KR100453719B1 - Recombinant vector series efficient for molecular research in Candida albicans - Google Patents
Recombinant vector series efficient for molecular research in Candida albicans Download PDFInfo
- Publication number
- KR100453719B1 KR100453719B1 KR10-2001-0065907A KR20010065907A KR100453719B1 KR 100453719 B1 KR100453719 B1 KR 100453719B1 KR 20010065907 A KR20010065907 A KR 20010065907A KR 100453719 B1 KR100453719 B1 KR 100453719B1
- Authority
- KR
- South Korea
- Prior art keywords
- recombinant vector
- gene
- vector
- candida
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000013598 vector Substances 0.000 title claims abstract description 208
- 241000222122 Candida albicans Species 0.000 title abstract description 20
- 229940095731 candida albicans Drugs 0.000 title abstract description 20
- 238000011160 research Methods 0.000 title description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 112
- 238000010367 cloning Methods 0.000 claims abstract description 64
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims abstract description 56
- 230000014509 gene expression Effects 0.000 claims abstract description 52
- 230000035897 transcription Effects 0.000 claims abstract description 33
- 238000013518 transcription Methods 0.000 claims abstract description 33
- 239000003550 marker Substances 0.000 claims abstract description 24
- 230000001580 bacterial effect Effects 0.000 claims abstract description 21
- 230000001939 inductive effect Effects 0.000 claims abstract description 13
- 108091006047 fluorescent proteins Proteins 0.000 claims abstract description 8
- 230000003115 biocidal effect Effects 0.000 claims abstract description 4
- 101150068888 MET3 gene Proteins 0.000 claims description 38
- 101100022915 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cys-11 gene Proteins 0.000 claims description 38
- 101100022918 Schizosaccharomyces pombe (strain 972 / ATCC 24843) sua1 gene Proteins 0.000 claims description 38
- 230000010076 replication Effects 0.000 claims description 18
- 241000233866 Fungi Species 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 10
- 102000005469 Chitin Synthase Human genes 0.000 claims description 7
- 108700006921 chitin synthase 2 Proteins 0.000 claims description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical group C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 238000010353 genetic engineering Methods 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 5
- 108020004414 DNA Proteins 0.000 description 43
- 101150050575 URA3 gene Proteins 0.000 description 38
- 231100000350 mutagenesis Toxicity 0.000 description 22
- 230000035772 mutation Effects 0.000 description 20
- 230000002779 inactivation Effects 0.000 description 17
- 239000002609 medium Substances 0.000 description 15
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 14
- 238000002703 mutagenesis Methods 0.000 description 13
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 10
- 230000006698 induction Effects 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 7
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 7
- 229940045145 uridine Drugs 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 244000053095 fungal pathogen Species 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 5
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 229930182817 methionine Natural products 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 101100148606 Caenorhabditis elegans pst-1 gene Proteins 0.000 description 4
- 101710140859 E3 ubiquitin ligase TRAF3IP2 Proteins 0.000 description 4
- 102100026620 E3 ubiquitin ligase TRAF3IP2 Human genes 0.000 description 4
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 229940035893 uracil Drugs 0.000 description 4
- 102000007469 Actins Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229940121375 antifungal agent Drugs 0.000 description 3
- 239000003429 antifungal agent Substances 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000005030 transcription termination Effects 0.000 description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 2
- 101000906865 Candida albicans Chitin synthase 2 Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 101000740205 Homo sapiens Sal-like protein 1 Proteins 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 102100037204 Sal-like protein 1 Human genes 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- XDQFBMPFEBUDIC-UHFFFAOYSA-N 1h-pyrimidine-2,4-dione Chemical compound O=C1C=CNC(=O)N1.O=C1C=CNC(=O)N1 XDQFBMPFEBUDIC-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- VWEWCZSUWOEEFM-WDSKDSINSA-N Ala-Gly-Ala-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(O)=O VWEWCZSUWOEEFM-WDSKDSINSA-N 0.000 description 1
- 101100022254 Candida albicans MAL2 gene Proteins 0.000 description 1
- 108700040089 Chitin synthases Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 101150094690 GAL1 gene Proteins 0.000 description 1
- 102100028501 Galanin peptides Human genes 0.000 description 1
- 101100121078 Homo sapiens GAL gene Proteins 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100446038 Mus musculus Fabp5 gene Proteins 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- KYOBSHFOBAOFBF-UHFFFAOYSA-N UMP Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1C(O)=O KYOBSHFOBAOFBF-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- DNGBZXWZIKPLIT-UHFFFAOYSA-L [Li+].[Li+].CC([O-])=O.CC([O-])=O Chemical compound [Li+].[Li+].CC([O-])=O.CC([O-])=O DNGBZXWZIKPLIT-UHFFFAOYSA-L 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 101150085125 apr gene Proteins 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000005868 electrolysis reaction Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 101150083490 mal1 gene Proteins 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- KYOBSHFOBAOFBF-ZAKLUEHWSA-N orotidine-5'-monophosphate Chemical compound O[C@@H]1[C@@H](O)[C@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1C(O)=O KYOBSHFOBAOFBF-ZAKLUEHWSA-N 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
본 발명은 캔디다 알비칸스에서 유전자 조작의 효율적 연구를 위한 일련의 벡터에 관한 것이다.The present invention relates to a series of vectors for the efficient study of genetic manipulation in Candida albicans.
본 발명의 재조합 벡터들은 박테리아의 선택 표지 유전자를 제거하고 박테리아의 자동복제서열과 항생제 내성 유전자를 남겨 놓은 박테리아용 벡터 안에, 캔디다의 자동복제서열 발현에 필요한 최소 서열, 선택 표지 유전자의 발현에 필요한 최소 서열 및 박테리아용 벡터로부터 유래한 다중 클로닝 부위만이 삽입되어 크기를 최소화한 재조합 벡터를 기본 구조로 하여, 필요에 따라 형광 단백질 유전자, 유도성 프로모터 및 전사종결인자 등의 인자를 첨가한 재조합 벡터이다.Recombinant vectors of the present invention remove the bacterial selection marker genes and leave the bacterial clones and antibiotic resistance genes in the bacterial vector, the minimum sequence necessary for the expression of Candida's automatic cloning sequence, the minimum required for the expression of the selection marker gene It is a recombinant vector having a basic structure based on a recombinant vector having a minimal size by inserting only multiple cloning sites derived from sequences and bacterial vectors, and adding factors such as fluorescent protein genes, inducible promoters and transcription terminators as necessary. .
본 발명의 재조합 벡터들은 기존의 벡터에 비해 크기가 작기 때문에 유전자 조작이 용이하고, 캔디다 내에서 발현율이 높아 이후의 실험 단계가 수월하게 진행될 수 있으며, 여러 가지 유용한 인자들을 갖추고 있어 유전자 라이브러리 제작, 연구 대상 유전자의 생리적 역할 및 연구 대상 단백질의 세포 내 위치·기능에 대한 연구에 유용하게 사용될 수 있다.Since the recombinant vectors of the present invention are smaller in size than conventional vectors, they are easy to genetically manipulate, have high expression rates in Candida, and can be easily used in subsequent experiments. It can be useful for the study of the physiological role of the gene of interest and the location and function of the protein of study.
Description
본 발명은 캔디다 알비칸스(Candida albicans)에서 유전자 조작의 효율적 연구를 위한 일련의 재조합 벡터에 관한 것이다.The present invention relates to a series of recombinant vectors for efficient study of genetic manipulation in Candida albicans .
병원에서 발생하는 주요 2차 감염증에 대한 조사 결과, 주된 감염원이 박테리아나 바이러스가 아닌 곰팡이에 의한 것으로 밝혀졌다. 2차 감염으로 인한 사망의 40 %가 병원성 곰팡이에 의한 것으로, 이 중 80%는 캔디다 알비칸스 한 종에 의한 것이다. 캔디다 알비칸스는 온혈 동물에 상주균으로 기생하는 병원성 곰팡이의 일종으로, 이들 병원성 곰팡이에 의한 질환은 장기간의 항암 치료, 항생제의 남용 및 후천성면역결핍증(AIDS) 등으로 인해 면역 기능이 저하된 환자가 급증함에 따라 더욱 심각한 문제로 대두되고 있다.Investigations of major secondary infections occurring in hospitals revealed that the main source of infection was mold, not bacteria or viruses. 40% of deaths from secondary infections are caused by pathogenic fungi, 80% of which are caused by one species of Candida albicans. Candida albicans is a type of pathogenic fungus that resides as a resident bacterium in warm-blooded animals.The disease caused by pathogenic fungi is caused by patients with impaired immune function due to long-term chemotherapy, abuse of antibiotics, and AIDS. As it increases, it is becoming a more serious problem.
캔디다를 비롯한 병원성 곰팡이에 의한 질환을 치료하기 위해 여러가지 항진균제들이 개발되어 왔다. 그러나 곰팡이는 숙주인 온혈 동물과 마찬가지로 진핵 생물이기 때문에 숙주와 많은 생화학적 성질을 공유하고 있어, 기존의 항진균제들은 숙주에 많은 부작용을 나타냈다. 따라서 곰팡이에 대해서만 선택적으로 치료 효과를 나타내는 항진균제 및 치료 방법의 개발을 위해, 곰팡이에 대한 분자생물학적 ·유전학적 연구가 절대적으로 필요하다.Various antifungal agents have been developed to treat diseases caused by pathogenic fungi, including Candida. However, fungi are eukaryotes, just like their warm-blooded counterparts, so they share many biochemical properties with the host, and conventional antifungal agents have many side effects. Therefore, in order to develop antifungal agents and therapeutic methods that selectively treat only fungi, molecular biological and genetic studies on the fungus are absolutely necessary.
다양한 방면에서 효율적인 분자생물학적 연구가 이루어지기 위해서, 연구 대상 유전자를 용이하게 조작할 수 있게 하는 수단인 벡터(vector)가 필수적으로 요구된다. 캔디다 등 병원성 곰팡이의 연구에 사용될 벡터가 갖추어야 할 요건으로, 곰팡이 내에서 벡터 스스로 복제가 가능하게 하는 자동복제서열(autonomous replicative sequence; ARS), 벡터의 발현 여부를 파악할 수 있게 하는 선택 표지유전자(selective marker gene), 다양한 클로닝(cloning) 부위 등의 기본적 구성 요소 뿐 아니라, 배지 조성 등의 환경에 따라 유전자 발현을 조절할 수 있는 유도성 프로모터(promoter)나 세포생물학적 연구에 유용하게 사용될 수 있는 발현 에피토프(epitope) 등이 필요하다.In order for efficient molecular biology research to be conducted in various aspects, a vector, which is a means for easily manipulating the gene to be studied, is essential. A requirement for vectors to be used in the study of pathogenic fungi, such as Candida, is an autonomous replicative sequence (ARS) that allows the vector to replicate itself in the fungus, and a selective marker gene that allows the expression of the vector to be identified. In addition to the basic components such as marker genes and various cloning sites, as well as inducible promoters that can regulate gene expression according to the environment, such as media composition, or expression epitopes that can be usefully used for cell biological research ( epitope).
캔디다에 비해 연구가 많이 진척된 곰팡이인 사카로마이세스 세레비재 (Saccharomyces cerevisiae)의 경우, 상기 요건을 갖춘 여러 종류의 벡터가 제공되고 있어서, 다양한 강도와 선택성을 지닌 프로모터를 이용하여 연구 대상 유전자의 발현을 조절할 수 있으며, 여러 종류의 표식 에피토프를 이용하여 연구 대상 유전자의 세포 내 위치 및 기능에 대한 연구가 가능하다.In the case of Saccharomyces cerevisiae , a fungus that has been studied much more than candida, various types of vectors having the above requirements are provided. Expression can be regulated, and various marker epitopes can be used to study the location and function of cells of the gene under study.
그러나, 캔디다 연구에 사용되는 벡터는 그 종류가 다양하지 못하며 사용될 수 있는 분야도 한정되어 있다.However, the vectors used in the study of Candida are not diverse and the fields that can be used are limited.
현재 자동복제서열, 선택 표지 유전자, 클로닝 부위 등을 갖춘 캔디다 벡터는 몇몇 종류가 개발되었으나, 캔디다 내에서 그 발현율이 매우 낮아 이후의 실험 단계에 많은 제약을 주어왔으며, 클로닝할 수 있는 부위도 두세 개 정도에 그쳐 클로닝에 많은 어려움이 있다.Currently, some types of Candida vectors with autoreplicating sequences, selectable marker genes, and cloning sites have been developed. However, their expression rate is very low in Candida, which has placed many limitations on subsequent experiments. There are many difficulties in cloning.
또한 기존의 캔디다 벡터들에 삽입된 선택 표지 유전자 및 에피토프 등은 한 두 종류로 제한되어 있어 다양한 목적의 실험에 사용되기 어렵고, 대부분 크기가 10 Kb에 달하기 때문에 2 Kb 이상의 유전자를 클로닝하는 것이 기술적으로 매우 어려워 실제 유전자 조작에 있어 많은 한계를 안고 있다.In addition, since the selectable marker gene and epitope inserted into existing Candida vectors are limited to one or two types, it is difficult to be used for various purpose experiments, and since most of them reach a size of 10 Kb, cloning genes of 2 Kb or more is technical. As it is very difficult, it has many limitations in actual genetic manipulation.
그러므로 캔디다 알비칸스 등의 병원성 곰팡이에 대한 효율적인 연구를 위해, 높은 발현율과 함께 다양한 방면의 연구에 사용될 수 있는 요건들을 갖추고 있으며, 유전자 조작에 적절한 크기를 가진 재조합 벡터에 대한 개발이 절실히 요구되고 있다.Therefore, in order to efficiently study pathogenic fungi such as Candida albicans, it has a high expression rate and a requirement to be used for various fields of research, and there is an urgent need for the development of a recombinant vector having a size suitable for genetic manipulation.
본 발명의 목적은 높은 발현율을 나타내며 다양한 목적의 연구에 사용될 수 있는 인자들을 갖춘 적절한 크기의 캔디다 벡터를 제공하는데 있다.It is an object of the present invention to provide an appropriate size Candida vector with high expression rates and with factors that can be used for various purpose studies.
상기 과제를 달성하기 위해 본 발명에서는 박테리아 벡터에 캔디다 알비칸스의 자동복제서열, 캔디다 알비칸스에서 발현되는 선택 표지 유전자 및 박테리아용 벡터로부터 유래한 다중 클로닝 부위(multicloning site; MCS)를 삽입하고, 필요에 따라 발현 에피토프, 유도성 프로모터 및 전사종결인자 등을 삽입한 일련의 재조합 벡터들을 제공하고자 한다.In order to achieve the above object, the present invention inserts an automatic replication sequence of Candida albicans, a selection marker gene expressed in Candida albicans, and a multicloning site (MCS) derived from a bacterial vector. According to the present invention, a series of recombinant vectors in which an expression epitope, an inducible promoter, and a transcription terminator are inserted are provided.
도 1은 본 발명의 재조합 벡터 pNH2a, pNH2b, pNH4, pNH5, pNH6a 및 pNH6b의 구조를 나타낸 도이다.1 is a diagram showing the structure of the recombinant vectors pNH2a, pNH2b, pNH4, pNH5, pNH6a and pNH6b of the present invention.
도 2는 본 발명의 재조합 벡터 pNH2b, pNH4, pNH5와 각 벡터의 돌연변이 유도 전단계 벡터들이 각각 도입된 형질전환체에 대해URA3유전자의 발현율을 측정한 도이다.Figure 2 is a diagram measuring the expression rate of the URA3 gene for the recombinant vector pNH2b, pNH4, pNH5 of the present invention and the transformants to which the mutation-inducing pre-step vectors of each vector are introduced.
도 3은 본 발명의 재조합 벡터 pNH2a가 도입된 형질전환체에 대해, 선택 압력이 가해지거나 가해지지 않은 조건에서URA3유전자의 발현율을 측정한 도이다.3 is a diagram measuring the expression rate of the URA3 gene for a transformant to which the recombinant vector pNH2a of the present invention is introduced, under a condition in which selection pressure is applied or not.
도 4는 본 발명의 재조합 벡터 pNH5와 이 벡터의 돌연변이 유도 전단계 벡터들이 각각 도입된 형질전환체에 대해,MET3프로모터의 유도 조건과 비유도 조건에서yEGFP3유전자의 발현을 확인함으로써MET3프로모터의 기능을 확인하기 위해 한 도이다.Figure 4 confirms the function of the MET3 promoter by confirming the expression of the yEGFP3 gene in the conditions of induction and non-induction of the MET3 promoter for the recombinant vector pNH5 of the present invention and the transformant pre-induction vector of the vector, respectively. It is one degree to do.
본 발명은 캔디다 알비칸스의 효율적 연구를 위한 일련의 재조합 벡터에 관한 것이다.The present invention relates to a series of recombinant vectors for the efficient study of Candida albicans.
본 발명의 재조합 벡터들은 박테리아의 선택 표지 유전자를 제거하고 박테리아의 자동복제서열과 항생제 내성 유전자를 남겨 놓은 박테리아용 벡터 안에, 캔디다의 자동복제서열 발현에 필요한 최소 서열, 선택 표지 유전자의 발현에 필요한 최소 서열 및 박테리아용 벡터로부터 유래한 다중 클로닝 부위만이 삽입되어 크기를 최소화한, 박테리아와 캔디다 양쪽에서 발현될 수 있는 셔틀 벡터(shuttle vector)를 기본 구조로 한다.Recombinant vectors of the present invention remove the bacterial selection marker genes and leave the bacterial clones and antibiotic resistance genes in the bacterial vector, the minimum sequence necessary for the expression of Candida's automatic cloning sequence, the minimum required for the expression of the selection marker gene The basic structure is a shuttle vector that can be expressed in both bacteria and candida, in which only multiple cloning sites derived from sequences and bacterial vectors are inserted to minimize size.
본 발명의 기본 벡터 제조시, 각 구성 요소들이 삽입되는 박테리아용 벡터는 재조합 벡터의 골격 역할을 하게 되며, 벡터의 크기를 최소화할 수 있도록 복제개시서열인OriC와 항생제 내성 유전자인Amp r 등 박테리아 내에서의 발현에 필수적인 요소만을 남기고, 선택 표지 유전자인LacZ ·LacI및 기존의 클로닝 부위 등 캔디다 연구에 불필요한 부분을 삭제한다. 본 발명에서 사용가능한 박테리아용 벡터는, 클로닝에 널리 이용되어 온 pUC18 등 공지의 여러 가지 박테리아용 벡터를 사용할 수 있다.In the preparation of the basic vector of the present invention, the bacterial vector into which each component is inserted serves as a skeleton of the recombinant vector, and a replication initiation sequence is used to minimize the size of the vector.OriCAnd antibiotic resistance genesAmp r Leaving only elements essential for expression in bacteria,LacZLacIAnd unnecessary parts of the Candida study, such as the existing cloning site. As the bacterial vector usable in the present invention, various known bacterial vectors such as pUC18 which have been widely used for cloning can be used.
자동복제서열은 원래 곰팡이의 게놈 DNA에 있는 서열의 일부로 곰팡이의 DNA 복제가 일어날 수 있도록 하는 부위로, 복제에 관여하는 단백질들이 이 부위에 결합함으로써 복제가 시작된다. 이 자동복제서열을 벡터에 삽입시킴으로써, 벡터는 곰팡이 세포 내에서 게놈 복제와 상관없이 스스로 복제할 수 있는 능력을 지니게 된다.Autoreplication sequences are parts of the sequence in the original genomic DNA of the fungus that allow DNA replication of the fungus to occur, and replication begins by binding proteins involved in the replication. By inserting this autoreplication sequence into the vector, the vector has the ability to replicate itself in the fungal cell, regardless of genomic replication.
선택 표지 유전자는 벡터 내의 유전자들이 곰팡이 내에서 제대로 발현되었는지를 판별할 수 있게 하는 표식(marker) 역할을 한다. 예를 들어, 실험에 널리 사용되는 캔디다 균주는 게놈 DNA 중 유라실(uracil) ·유리딘(uridine)계 염기의 합성에 관여하는URA3유전자에 인위적으로 돌연변이를 일으킨 것을 사용하는데, 이 균주에 정상URA3유전자를 함유한 벡터를 도입하여 형질전환시킬 경우, 성공적으로 형질전환된 균은 유라실이 없는 배지에서 생장할 수 있으나 형질전환되지 않은 균은 죽게 되므로, 이 경우URA3유전자는 외부에서 도입된 유전자들이 잘 발현되었는지를 선별하게 하는 표지로서 작용하게 된다. 본 발명에서 선택 표지 유전자는URA3등 공지의 여러 가지 선택 표지 유전자 중 어느 것을 사용해도 무방하나, 바람직하게는 다른 표지 유전자에 비해 보다 정확하게 벡터의 발현 여부를 판별할 수 있게 하는URA3유전자를 사용한다.Selection marker genes serve as markers to determine whether the genes in the vector are properly expressed in the fungus. For example, Candida strains are widely used in the experiment are the genomic DNA of uracil (uracil) · uridine (uridine) uses that caused the artificially mutated URA3 gene involved in the synthesis of total bases in the strain normal URA3 In the case of transformation by introducing a vector containing a gene, the transformed bacteria can grow in a medium without uracil, but the untransformed bacteria die, so the URA3 gene is It acts as a label to select whether it is well expressed. As the selection marker gene in the present invention, any one of various known selection marker genes such as URA3 may be used, but preferably, the URA3 gene is used to determine whether the vector is expressed more accurately than other marker genes.
다중 클로닝 부위는 제한 효소(restriction enzyme)들이 인식할 수 있는 부위를 여러 개 가지고 있는 부분으로, 다중 클로닝 부위에 많은 제한 효소 부위가 존재해야 유전자 조작이 용이해진다. 본 발명에서는 다양한 제한 효소 부위를 가진 pBluescriptⅡKS(+) 등 공지의 여러 가지 박테리아용 벡터의 다중 클로닝 부위 중 어느 것을 사용해도 무방하다.The multiple cloning site is a part having several sites that can be recognized by restriction enzymes. Genetic manipulation is easy when there are many restriction enzyme sites in the multiple cloning site. In the present invention, any of multiple cloning sites of various known bacterial vectors such as pBluescript IIKS (+) having various restriction enzyme sites may be used.
상기 요소들을 갖춘 기본 벡터에, 여러 가지 유용한 인자들을 삽입하여 본 발명의 재조합 벡터들을 제조한다. 본 발명의 기본 벡터에 추가로 삽입될 수 있는 유용한 인자들은 목적에 따라 형광 단백질, 유도성 프로모터, 전사종결인자 등을 사용할 수 있다.In the base vector with the above elements, various useful factors are inserted to prepare the recombinant vectors of the present invention. Useful factors which can be inserted further into the basic vector of the present invention may use fluorescent proteins, inducible promoters, transcription terminators and the like depending on the purpose.
형광 단백질은 곰팡이의 살아있는 세포에서 유전자의 발현 여부, 단백질의 기능, 위치 및 이동 경로를 연구하는데 매우 유용한 발현 에피토프이다. 본 발명에서 사용 가능한 형광 단백질 유전자는yEGFP(enhanced green fluoroscence protein),yGFP(green fluoroscence protein),yCFP(cyan fluoroscence protein) 등이 있으며, 바람직하게는 형광 현미경 기법을 이용한 연구에 가장 널리 사용되고 있는yEGFP3유전자를 사용한다.Fluorescent proteins are expression epitopes that are very useful for studying gene expression, function, location and migration pathways in fungal living cells. Fluorescent protein genes that can be used in the present invention include yEGFP (enhanced green fluoroscence protein), yGFP (green fluoroscence protein), yCFP (cyan fluoroscence protein) and the like, preferably yEGFP3 gene that is most widely used in research using fluorescence microscopy Use
유도성 프로모터는 배지에 함유된 영양소의 유무에 따라 대상 유전자의 발현을 조절하므로, 그 대상 유전자의 산물이 어떤 기능을 하는지 알 수 있게 한다. 예를 들어MET3프로모터의 경우, 메티오닌(methionine)이 배지에 존재하면 유전자의 발현을 억제하고, 배지에 존재하지 않으면 유전자가 발현되도록 한다. 본 발명에서 사용 가능한 프로모터는MET3프로모터를 비롯하여 갈락토오스(galactose), 말토오스(maltose) 등의 탄소원에 따라 유전자 발현을 조절하는GAL1프로모터,MAL1프로모터 등이 있으며, 바람직하게는 탄소원을 바꿀 필요가 없어 캔디다의 대사에 영향을 미치지 않으며 다른 프로모터에 비해 보다 강력하게 유전자 발현을 통제하는MET3프로모터를 사용한다.Inducible promoters regulate the expression of a target gene depending on the presence or absence of nutrients contained in the medium, so that the function of the product of the target gene can be known. For example, in the case of the MET3 promoter, if methionine is present in the medium, the expression of the gene is suppressed, and if not, the gene is expressed. Promoters usable in the present invention include MET3 promoter, GAL1 promoter, MAL1 promoter and the like to regulate gene expression according to carbon sources such as galactose, maltose, maltose, etc. Preferably, there is no need to change the carbon source of Use the MET3 promoter, which does not affect metabolism and controls gene expression more strongly than other promoters.
전사종결인자는 전사체 말단 또는 프로모터 영역에 인접하여 위치하는 DNA 서열로서, RNA 중합효소가 전사를 종료하도록 하는 서열이다. 별도로 전사종결인자의 서열이 없는 유전자를 발현시키고자 할 경우에, 이 전사종결인자를 그 유전자의 3'에 삽입함으로써 제대로 발현이 되도록 할 수 있다. 본 발명에서 사용가능한 전사종결인자는 캔디다의 키틴 합성 효소(chitin synthase)를 비롯하여 공지된 캔디다 유전자의 전사종결인자들 중 어느 것을 사용해도 무방하나, 바람직하게는 캔디다의 키틴 합성 효소 2의 전사종결인자를 사용한다.A transcription terminator is a DNA sequence located adjacent to a transcript end or promoter region and is a sequence that causes RNA polymerase to terminate transcription. In the case where a gene having no sequence of transcription terminator is to be expressed separately, the transcription terminator can be properly expressed by inserting the transcription terminator into 3 'of the gene. The transcription terminators usable in the present invention may be any of the known transcription terminators of the Candida gene, including Candida chitin synthase, but preferably the transcription terminator of Candida chitin synthase 2 Use
본 발명의 재조합 벡터에는 기본 벡터인 pNH2a, pNH2b와, 이들 기본 벡터에 추가로 여러 가지 인자를 삽입한 pNH4, pNH5, pNH6a 및 pNH6b의 6 종류가 있다.There are six kinds of recombinant vectors of the present invention, pNH2a and pNH2b, which are basic vectors, and pNH4, pNH5, pNH6a, and pNH6b, in which various factors are inserted in addition to these basic vectors.
본 발명의 재조합 벡터들의 구조는 도 1에 나타나 있으며, 그 각각의 특성은 다음과 같다.The structure of the recombinant vectors of the present invention is shown in Figure 1, the characteristics of each are as follows.
1) pNH2a와 pNH2b1) pNH2a and pNH2b
본 발명의 기본 벡터인 pNH2a(도 1a)와 pNH2b(도 1b)는 캔디다의 자동복제서열 발현에 필요한 최소 서열인 GenBank 등록번호 X16634의 1번부터 1203번까지의 서열(서열번호 1)과, 선택 표지 유전자URA3유전자의 발현에 필요한 최소 서열인 GenBank 등록번호 X14198의 11번부터 1365번까지의 서열(서열번호 2)을 박테리아용 벡터인 pUC18에 삽입한 후, pUC18의LacZ유전자에서부터 클로닝 부위,LacI유전자까지의 서열을 제거하고 대신yEGFP3유전자와 박테리아용 벡터 pBluescriptⅡKS(+)로부터 얻은 다중 클로닝 부위가 융합된 서열을 삽입한 후, 다시yEGFP3유전자를 제거하여 얻는다.PNH2a (FIG. 1A) and pNH2b (FIG. 1B), which are the basic vectors of the present invention, are selected from the sequences 1 to 1203 (SEQ ID NO: 1) of GenBank accession no. Insert the sequence 11-11365 (SEQ ID NO: 2) of GenBank accession number X14198, the minimum sequence required for the expression of the marker gene URA3 gene, into the bacterial vector pUC18, and then the cloning site, LacI gene from the LacZ gene of pUC18. The sequence is removed by inserting a sequence in which the fusion sequence of the yEGFP3 gene and the multiple cloning site obtained from the bacterial vector pBluescriptIIKS (+) is inserted, followed by removal of the yEGFP3 gene.
본 발명의 pNH2a와 pNH2b를 제조할 때, 캔디다의 자동복제서열과 선택 표지 유전자URA3유전자의 발현에 필요한 최소 서열은 각각의 서열을 증폭할 수 있도록 고안된 프라이머를 이용하여 PCR(polymerized chain reaction)로 얻는다.When preparing pNH2a and pNH2b of the present invention, the minimum sequence necessary for the automatic replication of Candida and the expression of the selection marker gene URA3 gene is obtained by PCR (polymerized chain reaction) using primers designed to amplify the respective sequences. .
본 발명의 pNH2a와 pNH2b를 제조할 때, 다양한 방법으로 클로닝을 할 수 있도록 다중 클로닝 부위를 각각 다른 방향으로 삽입한다. pNH2a의 경우 다중 클로닝 부위가 pBluescriptⅡKS(+)의 다중 클로닝 부위와 같은 방향으로 삽입되었으며, pNH2b는 다중 클로닝 부위가 그 반대 방향으로 삽입된다.When preparing pNH2a and pNH2b of the present invention, multiple cloning sites are inserted in different directions to allow cloning in various ways. In the case of pNH2a, multiple cloning sites were inserted in the same direction as the multiple cloning sites of pBluescriptIIKS (+), while in pNH2b, multiple cloning sites were inserted in the opposite direction.
본 발명의 pNH2a와 pNH2b를 제조할 때, pBluescriptⅡKS(+)의 다중 클로닝 부위만을 삽입하기에는 그 크기가 너무 작기 때문에, 실험상의 편의를 도모하기 위해yEGFP3를 함께 융합하여 삽입한 후, 나중에yEGFP3유전자는 제거한다.When preparing pNH2a and pNH2b of the present invention, since the size is too small to insert only the multiple cloning site of pBluescriptIIKS (+), the yEGFP3 gene is removed after fusion and insertion of yEGFP3 for later experimental convenience. do.
이와 같이, 본 발명의 기본 벡터 pNH2a와 pNH2b는 각 구성 요소들이 캔디다와 박테리아 둘 다에서 발현되는데 꼭 필요한 유전자만을 포함하고 필요없는 부분은 제거함으로써, 필수 구성 요소를 다 갖추면서도 최소 크기를 가질 수 있게 한 것이다.As such, the basic vectors pNH2a and pNH2b of the present invention contain only the genes necessary for each component to be expressed in both Candida and bacteria, and remove unnecessary portions, thereby having a minimum size while having all the necessary components. It is.
따라서, 본 발명의 pNH2a와 pNH2b는, 이들과 마찬가지로 자동복제서열과URA3를 가진 기존의 캔디다 벡터에 비해 훨씬 크기가 작아(∼ 5Kb), 연구 대상 유전자의 크기에 상관없이 클로닝을 용이하게 할 수 있으며, 필요에 따라 프로모터나 에피토프 등의 인자를 추가로 삽입할 수 있다.Therefore, pNH2a and pNH2b of the present invention are much smaller (~ 5Kb) than conventional Candida vectors with autoreplicating sequence and URA3 like these, and can facilitate cloning regardless of the size of the gene under study. If necessary, additional factors such as a promoter or epitope can be inserted.
또한 본 발명의 pNH2a와 pNH2b는 다중 클로닝 부위가 각각 다른 방향으로 삽입되어 있어, 연구 대상 유전자에 따라 클로닝하기에 적절한 벡터를 둘 중에서 선택하여 사용할 수 있다.In addition, pNH2a and pNH2b of the present invention have multiple cloning sites inserted in different directions, so that a vector suitable for cloning according to the gene of interest can be selected and used.
2) pNH42) pNH4
본 발명의 pNH4(도 1c)는 pNH2a의 다중 클로닝 부위의 3' 방향에 형광 단백질 유전자인yEGFP3와 캔디다의 키틴 합성 효소 2의 전사종결인자를 순차적으로 삽입한 벡터이다.PNH4 (FIG. 1C) of the present invention is a vector in which the transcription terminators of the fluorescent protein gene yEGFP3 and Candida chitin synthase 2 are sequentially inserted in the 3 'direction of the multiple cloning site of pNH2a .
본 발명의 pNH4 제조시,yEGFP3유전자는 Dr. Cormack 실험실에서 얻은 pUC19/yEGFP3로부터 제한효소로 절단하여 얻는다. 본 발명의yEGFP3유전자에는 별도의 전사종결인자가 없으므로,yEGFP3유전자의 전사가 제대로 이루어지도록 캔디다의 키틴 합성 효소 2를 주형으로 하여 PCR 반응을 통해 얻은 전사종결인자의 최소 크기 서열을yEGFP3유전자의 3'에 삽입한다.In the production of pNH4 of the present invention, the yEGFP3 gene is represented by Obtained by restriction enzyme digestion from pUC19 / yEGFP3 obtained from Cormack Laboratories. YEGFP3 gene of the present invention has a separate transcription termination factor is not, the transfer of yEGFP3 gene to occur properly, the minimum size sequence of the transcription termination factor obtained by the PCR reaction, the chitin synthase 2 in Candida as a template for yEGFP3 gene 3 ' Insert in
본 발명의 pNH4에는 별도의 프로모터가 없어, 연구 대상 유전자와 그 유전자의 프로모터를 함께 클로닝하게 되어 있으므로, 유전자 자신의 프로모터에 의한 조절 하에 그 유전자에 대해 연구할 수 있다.Since pNH4 of the present invention does not have a separate promoter, and the gene to be studied and the promoter of the gene are cloned together, the gene can be studied under the control of the gene itself.
또한 본 발명의 pNH4에는 곰팡이 내에서 녹색 형광 단백질을 발현시키는yEGFP3유전자가 있어, 이 녹색 형광 단백질이 연구 대상 단백질의 C-말단(C-terminal)에 융합되므로, 형광 신호를 이용한 세포 내에서의 대상 단백질 위치 및 기능에 대한 연구에 유용하게 사용될 수 있다.In addition, pNH4 of the present invention has a yEGFP3 gene that expresses a green fluorescent protein in a fungus, and since the green fluorescent protein is fused to the C-terminal of the protein of interest, the target in the cell using a fluorescent signal It can be usefully used for the study of protein location and function.
3) pNH53) pNH5
본 발명의 pNH5(도 1d)는 pNH4의 다중 클로닝 부위의 5' 방향에MET3프로모터를 삽입한 벡터이다.PNH5 (FIG. 1D) of this invention is a vector which inserted the MET3 promoter in the 5 'direction of the multiple cloning site | part of pNH4 .
본 발명의 pNH5 제조시,MET3프로모터의 최소 크기 서열은 PCR을 통해 얻는다.In preparing pNH5 of the present invention, the minimum size sequence of the MET3 promoter is obtained by PCR.
본 발명의 pNH5를 사용하면 형광 단백질에 의한 연구가 가능하다는 것은 pNH4와 마찬가지이나, 본 발명의 pNH5에는yEGFP3유전자의 5' 쪽에MET3프로모터가 있어, 배지에 메티오닌을 첨가 또는 배제함으로써 연구자의 의도대로 대상 유전자의 발현을 조절할 수 있다.The use of pNH5 of the present invention enables the study by fluorescent proteins, but the pNH5 of the present invention has a MET3 promoter on the 5 'side of the yEGFP3 gene. Can regulate gene expression.
4) pNH6a와 pNH6b4) pNH6a and pNH6b
본 발명의 pNH6a(도 1e)와 pNH6b(도 1f)는 각각 기본 벡터 pNH2a와 pNH2b의다중 클로닝 부위의 5'에MET3프로모터를, 다중 클로닝 부위의 3'에 전사종결인자를 삽입한 벡터이다.PNH6a (FIG. 1E) and pNH6b (FIG. 1F) of the present invention are vectors in which the MET3 promoter is inserted into 5 'of the multiple cloning sites of the base vectors pNH2a and pNH2b, and the transcription terminator is inserted into 3' of the multiple cloning sites, respectively.
본 발명의 pNH6a와 pNH6b를 이용하면MET3프로모터의 조절 하에 연구 대상 유전자의 기능을 관찰할 수 있다.Using pNH6a and pNH6b of the present invention, the function of the gene of study can be observed under the control of the MET3 promoter.
또한 본 발명의 pNH6a와 pNH6b에서는 다중 클로닝 부위의 3'에 전사종결인자가 위치하므로, 별도의 전사종결인자 서열이 없는 유전자도 클로닝할 수 있어 클로닝의 유용성을 도모할 수 있다.In addition, in pNH6a and pNH6b of the present invention, since the transcription terminator is located at 3 'of the multiple cloning site, genes without a separate transcription terminator sequence can be cloned, thereby facilitating cloning utility.
본 발명의 재조합 벡터에서 다중 클로닝 부위 내의 제한 효소 절단 부위 중 몇몇 부위가 다중 클로닝 부위 이외의 다른 부분에도 존재하기 때문에, 연구 대상 유전자를 이들 부위에 클로닝하는 것이 곤란하다. 따라서 본 발명의 재조합 벡터 각각에 대해 다중 클로닝 이외의 다른 부분에 존재하는 제한 효소 절단 부위를 불활성화시킨 돌연변이 벡터를 제조하였다.Since some of the restriction enzyme cleavage sites in the multiple cloning site in the recombinant vector of the present invention are also present in portions other than the multiple cloning site, it is difficult to clone the gene of interest into these sites. Thus, a mutant vector was prepared that inactivated the restriction enzyme cleavage site present in a portion other than multiple cloning for each of the recombinant vectors of the present invention.
모든 재조합 벡터에 대해 자동복제서열에 존재하는 두 개의 PstⅠ부위와 한 개의 BamHⅠ부위, 그리고URA3유전자에 존재하는 EcoRⅠ부위와 XbaⅠ부위가 각각 불활성화되었다. pNH5, pNH6a, pNH6b에서는MET3프로모터에 존재하는 XbaⅠ부위와 SalⅠ부위도 각각 불활성화되었다.For all recombinant vectors, two PstI sites, one BamHI site, and EcoRI and XbaI sites in the URA3 gene were inactivated, respectively. In pNH5, pNH6a and pNH6b, the XbaI and SalI sites in the MET3 promoter were also inactivated, respectively.
본 발명의 재조합 벡터들은 기본 벡터의 경우 기존의 캔디다 벡터에 비해 훨씬 크기가 작다. 또한 기본 벡터에 여러 가지 유용한 인자가 추가로 삽입된 재조합벡터의 경우에도 필수 구성 요소만을 갖춘 기존의 캔디다 벡터와 크기가 비슷하므로, 유전자 조작에 용이하게 사용될 수 있다.The recombinant vectors of the present invention are much smaller in size than the conventional Candida vector in the case of the base vector. In addition, even in the case of a recombinant vector in which various useful factors are added to the basic vector, the size is similar to that of a conventional Candida vector having only essential components, and thus can be easily used for genetic manipulation.
본 발명의 재조합 벡터들은 캔디다에서 발현시켰을 때, 다중카피(multicopy) 벡터와 비슷한 수의 카피로 존재한다. 따라서 본 발명의 재조합 벡터들을 사용하면 기존의 벡터보다 많은 양의 플라스미드를 얻을 수 있어 이후의 실험 단계를 수월하게 진행할 수 있다.Recombinant vectors of the present invention, when expressed in Candida, exist in a similar number of copies as a multicopy vector. Therefore, the use of the recombinant vectors of the present invention can obtain a larger amount of plasmid than the existing vector can facilitate the subsequent experimental step.
본 발명의 재조합 벡터들은 유라실·유리딘이 포함된 배지에서 오랫동안 배양해도URA3유전자를 소실하지 않고 오랫동안 보존하는 등 캔디다 내에서 안정적으로 존재하므로, 연구 과정 중 벡터가 불안정해짐에 따라 도중에 발생할 수 있는 문제점들이 해결될 수 있다.Recombinant vectors of the present invention stably exist in the candida, such as long-term preservation without loss of the URA3 gene even when cultured in a medium containing uracil and uridine, which may occur during the study as the vector becomes unstable. Problems can be solved.
본 발명의 재조합 벡터들은 상기한 공통의 장점 외에, 각기 다른 실험 목적에 따라 유용하게 사용될 수 있다.Recombinant vectors of the present invention can be usefully used according to different experimental purposes, in addition to the common advantages described above.
pNH2a와 pNH2b는 발현율이 높으면서도 크기가 작으므로, 캔디다를 비롯하여 곰팡이나 박테리아의 유전자 라이브러리(library)를 제작하는데 사용될 수 있다.pNH2a and pNH2b are high in expression and small in size, and thus can be used to produce gene libraries of candida and fungi or bacteria.
MET3 프로모터를 가진 pNH5, pNH6a, pNH6b는 프로모터의 조절에 따라 연구 대상 유전자의 발현이 활성화 또는 불활성화될 때 각각 나타나는 생리적 활성의 차이를 관찰할 수 있으므로, 기능을 알지 못하는 유전자의 역할을 규명하는데 유용하게 사용될 수 있다.PNH5, pNH6a, and pNH6b with the MET3 promoter can be used to identify the role of genes whose function is unknown because they can observe differences in physiological activity that occur when expression of the gene under study is activated or inactivated, depending on the regulation of the promoter. Can be used.
yEGFP3유전자를 가진 pNH4와 pNH5는 형광 현미경 기법을 이용하여 연구 대상 단백질의 세포 내 위치 및 기능을 파악하는데 사용될 수 있다. pNH4 and pNH5 with the yEGFP3 gene can be used to determine the intracellular location and function of the protein of interest using fluorescence microscopy.
이하 본 발명을 하기 실시예에 의해 더욱 상세히 설명하되, 본 발명이 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
[실시예 1] pNH2a와 pNH2b의 제조Example 1 Preparation of pNH2a and pNH2b
본 발명에서 제공하는 일련의 재조합 벡터들을 제조하기 위해, 우선 이들 벡터들의 기본 구조가 되는 pNH2a와 pNH2b를 제조하였다.In order to prepare a series of recombinant vectors provided by the present invention, first, pNH2a and pNH2b, which are the basic structures of these vectors, were prepared.
우선, 캔디다의 게놈(genome) DNA로부터 PCR을 수행하여 캔디다의 자동복제서열과 선택 표지 유전자URA3의 최소 필요 서열을 분리하였다. 사용된 프라이머는 각각 CaARS2-5', CaARS2-3' 프라이머와 CaURA3-3', CaURA3-5' 프라이머로 그 서열은 다음과 같다.First, PCR was performed from the genomic DNA of Candida to separate the Candida autoreplication sequence and the minimum required sequence of the selection marker gene URA3 . The primers used were CaARS2-5 ', CaARS2-3' primers and CaURA3-3 'and CaURA3-5' primers, respectively. The sequence is as follows.
CaARS2 (정방향): 5' - CATATGGATCATACCAAAAAAAAATCCGGGG - 3' (서열번호 3)CaARS2 (forward): 5 '-CATATGGATCATACCAAAAAAAAATCCGGGG-3' (SEQ ID NO: 3)
CaARS2 (역방향): 5' - CATATGGATCCCAGCTTCTCCTTATT - 3' (서열번호 4)CaARS2 (reverse): 5 '-CATATGGATCCCAGCTTCTCCTTATT-3' (SEQ ID NO: 4)
CaURA3 (정방향): 5' - GACGTCGGAATTGATTTGGATGGTAT - 3' (서열번호 5)CaURA3 (forward): 5 '-GACGTCGGAATTGATTTGGATGGTAT-3' (SEQ ID NO: 5)
CaURA3 (역방향): 5' - GACGTCTAGAAGGACCACCTTTGA - 3' (서열번호 6)CaURA3 (reverse): 5 '-GACGTCTAGAAGGACCACCTTTGA-3' (SEQ ID NO: 6)
PCR 결과 각각 1.2 Kb와 1.3 Kb의 유전자를 얻고, 염기 서열 분석을 통해 이들 유전자가 각각 캔디다의 자동복제서열 및URA3유전자임을 확인하였다. PCR로 얻은 두 유전자를 각각 pUC18의 NdeI 부위와 AatⅡ 부위에 삽입하여 자동복제서열 및URA3를 포함하는 재조합 벡터를 얻었다.As a result of PCR, 1.2 Kb and 1.3 Kb genes were obtained, and sequencing analysis confirmed that these genes were Candida's autoreplicating sequence and URA3 gene, respectively. Two genes obtained by PCR were inserted into the NdeI site and the AatII site of pUC18, respectively, to obtain a recombinant vector containing an autoreplication sequence and URA3 .
상기 재조합 벡터에 다중 클로닝 부위를 삽입하기 위해, pBluescriptⅡ KS(+)(이하 pBS)의 다중 클로닝 부위를 택하였으나 이 부분만을 클로닝하기에는 그크기가 너무 작으므로, 클로닝의 유용성을 도모하기 위해yEGFP3를 삽입하기로 하였다.yEGFP3는 Dr. Cormack 실험실에서 얻은 pUC19/yEGFP3을 주형으로 하여 PCR 후 얻은 산물을 사용하였으며, 이때 사용된 프라이머와 그 염기 서열은 다음과 같다.In order to insert multiple cloning sites into the recombinant vector, a multiple cloning site of pBluescriptII KS (+) (hereinafter referred to as pBS) was selected, but its size was too small to clone only this part, so that yEGFP3 was inserted to facilitate the cloning. It was decided as follows. yEGFP3 is Dr. The product obtained after PCR using pUC19 / yEGFP3 obtained from Cormack Laboratories as a template was used, and the primers and their base sequences used were as follows.
yEGFP (정방향): 5' - GAGCTCATGTCTAAAGGTGAAG - 3' (서열번호 7)yEGFP (forward): 5 '-GAGCTCATGTCTAAAGGTGAAG-3' (SEQ ID NO: 7)
yEGFP (역방향): 5' - GAGCTCCTGCAGTTATTTGTAC - 3' (서열번호 8)yEGFP (reverse): 5 '-GAGCTCCTGCAGTTATTTGTAC-3' (SEQ ID NO: 8)
PCR 결과 0.7 Kb 크기의 DNA를 얻어 pBS의 SacI 부위에 클로닝하였다. 이 벡터를 주형으로 하고 T3/T7 프라이머를 사용하여 PCR을 수행하였다. PCR 결과 pBS의 다중 클로닝 부위와yEGFP3유전자가 융합된 약 800 bp의 DNA를 얻었다.PCR resulted in 0.7 Kb of DNA was cloned into the SacI site of pBS. PCR was performed using this vector as a template and T3 / T7 primer. PCR yielded approximately 800 bp of DNA in which the multiple cloning site of pBS and the yEGFP3 gene were fused.
한편, 앞서 얻은 재조합 벡터, 즉 캔디다의 자동복제서열과URA3유전자를 포함하는 pUC18를 PvuⅡ로 절단하였으며, 이 과정에서 pUC18의LacZ유전자에서부터LacI유전자까지의 서열이 제거되었다. 절단된 4.9 Kb 크기의 벡터와 800 bp의 PCR 산물의 말단을 각각 클레노우(Klenow) 효소로 처리하여 평활 말단(blunt end)을 만든 후 이들을 연결(ligation)하였다. 그 결과 다중 클로닝 부위와yEGFP3유전자가 융합되어 있으며, 다중 클로닝 부위의 방향이 서로 다른 두 종류의 벡터를 얻고 이를 각각 pNH1a와 pNH1b로 명명하였다.On the other hand, pUC18 including the recombinant vector and the URA3 gene of the recombinant vector obtained above was cut with PvuII , and in this process, the sequences from LacZ gene to LacI gene of pUC18 were removed. The cleaved 4.9 Kb sized vector and the end of 800 bp PCR product were treated with Klenow enzyme, respectively, to make blunt ends and then ligation. As a result, the multiple cloning site and the yEGFP3 gene were fused, and two kinds of vectors having different directions of the multiple cloning site were obtained and named as pNH1a and pNH1b, respectively.
상기 벡터들로부터, 자동복제서열과URA3유전자와 다중 클로닝 부위만을 포함하는 기본 벡터들을 얻기 위해, pNH1a와 pNH1b를 SacI으로 절단하여yEGFP3를 제거하였다. 그 결과 남은 5 Kb의 DNA만을 자가 연결(self ligation)시킴으로써 본 발명의 기본 벡터가 되는 재조합 벡터들을 얻었다.From these vectors, yEGFP3 was removed by cleaving pNH1a and pNH1b with SacI to obtain autorepeat sequences and base vectors containing only the URA3 gene and multiple cloning sites. As a result, only the remaining 5 Kb of DNA by self ligation (self ligation) to obtain a recombinant vector to become the basic vector of the present invention.
본 발명의 재조합 벡터에서 다중 클로닝 부위 내의 제한 효소 절단 부위 중 몇몇 부위가 다중 클로닝 부위 이외의 다른 부분에도 존재하므로, 이들 부위를 이용하여 클로닝할 수가 없다는 문제점이 있었다. 따라서 다중 클로닝 이외의 다른 부분에 존재하는 제한 효소 절단 부위를 불활성화시켜, 다중 클로닝 부위의 모든 부위를 확보하기로 하였다.In the recombinant vector of the present invention, some of the restriction enzyme cleavage sites in the multiple cloning site exist in other parts besides the multiple cloning site, so there is a problem in that they cannot be cloned using these sites. Therefore, the restriction enzyme cleavage site present in other portions than the multiple cloning was inactivated to secure all sites of the multiple cloning site.
자동복제서열의 경우 두 개의 PstⅠ부위와 한 개의 BamHⅠ부위를 불활성화시켜야 했으므로, 특정 부분의 돌연변이 유도(site-directed mutagenesis)를 수행하였다. 각 재조합 벡터에 대해, 두 개의 PstⅠ부위를 불활성화시킬 수 있는 프라이머를 사용하여 PCR 반응을 수행하였다. 그러나 BamHⅠ부위는 알 수 없는 원인에 의해 돌연변이 유도가 제대로 이루어지지 않았으므로, 각 재조합 벡터에 대해 BamHⅠ효소를 단시간만 처리하여 부분적인 절단이 일어나게 하였다. 절단된 양 끝을 평활 말단으로 만들어 연결시킨 후, 박테리아에 도입하여 얻은 형질전환체 중 다중 클로닝 부위에만 BamHⅠ부위를 가지는 것을 선별하였다.In the case of the automatic replication sequence, two PstI sites and one BamHI site had to be inactivated. Therefore, site-directed mutagenesis was performed. For each recombinant vector, PCR reactions were performed using primers capable of inactivating two PstI sites. However, because the BamH I site was not properly induced by mutation due to unknown causes, partial cleavage occurred by treating BamH I enzyme for each recombinant vector for a short time. After the two ends were cut into smooth ends and connected, the BamHI sites were selected from only the multiple cloning sites of the transformants obtained by introducing the bacteria.
URA3유전자의 경우 각 재조합 벡터에 대해 EcoRⅠ부위와 XbaⅠ부위를 각각 불활성화하였다. In the case of URA3 gene, EcoRI site and XbaI site were inactivated for each recombinant vector.
각각의 돌연변이 유도에 사용된 프라이머의 염기 서열과 돌연변이 유도로 인한 결과를 표 1에 나타내었다.The base sequence of the primers used for inducing each mutation and the results due to the mutation induction are shown in Table 1.
이렇게 하여 얻은 재조합 벡터들은 캔디다의 자동복제서열과 캔디다의URA3유전자와 pBS의 다중 클로닝 부위의 발현에 필요한 최소 서열만을 포함하는 기본 벡터이며, 이들을 각각 pNH2a와 pNH2b로 명명하였다.The recombinant vectors thus obtained were basic vectors containing only the autoclone sequence of Candida and the minimal sequence necessary for the expression of Candida's URA3 gene and the multiple cloning site of pBS, which were named pNH2a and pNH2b, respectively.
[실시예 2] pNH4의 제조Example 2 Preparation of pNH4
본 발명의 기본 벡터를 이용하여,yEGFP3유전자와 전사종결인자를 추가로 삽입한 벡터를 얻기 위해 다음의 과정을 수행하였다.Using the basic vector of the present invention, the following procedure was performed to obtain a vector additionally inserted with the yEGFP3 gene and transcription terminator.
pNH2a의 제조 과정 중 얻은 벡터인 pNH1a에서,yEGFP3유전자의 염기 서열을 분석한 결과, 이 벡터 내의yEGFP3유전자에 중 네 개의 염기에 돌연변이가 일어났음을 확인하였다. 이러한 현상은yEGFP3유전자의 PCR 과정 중에 일어난 것으로, 다시 돌연변이가 일어날 가능성을 배제하기 위해 이 유전자를 pUC19에 클로닝되어 있는 원래의yEGFP3유전자로 대체하기로 하였다.In pNH1a, a vector obtained during the preparation of pNH2a, the nucleotide sequence of the yEGFP3 gene was analyzed, and it was confirmed that mutations occurred in four bases of the yEGFP3 gene in the vector. This phenomenon occurred during the PCR process of the yEGFP3 gene, and to eliminate the possibility of mutation again, the gene was replaced with the original yEGFP3 gene cloned into pUC19.
이를 위해 pNH2a를 SacI으로 잘라 돌연변이를 일으킨yEGFP3유전자 부분을 제거하였다. 돌연변이가 일어나지 않은 원래의yEGFP3유전자를 얻기 위해, pUC19/yEGFP3플라스미드를 HindⅢ와 PstI으로 잘라 720 bp의yEGFP3유전자를 얻었다. 절단된 pNH2a와yEGFP3유전자를 각각 평활 말단으로 만들어 연결시켰다.To this end, pNH2a was cut with SacI to remove the mutated yEGFP3 gene portion. To obtain the original yEGFP3 gene without mutation, the pUC19 / yEGFP3 plasmid was cut with HindIII and PstI to obtain a 720 bp yEGFP3 gene. The truncated pNH2a and yEGFP3 genes were linked to the blunt ends, respectively.
그런데yEGFP3유전자 내에는 별도로 전사의 종결을 지시하는 인자가 없으므로,yEGFP3유전자의 3'에 전사종결인자를 삽입하기로 하였다. 전사종결인자 서열은 캔디다의 키틴 합성 효소 2로부터 PCR을 통해 얻었으며, 이때 사용된 프라이머와 그 염기 서열은 다음과 같다.However, since there is no factor which indicates the transcription terminator of the gene separate from within yEGFP3, and to insert a transcriptional termination factor to the 3 'of the gene yEGFP3. Transcription terminator sequences were obtained by PCR from chitin synthase 2 of Candida, and the primers and their base sequences used were as follows.
CaCHS2T (정방향): 5'- GGCGCCCCAGCACCTCCAGAAGAG -3' (서열번호 17)CaCHS2T (forward): 5'- GGCGCCCCAGCACCTCCAGAAGAG -3 '(SEQ ID NO: 17)
CaCHS2T (역방향): 5'- GGCGCCCAAAAGAATAGTATGGGC -3' (서열번호 18)CaCHS2T (reverse): 5'- GGCGCCCAAAAGAATAGTATGGGC -3 '(SEQ ID NO: 18)
PCR 결과 얻은 469 bp의 PCR 산물을 T 벡터에 클로닝한 후 NarI과 PstI으로 잘라 전사종결인자의 최소 서열인 200 bp의 DNA를 얻고,yEGFP3가 삽입되어 있는 재조합 벡터는 NarI으로 절단한 뒤 각각의 양끝을 평활 말단으로 만들었다. 두 DNA를 연결하여 클로닝 후 얻어진 플라스미드에 대해, 염기 서열 분석을 통해 전사종결인자의 방향을 확인하여yEGFP3와 같은 방향으로 연결된 클론을 선택하였다.PCR result after a PCR product of 469 bp obtained by cloning the T vector to obtain a minimum sequence of DNA of 200 bp of the transcription termination factor cut with NarI and PstI, a recombinant vector that yEGFP3 is inserted, is then cut with NarI respective ends Was made to the blunt end. For the plasmid obtained by linking the two DNAs, the sequence of the transcription terminator was confirmed by sequencing to select the linked clone in the same direction as yEGFP3 .
이렇게 하여 얻은 재조합 벡터는 다중 클로닝 부위의 3'에yEGFP3유전자와 전사종결인자가 차례로 존재하며, 이 벡터를 pNH4로 명명하였다.The recombinant vector thus obtained has a yEGFP3 gene and a transcription terminator at 3 'of the multiple cloning site, and this vector was named pNH4.
[실시예 3] pNH5의 제조Example 3 Preparation of pNH5
yEGFP3유전자와 전사종결인자를 가질 뿐 아니라, MET3 프로모터의 조절을 받는 재조합 벡터를 얻기 위해 다음의 과정을 수행하였다. In addition to the yEGFP3 gene and transcription terminator, the following procedure was performed to obtain a recombinant vector regulated by the MET3 promoter.
우선 캔디다의 MET3 프로모터를 얻기 위해, 캔디다의 게놈 DNA을 주형으로 하여 PCR을 수행하였다. 이때 사용된 프라이머와 그 염기 서열은 다음과 같다.First, in order to obtain the Candida MET3 promoter, PCR was performed using the genomic DNA of Candida as a template. The primers used and their base sequences are as follows.
CaMet3p (정방향): 5' - CTGCAGGCGCTGAAAAACTACGAA - 3' (서열번호 19)CaMet3p (forward): 5 '-CTGCAGGCGCTGAAAAACTACGAA-3' (SEQ ID NO: 19)
CaMet3p (역방향): 5' - CTGCAGGTTTTCTGGGGAGGGTAT - 3' (서열번호 20)CaMet3p (reverse): 5 '-CTGCAGGTTTTCTGGGGAGGGTAT-3' (SEQ ID NO: 20)
그 결과 얻은 1.3 Kb의 DNA의 양끝을 평활 말단으로 만들고, pNH4을 KpnI으로 절단한 후 양끝을 평활 말단으로 만들어 이들을 연결하였다. 클로닝으로 얻어진 플라스미드의 염기 서열을 분석하여MET3프로모터의 방향을 확인하고,yEGFP3및 전사종결인자와 같은 방향으로 삽입된 클론을 선택하였다.Both ends of the resulting 1.3 Kb DNA were made with smooth ends, pNH4 was cut with KpnI, and both ends were made with smooth ends to connect them. The nucleotide sequence of the plasmid obtained by cloning was analyzed to confirm the orientation of the MET3 promoter, and clones inserted in the same direction as yEGFP3 and transcription terminator were selected.
상기 벡터에 대해 다중 클로닝 부위의 모든 부위를 확보하기로 하기 위해, 실시예 1에서와 같이 자동복제서열과URA3유전자에서 돌연변이 유도를 수행하였다.MET3프로모터의 경우 pNH5, pNH6a, pNH6b에 대해 XbaⅠ부위와 SalⅠ부위를 각각 불활성화하였으며 이때 사용된 프라이머는 다음 표 2과 같다.In order to secure all sites of the multiple cloning site for the vector, mutation induction was performed in the autoreplication sequence and the URA3 gene as in Example 1. In the case of the MET3 promoter, the XbaI site and the SalI site were inactivated with respect to pNH5, pNH6a, and pNH6b, respectively.
이렇게 하여 얻은 재조합 벡터는 다중 클로닝 부위의 5'에MET3프로모터가 존재하고, 다중 클로닝 부위의 3'에yEGFP3유전자와 전사종결인자가 차례로 존재하며, 이 벡터를 pNH5로 명명하였다. 본 발명의 pNH5는 2001년 10월 12일 생명공학연구소 유전자은행에 기탁번호 KCTC 10090BP로 기탁되었다.The recombinant vector thus obtained had a MET3 promoter at 5 'of the multiple cloning site, a yEGFP3 gene and a transcription terminator at 3' of the multiple cloning site, and this vector was named pNH5. PNH5 of the present invention was deposited with the accession number KCTC 10090BP to the Biotechnology Research Institute Gene Bank on October 12, 2001.
[실시예 4] pNH6a와 pNH6a의 제조Example 4 Preparation of pNH6a and pNH6a
MET3프로모터와 전사종결인자를 가진 재조합 벡터를 얻기 위해, 다음의 과정을 수행하였다. In order to obtain a recombinant vector having a MET3 promoter and a transcription terminator, the following procedure was performed.
기본 벡터 pNH2a는 KpnI으로, pNH2b는 SacI으로 각각 절단하고, 전사종결인자는 pNH4를 제조할 때와 같은 방법으로 준비한 후, 각 DNA의 양끝을 평활 말단으로 만들어 각 벡터에 전사종결인자를 연결하였다.The base vector pNH2a was cut into KpnI, pNH2b into SacI, and the transcription terminator was prepared in the same manner as in the preparation of pNH4, and both ends of each DNA were smoothed to connect the transcription terminator to each vector.
연결 후, pNH2a를 벡터로 하는 경우는 SacI으로, pNH2b를 벡터로 하는 경우는 KpnI으로 각각 절단하고,MET3프로모터는 PstI으로 절단한 후, 각 DNA의 양끝을 평활 말단으로 만들어 연결시켰다. 클로닝으로 얻어진 플라스미드는 염기 서열분석을 통해 삽입된 방향을 확인하였다.After ligation, pNH2a as a vector was cut into SacI, and pNH2b as a vector was cut into KpnI, and MET3 promoter was cut into PstI, and then both ends of each DNA were smoothed and joined. The plasmid obtained by cloning confirmed the inserted direction through sequencing.
상기 벡터에 대해 다중 클로닝 부위의 모든 부위를 확보하기로 하기 위해, 실시예 3에서와 같이 자동복제서열,URA3유전자,MET3프로모터에서 돌연변이 유도를 수행하였다.In order to secure all sites of the multiple cloning site for the vector, mutation induction was performed in the autoreplication sequence, the URA3 gene, and the MET3 promoter as in Example 3.
이렇게 하여 얻어진 재조합 벡터들은 다중 클로닝 부위의 5'에MET3프로모터가 존재하고, 다중 클로닝 부위의 3'에 전사종결인자가 존재하며, 이를 각각 pNH6a와 pNH6b로 명명하였다.The recombinant vectors thus obtained had a MET3 promoter at 5 'of the multiple cloning site and a transcription terminator at 3' of the multiple cloning site, which were named pNH6a and pNH6b, respectively.
[실험예 1] 본 발명의 재조합 벡터의 자동복제서열의 기능성 및 안정도 확인Experimental Example 1 Confirmation of Functionality and Stability of Automatic Replication Sequence of Recombinant Vectors of the Present Invention
1) 본 발명의 재조합 벡터의 자동복제서열의 기능성 확인 - 11) Functional check of the automatic replication sequence of the recombinant vector of the present invention-1
본 발명의 재조합 벡터의 자동복제서열의 기능성을 확인하기 위해, 본 발명의 재조합 벡터를 실제로 캔디다에 도입한 후 선택 표지 유전자의 발현율을 측정하였다. 본 발명의 재조합 벡터 제조시, 다중 클로닝 부위의 확보를 위해 모든 벡터에서 자동복제서열과URA3유전자의 일부분을 돌연변이시켰기 때문에, 최종 산물인 재조합 벡터와 돌연변이 유도를 수행하기 전단계의 벡터에 대해 함께 실험을 수행하였다.In order to confirm the functionality of the automatic replication sequence of the recombinant vector of the present invention, the recombinant vector of the present invention was actually introduced into Candida and the expression rate of the selected marker gene was measured. In the production of the recombinant vector of the present invention, since the autoreplicating sequence and a portion of the URA3 gene were mutated in all vectors to secure multiple cloning sites, experiments were performed together with the final product, the recombinant vector and the vector before performing mutation induction. Was performed.
본 발명의 재조합 벡터 중 pNH2b, pNH4, pNH5와 각 벡터에 대해 돌연변이 유도 전단계의 벡터들, 그리고 액틴(actin)이 삽입된 플라스미드를 각각 전기충격법 (electroporation)을 이용하여 캔디다에 도입시켰다. 형질전환시킬 균주로 캔디다의 야생형(wild type)인 CAI4 균주(ura3Δ::imm34/ura3Δ::imm34)를 사용하였으며, 전기충격에 민감해지도록 저온 상태를 유지하면서 캔디다 세포를 100 mM 리튬 아세테이트(lithium acetate)와 10 mM DTT (dithiothreitol)를 함유하는 TE 완충액으로 처리하였다. 2 ㎜의 홈을 가진 전기충격용 큐벳(cuvette)에 캔디다 세포 40 ㎕와 DNA 1 ㎍을 넣고 얼음에 5분간 두었다. 큐벳을 전기충격기(Easyject Plus, Eurogentec)에 고정시킨 후, 1.8 ㎸, 25 ㎌, 99 Ω의 조건으로 전기충격을 가했다. 이렇게 하여 각각의 플라스미드로 형질전환된 캔디다를 얻었다.Among the recombinant vectors of the present invention, pNH2b, pNH4, and pNH5, the vectors before mutation induction for each vector, and the plasmid into which the actin was inserted were introduced into the candida using electroporation, respectively. Candida wild type CAI4 strain ( ura3Δ :: imm34 / ura3Δ :: imm34 ) was used as the strain to be transformed, and the Candida cells were 100 mM lithium acetate (lithium) while maintaining a low temperature to be sensitive to electric shock. acetate) and 10 mM DTT (dithiothreitol). 40 μl of Candida cells and 1 μg of DNA were placed in an electric shock cuvette having a 2 mm groove and placed on ice for 5 minutes. The cuvette was fixed to an electric shock (Easyject Plus, Eurogentec) and then subjected to an electric shock under conditions of 1.8 kW, 25 kW and 99 kW. This gave Candida transformed with each plasmid.
이때 각 재조합 벡터의 발현율을 측정하기 위해, 단일카피 벡터인 YCp50과 다중카피 벡터인 YEp24를 마찬가지의 방법으로 도입하여 형질전환시킨 사카로마이세스 세레비재를 함께 준비하였다.At this time, in order to measure the expression rate of each recombinant vector, the transformed Saccharomyces cerevisiae was prepared by introducing a single copy vector YCp50 and a multicopy vector YEp24 in the same manner.
각 형질전환체로부터 전체 RNA를 추출한 후(Domdey H. et al., Cell, 39:611-621, 1984), 슬롯 블롯 분석법(slot blot analysis)을 수행하였다. 각각의 전체 RNA 40 ㎍을 RNA 분해 효소가 없는 물 10 ㎕에 녹인 후, RNA 변성화 용액(RNA denaturation solution; 포름아미드(formamide) 660 ㎕, 37% 포름알데히드 (formaldehyde) 210 ㎕, 10 ×MOPS 전기영동 완충액 130 ㎕) 30 ㎕와 섞어 65 ℃에서 5분간 항온처리(incubation)하였다. 상기 반응액을 얼음 상에서 식힌 후, 동일 부피의 20 ×SSC 완충액을 처리하였다.After extracting total RNA from each transformant (Domdey H. et al., Cell, 39: 611-621, 1984), a slot blot analysis was performed. 40 μg of each total RNA was dissolved in 10 μl of water without RNAase, then RNA denaturation solution (660 μl of formamide, 210 μl of 37% formaldehyde, 10 × MOPS electrolysis 130 µl of the lyophoretic buffer) and 30 µl were incubated at 65 ° C for 5 minutes. The reaction was cooled on ice and then treated with the same volume of 20 × SSC buffer.
한편, RNA를 전이(transfer)시킬 멤브레인(membrane)은 적절한 크기로 잘라 20 ×SSC 완충액에 1시간 동안 담가두었다가, 블롯팅할 기기(blotting manifold)의 반응구멍(well)에 멤브레인을 위치시킨 후, 10 ×SSC 완충액으로 멤브레인을 세척하였다. RNA를 변성시킨 반응액을 멤브레인 위에 올린 후 10 ×SSC 완충액으로 세척하고, 5분간 진공을 이용하여 멤브레인을 건조시켰다.Meanwhile, the membrane to transfer RNA is cut to an appropriate size, soaked in 20 x SSC buffer for 1 hour, and then the membrane is placed in a reaction well of a blotting manifold. The membrane was washed with 10 x SSC buffer. The RNA-modified reaction solution was placed on the membrane, washed with 10 x SSC buffer, and dried for 5 minutes under vacuum.
RNA가 전이된 멤브레인을 자외선으로 고정(cross-linking)시킨 후, 전혼성화 용액(prehybridization solution; 포름아미드 5 ㎖, 물 2 ㎖, 50% 덱스트란 설페이트(dextran sulfate) 2 ㎖, 10% SDS 1 ㎖, 0.58 g 염화나트륨, 연어의 정자(salmon sperm) DNA 100 ㎕)에 넣어 42 ℃에서 3시간 동안 항온처리하였다. 전혼성화 용액에 동위원소로 표지한 프로브(probe)를 20 ㎕ 넣어 42 ℃에서 16시간 이상 혼성화하였다. 이때 프로브는 선택 표지 유전자인URA3유전자를 PCR 반응으로 얻어 사용하였으며, 재조합 벡터 제조시 사용된 서열번호 3, 4의 염기 서열을 가진 프라이머를 이용하였다.After cross-linking the RNA-transferred membranes with UV light, prehybridization solution (5 ml of formamide, 2 ml of water, 2 ml of 50% dextran sulfate, 1 ml of 10% SDS) , 0.58 g sodium chloride, salmon sperm (100 μl of salmon sperm DNA) was added thereto and incubated at 42 ° C. for 3 hours. 20 μl of an isotopically labeled probe was added to the prehybridization solution and hybridized at 42 ° C. for at least 16 hours. At this time, the probe was used to obtain the URA3 gene, which is a selection marker gene, by PCR reaction, and used primers having the nucleotide sequences of SEQ ID NOs: 3 and 4 used in the preparation of the recombinant vector.
혼성화가 끝난 후, 0.1 % SDS가 각각 함유된 2 ×SSC, 1 ×SSC 및 0.1 ×SSC의 용액을 사용하여 순서대로 세척한 후, 자기방사표지법(autoradiography)에 의해 결과를 분석하였다. 슬롯 블롯 분석시, 각 경우의URA3mRNA 밀도를 비교하기 위해 곰팡이에서 항상 일정하게 발현되는 actin mRNA의 양을 함께 분석하고, 그 결과를 도 2에 나타내었다.After hybridization, washing was performed sequentially using a solution of 2xSSC, 1xSSC and 0.1xSSC containing 0.1% SDS, respectively, and the results were analyzed by autoradiography. In slot blot analysis, in order to compare the URA3 mRNA density in each case, the amount of actin mRNA constantly expressed in the fungus was analyzed together, and the results are shown in FIG. 2.
도 2에서, 항상 일정하게 발현되는 유전자인 액틴의 mRNA의 양에 비추어볼 때 pNH2b(c), pNH4(e), pNH4의 돌연변이 유도 전단계 벡터(f)는 YEp24(b)와 비슷한 정도의 높은URA3발현율을 나타냈으며, pNH5(g), pNH2b의 돌연변이 유도 전단계 벡터(d), pNH5의 돌연변이 유도 전단계 벡터(h)는 그보다 낮은 발현율, 즉 YCp50(a)과 비슷한 정도의 발현율을 나타냈다.In Fig. 2, in view of the amount of mRNA of actin, which is a gene that is constantly expressed, pNH2b (c), pNH4 (e), and pNH4 mutation-induced preparatory vectors (f) have high levels of URA3 similar to YEp24 (b). The expression rate was shown, pNH5 (g), pNH2b mutagenesis pre-step vector (d), pNH5 mutagenesis-preliminary vector (h) showed a lower expression rate, that is, similar to YCp50 (a).
따라서 본 발명의 재조합 벡터들은 대부분 다중카피 벡터만큼의 높은 발현율을 나타내므로, 발현율이 단일카피 벡터의 수준에 그쳤던 기존의 캔디다 벡터보다 훨씬 높은 활성을 가진 자동복제서열을 지니고 있음을 알 수 있다.Therefore, since most of the recombinant vectors of the present invention exhibit the same expression rate as that of the multicopy vector, it can be seen that the expression rate has an autoreplication sequence having much higher activity than the conventional Candida vector, which was only the level of the single copy vector.
또한 돌연변이 유도를 하기 전단계의 벡터와 돌연변이 유도를 수행하여 얻은 최종 산물인 재조합 벡터의 발현율에 별다른 차이가 없는 것으로 보아, 본 발명의 재조합 벡터에 포함된 자동복제서열과URA3유전자는 제조 과정 중 발생한 몇 개의 염기 돌연변이에 상관없이 캔디다 내에서 제대로 작용함을 알 수 있다.2) 본 발명의 재조합 벡터의 자동복제서열의 기능성 확인 - 2상기 결과를 재차 확인하기 위해, 상기와 마찬가지의 방법에 의해 본 발명의 벡터 pNH2a, pNH2b, pNH4, pNH5, pNH6a 및 pNH6b를 CAI4 균주에 도입시킨 후, 각 형질전환체로부터 게놈 DNA 및 plasmid DNA를 추출하여(Hoffman and Winston, 1987), 서던 분석법(Southern analysis)을 수행하였다.추출된 DNA를 sacI, AhdI, XmnI를 동시에 사용하여 37℃에서 1시간 동안 절단하였다. 절단된 DNA를 0.8% 아가로스 젤에 걸어 전기영동을 수행하였다. 마커를 잘라낸 뒤, EtBr로 젤을 염색하고, 변성화 용액(denaturation solution, 1.5M 염화나트륨. 0.5M 수산화나트륨)에 30분간 담가 흔들어준 후, 증류수로 세척하였다. 젤을 중성화 용액(neutralization solution, 0.5M 트리스-Cl(pH 8.0), 1.5M 염화나트륨)에 30분간 담가 흔들어주고, 증류수로 3번 세척하였다.상기와 같이 처리된 젤 상의 DNA들을 멤브레인으로 전이시킨 후, 자외선으로 고정시켰다. 멤브레인을 전혼성화 용액에 담가 42℃에서 약 4시간 정도 흔들어주었다.그 후 프로브를 첨가하고 하룻밤 동안 혼성화 반응을 수행하였다. 이때 프로브로 액틴 유전자 ACT1과 암피실린 내성 유전자 APR를 사용하였다. 혼성화 반응 후, 0.1 % SDS가 각각 함유된 2 ×SSC, 1 ×SSC 및 0.1 ×SSC의 용액을 사용하여 65℃에서 약 30분씩 멤브레인을 세척하였다. 상기 멤브레인에 대해 자기방사표지법을 수행하고, 그 결과를 도 3에 나타내었다.도 3에 나타난 바와 같이, 본 발명의 재조합 벡터 중 pNH2a(2열), pNH2b(3열) 및 pNH4(4열)로 형질전환된 캔디다에서는, 상기 벡터들에 의해 발현되는 APR이 캔디다에서 일상적으로 발현되는 유전자인 ACT1보다 3∼5배 이상 높은 밀도의 카피 수를 지니는 것으로 나타났다. 그러나 액틴 유전자(1열)가 APR 유전자보다 3배 이상의 크기를 지닌다는 점을 감안할 때, 실제 카피 수는 APR이 ACT1의 5∼8배 정도가 된다.마찬가지로 본 발명의 재조합 벡터 중 pNH5(5열), pNH6a(6열) 및 pNH6b(7열)로 형질전환된 캔디다에서도, 유전자 크기를 감안할 때 APR의 카피 수는 ACT1의 카피 수와 비슷할 것으로 보인다.따라서 본 발명의 재조합 벡터들은 캔디다 내에서 일상적으로 발현되는 유전자와 비슷하거나 또는 그보다 훨씬 많은 카피 수를 지니므로, 발현율이 단일카피 벡터의 수준에 그쳤던 기존의 캔디다 벡터보다 훨씬 높은 활성을 가진 자동복제서열을 지니고 있음을 알 수 있다.In addition, since there is no difference in the expression rate of the vector before the mutation induction and the recombinant vector, which is the final product obtained by performing the mutation induction, the autoreplication sequence and the URA3 gene included in the recombinant vector of the present invention are generated during the manufacturing process. Regardless of the dog's base mutation, it works well in Candida. 2) Confirmation of Functionality of Automatic Replication Sequences of Recombinant Vectors of the Present Invention -2 To confirm the results above, the vectors pNH2a, pNH2b, pNH4, pNH5, pNH6a and pNH6b of the present invention were transferred to the CAI4 strain by the same method as above. After introduction, genomic DNA and plasmid DNA were extracted from each transformant (Hoffman and Winston, 1987) and Southern analysis was performed. The extracted DNA was simultaneously subjected to 37 ° C. using sacI, AhdI, and XmnI. Cut for 1 h at. The cleaved DNA was hung on 0.8% agarose gel to perform electrophoresis. After cutting off the marker, the gel was stained with EtBr, soaked in a denaturation solution (1.5 M sodium chloride, 0.5 M sodium hydroxide) for 30 minutes, shaken, and washed with distilled water. The gel was immersed in a neutralization solution (0.5M Tris-Cl (pH 8.0), 1.5M sodium chloride) for 30 minutes, shaken and washed three times with distilled water. After transferring the DNA on the treated gel to the membrane, And fixed with ultraviolet rays. The membrane was immersed in the prehybridization solution and shaken at 42 ° C. for about 4 hours. The probe was then added and the hybridization reaction was performed overnight. Actin gene ACT1 and ampicillin resistance gene APR were used as probes. After the hybridization reaction, the membrane was washed at 65 ° C. for about 30 minutes using a solution of 2 × SSC, 1 × SSC and 0.1 × SSC containing 0.1% SDS, respectively. Self-radiolabelling was performed on the membrane and the results are shown in FIG. 3. As shown in FIG. 3, pNH2a (column 2), pNH2b (column 3) and pNH4 (column 4) of the recombinant vector of the present invention. In Candida transformed with, the APR expressed by the vectors was found to have a copy number of 3 to 5 times higher density than ACT1, a gene commonly expressed in Candida. However, given that the actin gene (column 1) is three times larger than the APR gene, the actual copy number is about 5 to 8 times the APR of ACT1. Similarly, pNH5 (column 5) ), even in Candida transformed with pNH6a (column 6) and pNH6b (column 7), given the gene size, the copy number of APR appears to be comparable to that of ACT1. Thus, the recombinant vectors of the present invention are routinely used in Candida. Since it has a number of copies that are similar to or much higher than that of the gene expressed by, it can be seen that the expression rate has an autoreplication sequence having a much higher activity than the conventional Candida vector, which was only at the level of a single copy vector.
3) 본 발명의 재조합 벡터의 자동복제서열의 안정도 확인 - 13) Check the stability of the automatic replication sequence of the recombinant vector of the present invention-1
본 발명의 재조합 벡터의 자동복제서열의 안정도를 확인하기 위해, 본 발명의 재조합 벡터를 실제로 캔디다에 도입한 후 선택 압력(selection pressure)이 가해지는 상황에서 선택 표지 유전자의 발현 안정성을 측정하였다.In order to confirm the stability of the automatic replication sequence of the recombinant vector of the present invention, the expression stability of the selection marker gene was measured in the situation where selection pressure was applied after the recombinant vector of the present invention was actually introduced to Candida.
본 발명의 재조합 벡터 중 pNH2a를 택하여, 발현율을 확인할 때와 마찬가지의 방법으로 전기 충격법을 이용하여 CAI4 균주에 도입하였다. 정상적인 발현율을 확인하기 위해서, 형질전환된 CAI4를 대수성장기(log phase)까지 배양하여 전체 RNA를 얻었다.Among the recombinant vectors of the present invention, pNH2a was selected and introduced into the CAI4 strain using an electric shock method in the same manner as when the expression rate was confirmed. In order to confirm the normal expression rate, the transformed CAI4 was cultured to the log phase to obtain total RNA.
선택 압력을 가한 조건에서의 발현율을 확인하기 위해서, 형질전환된 CAI4를 대수성장기 이후까지 계속 배양하여 성장정지기(stationery phase)까지 키운 후, 각각 유라실 또는 유리딘이 함유된 배지로 옮겼다. 이때 선택 압력이 가해지지 않은 조건에서의 발현율을 함께 비교하기 위해 아무런 염기도 함유되지 않은 배지로옮긴 조건도 준비하였다. 각 조건마다 하루에 한번씩 새 배지로 갈아주면서 3일간 배양한 후, 전체 RNA를 추출하였다.In order to confirm the expression rate under the conditions of selection pressure, the transformed CAI4 was continuously cultured until the logarithmic growth period, grown to the stationary phase, and transferred to a medium containing uracil or uridine, respectively. At this time, conditions for transferring to a medium containing no base were also prepared in order to compare the expression rates under conditions under which no selection pressure was applied. After culturing for 3 days while changing to fresh medium once a day for each condition, the total RNA was extracted.
각각의 조건으로부터 발현율을 확인할 때와 마찬가지의 방법으로 슬롯 블롯 분석법을 수행하여, 그 결과를 도 4에 나타내었다.Slot blot analysis was performed in the same manner as when confirming the expression rate from each condition, and the results are shown in FIG. 4.
도 4에서, pNH2a로 형질전환된 캔디다는 유라실(b)이나 유리딘(c) 등 염기가 함유된 배지에 여러 세대 동안 배양했을 때에도URA3유전자를 소실하지 않았으며, 대수성장기까지 배양한 캔디다(a)나 염기를 공급해주지 않은 배지에서 배양한 캔디다(d)에서와 마찬가지로 높은URA3발현율을 나타내었다.4) 본 발명의 재조합 벡터의 자동복제서열의 안정도 확인 - 2
[실험예 2] 본 발명의 재조합 벡터의Experimental Example 2 of the Recombinant Vector of the Present Invention MET3MET3 프로모터와Promoter and yEGFP3yEGFP3 유전자의 기능성 확인Functional check of gene
본 발명의 재조합 벡터에 대해MET3프로모터와yEGFP3유전자의 기능성을 확인하기 위해, 다중 클로닝 부위의 5'에MET3프로모터를, 3'에yEGFP3유전자를 지니고 있는 pNH5를 대표로 택하였다.To verify the functionality of the MET3 promoter and yEGFP3 genes for the recombinant vector of the present invention, a multi-cloning site 5 'to the MET3 promoter, 3' were chosen to represent the pNH5 which has a yEGFP3 gene.
pNH5의MET3프로모터 부분에서 다중 클로닝 부분의 확보를 위해 XabⅠ과 SalⅠ 부위를 돌연변이시켰으므로, 돌연변이가 일어난 벡터에서MET3프로모터가 제대로 기능할 수 있는지를 확인할 필요가 있었다. 돌연변이시키기 전의 원래 벡터, XabⅠ부위에 돌연변이를 일으킨 벡터, SalⅠ부위에 돌연변이를 일으킨 벡터 및XabⅠ과 SalⅠ부위에 모두 돌연변이를 일으킨 벡터를 실험예 1과 마찬가지의 방법으로 CAI4에 각각 도입하였다.Since the Xab I and Sal I sites were mutated to secure multiple cloning sites in the MET3 promoter region of pNH5 , it was necessary to confirm whether the MET3 promoter could function properly in the mutated vector. The original vector before mutation, the vector which mutated the XabI site, the vector which mutated the SalI site, and the vector which mutated both XabI and SalI site were introduced into CAI4 in the same manner as in Experimental Example 1.
각 형질전환체를 메티오닌이 첨가되지 않았거나 첨가된 배지에서 배양한 후, 형광 현미경 하에서yEGFP3유전자의 발현을 관찰하였다(도 5).Each transformant was cultured in medium without or without methionine, and then the expression of the yEGFP3 gene was observed under a fluorescence microscope (FIG. 5).
도 5에서, 메티오닌이 첨가되지 않은 배지에서 배양된 형질전환체에서는 모두 형광을 나타내는 세포들을 관찰할 수 있었으므로,MET3프로모터에 의해yEGFP3유전자가 제대로 발현되었음을 알 수 있다. 반면 메티오닌이 첨가된 배지에서 배양된 형질전환체들에서는 모두 형광을 나타내는 세포가 관찰되지 않았다.In Figure 5, because there was methionine in the transformant cultured on medium with no added both to observe the cells exhibiting fluorescence, it can be seen that the expression of properly yEGFP3 gene by the MET3 promoter. On the other hand, no fluorescence was observed in the transformants cultured in the medium containing methionine.
따라서 본 발명의 재조합 벡터의MET3프로모터는 메티오닌의 존재 유무에 따라 정확하게 유전자의 발현을 조절할 수 있으며,yEGFP3유전자 또한 정상적으로 발현됨을 알 수 있다.Therefore, the MET3 promoter of the recombinant vector of the present invention can accurately control the expression of the gene according to the presence or absence of methionine, it can be seen that the yEGFP3 gene is also normally expressed.
본 발명의 재조합 벡터들은 기존의 벡터에 비해 크기가 작기 때문에 유전자 조작이 용이하다.The recombinant vectors of the present invention are easy to genetically manipulate because they are smaller in size than conventional vectors.
본 발명의 재조합 벡터들은 캔디다 내에서 발현율이 높으며 안정성을 갖추고 있으므로, 이후의 실험 단계가 수월하게 진행될 수 있다.Since the recombinant vectors of the present invention have high expression rate and stability in Candida, subsequent experimental steps can be easily performed.
본 발명의 재조합 벡터들은 여러 가지 유용한 인자들을 갖추고 있으므로, 유전자 라이브러리 제작, 연구 대상 유전자의 생리적 역할 및 연구 대상 단백질의 세포 내 위치·기능에 대한 연구에 유용하게 사용될 수 있다.Since the recombinant vectors of the present invention have various useful factors, they can be usefully used for the preparation of gene libraries, the physiological role of the genes to be studied, and the study of the location and function of cells of the proteins to be studied.
<110> CHOI, Won Ja <120> Recombinant vector series efficient for research about Candida albicans <130> 01P-68 <160> 24 <170> KopatentIn 1.71 <210> 1 <211> 1203 <212> DNA <213> Candida albicans <220> <221> mRNA <222> (1)..(1203) <223> Candida albicans DNA of an autonomously replicating sequence (ARS) <300> <303> GenBank <308> X16634 <309> 1991-05-14 <400> 1 ggatcatacc aaaaaaaaat ccggggtagc gatgaggtag tgcaagttat accagagagc 60 ctcaatttga acgtggtact gcacacaaac cgtagcccta accctaatta aatgcacgtg 120 acccacacaa ttttcaacca ccaacaacac accaaaaatg tatgtacact cggagtggaa 180 aatatttccc caggcataat tgtggttgcc aattatagtg gagtgtttgt tattagtata 240 ggagtactcc ttttatgtgg aaaaatcgaa aaacataaaa cagtgcagct atacactaac 300 aaatggtata cccatggtca gacaacacca aacaacacac ccgtaatggt tggtttggct 360 aaatagagca tctcattttg tgtattattt tatgttttgg atatttttag tttgaaattt 420 atataaaaat attttactcc aattttcttg ccaaattttg tacaaaaagt aaaaaataga 480 acttccaatt tttgttaaac aaacttcaat ctaacaaccg ggatagtgtt tggggggctg 540 tgtaacacca ctggtgtaat aaaagtgcca atatttggaa attaattttt tggtggtgga 600 cgtttcttcc taaaacacct agatgtgcta gtataattgc acaaccagca cgcacactgc 660 gtctggccgg tcctggacta cattttgtct ctaacattcg tagctgcagc actgctacaa 720 cccatcaatg gccacccgga catacatatt cccgttttgt gccacagcca cccaactttc 780 cggctcaaaa aatggctcca cacaatttgg gcaccccgat tacccctccc ctgcagcgac 840 agcacataca ctttggtggt tgtggggcta gagccctaaa agtgaatttg gtcacgtgag 900 agacagcaga taaggttcgc atttttttat tttttcggtt gcggccatat ctagcagaaa 960 gcaccgttcc ccgttcgatc aaccgtagtt aagctgctaa gagcaatacc gagtagtgta 1020 gtgggagacc atacgcgaaa ctattgtgct gcaatctatt tttttttagt aacgtatatt 1080 ttttttttgc atgaaccaag ggtaatcgac cctgctctat ccagtgatgg tgataaagtt 1140 gggtggtgtc taacaaaaag ggggagaacc agagggtgta taaataagga gaagctggga 1200 tcc 1203 <210> 2 <211> 1365 <212> DNA <213> Candida albicans <220> <221> mRNA <222> (11)..(1365) <223> Candida albicans URA3 gene for orotidine-5'-monophosphate decarboxylase <300> <303> GenBank <308> X14198 <309> 1993-09-12 <400> 2 agtactaata ggaattgatt tggatggtat aaacggaaac aaaaaaaaga gctggtacta 60 ctttctttaa aattatttta ttatttgatt ttatttaata gtatatatta tattttgaac 120 gtagattatt ttgttgaaag ttgctgtagt gccattgatt cgtaacacta attctgtatt 180 agtcattcct cttgtttgat agtatccaaa aaaacggcta tttttttgca atcttatttc 240 ctgcatatta tacagataac ataatgaaag aaaaaatctt tttttttgtt cttcaatgat 300 gatttcaacc attcttttaa acattgatca attcctgagc aacaacccca tacacactgg 360 tttatatacc gcccctttta cagttgaaga aagaaataga aatagaaata gcaaacaaaa 420 gatatgacag tcaacactaa gacctatagt gagagagcag aaactcatgc ctcaccagta 480 gcacagcgat tatttcgatt aatggaactg aagaaaacca atttatgtgc atcaattgac 540 gttgatacca ctaaggaatt ccttgaatta attgataaat taggtcctta tgtatgctta 600 atcaagactc atattgatat aatcaatgat ttttcctatg aatccactat tgaaccatta 660 ttagaacttt cacgtaaaca tcaatttatg atttttgaag atagaaaatt tgctgatatt 720 ggtaataccg taaagaaaca atatattggt ggagtttata aaattagtag ttgggcagat 780 attaccaatg ctcatggtgt cactgggaat ggagtggttg aaggattaaa acagggagct 840 aaagaaacca ccaccaacca agagccaaga gggttattga tgttagctga attatcatca 900 gtgggatcat tagcatatgg agaatattct caaaaaactg ttgaaattgc taaatccgat 960 aaggaatttg ttattggatt tattgcccaa cgtgatatgg gtggccaaga agaaggattt 1020 gattggctta ttatgacacc tggagttgga ttagatgata aaggtgatgg attaggacaa 1080 caatatagaa ctgttgatga agttgttagc actggaactg atattatcat tgttggtaga 1140 ggattgtttg gtaaaggaag agatccagat attgaaggta aaaggtatag aaatgctggt 1200 tggaatgctt atttgaaaaa gactggccaa ttataaatgt gaagggggag attttcactt 1260 tattagattt gtatatatgt agaataaata aataaataag ttaaataaat aattaaataa 1320 gggtggtaat tattactatt tacaatcaaa ggtggtcctt ctaga 1365 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying ARS of Candida albicans <400> 3 catatggatc ataccaaaaa aaaatccggg g 31 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying ARS of Candida albicans <400> 4 catatggatc ccagcttctc cttatt 26 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying URA3 gene of Candida albicans <400> 5 gacgtcggaa ttgatttgga tggtat 26 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying URA3 gene of Candida albicans <400> 6 gacgtctaga aggaccacct ttga 24 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying yeast EGFP3 gene <400> 7 gagctcatgt ctaaaggtga ag 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying yeast EGFP3 gene <400> 8 gagctcctgc agttatttgt ac 22 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of first Pst1 site in ARS <400> 9 cgtagctgcc gcactgctac aacccatc 28 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of first Pst1 site in ARS <400> 10 gggttgtagc agtgcggcag ctacgaatg 29 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of second Pst1 site in ARS <400> 11 gtatgtgctg tcgcggcagg ggaggg 26 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of second Pst1 site in ARS <400> 12 gattacccct cccctgccgc gacagc 26 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of EcoR1 site in URA3 gene <400> 13 caaggaactc cttagtggta tcaac 25 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of EcoR1 site in URA3 gene <400> 14 ccactaagga gttccttgaa ttaattg 27 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of Xba1 site in URA3 gene <400> 15 cgtctaggag gaccaccttt gattg 25 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse forward primer for mutagenesis resulting inactivation of Xba1 site in URA3 gene <400> 16 caaaggtggt cctcctagac gtc 23 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying terminator sequence of Candida albicans chitin synthase 2 <400> 17 ggcgccccag cacctccaga agag 24 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying terminator sequence of Candida albicans chitin synthase 2 <400> 18 ggcgcccaaa agaatagtat gggc 24 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying MET3 promoter sequence of Candida albicans <400> 19 ctgcaggcgc tgaaaaacta cgaa 24 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying MET3 promoter sequence of Candida albicans <400> 20 ctgcaggttt tctggggagg gtat 24 <210> 21 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of Xba1 site in MET3 promoter sequence <400> 21 cgatgtttaa atctagctac ccaatatg 28 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of Xba1 site in MET3 promoter sequence <400> 22 ggggatttca tattgggtag ctagatttaa ac 32 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of Sal1 site in MET3 promoter sequence <400> 23 gttcattgtc gtggcgacat tctaac 26 <210> 24 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of Sal1 site in MET3 promoter sequence <400> 24 ctttagttag aatgtcgcca cgacaatg 28<110> CHOI, Won Ja <120> Recombinant vector series efficient for research about Candida albicans <130> 01P-68 <160> 24 <170> KopatentIn 1.71 <210> 1 <211> 1203 <212> DNA <213> Candida albicans <220> <221> mRNA <222> (1) .. (1203) <223> Candida albicans DNA of an autonomously replicating sequence (ARS) <300> <303> GenBank <308> X16634 <309> 1991-05 -14 <400> 1 ggatcatacc aaaaaaaaat ccggggtagc gatgaggtag tgcaagttat accagagagc 60 ctcaatttga acgtggtact gcacacaaac cgtagcccta accctaatta aatgcacgtg 120 acccacacaa ttttcaacca ccaacaacac accaaaaatg tatgtacact cggagtggaa 180 aatatttccc caggcataat tgtggttgcc aattatagtg gagtgtttgt tattagtata 240 ggagtactcc ttttatgtgg aaaaatcgaa aaacataaaa cagtgcagct atacactaac 300 aaatggtata cccatggtca gacaacacca aacaacacac ccgtaatggt tggtttggct 360 aaatagagca tctcattttg tgtattattt tatgttttgg atatttttag tttgaaattt 420 atataaaaat attttactcc aattttcttg cca aattttg tacaaaaagt aaaaaataga 480 acttccaatt tttgttaaac aaacttcaat ctaacaaccg ggatagtgtt tggggggctg 540 tgtaacacca ctggtgtaat aaaagtgcca atatttggaa attaattttt tggtggtgga 600 cgtttcttcc taaaacacct agatgtgcta gtataattgc acaaccagca cgcacactgc 660 gtctggccgg tcctggacta cattttgtct ctaacattcg tagctgcagc actgctacaa 720 cccatcaatg gccacccgga catacatatt cccgttttgt gccacagcca cccaactttc 780 cggctcaaaa aatggctcca cacaatttgg gcaccccgat tacccctccc ctgcagcgac 840 agcacataca ctttggtggt tgtggggcta gagccctaaa agtgaatttg gtcacgtgag 900 agacagcaga taaggttcgc atttttttat tttttcggtt gcggccatat ctagcagaaa 960 gcaccgttcc ccgttcgatc aaccgtagtt aagctgctaa gagcaatacc gagtagtgta 1020 gtgggagacc atacgcgaaa ctattgtgct gcaatctatt tttttttagt aacgtatatt 1080 ttttttttgc atgaaccaag ggtaatcgac cctgctctat ccagtgatgg tgataaagtt 1140 gggtggtgtc taacaaaaag ggggagaacc agagggtgta taaataagga gaagctggga 1200 tcc 1203 <210> 2 <211> 1365 <212> DNA <213> Candida albicans <220> <221> mRNA <222> (11). (1365) <223> Candida albicans URA3 gene for orotidine-5'-monophosphate decarboxylase <300> <303> GenBank <308> X14198 <309> 1993-09-12 <400> 2 agtactaata ggaattgatt tggatggtat aaacggaaac aaaaaaaaga gctggtacta 60 ctttctttaa aattatttta ttatttgatt ttatttaata gtatatatta tattttgaac 120 gtagattatt ttgttgaaag ttgctgtagt gccattgatt cgtaacacta attctgtatt 180 agtcattcct cttgtttgat agtatccaaa aaaacggcta tttttttgca atcttatttc 240 ctgcatatta tacagataac ataatgaaag aaaaaatctt tttttttgtt cttcaatgat 300 gatttcaacc attcttttaa acattgatca attcctgagc aacaacccca tacacactgg 360 tttatatacc gcccctttta cagttgaaga aagaaataga aatagaaata gcaaacaaaa 420 gatatgacag tcaacactaa gacctatagt gagagagcag aaactcatgc ctcaccagta 480 gcacagcgat tatttcgatt aatggaactg aagaaaacca atttatgtgc atcaattgac 540 gttgatacca ctaaggaatt ccttgaatta attgataaat taggtcctta tgtatgctta 600 atcaagactc atattgatat aatcaatgat ttttcctatg aatccactat tgaaccatta 660 ttagaacttt cacgtaaaca tcaatttatg atttttgaag atagaaaatt tgctgatatt 720 ggtaataccg taaagaaaca atatattggt ggagtttata aaattagtag ttgggcagat 780 attaccaatg ctcatggtgt cactgggaat ggagtggttg aaggattaaa acagggagct 840 aaagaaacca ccaccaacca agagccaaga gggttattga tgttagctga attatcatca 900 gtgggatcat tagcatatgg agaatattct caaaaaactg ttgaaattgc taaatccgat 960 aaggaatttg ttattggatt tattgcccaa cgtgatatgg gtggccaaga agaaggattt 1020 gattggctta ttatgacacc tggagttgga ttagatgata aaggtgatgg attaggacaa 1080 caatatagaa ctgttgatga agttgttagc actggaactg atattatcat tgttggtaga 1140 ggattgtttg gtaaaggaag agatccagat attgaaggta aaaggtatag aaatgctggt 1200 tggaatgctt atttgaaaaa gactggccaa ttataaatgt gaagggggag attttcactt 1260 tattagattt gtatatatgt agaataaata aataaataag ttaaataaat aattaaataa 132 0 gggtggtaat tattactatt tacaatcaaa ggtggtcctt ctaga 1365 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying ARS of Candida albicans <400> 3 catatggatc ataccaaaaa aaaatccggg g 31 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying ARS of Candida albicans <400> 4 catatggatc ccagcttctc cttatt 26 <210> 5 <211> 26 <212> DNA <213 > Artificial Sequence <220> <223> forward primer for amplifying URA3 gene of Candida albicans <400> 5 gacgtcggaa ttgatttgga tggtat 26 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying URA3 gene of Candida albicans <400> 6 gacgtctaga aggaccacct ttga 24 <210> 7 <211> 2 2 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying yeast EGFP3 gene <400> 7 gagctcatgt ctaaaggtga ag 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220 > <223> reverse primer for amplifying yeast EGFP3 gene <400> 8 gagctcctgc agttatttgt ac 22 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of first Pst1 site in ARS <400> 9 cgtagctgcc gcactgctac aacccatc 28 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of first Pst1 site in ARS <400> 10 gggttgtagc agtgcggcag ctacgaatg 29 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <22 3> forward primer for mutagenesis resulting inactivation of second Pst1 site in ARS <400> 11 gtatgtgctg tcgcggcagg ggaggg 26 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of second Pst1 site in ARS <400> 12 gattacccct cccctgccgc gacagc 26 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of EcoR1 site in URA3 gene <400> 13 caaggaactc cttagtggta tcaac 25 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of EcoR1 site in URA3 gene <400> 14 ccactaagga gttccttgaa ttaattg 27 <210> 15 <211> 25 <212> DN A <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of Xba1 site in URA3 gene <400> 15 cgtctaggag gaccaccttt gattg 25 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence < 220> <223> reverse forward primer for mutagenesis resulting inactivation of Xba1 site in URA3 gene <400> 16 caaaggtggt cctcctagac gtc 23 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying terminator sequence of Candida albicans chitin synthase 2 <400> 17 ggcgccccag cacctccaga agag 24 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying terminator sequence of Candida albicans chitin synthase 2 <400> 18 ggcgcccaaa agaatagtat gggc 24 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying MET3 promoter sequence of Candida albicans <400> 19 ctgcaggcgc tgaaaaacta cgaa 24 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying MET3 promoter sequence of Candida albicans <400> 20 ctgcaggttt tctggggagg gtat 24 <210> 21 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of Xba1 site in MET3 promoter sequence <400> 21 cgatgtttaa atctagctac ccaatatg 28 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of Xba1 site in MET3 promoter sequence <400> 22 gggg atttca tattgggtag ctagatttaa ac 32 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mutagenesis resulting inactivation of Sal1 site in MET3 promoter sequence <400> 23 gttcattgtc gtggcgacat tctaac 26 < 210> 24 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mutagenesis resulting inactivation of Sal1 site in MET3 promoter sequence <400> 24 ctttagttag aatgtcgcca cgacaatg 28
Claims (25)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2001-0065907A KR100453719B1 (en) | 2001-10-25 | 2001-10-25 | Recombinant vector series efficient for molecular research in Candida albicans |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2001-0065907A KR100453719B1 (en) | 2001-10-25 | 2001-10-25 | Recombinant vector series efficient for molecular research in Candida albicans |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20030033782A KR20030033782A (en) | 2003-05-01 |
| KR100453719B1 true KR100453719B1 (en) | 2004-10-20 |
Family
ID=29566303
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR10-2001-0065907A Expired - Fee Related KR100453719B1 (en) | 2001-10-25 | 2001-10-25 | Recombinant vector series efficient for molecular research in Candida albicans |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100453719B1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0982401A2 (en) * | 1998-08-14 | 2000-03-01 | Janssen Pharmaceutica N.V. | Drug targets in Candida albicans |
-
2001
- 2001-10-25 KR KR10-2001-0065907A patent/KR100453719B1/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0982401A2 (en) * | 1998-08-14 | 2000-03-01 | Janssen Pharmaceutica N.V. | Drug targets in Candida albicans |
Non-Patent Citations (5)
| Title |
|---|
| Curr Genet. 1995 Feb;27(3):243-8, Myers KK * |
| Infect Immun. 1992 Mar;60(3):876-84, Goshorn AK * |
| Mol Gen Genet. 1992 Nov;235(2-3):453-7, Cannon RD * |
| Mol Microbiol. 1999 Nov;34(4):792-8, Care RS * |
| Yeast. 2000 Sep 15;16(12):1121-9, Backen AC * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20030033782A (en) | 2003-05-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105331627B (en) | A method of prokaryotic gene group editor is carried out using endogenous CRISPR-Cas system | |
| Benton et al. | Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes | |
| US4808537A (en) | Methanol inducible genes obtained from pichia and methods of use | |
| US4855231A (en) | Regulatory region for heterologous gene expression in yeast | |
| US6080540A (en) | Cloning of mammalian genes in microbial organisms and methods for pharmacological screening | |
| Ishiura et al. | Phage particle-mediated gene transfer to cultured mammalian cells | |
| Donohue et al. | Cloning and expression of the Rhodobacter sphaeroides reaction center H gene | |
| Rosenfeld et al. | Cloning and characterization of the xyl genes from Escherichia coli | |
| Lovett et al. | Nucleotide sequence of the Escherichia coli recJ chromosomal region and construction of recJ-overexpression plasmids | |
| Ny et al. | Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12 | |
| WO1993000353A1 (en) | Sequences characteristic of human gene transcription product | |
| FI104264B (en) | Process for improving the production of secondary metabolites by the use of grouped biosynthetic genes | |
| Leiting et al. | Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum | |
| Suto et al. | A double-strand break repair component is essential for S phase completion in fission yeast cell cycling | |
| Hansen et al. | The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli | |
| Cossart et al. | Construction and expression of a hybrid plasmid containing the Escherichia coli thrA and thrB genes | |
| Taguchi et al. | The cloning and mapping of ADR6, a gene required for sporulation and for expression of the alcohol dehydrogenase II isozyme from Saccharomyces cerevisiae | |
| KR100453719B1 (en) | Recombinant vector series efficient for molecular research in Candida albicans | |
| JP3089036B2 (en) | Overexpression of single-stranded molecules | |
| JPH11514885A (en) | Chitin synthase 1 | |
| US5389526A (en) | Plasmid vectors for cellular slime moulds of the genus dictyostelium | |
| Chen et al. | Multiple-copy-gene integration on chromosome of Escherichia coli for beta-galactosidase production | |
| ZA200302080B (en) | Expression vectors with modified Co1E1 origin of replication for control of plasmid number. | |
| US5565333A (en) | Plasmid replication origin increasing the copy number of the plasmid containing the said origin | |
| WO1994020639A9 (en) | Over expession of single-stranded molecules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| PA0109 | Patent application |
Patent event code: PA01091R01D Comment text: Patent Application Patent event date: 20011025 |
|
| PA0201 | Request for examination | ||
| PG1501 | Laying open of application | ||
| E902 | Notification of reason for refusal | ||
| PE0902 | Notice of grounds for rejection |
Comment text: Notification of reason for refusal Patent event date: 20040228 Patent event code: PE09021S01D |
|
| E701 | Decision to grant or registration of patent right | ||
| PE0701 | Decision of registration |
Patent event code: PE07011S01D Comment text: Decision to Grant Registration Patent event date: 20040907 |
|
| GRNT | Written decision to grant | ||
| PR0701 | Registration of establishment |
Comment text: Registration of Establishment Patent event date: 20041011 Patent event code: PR07011E01D |
|
| PR1002 | Payment of registration fee |
Payment date: 20041011 End annual number: 3 Start annual number: 1 |
|
| PG1601 | Publication of registration | ||
| PR1001 | Payment of annual fee |
Payment date: 20070821 Start annual number: 4 End annual number: 4 |
|
| PR1001 | Payment of annual fee |
Payment date: 20080828 Start annual number: 5 End annual number: 5 |
|
| PR1001 | Payment of annual fee |
Payment date: 20091009 Start annual number: 6 End annual number: 6 |
|
| PR1001 | Payment of annual fee |
Payment date: 20101006 Start annual number: 7 End annual number: 7 |
|
| PR1001 | Payment of annual fee |
Payment date: 20110930 Start annual number: 8 End annual number: 8 |
|
| FPAY | Annual fee payment |
Payment date: 20120828 Year of fee payment: 9 |
|
| PR1001 | Payment of annual fee |
Payment date: 20120828 Start annual number: 9 End annual number: 9 |
|
| FPAY | Annual fee payment |
Payment date: 20130930 Year of fee payment: 10 |
|
| PR1001 | Payment of annual fee |
Payment date: 20130930 Start annual number: 10 End annual number: 10 |
|
| LAPS | Lapse due to unpaid annual fee | ||
| PC1903 | Unpaid annual fee |
Termination category: Default of registration fee Termination date: 20150909 |