WO2010077005A2 - Polypeptide et polynucléotide issus de mycobactérie et procédé de criblage d'agent anti-mycobactérien - Google Patents

Polypeptide et polynucléotide issus de mycobactérie et procédé de criblage d'agent anti-mycobactérien Download PDF

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WO2010077005A2
WO2010077005A2 PCT/KR2009/007702 KR2009007702W WO2010077005A2 WO 2010077005 A2 WO2010077005 A2 WO 2010077005A2 KR 2009007702 W KR2009007702 W KR 2009007702W WO 2010077005 A2 WO2010077005 A2 WO 2010077005A2
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mycobacterium
mycobacteria
strain
derived
cuti
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WO2010077005A3 (fr
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Young Min Kim
Sae Woong Park
Taeksun Song
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Industry Academic Cooperation Foundation of Yonsei University
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Industry Academic Cooperation Foundation of Yonsei University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)

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  • the present invention relates to mycobacteria-derived polypeptide and polynucleotide, and a method of screening an anti-mycobacterial agent.
  • Tuberculosis is one of the most common infectious diseases in the world and continues to be a major health problem due to very low protective efficiency of the BCG vaccine, increase in opportunistic infections of AIDS patients, and increase in multi-drug-resistant tuberculosis (MDR-TB) infection that cannot be treated using conventional drugs.
  • MDR-TB multi-drug-resistant tuberculosis
  • CO-DH Carbon monoxide dehydrogenase
  • the present invention provides mycobacteria-derived polypeptide and polynucleotide.
  • the present invention provides a primer or probe specifically hybridized to a mycobacteria-derived nucleotide molecule.
  • the present invention provides an antibody specifically binding to a mycobacteria- derived polypeptide.
  • the present invention provides a recombinant vector including a mycobacteria- derived nucleotide sequence and promoter and a cell transformed thereby.
  • the present invention provides a method of screening an anti-mycobacterial agent.
  • a primer or probe specifically hybridized to a mycobacteria-derived nucleotide molecule.
  • an antibody specifically binding to a mycobacteria-derived polypeptide.
  • a recombinant vector including a mycobacteria-derived nucleotide sequence and promoter and a cell transformed thereby.
  • a recombinant vector including a method of screening an anti-mycobacterial agent.
  • the mycobacteria-derived polypeptide and polynucleotide and the method of screening an anti-mycobacterial agent according to the present invention may be applied to develop an anti-mycobacterial agent that does not have side effects in the human body.
  • FIG. 1 shows a method of obtaining Mycobacterium sp. strain JCl cutlmut&nt. Mycobacterium sp. strain JCl cwt/"mutant was selected using pSW60 having a mutated cutl gene having a middle portion deleted and a sacB gene and hygR gene for selecting a mutant.
  • FIG. 2 shows PCR results to identify Mycobacterium sp. strain JCl c ⁇ ?/mutant.
  • Primers for analysis were cutI-F(5'-CCGAGACCATCGACTGGGTG-3') and cutl- R(5-GCGCATTACCGTGCGACGTG-3').
  • a PCR product having the length of 1,772-bp was obtained from Mycobacterium sp. strain JCl wild type, and a PCR product having the length of 1,183-bp was obtained from Mycobacterium sp. strain JCl c ⁇ ?/mutant.
  • Lane 1 is a size marker to identify the size of the PCR products
  • Lanes 2 to 7 show the PCR results of Mycobacterium sp. strain JCl cutlmutant candidates.
  • candidates of lanes 2, 4, and 6 were identified as My- cobacterium sp. strain JCl c ⁇ /mutants.
  • FIG. 3 is a graph showing the growth of Mycobacterium sp. strain JCl cwt/"mutant using glucose.
  • Mycobacterium sp. strain JCl wild type and Mycobacterium sp. strain JCl cutl mutant were cultured in a SMB-glucose medium to obtain growth curves. There was no difference between Mycobacterium sp. strain JCl wild type and Mycobacterium sp. strain JCl cutlmutant with glucose.
  • FIG. 4 is a graph showing the growth of Mycobacterium sp. strain JCl cutlmut&nt using CO.
  • Mycobacterium sp. strain JCl cutlmutant Mycobacterium sp. strain JCl wild type and Mycobacterium sp. strain JCl cutlmut&nt were cultured in a SMB-CO medium to obtain growth curves.
  • Mycobacterium sp. strain JCl cutlmut&nt could not grow with CO.
  • FIG. 5 is a graph showing a complementation test of Mycobacterium sp. strain JCl cutlmut&nt. As a result of the complementation test of Mycobacterium sp. strain JCl cutlmutant, it was identified that Mycobacterium sp. strain JCl cutlmutant could not grow with CO because of cutl mutation.
  • FIG. 6 shows that CO-DH protein is not expressed in Mycobacterium sp. strain JCl cutlmutant.
  • Lane 1 and 2 show CBB staining results. It was identified that other proteins were similarly expressed in Mycobacterium sp. strain JCl cutlmut&nt (Lane 2, 4 and 6) but CO-DH was not expressed in Mycobacterium sp. strain JCl cutlmutant when compared with Mycobacterium sp. strain JCl wild type(Lane 1, 3 and 5).
  • Lane 3 and 4 show CO-DH active staining results. It was identified that Mycobacterium sp. strain JCl cutlmut&nt did not have CO-DH activity since CO-DH was not expressed in Mycobacterium sp. strain JCl cutlmutant according to the western blot results using Mycobacterium sp. strain JCl CO-DH antibody(Lane 5 and 6).
  • FIG. 7 shows the transcription of cutBCA in Mycobacterium sp. strain JCl cutl mutant. It was identified that transcription of cutBCA did not occur in Mycobacterium sp. strain JCl cutlmutant according to northern blot using cutB as a probe. Lane 1 and 2 show the total RNA extracted. Lane 3 and 4 show the northern blot results using cutB as a probe. Lane 1 shows the total RNA extracted from Mycobacterium sp. strain JCl. Lane 2 shows the total RNA extracted from Mycobacterium sp. strain JCl c ⁇ ?/mutant. The RNA was extracted after Mycobacterium sp. strain JCl and Mycobacterium sp. strain JCl cutlmut&nt were cultured to an exponential growth phase in a SMB-glucose medium.
  • FIGS. 8 to 11 are graphs showing activity of cutBCA promoter in Mycobacterium sp. strain JCl cutlmut&nt.
  • FIGS. 8 to 11 are graphs showing activity of cutBCA promoter in Mycobacterium sp. strain JCl cutlmut&nt.
  • FIG. 12 is a graph showing inhibition of growth of Mycobacterium sp. strain JCl cutl mutant by NO.
  • the survival rate of Mycobacterium sp. strain JCl c ⁇ ?/mutant(-O-) was reduced by about 80% compared to that of Mycobacterium sp. strain JCl wild type(-#-)as a result of measuring resistance of Mycobacterium sp. strain JCl and My- cobacterium sp. strain JCl cwt/"mutant against NO using SNP generating NO. Best Mode for Carrying out the Invention
  • a macrophage is an immune cell eliminating pathogens invading a human body.
  • the macrophage swallows the pathogens and produces various compounds, e.g., nitric oxide (NO), to digest and break down the pathogens.
  • NO nitric oxide
  • some pathogenic bacteria may live and proliferate in the macrophage by inhibiting the functions of NO or avoiding the influence of NO using a natural NO inhibitor produced by the macrophage or by self -producing an enzyme detoxifying NO.
  • pathogenic mycobacteria contain an enzyme such as NO dioxygenase or perox- ynitritase which oxidize NO in the macrophage.
  • carbon monoxide dehydrogenase according to an embodiment of the present invention oxides NO.
  • the "mycobacteria” used herein are classified as aerobic acid fast gram-positive bacteria with a thick cell wall that is waxy, hydrophobic, and rich in mycolic acid. Mycobacteria may be classified into non-pathogenic mycobacteria that grow fast and pathogenic mycobacteria that grow slow.
  • the non-pathogenic mycobacteria may be Mycobacterium fortuitum, Mycobacterium parafortuitum, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium phlei, Mycobacterium cuneatum, Myco- baterium gastri, Mycobacterium ID-Y, Mycobacterium flavescens, Mycobacterium neoaurum, Mycobacterium peregrinum, Mycobacterium diernhoferi, Mycobacterium smegmatis, Mycobacterium wolinsky, Mycobacterium sp. strain JCl, or the like.
  • the pathogenic mycobacteria may be Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium avium, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium asiaticum, Mycobacterium porcinum, or the like. It has been reported that quite a large number of these mycobacteria use carbon monoxide as an only carbon source and energy source. researchers have found features of mycobacteria based on the non-pathogenic mycobacteria having genetic features similar to those of the pathogenic mycobacteria for convenience of cultivation and safety.
  • the "carboxydobacteria” used herein indicates bacteria that grow using carbon monoxide as a sole carbon source and energy source.
  • the carbon monoxide dehydrogenase (CO-DH) is a key enzyme that oxidizes carbon monoxide into carbon dioxide in carboxydobacteria according to the following reaction: CO + H 2 O ⁇ CO 2 +2H + - ⁇ -2e ⁇
  • the carbon monoxide dehydrogenase oxidizes toxic nitrogen monoxide into non-toxic nitrogen dioxide in mycobacteria. According to this mechanism, pathogenic mycobacteria may be alive in a macrophage.
  • the carbon monoxide dehydrogenase may be used together with nitric oxide dehydrogenase (NO-DH).
  • NO-DH nitric oxide dehydrogenase
  • strain JCl may be represented by "cutBCA".
  • the " Mycobacterium sp. strain JCT'that will be used in the following examples belongs to mycobacteria and also belongs to carboxydobacteria.
  • 16S rRNA gene of My- cobacterium sp. strain JCl shares at least 96% similarity with 16S rRNA gene of the other mycobacteria and up to 99% similarity with 16S rRNA of some mycobacteria, e.g., M. peregrinum, M. wolinsky, etc.
  • Mycobacterium sp. strain JCl also contains 10-methyl Ci 8 0 that is also referred to as tuberculostearic acid and has fatty acid distribution that is similar to that of the other mycobacteria.
  • the mycobacteria-derived polypeptide according to the present embodiment has an amino acid sequence SEQ ID NO: 2.
  • the polypeptide having an amino acid sequence of SEQ ID NO: 2 is a protein activating the transcription of carbon monoxide dehydrogenase gene or the expression of the carbon monoxide dehydrogenase.
  • the mycobacteria-derived polypeptide according to the present embodiment may have an amino acid sequence representing substantial identity to the amino acid sequence.
  • the substantial identity indicates at least 60% of homology, preferably 80% of homology, and more preferably 90% of homology, when the amino acid sequence and another sequence are aligned so as to maximally correspond to each other and the aligned sequences are analyzed using an algorithm that is commonly used in the art. Aligning methods to compare sequences are well known in the art. Various methods and algorithms for alignment are disclosed by Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. MoI. Bio.
  • NCBI Basic Local Alignment Search Tool may be accessed at ,the webpage of the National Center for Biological Information(NCBI) and may be used with a sequence analysis program such as blastp, blasm, blastx, tblastn, and tblastx. A method of comparing sequence homology using this program may be found at http://www.ncbi.nlm.nih.gOv/B LAST/blast_help-html.
  • the mycobacteria-derived polypeptide may also include amino acid sequence variants in addition to those having natural amino acid sequence.
  • the mycobacteria- derived polypeptide variants refer to proteins having a different sequence since by deletion, insertion, non-conservative substitution, or conservative substitution of at least one amino acid residue of the natural amino acid sequence of the mycobacteria- derived polypeptide or a combination thereof. Amino acid substitutions in proteins and peptides which do not change activities of molecules are known in the art.
  • the common amino acid substitutions are found between Ala/Ser, Val/Ile, Asp/Glu, Thr/ Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/ Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
  • the amino acid may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation.
  • the mycobacteria-derived polypeptide or their variants may be extracted from natural resources, synthesized, or prepared using a recombination process based on DNA sequences.
  • a mycobacteria-derived polynucleotide having (a) a nucleotide sequence coding the amino acid having SEQ ID NO: 2; a nucleotide sequence complementary to the nucleotide sequence of (a); or a nucleotide sequence obtained by hybridizing the nucleotides of (a) and (b) under stringent conditions.
  • nucleotide used herein is a double- stranded or single- stranded deoxyribonu- cleotide or ribonucleotide.
  • the nucleotide may include a RNA genome sequence, a cDNA, a RNA sequences transcribed thereby, and analogs of natural nucleotides unless specifically stated otherwise.
  • the nucleotide according to the present embodiment includes not only a nucleotide coding the amino acid having SEQ ID NO: 2 but also its complementary sequence.
  • the complementary sequence may include a perfectly complementary sequence and a substantially complementary sequence.
  • the "substantially complementary sequence” used herein is a sequence that may be hybridized to the nucleotide of SEQ ID NO: 1 under stringent conditions that are known in the art.
  • the "stringent conditions” used herein are disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Haymes, B.
  • the stringent conditions may be obtained by a) washing with 0.015 M sodium chloride/0.0015 M sodium citrate/ 0.1% sodium dodecyl sulfate at 5O 0 C, or b) hybridizing in a hybridization buffer solution (50% formamide, 2 x SSC and 10% dextran sulfate) at 55 0 C and washing with EDTA-containing 0.1 x SSC at 55 0 C.
  • the nucleotide sequence may include a nucleotide sequence coding the amino acid having SEQ ID NO: 2 or a nucleotide sequence hybridized with a nucleotide sequence complementary to the nucleotide sequence coding the amino acid having SEQ ID NO: 2 under stringent conditions.
  • the stringent conditions may be obtained by hybridizing in a hybridization buffer solution (50% formamide, 2 x SSC and 10% dextran sulfate) at 55 0 C and washing with EDTA-containing 0.1 x SSC at 55 0 C.
  • the nucleotide sequence may include a part, i.e., a fragment, of the nucleotide sequence coding the amino acid having SEQ ID NO: 2.
  • the my- cobacteria-derived nucleotide may include a nucleotide having SEQ ID NO: 1.
  • a primer or probe specifically hybridized to the mycobacteria-derived nucleotide.
  • the "primer” used herein is a single-stranded oligonucleotide functioning as an origin of polymerization of template DNA under an appropriate conditions (i.e., 4 types of different nucleoside triphosphates and polymerases) at a suitable temperature and in a suitable buffer solution.
  • the length of the primer may vary according to various factors, for example, temperature and the use of the primer, but the primer may have 15 to 30 nucleotides. Generally, a short primer molecule may form sufficiently stable hybrid complexes with a template at a low temperature.
  • the sequence of the primer is not required to be perfectly complementary to a part of the sequence of the template.
  • the primer may have sufficient complementarity to be hybridized with the template and perform intrinsic functions of the primer.
  • a primer set is not required to be perfectly complementary to the sequence of the nucleotide that is a template.
  • the primer set may have sufficient complementarity to be hybridized with the sequence and perform intrinsic functions of the primer.
  • the primer may be designed based on cDNA sequence of the nucleotide, for example, using a program for designing primers (PRIMER 3 program).
  • the primer according to the present embodiment may be hybridized or annealed to a part of the template to form a double-strand structure.
  • Conditions for hybridizing nucleotides suitable for forming the double- stranded structure are disclosed by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Haymes, B. D.,et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C.(1985).
  • the "probe” used herein is a deoxyribonucleotide or ribonucleotide which may be hybridized to a specific nucleotide sequence.
  • the probe may be a single strand for increasing efficiency of hybridization.
  • the probe may be a deoxyribonucleotide.
  • the probe according to the present embodiment may include a perfectly complementary sequence to the nucleotide or a substantially complementary sequence that does not inhibit specific hybridization. Conditions suitable for the hybridization are described above.
  • an antibody specifically binding to a mycobacteria-derived polypeptide is provided.
  • the antibody specifically binding to a mycobacteria-derived polypeptide may be a polyclonal or monoclonal antibody, and preferably a monoclonal antibody.
  • the antibody specifically binding to a mycobacteria-derived polypeptide may be prepared using a method that is commonly used in the art, for example, fusion, recombination of DNA, or phage antibody library. Methods of preparing antibody are disclosed by Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, (1999); and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY(1991), which are incorporated herein by reference.
  • hybridoma cells producing a monoclonal antibody may be prepared by fusing an immortal cell line with an antibody-producing lymphocyte, which is well known in the art.
  • the polyclonal antibody may be prepared by administering the mycobacteria-derived polypeptide antigen to an animal by injection, collecting an anti-serum from the animal, and isolating an antibody from the anti-serum using affinity chromatography.
  • a recombinant vector having a nucleotide sequence coding the mycobacteria-derived polypeptide and a promoter operatively linked to the mycobacteria-derived polynucleotide.
  • the mycobacteria-derived nucleotide is operatively linked to the promoter.
  • the "operatively linked” used herein indicates a functional binding between a nucleotide expression controlling sequence (e.g., promoter sequence) and other nucleotide sequence, and the nucleotide expression controlling sequence may control transcription and/or translation of the other nucleotide sequence.
  • the vector may be a vector for cloning or expression.
  • a prokaryotic cell or a eukaryotic cell may be used as a host cell for the vector. Since the polynucleotide is derived from bacteria, a prokaryotic cell may be used as a host cell for the convenience of cultivation.
  • the vector may include a promoter capable of initiating transcription, such as tac promoter, lac promoter, / ⁇ cUV5 promoter, lpp promoter, p L ⁇ promoter, p R ⁇ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, a ribosome-binding site to initiate translation, and a transcription/ translation termination sequence.
  • a promoter and operator sites of biosynthetic pathway of E. coli tryptophan and left-side promoter of phage ⁇ (e.g.,p L ⁇ promoter) may be used as a controlling site.
  • the recombinant vector according to the present embodiment may be prepared using plasmid, such as pSClOl, CoIEl, pBR322, pUC8/9, pHC79, pUC19, and pET, phage, such as ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ zl and M 13, or virus, such as SV40, which are widely used in the art.
  • plasmid such as pSClOl, CoIEl, pBR322, pUC8/9, pHC79, pUC19, and pET
  • phage such as ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ zl and M 13, or virus, such as SV40, which are widely used in the art.
  • the recombinant vector according to the present embodiment may be fused to another sequence in order to facilitate purification of mycobacteria-derived protein expressed from the recombinant vector.
  • the fused sequence may be glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA) and hexahistidine (6x His, Qiagen, USA), and preferably 6x His. Due to the additional sequence for the purification, protein expressed in the host cell may be quickly and efficiently purified through affinity chramoatography.
  • the expression vector is a selectable marker that may include an antibiotic-resistant gene commonly used in the art.
  • the expression vector may include genes resistant to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, hygromycin and tetracycline.
  • a genome DNA and its transcripts may be used.
  • a method of preparing transcripts is known in the art.
  • the vector is required to be linearized.
  • the host cell for transcription may be any host cell that is known in the art.
  • the vector may be transported to the host cell during the transformation using a CaCl 2 method, a Hanahan method, and electro- poration.
  • a method of screening an anti-mycobacterial agent including: (a) contacting a sample to be assayed to a cell including the mycobacteria-derived polypeptide or the my- cobacteria-derived polynucleotide; and (b) measuring the activity of the polypeptide, the amount of transcribed or expressed polynucleotide, wherein if the activity of the polypeptide, or the amount of transcribed or expressed polynucleotide is down- regulated, the sample is determined to be an anti-mycobacterial agent.
  • a substance to be screen is contacted to a cell containing the mycobacteria-derived polypeptide or the mycobacteria-derived polynucleotide.
  • sample used herein is an unknown substance to be screened in order to detect whether the sample influences the activity of the polypeptide and the amount of transcribed or expressed polynucleotide.
  • the sample may include chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural extract, but is not limited thereto.
  • the cell may include any cell containing the mycobacteria-derived polypeptide or the mycobacteria-derived polynucleotide, for example, transformed E. coli or wide type mycobacteria.
  • Examples of the transformed microorganism or wild type mycobacteria used for the screening are as follows.
  • the mycobacteria used in the present embodiment may include Mycobacterium fortuitum, Mycobacterium parafortuitum, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium phlei, Mycobacterium cuneatum, Mycobaterium gastri, Mycobacterium ID-Y, Mycobacterium flavescens, Mycobacterium neoaurum, Mycobacterium peregrinum, Mycobacterium diernhoferi, Mycobacterium smegmatis, Mycobacterium wolinsky, Mycobacterium sp.
  • strain JCl Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium avium, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium asiaticum or Mycobacterium porcinum, preferably, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium phlei, Mycobaterium gastri, Mycobacterium flavescens, Mycobacterium neoaurum, Mycobacterium smegmatis, Mycobacterium sp.
  • strain JCl Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium avium, and more preferably, Mycobacterium smegmatis, Mycobacterium sp. strain JCl or Mycobacterium tuberculosis.
  • the sample that is assayed by the screening may be a single compound or a mixture of compounds, for example, natural extract or cell or tissue culture.
  • the sample may be obtained from a library of synthesized or natural compounds.
  • a method of obtaining the library is well known in the art.
  • the library of the synthesized compounds may be purchased from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma- Aldrich (USA), and the library of the natural compounds may be purchased from Pan Laboratories (USA) and My- coSearch (USA).
  • the activity of the polypeptide and the amount of transcribed or expressed polynucleotide in the cell are measured. If the activity of the polypeptide or the amount of transcribed or expressed polynucleotide is down-regulated, the substance may be determined as an anti-mycobacterial agent.
  • the amount of polypeptide may be measured using various analytical methods.
  • the amount of polypeptide may be measured by various immunoanalytical formats using antibodies specifically binding to the polypeptide.
  • the immunoanalysis may be conducted by various quantitative immunoanalytical protocols.
  • the immunoanalytical format may include a radioimmunoassay, radioimmunoprecipitation, im- munoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, sandwich assay, immunofluorescence and immunoaffinity purification, but is not limited thereto.
  • an antibody marked with a radioisotope such as C 14 ,I 125 ,P 32 and S 35 may be used to detect the polypeptide.
  • the method may include: coating degradation products of an unknown cell on the surface of a solid substrate; reacting a polypeptide-specific antibody, as a primary antibody, with the degradation products of the cell; reacting the resultants with a secondary antibody binding to an enzyme; and measuring the activity of the enzyme.
  • the solid substrate may be a hydrocarbon polymer (for example, polystyrene and polypropylene), glass, metal or gel, and preferably microtiter plate.
  • hydrocarbon polymer for example, polystyrene and polypropylene
  • glass for example, glass, metal or gel, and preferably microtiter plate.
  • the enzyme bound to the secondary antibody may include an enzyme catalyzing color development, fluorescence, luminescence, or infrared light interactance, but is not limited thereto.
  • alkaline phosphatase ⁇ -galactosidase, horseradish peroxidase, luciferase, and cytochrome P 450 may be used as the enzyme.
  • the substrate may be a color developing substrate such as bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol- AS-B 1 -phosphate, and enhanced chemifluorescence (ECF).
  • BCIP bromochloroindolyl phosphate
  • NBT nitro blue tetrazolium
  • ECF enhanced chemifluorescence
  • the substrate may be chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, bis-N-methylacryidinium nitrate (lucigenin), resorufin benzyl ether, luminol, 10-acetyl-3,7-dihydroxyphenoxazine (amflex red), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), 2,2'-Azine-di[3-ethylbenzthiazoline sulfonate] (ABTS), o - phenylenediamine(OPD) and naphthol/pyronine, glucose oxidase, nitroblue tetrazolium(t-NBT) and phenzaine methosulfate(m-PMS).
  • chloronaphthol aminoethylcarbazole
  • diaminobenzidine D-
  • the method may include: coating an antibody of the polypeptide, as a capturing antibody, on the surface of a solid substrate; reacting the capturing antibody with a cell sample; reacting the resultants with a detecting antibody binding to a label generating a signal and specifically reacting with the polypeptide; and measuring the signal generated by the label.
  • the detecting antibody includes a label generating a detectable signal.
  • the label may include chemicals (e.g., biotin), enzymes (alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, luciferase, and cytochrome P 450 ),radio active materials(C 14 ,I 125 ,P 32 and S 35 ), fluorescent materials(fluorescein), luminescent materials, chemilu- minescent materials and fluorescence resonance energy transfer(FRET) material,but is not limited thereto.
  • chemicals e.g., biotin
  • enzymes alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, luciferase, and cytochrome P 450
  • radio active materials C 14 ,I 125 ,P 32 and S 35
  • fluorescent materials fluorescein
  • luminescent materials chemilu- minescent materials
  • FRET fluorescence resonance energy
  • the activity of the enzyme or the signal may be measured using a method that is well known in the art.
  • the polypeptide may be quantitatively analyzed. If biotin is used as the label, the signal may be efficiently detected using streptavidin. If luciferase is used, the signal may be efficiently detected using luciferin.
  • the sample is determined as an anti-mycobacterial agent.
  • the amount of transcribed or expressed polynucleotide may be measured using methods that are well known in the art.
  • the amount of transcribed or expressed polynucleotide may be measured by hybridization using RT-PCR, northern blotting, cDNA microarray or ms/ft/hybridization.
  • the total RNA is isolated from the cell, and a single- stranded cDNA using an oligo dT primer (e.g., prokaryotic cell) or a primer specific to a gene to be identified (e.g., eukaryotic cell), and a reverse transcriptase. Then, a PCR is conducted using the single-stranded cDNA as a template and a polynucleotide- specific primer set. Then, PCR products are subjected to electrophoresis, and bands obtained thereby are analyzed to measure the change of the amount of expressed cutl gene.
  • an oligo dT primer e.g., prokaryotic cell
  • a primer specific to a gene to be identified e.g., eukaryotic cell
  • the sample may be determined to be an anti-mycobacterial agent.
  • the anti-mycobacterial agent may be a substance for preventing or treating Buruli ulcer, lymphadenitis, or tuberculosis.
  • M. tuberculosis H37Rv A variety of pathogenic mycobacteria including M. tuberculosis H37Rv that is a my- cobacterium tuberculosis have CO-DH(e.g.,M. tuberculosis C, M. tuberculosis CDC 1551, M. tuberculosis FI l, M. bovis, M. tuberculosis sp. Haarlem, M. marinum, M. ulcerous, or the like).
  • some bacteria for example, M. marinum, M. ulcerans, etc., may induce another disease such as Buruli ulcer and lymphadenitis as well as tuberculosis.
  • the anti-mycobacterial agent may be used to prevent or treat Buruli ulcer and tuberculosis, and preferably to prevent or treat tuberculosis.
  • the tuberculosis screened by the method according to the present embodiment may include ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, tuberculosis of epididymis, tuberculosis of lymph nodes, laryngeal tuberculosis, tuberculosis of middle ear, intestinal tuberculosis and pulmonary tuberculosis, but is not limited thereto.
  • the anti-mycobacterial agent developed according to the screening method may be efficiently used to treat diseases related to mycobacteria with conventional anti- mycobacterial agents.
  • Bacterium used herein was Mycobacterium sp. strain JCl. Bacteria were cultured in a standard mineral base (SMB) medium (Kim et al., J Bacteriol 148,904-911(1981)) used to culture carboxydobacteria while supplying carbon monoxide(30%, v/v) or glucose(0.2%, w/v) at 37 0 C. E. coli was cultured in a Luria-Bertani(LB) medium for gene manipulation.
  • SMB standard mineral base
  • LB Luria-Bertani
  • Mycobacterium sp. strain JCl was shaking-cultured to an exponential growth phase in a SMB medium containing glucose at 37 0 C at 200 rpm, and 5 m# of the culture solution was centrifuged (18,000 x g, for 15 minutes, at 4 0 C) to obtain a precipitate. 567 j ⁇ of a TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0]) was added to the precipitate to prepare a suspension.
  • a TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0]
  • a phenol-chloroform-isoamyl alcohol (25:24:1, v/v/v) solution was added thereto, and the resultant was centrifuged (18,000 x g, for 15 minutes, at 4 0 C) to obtain a supernatant.
  • 3 M sodium acetate (pH 5.2) was added to the supernatant such that the concentration of sodium acetate was set to 0.3 M, and ethanol having twice amount of the supernatant was added thereto.
  • the resultant was maintained at -2O 0 C for 30 minutes and centrifuged (18,000 x g, for 15 minutes, at 4 0 C) to obtain a precipitate.
  • the precipitate was washed with 70% (v/v) ethanol, dried, and dissolved in 20 j ⁇ of RNase (10 ⁇ g/m-6)-TE buffer.
  • the resultant was centrifuged (18,000 x g, for 10 minutes, at 4 0 C), and a supernatant was carefully collected and passed through a column filled with a silica bead membrane (Intron) while centrifuging (18,000 x g, for 1 minute, at 25 0 C).
  • the supernatant passed through the column was removed and 700 j ⁇ of a washing buffer was passed through the column while centrifuging (18,000 x g, for 1 minute, at 25 0 C).
  • the washing buffer was completely removed, and the column was dried at room temperature for 2 minutes. 50 ⁇ Jl of distilled water was added thereto and maintained at room temperature for 1 minute.
  • the resultant was centrifuged (18,000 x g, for 1 minute, at 25 0 C) to obtain a solution containing plasmid.
  • a mega-spin kit (Intron) was used to isolate DNA fragments from agarose gel.
  • DNA sample processed with a restriction enzyme was subjected to electrophoresis in agarose gel to identify the site of a desired DNA fragment using UV transilluminator.
  • a desired portion of the agarose gel was cut and placed in a test tube, and 500 ⁇ Jl of a gel extraction buffer (Intron) was added thereto and placed in an oven for 5 minutes.
  • a gel extraction buffer Intron
  • the agarose gel is dissolved in the gel extraction buffer, they were mixed at room temperature, and then the mixture was passed through an elution column while cen- trifuging (18,000 x g, for 1 minute, at 25 0 C).
  • the mixture passed through the elution column was removed and 700 ⁇ Jl of a washing buffer was passed through the elution column while centrifuging (18,000 x g, for 1 minute, at 25 0 C).
  • the washing buffer passed through the elution column was removed and the elution column was dried at room temperature for 1 minute.
  • 30 j ⁇ of distilled water was dropped at the center of the elution column and dried at room temperature for 2 minutes.
  • the collecting tube was replaced with a new collecting tube and centrifuged (18,000 x g, for 1 minute, at 25 0 C) to obtain isolated DNA fragments.
  • E. coli was transformed using a CaCl 2 method, and Mycobacterium sp. strain JCl was transformed using electroporation, methods which were modified from Parish and Stacker methods. For the electroporation, Gene PulserTMand Pulse Controller of Bio- Rad Laboratories, Inc. were used.
  • E. coli competent cells were prepared by inoculating E. coli into a LB liquid culture medium and cultured at 37 0 C overnight, inoculating the cultured E. coli into 100 m# of a fresh LB liquid culture medium, shaking-culturing the E. coli for 3 hours, transferring the E.
  • the competent cell was centrifuged (4,000 x g, for 10 minutes, at 4 0 C), and E. coli collected and re-suspended with 10 m ⁇ , of a cold 0.1 M CaCl 2 solution and placed in ice for 30 minutes.
  • the suspension was centrifuged (4,000 x g, for 10 minutes, at 4 0 C) to collect E. coli, a supernatant was removed. 2 m ⁇ , of cold 0.1 M CaCl 2 solution containing 15%(v/v) glycerol was added to the remaining solution, and 50 ⁇ Jl of each E.
  • coli was divided into sterilized test tubes and stored at -7O 0 C before use.
  • DNA was mixed with the competent cell and the mixture was placed in ice for 20 minutes, heat- treated at 42 0 C for 90 seconds, and cooled in ice for 1 minute. Then, 200 j ⁇ of a LB liquid culture medium was added thereto, and the DNA and the competent cell were cultured at 37 0 C for 1 hour. The culture solution was spread onto a LB solid culture medium containing antibiotics and cultured at 37 0 C. 40 ⁇ Jl of
  • My- cobacterium sp. strain JCl was shaking-cultured in a SMB medium containing glucose to an exponential growth phase (OD 6 oo:0.5), the culture solution was placed in ice for 2 hours, and the resultant was centrifuged(4,000xg, for 10 minutes, at 4 0 C). The resultant was washed with the same amount of sterilized cold 10% (v/v) glycerol solution and washed twice by reducing the volume by 1/3. The resultant was added to a 10% (v/v) glycerol solution and stored at -7O 0 C to be used as the competent cell.
  • a restriction enzyme was purchased from KOSCO (Seoul, Korea), and T4 DNA ligase was purchased from Invitrogen (Carlsbad, California). The use of the restriction enzyme, the DNA ligation, and DNA recombination are performed based on manufacturer's instructions. Ampicillin was purchased from Gemini Bio-Product Co. (Calabasas, CA), and hygromycin was purchased from Roche (Mannheim, Germany). The other reagents were purchased from Sigma (St. Louis, MO).
  • pJK53 including cutl gene was cleaved by Kpnl to obtain fragments having the lengths of 4,020-bp, 2,739-bp, and 589-bp. Among them, fragments having the lengths of 4,020-bp and 2,739-bp were ligated to prepare pSW58 vector including c ⁇ ?/-deleted gene.
  • pSW58 vector including c ⁇ ?/-deleted gene.
  • pKO Korean, D. R. M. Voskuil, D. Schnappinger,R. Liao, M. I. Harrell, and G. K. Schoolnik., Proc. Natl. Acad. Sci.
  • PCR was performed using a Master gradient (Eppendorf, Hamburg, Germany).
  • 50 ⁇ i of PCR solution includes 2.5 niM MgCl 2 , dNTP, a 10x buffer, 20 pmol of primers, an Ex Taq DNA polymerase(Takara, Shiga, Japan) and 100 ng of a DNA template.
  • PCR conditions are as follows: pre-denaturation at 94 0 C for 10 minutes, denaturation at 94 0 C for 1 minute, annealing at 65 0 C for 1 minute, elongation 30 times at 72 0 C for 2 minutes, and post-elongation at 72 0 C for 10 minutes.
  • CT-F (5'-AAGCTTAGTCCAGTCCGAACCCGAAC-3';underline, Hmdm recognition site) and cutI-CT-R(5'-GGATCCCGAAT AGGAAGCC AGCTTTC-3' underline, Bam HI recognition site) primers and ligated to pNBVI cleaved using HindJR and BamHI to prepare pSW98 vector(Table 1).
  • the prepared pSW98 was subjected to electroporation for the complementation test of Mycobacterium sp. strain JCl cutl mutant.
  • Mycobacterium sp. strain JCl cultured in a SMB-glucose medium was cen- trifuged(18,000xg, for 10 minutes, at 4 0 C) (Eppendorf centrifuge-5403, Hamburg, Germany) and collected, washed twice with a 50 mM Tris-HCl (pH 7.5) buffer solution, and re-suspended with 3 m# of the 50 mM Tris-HCl (pH 7.5) buffer solution.
  • strain JCl was sonicated at O 0 C using a sonicator (Sonics & Materials Inc., Danbury, CT) at 20% amplitude for 3 seconds and paused for 10 seconds. This process was repeated 20 times to homogenize Mycobacterium sp. strain JCl. The homogenized culture solution was centrifuged (18,000 x g, for 30 minutes, at 4 0 C) (Eppendorf centrifuge-5403) and collected, and a supernatant was used as an enzyme extract.
  • a sonicator Sonics & Materials Inc., Danbury, CT
  • Electrophoresis was performed using a Mighty Small SE245 vertical slab gel device (Amersham Pharmacia Biotech, Arlington Heights, IL) using a modified Laemmli method. In order to analyze a native enzyme, electrophoresis was performed without using SDS (Sigma, St. Louis, MO). A 7.5% (w/v) of acrylamide gel was prepared using a stock solution including 30% (w/v) acrylamide (Sigma) and 0.8% (w/v) N, N' - bis-methylene acrylamide(Sigma)7.5%(w/v). Protein mixed with a buffer was loaded, and the electrophoresis was performed while cooling using an ice water circulation device.
  • Protein was stained using a solution including 45% (v/v) methanol, 10% (v/v) acetic acid, and 0.25% (w/v) CBB R-250 for 30 minutes. The stained gel was washed several times with a destaining solution (30% [v/v] methanol and 10% [v/v] acetic acid).
  • the enzyme extract was subjected to electrophoresis in acrylamide gel not having SDS, and the acrylamide gel was added to a glass tube including a 50 mM Tris-HCl (pH 7.5) buffer solution and maintained therein for 5 minutes to saturate with CO. 1 m ⁇ , of a solution including 0.05% (w/v) phenazine methosulfate (PMS, Sigma) and 0.25% (w/v) nitroblue tetrazolium (NBT, Sigma) was added thereto, and the resultant was maintained until a clear active staining band was observed. This process was performed while light was blocked.
  • a solution including 0.05% (w/v) phenazine methosulfate (PMS, Sigma) and 0.25% (w/v) nitroblue tetrazolium (NBT, Sigma
  • the enzyme extract was subjected to electrophoresis in acrylamide gel not containing SDS, and protein in the acrylamide gel was transferred to a membrane using a Western blotter (Amersham Pharmacia Biotech.) by electricity.
  • the membrane was dipped in Ix PBS (13.7 mM NaCl, 0.27 mM KCl, 0.43 mM Na 2 HPO 4 -7H 2 O and 0.14 mM KH 2 PO 4 ) for 1 minute and shaken using a 10% blocking solution prepared by dissolving 10%[w/v] skim milk in IxPBS for 1 hour.
  • strain JCl obtained from a rabbit was added to the above solution in a ratio of 1:5,000, and the mixture was slowly shaken for 1 hour.
  • the mixture was washed twice with Ix PBS for 15 minutes and slowly shaken using a 10% blocking solution including ZymaxTMGoatanti-Rabbit IgG(H+L)(Zymed, S. Sanfrancisco, California) in a ratio of 1:5,000 for 1 hour.
  • the resultant was washed three times with PBS for 15 minutes, and a mixture of solution A and solution B of WEST-ZOLTM(Intron, Sungnam, Korea)in a ratio of 1 : 1 was sprayed to the membrane.
  • the membrane was photosensitized with an X-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan).
  • My- cobacterium sp. strain JCl was transformed using pCS6 including a promoter of a putative copyl CO-DH gene having the length of 720-bp that is from 12bp site from 'A' of start codon(ATG) of cutB in the opposite direction of transcription of cutB to 41 lbp site from 'G' of start codon(GTG) of cutR gene in the transcription direction of cutR, pCS7 including a promoter of a putative copy II CO-DH gene having the length of 760-bp that is from 12bp site from 'A' of start codon(ATG) of cutB in the opposite direction of transcription of cutB to 496bp site from 'A' of start codon(ATG) of cutD gene in the transcription direction of cutD, and pCS8 as a control without having a promoter using electroporation(Lee,Cho-soon, Master'
  • pNBVl that is used to prepare pCS6, pCS7 and pCS8 was disclosed by Howard et al, Gene 166:181-182(1995).
  • the inoculated culture solution was cultured at 37 0 C at 200 rpm, and re-suspended in 3 m# of 50 mM Tris-HCl (pH 7.5).
  • the suspended Mycobacterium sp. strain JCl was sonicated using a sonicator(Sonic & Materials, VCX600, Danbury, CT) at 20% amplitude for 3.0 seconds and paused for 9.9 seconds. This process was repeated for 5 minutes to homogenize Mycobacterium sp. strain JCl. Then, the homogenized Mycobacterium sp. strain JCl was centrifuged (18,000 x g, for 30 minutes, at 4 0 C) to obtain a supernatant.
  • the supernatant was used as an enzyme extract to measure the activity of ⁇ - galactosidase according to a Miller method.
  • 500 ⁇ i of a Z buffer 0.1 M sodium phosphate [pH 7.0], 10 mM KCl, 1 mM MgSO 4 , 50 mM ⁇ -mercaptoethanol
  • 200 ⁇ i of o -ni- trophenyl- ⁇ -D-galactoside(ONPG[4 mg/m-6]) was added thereto as a substrate.
  • Color change of the mixture was measured using absorbance at 420 nm.
  • ⁇ - galactosidase 1 unit of ⁇ - galactosidase was defined as an amount required to produce 1 nmole o-nitrophenol from ONPG per minute,and specific activity of enzyme was represented by ⁇ - galactosidase unit per mg of protein per minute.
  • a survival rate of Mycobacterium sp. strain JCl and cutl- mutants against SNP was measured using a modified Hernandez-Urzua method. 100 j ⁇ of each of My- cobacterium sp. strain JCl and cutl- mutants cultured to the mid-exponential growth phase in a SMB-glucose medium was added to a test tube. The survival rate thereof was measured after 0, 30, 60, and 90 minutes, three experimental groups to which 5 mM SNP was added and three control groups were prepared. The experimental groups and control groups were cultured, diluted, and smeared onto a SMB-glucose solid medium.
  • Colony forming units were calculated based on the number of colony to obtain a survival curve.
  • the Mycobacterium sp. strain JCl was cultured under sterile conditions with light since SNP is degraded by light to generate NO.
  • cutBCA that is a structural gene of carbon monoxide dehydrogenase of Mycobacterium sp. strain JCl and cutBCA homolog gene of pathogenic mycobacteria was analyzed using a ClustalW program. Referring to Table 2 below, cutBCA of Mycobacterium sp. strain JCl had very high identity of nucleotide sequence to cutBCA homolog gene of pathogenic mycobacteria such as M. tuberculosis. In addition, identity of cutl according to the present invention to homolog gene of various pathogenic mycobacteria was analyzed in the same manner as described above (Table 3).
  • Mycobacterium sp. strain JCl cwt/"mutant was induced by homologous recombination and screened using levan sucrase that is a sacB gene product which is widely used to prepare mutants of mycobacteria. It was identified that Mycobacterium sp. strain JCl cutlmutant grew well with glucose(FIG. 3) but could not grow with CO(FIG. 4).
  • CO-DH enzyme active staining and western blot were performed in order to identify whether Mycobacterium sp. strain JCl cutlmut&nt could not grow with CO since CO- DH protein was not expressed or since a cof actor was not properly acting even though the CO-DH protein was expressed. As a result of the CO-DH enzyme active staining and western blot, it was identified that Mycobacterium sp. strain JCl cutlmut&nt could not grow with CO since CO-DH protein was not expressed(FIG. 6).
  • strain JCl wild type increased in the late-exponential growth phase, but the activity of the promoter of Mycobacterium sp. strain JCl cutlmut&nt did not increase. Thus, it was identified that Cutl is an important factor influencing the transcription of CO-DH.

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Abstract

La présente invention porte sur un polypeptide issu de mycobactérie, sur un polynucléotide issu de mycobactérie et sur un procédé de criblage d'un agent anti-mycobactérien.
PCT/KR2009/007702 2008-12-29 2009-12-23 Polypeptide et polynucléotide issus de mycobactérie et procédé de criblage d'agent anti-mycobactérien Ceased WO2010077005A2 (fr)

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KR101291669B1 (ko) * 2011-05-09 2013-08-01 서울대학교산학협력단 미코박테리아―유래된 ｄｎａ 오류 복구 뉴클레오타이드 서열 및 이의 용도
KR101716239B1 (ko) 2015-02-12 2017-03-28 사단법인대한결핵협회 마이코박테리아의 성장 증진용 조성물 및 방법

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PARK, S. W. ET AL.: 'Carbon monoxide dehydrogenase in mycobacteria possesses a nitric oxide dehydrogenase activity.' BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. vol. 362, no. 2, 10 August 2007, pages 449 - 453 *
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Publication number Priority date Publication date Assignee Title
CN104830888A (zh) * 2015-03-30 2015-08-12 江南大学 一种新的新金色分枝杆菌表达体系及其在转化植物甾醇合成add中的应用
CN104830888B (zh) * 2015-03-30 2018-05-01 江南大学 一种新的新金色分枝杆菌表达体系及其在转化植物甾醇合成add中的应用

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