KR20130056542A - 말에서 플렉트린 상동성과 에프와이브이이 도메인을 가지는 신규 유전자 - Google Patents
말에서 플렉트린 상동성과 에프와이브이이 도메인을 가지는 신규 유전자 Download PDFInfo
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- KR20130056542A KR20130056542A KR1020110122196A KR20110122196A KR20130056542A KR 20130056542 A KR20130056542 A KR 20130056542A KR 1020110122196 A KR1020110122196 A KR 1020110122196A KR 20110122196 A KR20110122196 A KR 20110122196A KR 20130056542 A KR20130056542 A KR 20130056542A
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- protein
- arg
- nucleotide sequence
- nucleic acid
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Abstract
본 발명은 말에서 pleckstrin homology(PH)와 FYVE 도메인을 가지는 lysosome-associated apoptosis-inducing protein(LAPF)와 상동을 가지는 신규 유전자에 관한 것으로 경주마의 혈액과 근육 조직으로부터 RNA를 추출하여 RNA-seq기법을 이용하여 말 조직에서 발현하는 Fz9과 상동성을 가지는 신규 유전자를 동정하였다. 그 염기서열 분석 및 생물정보학 분석을 통하여 이 유전자가 말에서 LPAF 와 상동성을 가지는 신규 단백질을 코딩함을 밝혔다.
Description
본 발명은 말에서 pleckstrin homology(PH)와 FYVE 도메인을 가지는 Lysosome-associated apoptosis-inducing protein(LAPF)와 상동성을 가지는 신규 유전자에 관한 것으로 경주마의 혈액과 근육 조직으로부터 RNA를 추출하여 RNA-seq 기법을 이용하여 말 조직에서 발현하는 Fz9과 상동성을 가지는 신규 유전자를 동정하였다. 그 염기서열 분석 및 생물정보학 분석을 통하여 이 유전자가 말에서 LPAF와 상동성을 가지는 신규 단백질을 코딩함을 밝혔다.
LAPF는 PH(pleckstrin homoloy) 도메인과 FYVE 도메인을 동시에 가지는 단백질로서 L929세포에 과발현시켰을 때 세포 사멸을 유도하며 라이소좀으로 이동하여 TNF-alpha에 유도되는 세포사멸을 촉진한다. 그리고 상기 두 도메인에 유전자 돌연변이를 유도하면 라이소좀으로의 이동과 세포사멸이 저해된다. 이러한 세포사멸과정은 라이소좀-미토콘드리아 의존적 신호전달 경로를 통해서 일어난다고 알려져 있다(Chen W, Li N, Chen T, Han Y et al., 2005). 세포사멸을 유도하기 위해 LAPF와 인산화된 종양억제인자 p53(ser15/18)과 결합이 필요하며, 이 결합은 LAPF의 PH 도메인과 p53 전사 조절부위 사이에서 일어난다(Li N, Zheng Y, Chen W et al., 2007).
PH와 FYVE 도메인을 가지는 LAPF 단백질은 성장과 사멸을 조절하는 데 중요한 역할을 하는 신호전달 단백질로서 말에서는 보고된 바가 없으며, 또한 이 단백질에 의한 신호전달반응에 대한 정보는 아직 알려진 바가 없다.
PH와 FVYE 도메인에 관한 참고기술로는, 인간의 PH와 FVYE 도메인을 포함하는 유전자에 관한 기술(제 10-2009-0052521 호, 제 10-2010-0058420 호)이 있으나, 이 기술들은 인간의 PH와 FVYE 도메인을 포함하는 유전자에 관련된 기술로, 말에서의 PH와 FVYE 도메인 관련 유전자에 관한 기술은 전무한 실정이다.
본 발명은 상기 문제점을 해결하고, 상기 필요성에 의해 말의 PH와 FYVE 도메인을 가지는 LAPF 단백질 유전자를 대량 RNA-seq 기법을 이용하여 동정하여LAPF 단백질과 상동한 구조를 가지는 단백질의 구조를 밝혀 이 단백질의 기능 규명에 기여하고자 한다.
상기와 같은 기술적 과제를 달성하기 위한 본 발명은 말에서 PH와 FYVE 도메인을 가지는 신규 유전자와 관련된 기술을 제공하고자 한다.
첫째, 말의 혈액 또는 근육으로부터 분리된, 서열번호 1에 기재된 뉴클레오티드 서열로 이루어진 말의 신규 유전자를 제공한다.
둘째, 서열번호 1에 기재된 뉴클레오티드 서열을 포함하는 핵산 분자로 이루어진 군으로부터 선택되는 PH와 FYVE 도메인을 가지는 단백질을 코딩하는 핵산 분자를 제공한다.
셋째, 도 3에 기재된 아미노산 서열과 95% 이상의 상동성이 있는 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 단리된 핵산분자 또는 그의 상보체를 제공한다.
넷째, 서열번호 2에 기재된 아미노산 서열로 이루어지는 말의 PH와 FYVE 도메인을 가지는 단백질을 형성하는 폴리펩티드 또는 그의 일부를 제공한다.
다섯째, 상기 폴리펩티드 또는 그의 일부로부터 구성되는 말의 PH와 FYVE 도메인을 가지는 신규 단백질을 제공한다.
PH와 PYVE 도메인을 가지는 단백질의 구조 및 신호전달 능력을 규명하는데 기여함으로써 PH와 PYVE 도메인을 가지는 단백질에 의한 성장, 경주능력, 번식능력 및 면역조절능력 조절을 통한 말의 운동능력 향상에 기여할 수 있을 것이다.
도 1은, 말에서 PH와 FYVE 도메인을 가지는 단백질을 코딩하는 염기서열을 나타낸다.
도 2는, 말에서 PH와 FYVE 도메인을 가지는 유전자의 염기서열로부터 아미노산 서열의 번역과정을 보여준다.
도 3은, 말에서 PH와 FYVE 도메인을 가지는 단백질과 상동성을 보이는 단백질들 간의 상동성 분석 결과를 보여준다.
도 2는, 말에서 PH와 FYVE 도메인을 가지는 유전자의 염기서열로부터 아미노산 서열의 번역과정을 보여준다.
도 3은, 말에서 PH와 FYVE 도메인을 가지는 단백질과 상동성을 보이는 단백질들 간의 상동성 분석 결과를 보여준다.
실시예 1 : 경주마 샘플로부터 RNA-seq 분석
(1) RNA prep
6두의 경주마로부터 50ml의 혈액을 주사기로 채혈하여 항응고제가 들어있는 50 ml 튜브에 RNA 추출 전까지 보관하였다. 근육 조직은 삼두근으로부터 채취하였다. RNA는 Trizol(Invitrogen)과 RNeasy RNA 추출 키트(Qigen)을 이용하여 분리하였다. RNA정량은 BioAnalyzer 2000을 이용하여 실시하였다.
(2) cDNA
mRNA sequencing 라이브러리는 mRNA는 oligo-dT 비드를 이용하여 분리하였고, 분리된 mRNA를 조각내었고, random primed reverse transcription 반응에 이어 second strand 합성을 실시하였다. Double-stranded short cDNA 단편은 QiaQuick PCR 분리키트(Qiagen)로 분리하였다.
(3) Sequencing library and Sequencing
각 cDNA의 양 말단은 exonuclease를 처리하여 fill-in/blunt 반응을 실시하였다. 이어 각 blunt cDNA의 양 말단에 A-염기를 첨가하였고 이를 sequencing adaptor와 결합시켰다. Adaptor와 결합된 cDNA를 아가로즈 전기영동 후, QIAquick 젤 분리 키트(Qiagen)로 분리한 다음 농축하였다. 라이브러리의 정량과 정성분석은 각각 Qubit(BioRad)와 BioAnalyzer 2100를 이용하여 실시하였다. 라이브러리에 대한 염기서열 분석은 Illumina HiSeq2000를 이용하여 실시하였다.
[표 1] 경주마 6두로부터 획득한 transcriptome 서열과 reference genome에 대한 mapping rates에 대한 통계
실시예 2 : 생물정보학 분석
분석된 염기서열을 Blast 서버(NCBI, NIH, USA)를 이용하여 이미 발표된 말의 게놈 염기서열 및 유전자와 상동성 분석을 실시하여 기존 데이터베이스에 존재하지 않은 미지의 염기서열을 찾았다(도1). 이 염기서열을 아미노산으로 번역한 다음 ExPaSy 서버에 있는 Prosite 프로그램을 이용하여 추정된 단백질의 기능적 도메인을 스캔하였다. 다른 포유류에 존재하는 단백질과의 상동성 분석을 위하여 BlastP 프로그램을 이용한 분석으로부터 상동 단백질들을 찾아내어(말 ZFP658은XP_001917682.1, 사람 ZFP658은 NP_149350.3, 사람 ZFP850은 NP_001180481.1, 사람 ZFP836은 NP_001096127.1.) Clustal W 프로그램으로 상동성 분석을 실시하였다.
<110> Hankyong Industry Academic Cooperation Center
<120> A Horese Novel Gene containing PH(pleckstrin homology) and FYVE
domains
<130> P11505MG
<160> 2
<170> KoPatentIn 2.0
<210> 1
<211> 905
<212> DNA
<213> Equus caballus
<400> 1
ccgaggagat caggagagcc cccggccggg ctgcggcgcg cgagcccgaa ggcttcccgc 60
ccgccacctc cgagctgccc cggccgccgc gtcgaggtgg cctccgggct cgggcgggtt 120
gggcgcgcag ccagggctgt ctcagactct ggccccgagc agcggcgcgg agaagggcgc 180
ttgttgctgg ctggcggggt ggcggctgca cgggctccag gagagcgggt gagaacagcc 240
cagctgccgg agaacatggt ggactacctg gcaaacacgg agatcaacag ccagcgcatt 300
gctgccgttg agagctgctt cggggcttcg gggcagccgc tggccctgcc aggccgggtg 360
ctgctgggcg agggcgtgct gaccaaggag tgccgcaaga aggccaagcc gcgcatcttc 420
ttcctcttca acgacatcct ggtctacggc agcatcgtgc tcaacaagcg caggtaccgc 480
agccagcatg tcatccccct ggaggaggtg acgctggagc cgctgcccga gaccctgcag 540
gccaagaacc gctggatgat caagacggcc aagaagtcct tcgtggtgtc ggccgcctcc 600
accacggagc gccaggagtg gatcagccac attgaggagt gtgtgcggcg gcagctgctg 660
gccacaggcc gccagcccag cacggagcac gcggcgccct ggatcccgga caaggccacg 720
gacatctgca tgcggtgcac gcagacgcgc ttctcggcgc tcacgcggcg ccaccactgc 780
cgcaagtgtg gctttgtggt ctgtgccgag tgctcccgcg agcgcttcct cctgcctcgc 840
ctcgcgccca agcccctgcg cgtctgcaag cctctgctac cgtccagctg gtcgcccaat 900
aactg 905
<210> 2
<211> 301
<212> PRT
<213> Equus caballus
<400> 2
Pro Arg Arg Ser Gly Glu Pro Pro Ala Gly Leu Arg Arg Ala Ser Pro
1 5 10 15
Lys Ala Ser Arg Pro Pro Pro Pro Ser Cys Pro Gly Arg Arg Val Glu
20 25 30
Val Ala Ser Gly Leu Gly Arg Val Gly Arg Ala Ala Arg Ala Val Ser
35 40 45
Asp Ser Gly Pro Glu Gln Arg Arg Gly Glu Gly Arg Leu Leu Leu Ala
50 55 60
Gly Gly Val Ala Ala Ala Arg Ala Pro Gly Glu Arg Val Arg Thr Ala
65 70 75 80
Gln Leu Pro Glu Asn Met Val Asp Tyr Leu Ala Asn Thr Glu Ile Asn
85 90 95
Ser Gln Arg Ile Ala Ala Val Glu Ser Cys Phe Gly Ala Ser Gly Gln
100 105 110
Pro Leu Ala Leu Pro Gly Arg Val Leu Leu Gly Glu Gly Val Leu Thr
115 120 125
Lys Glu Cys Arg Lys Lys Ala Lys Pro Arg Ile Phe Phe Leu Phe Asn
130 135 140
Asp Ile Leu Val Tyr Gly Ser Ile Val Leu Asn Lys Arg Arg Tyr Arg
145 150 155 160
Ser Gln His Val Ile Pro Leu Glu Glu Val Thr Leu Glu Pro Leu Pro
165 170 175
Glu Thr Leu Gln Ala Lys Asn Arg Trp Met Ile Lys Thr Ala Lys Lys
180 185 190
Ser Phe Val Val Ser Ala Ala Ser Thr Thr Glu Arg Gln Glu Trp Ile
195 200 205
Ser His Ile Glu Glu Cys Val Arg Arg Gln Leu Leu Ala Thr Gly Arg
210 215 220
Gln Pro Ser Thr Glu His Ala Ala Pro Trp Ile Pro Asp Lys Ala Thr
225 230 235 240
Asp Ile Cys Met Arg Cys Thr Gln Thr Arg Phe Ser Ala Leu Thr Arg
245 250 255
Arg His His Cys Arg Lys Cys Gly Phe Val Val Cys Ala Glu Cys Ser
260 265 270
Arg Glu Arg Phe Leu Leu Pro Arg Leu Ala Pro Lys Pro Leu Arg Val
275 280 285
Cys Lys Pro Leu Leu Pro Ser Ser Trp Ser Pro Asn Asn
290 295 300
Claims (5)
- 말의 혈액 또는 근육으로부터 분리된, 서열번호 1에 기재된 뉴클레오티드 서열로 이루어진 말의 신규 유전자.
- 서열번호 1에 기재된 뉴클레오티드 서열을 포함하는 핵산 분자로 이루어진 군으로부터 선택되는 PH와 FYVE 도메인을 가지는 단백질을 코딩하는 핵산 분자.
- 제 2항에 있어서, 포유류의 PH와 FYVE 도메인을 가지는 단백질의 아미노산 서열과 95% 이상의 상동성이 있는 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 단리된 핵산분자.
- 서열번호 2에 기재된 아미노산 서열로 이루어지는 말의 PH와 FYVE 도메인을 가지는 단백질을 형성하는 폴리펩티드 또는 그의 일부.
- 상기 폴리펩티드 또는 그의 일부로부터 구성되는 말의 PH와 FYVE 도메인을 가지는 신규 단백질.
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| KR1020110122196A KR20130056542A (ko) | 2011-11-22 | 2011-11-22 | 말에서 플렉트린 상동성과 에프와이브이이 도메인을 가지는 신규 유전자 |
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