly Ala Thr 35 40 45 Cys Gly Gly Ala Gly Aly Ala Thr Thr Thr Gly Gly Cys 50 55 60 <210> 4 <211> 9 <212> PRT <213> Protein transduction domain <400> 4 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5

Claims

Myxovirus resistance 1 (Mx1) A cell permeable Mx1 recombinant protein with a protein transduction domain (PTD) attached to one side of the protein.

The method according to claim 1,
Wherein the protein transfer domain is a sequence consisting of arginine.

3. The method of claim 2,
Wherein the arginine is a sequence of 5 to 10 joined.

The method according to claim 1,
Wherein the protein transduction domain is a trans-activator of transcription (Tat).

The method according to claim 1,
Wherein the protein transduction domain is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

The method according to claim 1,
Wherein a recombinant protein is bound to a histidine-tag on one side of the recombinant protein.

The method according to claim 1,
Wherein the immunoglobulin Fc region gene is bound to one side of the recombinant protein.

8. The method of claim 7,
Wherein the immunoglobulin Fc gene is linked to a Tobacco etch virus nuclear inclusion endopeptase (TEV protease) sequence.

9. A polynucleotide encoding the cell permeable Mx1 recombinant protein of any one of claims 1 to 8.

9. A recombinant expression vector comprising the polynucleotide of claim 9.

11. The method of claim 10,
RTI ID = 0.0 > pCEFL. &Lt; / RTI >

11. The method of claim 10,
Wherein the recombinant expression vector comprises the nucleotide sequence of SEQ ID NO: 1.

9. A pharmaceutical composition for antiviral use, comprising the cell permeable Mx1 recombinant protein of any one of claims 1 to 8.

14. The method of claim 13,
Wherein said pharmaceutical composition is characterized in that it is for treating or preventing an infection in influenza virus.

14. The method of claim 13,
Wherein said pharmaceutical composition is administered intravenously by intravenous injection, intramuscular injection, intramuscular injection, subcutaneous injection, intraconjugate injection, transdermal delivery or airway inhalation.