CN117384906B - 一种来源于aanat基因的启动子元件及其应用 - Google Patents
一种来源于aanat基因的启动子元件及其应用 Download PDFInfo
- Publication number
- CN117384906B CN117384906B CN202311676378.9A CN202311676378A CN117384906B CN 117384906 B CN117384906 B CN 117384906B CN 202311676378 A CN202311676378 A CN 202311676378A CN 117384906 B CN117384906 B CN 117384906B
- Authority
- CN
- China
- Prior art keywords
- promoter element
- aanat
- promoter
- gene
- 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.)
- Active
Links
- 101150071927 AANAT gene Proteins 0.000 title claims description 23
- 230000014509 gene expression Effects 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 241000283707 Capra Species 0.000 claims description 29
- 210000004027 cell Anatomy 0.000 claims description 28
- 239000013604 expression vector Substances 0.000 claims description 14
- 230000001105 regulatory effect Effects 0.000 claims description 12
- 108700009124 Transcription Initiation Site Proteins 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 230000002103 transcriptional effect Effects 0.000 claims description 7
- 210000004962 mammalian cell Anatomy 0.000 claims description 6
- 108091026890 Coding region Proteins 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 abstract description 13
- 108091062157 Cis-regulatory element Proteins 0.000 abstract description 2
- 210000002950 fibroblast Anatomy 0.000 description 15
- 102100030547 Serotonin N-acetyltransferase Human genes 0.000 description 14
- 239000012634 fragment Substances 0.000 description 12
- 210000001161 mammalian embryo Anatomy 0.000 description 12
- 101000652292 Homo sapiens Serotonin N-acetyltransferase Proteins 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 9
- YJPIGAIKUZMOQA-UHFFFAOYSA-N Melatonin Natural products COC1=CC=C2N(C(C)=O)C=C(CCN)C2=C1 YJPIGAIKUZMOQA-UHFFFAOYSA-N 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 108060001084 Luciferase Proteins 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 6
- 229960003987 melatonin Drugs 0.000 description 6
- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 description 6
- 238000007480 sanger sequencing Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 150000007523 nucleic acids Chemical group 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000013519 translation Methods 0.000 description 5
- 108010074515 Arylalkylamine N-Acetyltransferase Proteins 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 108091092724 Noncoding DNA Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 108020003589 5' Untranslated Regions Proteins 0.000 description 2
- 102000012431 Acetylserotonin O-Methyltransferase Human genes 0.000 description 2
- 108010022539 Acetylserotonin O-methyltransferase Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108090000331 Firefly luciferases Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 2
- 235000011613 Pinus brutia Nutrition 0.000 description 2
- 241000018646 Pinus brutia Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000011013 endotoxin removal Methods 0.000 description 2
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 229940076279 serotonin Drugs 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- JTEJPPKMYBDEMY-UHFFFAOYSA-N 5-Methoxytryptamine Natural products COC1=CC=C2NC=C(CCN)C2=C1 JTEJPPKMYBDEMY-UHFFFAOYSA-N 0.000 description 1
- BNRWXKGBIMZFLK-UHFFFAOYSA-N 5-methoxytryptamine Chemical compound [CH]1C(OC)=CC=C2N=CC(CCN)=C21 BNRWXKGBIMZFLK-UHFFFAOYSA-N 0.000 description 1
- 229940097276 5-methoxytryptamine Drugs 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- MVAWJSIDNICKHF-UHFFFAOYSA-N N-acetylserotonin Chemical compound C1=C(O)C=C2C(CCNC(=O)C)=CNC2=C1 MVAWJSIDNICKHF-UHFFFAOYSA-N 0.000 description 1
- 241000269978 Pleuronectiformes Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241001493546 Suina Species 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical class IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000027288 circadian rhythm Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000003468 luciferase reporter gene assay Methods 0.000 description 1
- 238000007886 magnetic bead extraction Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000005495 thyroid hormone Substances 0.000 description 1
- 229940036555 thyroid hormone Drugs 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- 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
- C12N15/67—General methods for enhancing the expression
-
- 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
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01087—Aralkylamine N-acetyltransferase (2.3.1.87)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本公开提供了一种启动子元件及其应用,所述启动子元件包括如SEQ ID NO:19所示的核苷酸序列,或与其具有85%序列同一性的序列的至少一种或多种。本公开所述启动子元件能够作为表达目的基因的顺式作用元件,增加靶蛋白基因的表达效果,具有较广泛的应用前景和应用潜力。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种启动子元件及其应用。
背景技术
在哺乳动物中,AANAT基因编码的芳基烷基胺-N-乙酰基转移酶是一种重要的胞质酶,也是褪黑素合成的限速酶。褪黑素的体内合成分多个反应步骤,血清素在合成限速酶AANAT催化下生成N-乙酰血清素,而后在另外一个限速酶HIOMT的催化下发生甲基化,最终生成褪黑素。另一个途径则为,血清素先接受HIOMT催化生成5-甲氧基色胺(Methoxytryptamine),而后再由AANAT催化合成褪黑素。而在不同的物种中合成的通路可能不尽相同。在前人的研究中发现比目鱼松果体中的AANAT基因能够影响甲状腺激素分泌,与发育存在一定关系。AANAT的活性与MLT的分泌在节律上保持一致,AANAT基因通过调控褪黑素的合成和分泌量来参与调控哺乳动物的昼夜节律以及繁殖性能。研究发现AANAT基因的G619A突变与人的睡眠时相延迟综合症有关。
目前海南黑山羊的AANAT基因在NCBI和ensmbl数据库中序列还不完全,尤其是核心启动子区域仍未可知,进而影响了对海南黑山羊褪黑素分泌研究的发展。
发明内容
为了解决上述至少一个问题,本公开提供了一种启动子元件及其应用。
根据本公开的第一方面,提供了一种启动子元件,该启动子元件包括如SEQ IDNO:19所示的核苷酸序列,或与其具有85%序列同一性的序列中的至少一种或多种。
在一些实施方式中,所述启动子元件来源于哺乳动物。
在一些具体的实施方式中,所述哺乳动物为偶蹄目动物。
在一些具体的实施方式中,所述哺乳动物为黑山羊。
在一些具体的实施方式中,所述哺乳动物为海南黑山羊。
在一些实施方式中,所述启动子元件来源于海南黑山羊AANAT基因转录起始位点上游的-540bp~-958bp。
根据本公开的第二方面,提供了一种转录表达盒,所述转录表达盒包含第一方面所述启动子元件和靶蛋白编码序列,其中所述启动子元件可操纵连接所述靶蛋白编码序列。
在一些实施方式中,所述靶蛋白包括芳基烷基胺-N-乙酰基转移酶、羟基吲哚一氧一甲基转移酶或荧火虫荧光素酶。
在一些实施方式中,所述靶蛋白为芳基烷基胺-N-乙酰基转移酶。
在一些实施方式中,所述靶蛋白为海南黑山羊的芳基烷基胺-N-乙酰基转移酶。
根据本公开的第三方面,提供了一种表达载体,所述表达载体包含第一方面所述启动子元件或第二方面所述转录表达盒。
在一些实施方式中,所述表达载体选自原核表达载体、真核表达载体或病毒表达载体中的一种或多种。
根据本公开的第四方面,提供了一种宿主细胞,所述宿主细胞携带第一方面所述的启动子元件或第二方面所述转录表达盒或第三方面所述的表达载体中的一种或多种。
在一些实施方式中,所述宿主细胞选自原核细胞和真核细胞。
在一些具体的实施方式中,所述原核细胞包括细菌细胞、大肠杆菌、链霉菌。
在一些具体的实施方式中,所述真核细胞包括酵母细胞、哺乳动物细胞、昆虫细胞。
在一些具体的实施方式中,所述哺乳动物细胞包括人、猴、小鼠、大鼠、仓鼠、山羊、绵羊、牛、猪、犬、猫。
在一些具体的实施方式中,所述真核细胞包括人类细胞、小鼠细胞、大鼠细胞、仓鼠细胞、山羊细胞、绵羊细胞或任何其它宿主细胞。
在一些实施方式中,所述哺乳动物细胞包括A549,WEHI,3T3,10T1/2,BHK,MDCK,COS1,COS7,BSC1,BSC40,BMT10,VERO,WI38,HeLa,293细胞(可以表达功能性的腺病毒E1),Saos,C2C12,L细胞,HT1080,HepG2。
在一些实施方式中,所述哺乳动物细胞包括成纤维细胞、肝细胞和成肌细胞。
在一些实施方式中,所述成纤维细胞选自胚胎成纤维细胞。
在一些实施方式中,所述成纤维细胞选自山羊胚胎成纤维细胞。
根据本公开的第五方面,提供了第一方面所述启动子元件或第二方面所述转录表达盒在调控基因表达中的应用。
本公开成功利用双荧光素酶报告基因***鉴定海南黑山羊AANAT启动子核心区域,筛选得到一种启动子元件,本公开所述启动子元件能够作为表达目的基因的顺式作用元件,增加靶蛋白基因的表达效果,具有较广泛的应用前景和应用潜力。
附图说明
图1示出了AANAT基因5' RACE片段Sanger测序序列峰图第一部分。
图2示出了AANAT基因5' RACE片段Sanger测序序列峰图第二部分。
图3示出了AANAT基因3'RACE片段Sanger测序序列峰图第一部分。
图4示出了AANAT基因3' RACE片段Sanger测序序列峰图第二部分。
图5示出了AANAT基因从转录起始位点到转录终止位点的核苷酸序列。其中上端红色部分表示5'非编码区,下端红色部分表示3'非编码区,中间黄色部分表示蛋白质编码区。
图6示出了AANAT启动子区域电泳图。
图7示出了荧光素酶活性检测结果。
具体实施方式
为使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步的详细说明。此处所描述的具体实施例仅用于解释本发明,并不用于构成对本发明的任何限制。此外,在以下说明中,省略了对公知结构和技术的描述,以避免不必要地混淆本公开的概念。这样的结构和技术在许多出版物中也进行了描述。
定义
除非另有定义,否则本发明使用的所有技术术语和科技术语都具有如在本发明所属领域中通常使用的相同含义。出于解释本说明书的目的,将应用以下定义,并且在适当时,以单数形式使用的术语也将包括复数形式,反之亦然。
除非上下文另有明确说明,否则本文所用的表述“一种”和“一个”包括复数指代。
本文所使用的术语“启动子”或“启动子元件”在本文定义为这样的DNA序列,其结合RNA聚合酶并指导该聚合酶到达多核苷酸的正确下游转录起始位点以起始转录,所述多核苷酸编码具有生物学活性的多肽。RNA聚合酶有效地催化与编码区的适当DNA链互补的信使RNA装配。术语“启动子”或“启动子元件”还应理解为包括用于在转录成mRNA之后的翻译的5′非编码区(启动子和翻译起点之间),顺式作用转录调控元件例如增强子,和/或其它能够与转录因子相互作用的核苷酸序列。启动子或启动子元件可以是野生型启动子、变体启动子、杂合启动子体或共有启动子(consensus promoter)。
本文所使用的术语“核心启动子”指的是由启动子所包含的核酸序列。核心启动子通常是正确启动转录所需的启动子的最小部分。核心启动子通常包括转录起始位点和RNA聚合酶的结合位点。
本文所使用的术语“序列同一性”是指两个多核苷酸之间的“序列同一性百分比”或“同一性百分比”,即在考虑到了为了两个序列的最佳比对而必须引入的添加或缺失(即,空位)的情况下,比较窗口内序列所共有的相同匹配位置的数量。匹配位置是其中在靶序列和参考序列中都存在相同核苷酸的任何位置。由于空位不是核苷酸,所以靶序列中存在的空位不计在内。同样,由于计入靶序列核苷酸,而不计来自参考序列的核苷酸,所以不计参考序列中存在的空位。至少85%序列同一性包括所述序列全长的至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%或100%具有序列同一性的连续片段。
本文所使用的术语“表达载体”,在将上述分离的核酸分子连接到载体上时,可以将核酸序列与载体上的调控元件直接或者间接相连,只要这些调控元件能够调控该核酸分子的翻译和表达等即可。当然这些调控元件可以直接来自于载体本身,也可以是外源性的,即并非来自于载体本身。也就是说,核酸分子与调控元件可操作地连接。常用的表达载体例如原核表达载体、真核表达载体或病毒表达载体等等。
本文所使用的术语“可操作地连接”是指将外源基因连接到载体上,使得载体内的调控元件,例如转录调控序列和翻译调控序列等等,能够发挥其预期的调节外源基因的转录和翻译的功能。
下面提供实施例和附图以帮助理解本发明。但应理解,这些实施例和附图仅用于说明本发明,但不构成任何限制。本发明的实际保护范围在权利要求书中进行阐述。应理解,在不脱离本发明精神的情况下,可以进行任何修改和改变。
实施例
实施例1.海南黑山羊AANAT基因的RACE克隆
步骤1:对牛(Genbank No:NM_177509.2)、绵羊(Genbank No:NM_001009461.1)、鼠(Genbank No:NM_001009461.1)和山羊(Genbank No:XM_018064062.1)不同物种的AANAT氨基酸序列进行序列对比,针对蛋白质编码区中的保守序列设计2对引物,引物序列见表1。
步骤2:以海南黑山羊松果体组织提取到的总RNA为模版,使用表1所示的2对引物反转录成cDNA第一链作为模板,进行RACE克隆,PCR扩增后得到AANAT基因的3'和5'端序列片段。
将所获得的片段连接至pCE2-TA/Blunt-Zero载体(诺唯赞)并进行第一代测序(Sanger测序)。AANAT基因5'RACE片段Sanger测序峰图见图1和图2,AANAT基因3'RACE片段Sanger测序峰图见图3和图4。
将所得的3'和5'末端RACE片段分别去载体序列后,进行拼接,获得海南黑山羊AANAT基因全长序列(SEQ ID NO:13)。图5示出了山羊AANAT基因全长序列,其中5'UTR区域(图5上端红色标注)如SEQ ID NO:14所示序列,蛋白质编码区(图5中黄色标注)如SEQ IDNO:15所示序列,其中3'UTR区域(图5下端红色标注)如SEQ ID NO:16所示序列。
实施例2.使用双荧光素酶报告基因鉴定山羊AANAT启动子活性
1.双荧光素酶报告基因载体的构建
将实施例1测序得到的AANAT基因5'UTR序列与Ensembl数据库中的序列(chr19:54512968~chr19:54512546)进行比对,以mRNA链第一个核苷酸对应DNA链上的碱基为转录起始位点,并以转录起始位点上游2000bp以内的区域作为启动子区域设计引物,引物序列如表2所示。
使用表2所示引物扩增出的四种长度不等的扩增产物:AANAT-P0(SEQ ID NO:17)、AANAT-P1(SEQ ID NO:18)、AANAT-P2(SEQ ID NO:19)和AANAT-P3(SEQ ID NO:20),对扩增产物进行电泳鉴定产物条带长度,所述电泳图见图6。
取4周龄大山羊胎儿,取其部分皮肤组织剪碎后使用0.25%胰蛋白酶消化成单细胞悬液,使用含有10%胎牛血清的DMEM高糖培养基进行培养,每隔3天更换新鲜的培养基,直至培养细胞汇合度60%至70%,获得山羊胚胎成纤维细胞。使用诺唯赞的磁珠提取DNA试剂盒(VAMNE Magnetic Pathogen DNA Kit)对通过上述方法获得的山羊胚胎成纤维细胞进行DNA的提取,并扩增以上AANAT启动子片段,PCR扩增的体系每50μL含有2×Phanta FlashMaster Mix(Dye Plus) 25μL、10μM上游引物2μL、10μM下游引物2μL、200ng/μL模板1μL和胚胎水20μL。PCR扩增的程序为:98℃预变性30秒;98℃变性10秒;65℃退火5秒;72℃延伸10秒,30个循环;72℃终延伸1分钟。并使用诺唯赞的胶回收DNA试剂盒(FastPure Gel DNAExtraction Mini Kit)对其进行产物回收。
分别使用KpnI和Hind III限制性内切酶(TAKARA)双酶切处理pGL3-Basic质粒和扩增后的AANAT启动子片段,并使用T4连接酶(TAKARA)进行连接,连接的体系每10μL含有双酶切AANAT基因启动子7μL、双酶切pGL3-Basic质粒1μL、T4连接酶缓冲液1μL和T4连接酶1μL;所述连接的条件包括:16℃,4小时。连接产物转化至大肠杆菌DH5α感受态细胞(TAKARA),转化步骤按照大肠杆菌DH5α感受态细胞说明书进行,转化后挑取阳性克隆菌落送至上海生工生物工程公司进行第一代测序(Sanger测序),测序正确的重组蛋白表达载体分别命名为pGL3-AANAT-P0、pGL3-AANAT-P1、pGL3-AANAT-P2、pGL3-AANAT-P3。
2.AANAT基因核心启动子的鉴定
对测序正确的质粒菌液使用诺唯赞的质粒去内毒素小提试剂盒(FastPureEndoFree Plasmid Mini Kit)进行去内毒素质粒的提取。
对山羊胚胎成纤维细胞进行37℃解冻复苏,使用含有10%胎牛血清的DMEM高糖培养基进行培养,细胞汇合至70%至80%后传代,弃去原培养基,使用无菌PBS缓冲液冲洗两遍后,放置到37℃培养箱中经0.25%EDTA的胰蛋白酶消化,消化时间约2分钟取出后,加入20%的胎牛血清DMEM培养基中和胰蛋白酶,吸管吹打,使细胞脱落。按照比例为1:3接种于细胞培养板6孔板每孔,加入5mL,10%的胎牛血清DMEM培养基,37℃,5%CO2培养箱中培养。每3天可以进行一次细胞传代,按照1:3的比例传代。
成纤维细胞生长至70~90%汇合度时转染,使用Opti-MEM培养基稀释Lipofectamine™3000试剂(赛默飞)并充分混匀,再使用Opti-MEM培养基稀释去内毒素质粒,制备质粒预混液,实验组为pGL3-AANAT-P0、pGL3-AANAT-P1、pGL3-AANAT-P2、pGL3-AANAT-P3四种质粒分别与pRL-TK质粒共转染,阴性对照为pGL3-Basic和pRL-TK两种空质粒共转染,向管中添加P3000试剂并充分混匀。在每管已稀释的Lipofectamine™ 3000试剂中加入稀释的DNA(1:1比例)室孵育5分钟,加入DNA-脂质体复合物至细胞中,37℃孵育细胞2至4天,然后分析转染细胞。
3.荧光素酶活性鉴定
收集转染后的细胞,使用诺唯赞的双荧光素酶报告基因检测试剂盒(Duo-LiteLuciferase Assay System)对转染后细胞进行荧光素酶检测。其中萤火虫荧光素酶催化产生的荧光值以F表示,海肾荧光素酶催化产生的荧光值以R表示。以未转染的细胞作为空白对照(即背景F和背景R),用以背景扣除;转染细胞经实验化合物处理作为实验组细胞(即实验组F和实验组R),转染细胞不经处理,用以标准化结果(即对照组F和对照组R),
通过酶标仪测定实验结果发现,实验组pGL3-AANAT-P0相对活性为12.5376±1.21;pGL3-AANAT-P1相对活性为15.34±1.432;pGL3-AANAT-P2相对活性为35.1233±2.331;pGL3-AANAT-P3相对活性为17.322±1.306,对照组的荧光素酶相对活性值为0.12147±1.342。结果见图7。从图7的结果可以看出实验组pGL3-AANAT-P2的荧光素酶相对活性均显著高于对照组(P<0.05),由此可以判定AANAT启动子上游540bp至958bp为启动子核心区域,具有明显的转录起始的效果。
本发明的技术方案不限于上述具体实施例的限制,凡是根据本发明的技术方案做出的技术变形,均落入本发明的保护范围之内。
Claims (6)
1.一种启动子元件,其特征在于:
所述启动子元件的核苷酸序列如SEQ ID NO:19所示,所述启动子元件来源于海南黑山羊。
2.根据权利要求1所述启动子元件,其特征在于,所述启动子元件来源于海南黑山羊AANAT基因转录起始位点上游的-540bp ~ -958bp。
3.一种转录表达盒,其特征在于,所述转录表达盒包含权利要求1所述启动子元件和靶蛋白编码序列,其中所述启动子元件可操纵连接所述靶蛋白编码序列。
4.一种表达载体,其特征在于,所述表达载体包含权利要求1或2所述启动子元件或权利要求3所述转录表达盒。
5.一种宿主细胞,其特征在于,所述宿主细胞携带如权利要求1或2所述的启动子元件或权利要求3所述转录表达盒或携带如权利要求4所述的表达载体中的一种或多种,所述宿主细胞为哺乳动物细胞。
6.权利要求1或2所述启动子元件或权利要求3所述转录表达盒在调控哺乳动物细胞的基因表达中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311676378.9A CN117384906B (zh) | 2023-12-08 | 2023-12-08 | 一种来源于aanat基因的启动子元件及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311676378.9A CN117384906B (zh) | 2023-12-08 | 2023-12-08 | 一种来源于aanat基因的启动子元件及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117384906A CN117384906A (zh) | 2024-01-12 |
CN117384906B true CN117384906B (zh) | 2024-03-12 |
Family
ID=89437673
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311676378.9A Active CN117384906B (zh) | 2023-12-08 | 2023-12-08 | 一种来源于aanat基因的启动子元件及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117384906B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR19990042573A (ko) * | 1997-11-27 | 1999-06-15 | 이황희 | 흑염소의 인슐린 유사 성장인자 1 프로모터 2 |
WO2001087909A2 (en) * | 2000-05-18 | 2001-11-22 | Genaissance Pharmaceuticals, Inc. | Haplotypes of the aanat gene |
CN106852157A (zh) * | 2014-06-16 | 2017-06-13 | 约翰斯·霍普金斯大学 | 用于使用h1启动子表达crispr向导rna的组合物和方法 |
CN113278709A (zh) * | 2021-05-27 | 2021-08-20 | 贵州省种畜禽种质测定中心 | 贵州黑山羊多羔主效基因应用及引物对和试剂盒 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019060993A1 (en) * | 2017-09-28 | 2019-04-04 | The Governors Of The University Of Alberta | PROMOTER OF THE RETINOIC ACID-INDUCIBLE GENE I AND RELATED COMPOSITIONS AND METHODS |
-
2023
- 2023-12-08 CN CN202311676378.9A patent/CN117384906B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR19990042573A (ko) * | 1997-11-27 | 1999-06-15 | 이황희 | 흑염소의 인슐린 유사 성장인자 1 프로모터 2 |
WO2001087909A2 (en) * | 2000-05-18 | 2001-11-22 | Genaissance Pharmaceuticals, Inc. | Haplotypes of the aanat gene |
CN106852157A (zh) * | 2014-06-16 | 2017-06-13 | 约翰斯·霍普金斯大学 | 用于使用h1启动子表达crispr向导rna的组合物和方法 |
CN113278709A (zh) * | 2021-05-27 | 2021-08-20 | 贵州省种畜禽种质测定中心 | 贵州黑山羊多羔主效基因应用及引物对和试剂盒 |
Non-Patent Citations (2)
Title |
---|
Overexpression of ovine AANAT and HIOMT genes in switchgrass leads to improved growth performance and salt-tolerance;Yan-Hua Huang et al.;Sci Rep .;第7卷(第1期);文献号:12212,第1-13页 * |
Peng,O.K. et al..GenBank: KF220307.1.GenBank.2013,ORIGIN. * |
Also Published As
Publication number | Publication date |
---|---|
CN117384906A (zh) | 2024-01-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN115651927B (zh) | 编辑rna的方法和组合物 | |
Simonsen et al. | Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells | |
KR101038126B1 (ko) | 새로운 융합 프로모터 및 이를 포함하는 재조합 벡터 | |
Zhang et al. | Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats | |
CA2835428A1 (en) | Engineered nucleic acids and methods of use thereof for non-human vertebrates | |
US20140065666A1 (en) | CLDN5 Mini-Promoters | |
Wei et al. | Efficient CRISPR/Cas9-mediated gene editing in Guangdong small-ear spotted pig cells using an optimized electrotransfection method | |
CN117384906B (zh) | 一种来源于aanat基因的启动子元件及其应用 | |
US8404486B2 (en) | Recombination sequences | |
CN114990093B (zh) | 氨基酸序列小的蛋白序列mini rfx-cas13d | |
CN101289671B (zh) | 一种转基因动物的制备方法 | |
CN102643816B (zh) | 绵羊角蛋白31皮肤毛囊特异性启动子及其克隆 | |
EP1565562B1 (en) | Sequence specific dna recombination in eukaryotic cells | |
CN104975018B (zh) | 一种新型增强子及其应用 | |
CN102876698A (zh) | 抑制绵羊成纤维细胞生长因子5表达的试剂及其应用 | |
CN112779289A (zh) | 一种人类及哺乳动物细胞表达载体、表达***及其构建方法和应用 | |
CN102643817B (zh) | 绵羊k71皮肤毛囊特异性启动子及其克隆 | |
Zhou et al. | Construction of a recombinant human FGF1 expression vector for mammary gland-specific expression in human breast cancer cells | |
CN108611366A (zh) | 通过miRNA let-7b调控GHR基因表达的方法 | |
Liu et al. | Effect of QSOX1 on cattle carcass traits as well as apoptosis and triglyceride production in bovine fetal fibroblasts and mammary epithelial cells | |
CN114891791B (zh) | 特异性靶向犬Rosa26基因的sgRNA及其应用 | |
CN105695509B (zh) | 一种获得高纯度心肌细胞的方法 | |
Gao et al. | Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo | |
CN106609283A (zh) | 一种利用转座子进行多基因同时操作的方法 | |
MXPA06004121A (es) | Recombinacion mediada por flp. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |