具体实施方式
下面结合附图对本发明的具体实施方式做进一步说明。SEQ ID No.1是 mTET2CDT的编码DNA序列,SEQ ID No.2是mTET2CDTm的编码DNA序列, SEQ ID No.3是mTET2CDT的氨基酸序列,SEQ ID No.4是mTET2CDTm的氨基酸序列。
图1为mTET2、mTET2CDT、MBP-mTET2CDT氧化5mC效率示意图。所提供的实施例仅是对本发明方法的说明,而不以任何方式限制本发明揭示的其余内容。本实施例所使用的探针和引物序列及修饰如表1所示。
表1 探针及引物序列
实施例1:mTET2、mTET2CDT、mTET2CDTm酶比活性的测试。
使用Epigentek的Epigenase 5mC-羟化酶TET活性/抑制分析试剂盒(荧光法)按照说明书的流程来测定mTET2、mTET2CDT、mTET2CDTm将5mC转化成5hmC的酶比活性。
结果见图2,mTET2CDTm具有最高的酶比活性,5hmC产物占比最高, mTET2CDT次之,mTET2最低。
实施例2:mTET2、mTET2CDT、mTET2CDTm对5mC oligo的氧化能力的测定。
在本实施例中,测定了三种mTET2酶对5mC的氧化动力学曲线,具体实施方式如下:
表2
组分 |
用量 |
5mC oligo |
1-100 ng |
10× mTET buffer |
3 Μl |
3 mM Fe(NH4)2(SO4)2 |
1-10 μL |
10 μM mTET2酶突变体 |
2-10 μL |
补ddH<sub>2</sub>O至 |
30 μL |
37℃反应10-60 min。
反应结束后,加入0.3-3 μL 10 mg/mL 蛋白酶K,50℃反应10-30 min。使用磁珠法或者QIAquick Nucleotide Removal Kit (Qiagen)回收DNA进行LC-MS/MS分析。5mC、5hmC、5fC和5caC含量占比分析流程见Hideharu Hashimoto et al(Nature,2013)。
实验结果如图3所示,三种mTET2蛋白及突变体氧化5mC的效率从高到低排序分别是mTET2CDTm> mTET2CDT > mTET2。其中mTET2CDT氧化产物中5caC的占比最高,mTET2CDTm氧化产物中5hmC的占比最高。
实施例3: mTET2重组蛋白及突变体在MAPS-qPCR和MAPS-seq中的应用。
在本实施例中,利用MAPS-qPCR和MAPS-seq来验证mTET2重组蛋白及突变体在DNA甲基化检测应用中的效率。
1)PCR技术构建了DNA甲基化标准品:
表3
组分 |
用量 |
10 μM 5mC-Forward |
12 μL |
10 μM 5mC-Reverse |
12 μL |
10 μM 5mC-Template |
1 μL |
2×Hieff Canace® Gold PCR Master Mix(Yeasen) |
25 μL |
Total |
50 μL |
混匀后按照以下程序进行反应:
表4
PCR结束后,加入40 μL Agencourt AMPure XP beads(Beckman,A63881)混匀后,室温静置孵育5 min。将PCR管置于磁力架上,待溶液澄清后,吸去上清,加入200 μL新鲜配制的80%乙醇静置30 s,吸干净乙醇;再次加入200 μL新鲜配制的80%乙醇静置30 s,吸干净乙醇,室温静置晾干3 min。加入20 μL ddH2O悬浮磁珠后,室温静置5 min。将PCR管置于磁力架上,待溶液澄清后,取上清,Nanodrop测定回收的PCR产物浓度,Qsep测定PCR产物纯度,一代测序测定PCR产物序列。
2)mTET2重组蛋白及突变体处理:
表5
组分 |
用量 |
5mC oligo |
1-100 ng |
10× mTET buffer |
3 Μl |
3 mM Fe(NH4)2(SO4)2 |
1-10 μL |
300 mM 氨硼烷锂 |
1-3 μL |
10 μM mTET2重组蛋白或突变体 |
2-10 μL |
补ddH2O至 |
30 μL |
37℃反应10-60 min。
反应结束后,加入0.3-3 μL 10 mg/mL 蛋白酶K,50℃反应10-30 min。使用磁珠法回收处理后的DNA。
3)过氧钨酸盐处理:
表6
组分 |
用量 |
上述回收的DNA |
13 μL |
10× PBS缓冲液 |
2 μL |
200 mM 过氧钨酸钾 |
5 μL |
补ddH2O至 |
20 μL |
70℃处理3 h。反应结束后,使用磁珠法回收处理后的DNA。
4)qPCR验证mTET2-MAPS检测m5C修饰的效率。
表7
组分 |
用量 |
上述回收的DNA经1000倍稀释后 |
1 μL |
2× Hieff UNICON® qPCR SYBR Green Master Mix |
10 μL |
C-引物或T-引物(10 μM) |
1 μL |
引物R(10 μM) |
1 μL |
PCR阻断探针(10 μM) |
1 μL |
补ddH2O至 |
20 μL |
表8
5)DNA NGS验证mTET2-MAPS检测m5C修饰的效率。
回收的DNA使用翊圣生物的Hieff NGS® Ultima DNA Library Prep Kit forIllumina进行DNA文库构建,构建好的文库使用illumina的NovaSeq 6000进行测序。获得的数据分析5mC转化成T的效率。
MAPS-qPCR和MAPS-seq结果如表9、图4和图5所示,MAPS-qPCR和MAPS-seq能够有效检测DNA的甲基化,其中mTET2CDTm能够有效提高MAPS-qPCR和MAPS-seq对DNA甲基化的检测效率和准确性。
表9
条件 |
引物 |
CT值 |
MAPS- |
C引物 |
13.88 |
MAPS- |
T引物 |
28.35 |
mTET2 MAPS |
C引物 |
25.13 |
mTET2 MAPS |
T引物 |
13.15 |
mTET2CDT MAPS |
C引物 |
24.73 |
mTET2CDT MAPS |
T引物 |
14.26 |
mTET2CDTm MAPS |
C引物 |
26.87 |
mTET2CDTm MAPS |
T引物 |
12.03 |
实施例4:MAPS-seq验证mTET2重组蛋白及突变体对 Control DNA CpGmethylated pUC19甲基化水平的检测效率。
在本实施例中,利用MAPS验证mTET2重组蛋白及突变体对 Control DNA CpGmethylated pUC19甲基化水平的检测效率,具体实施方式见实施例3。
结果如图6所示, mTET2CDTm突变体能够有效提高MAPS-seq在Control DNA CpGmethylated pUC19标准品上DNA甲基化的检测效率。
综上,本发明提供了两种高活性的mTET2突变体mTET2CDT和 mTET2CDTm,显著提高了mTET2的氧化活性。此外,mTET2CDTm还提高了氧化5mC过程中5hmC中间产物的占比。与野生型TET2相比,这两个高活性的TET酶突变体明显提高了DNA甲基化检测灵敏度和准确性,可以应用于疾病诊断,尤其是在肿瘤早筛领域。
序列表
<110> 翌圣生物科技(上海)股份有限公司
<120> 高活性的mTET2酶突变体、其编码DNA及其应用
<141> 2021-07-21
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1341
<212> DNA
<213> Artificial Sequence
<400> 1
atgcagtctc agaacggcaa atgcgagggc tgcaaccctg acaaagatga agcgccgtac 60
tacacccatc tgggtgctgg tccagacgtg gcggctatcc gtaccctgat ggaagagcgt 120
tatggtgaaa aaggtaaagc aatccgcatc gaaaaagtta tctacactgg caaggaaggt 180
aaatcctctc agggttgtcc gatcgcaaaa tgggtttacc gtcgttcttc cgaggaagaa 240
aaactgctgt gcctggttcg cgtgcgtccg aaccatacct gcgaaaccgc cgtgatggtt 300
atcgccatta tgctgtggga tggcatcccg aagctgctgg cttctgaact gtacagcgaa 360
ctgactgata tcctgggtaa atgcggcatt tgtaccaacc gtcgttgcag ccagaacgaa 420
acccgtaact gctgctgtca gggcgaaaac ccggaaacct gtggcgcctc tttcagcttc 480
ggctgctctt ggtctatgta ttacaacggc tgcaaattcg cgcgtagcaa gaaaccgcgt 540
aaattccgtc tgcacggcgc tgaaccgaag gaagaagaac gtctgggtag ccatctgcag 600
aatctggcta ccgttattgc cccgatttac aagaaactgg caccagacgc ctacaacaac 660
caggtagagt tcgaacacca ggccccagac tgttgtctgg gcctgaaaga aggtcgtccg 720
ttctccggcg ttactgcatg tctggacttt tctgcccaca gccaccgtga ccagcagaat 780
atgccgaacg gctctaccgt agtggttacc ctgaaccgtg aagacaaccg tgaagtcggt 840
gcgaaacctg aagacgaaca gttccacgtc ctgcctatgt atattatcgc accggaagat 900
gaattcggtt ctaccgaagg tcaggaaaag aaaattcgta tgggttctat cgaagttctg 960
caaagcttcc gccgtcgtcg tgttatccgt atcggtgaac tgccgaaatc ctgtaaaaaa 1020
ggcggtggtg gtagcgtgtc cggtcaggat gctgcagccg tgcaggagat tgaatactgg 1080
tctgacagcg aacataattt ccaggacccg tgcatcggtg gtgttgcgat cgcaccgact 1140
cacggtagca tcctgattga atgcgctaaa tgcgaagtac acgctactac gaaagtgaac 1200
gacccggatc gcaatcaccc gacccgtatc tccctggttc tgtaccgcca caaaaacctg 1260
ttcctgccga aacactgcct ggcactgtgg gaagcgaaaa tggccgagaa agcgcgcaaa 1320
gaagaggagt gcggcaaaaa c 1341
<210> 2
<211> 1341
<212> DNA
<213> Artificial Sequence
<400> 2
atgcagtctc agaacggcaa atgcgagggc tgcaaccctg acaaagatga agcgccgtac 60
tacacccatc tgggtgctgg tccagacgtg gcggctatcc gtaccctgat ggaagagcgt 120
tatggtgaaa aaggtaaagc aatccgcatc gaaaaagtta tctacactgg caaggaaggt 180
aaatcctctc agggttgtcc gatcgcaaaa tgggtttacc gtcgttcttc cgaggaagaa 240
aaactgctgt gcctggttcg cgtgcgtccg aaccatacct gcgaaaccgc cgtgatggtt 300
atcgccatta tgctgtggga tggcatcccg aagctgctgg cttctgaact gtacagcgaa 360
ctgactgata tcctgggtaa atgcggcatt tgtaccaacc gtcgttgcag ccagaacgaa 420
acccgtaact gctgctgtca gggcgaaaac ccggaaacct gtggcgcctc tttcagcttc 480
ggctgctctt ggtctatgta ttacaacggc tgcaaattcg cgcgtagcaa gaaaccgcgt 540
aaattccgtc tgcacggcgc tgaaccgaag gaagaagaac gtctgggtag ccatctgcag 600
aatctggcta ccgttattgc cccgatttac aagaaactgg caccagacgc ctacaacaac 660
caggtagagt tcgaacacca ggccccagac tgttgtctgg gcctgaaaga aggtcgtccg 720
ttctccggcg ttactgcatg tctggacttt tctgcccaca gccaccgtga ccagcagaat 780
atgccgaacg gctctaccct ggtgtgtacc ctgaaccgtg aagacaaccg tgaagtcggt 840
gcgaaacctg aagacgaaca gttccacgtc ctgcctatgt atattatcgc accggaagat 900
gaattcggtt ctaccgaagg tcaggaaaag aaaattcgta tgggttctat cgaagttctg 960
caaagcttcc gccgtcgtcg tgttatccgt atcggtgaac tgccgaaatc ctgtaaaaaa 1020
ggcggtggtg gtagcgtgtc cggtcaggat gctgcagccg tgcaggagat tgaatactgg 1080
tctgacagcg aacataattt ccaggacccg tgcatcggtg gtgttgcgat cgcaccgact 1140
cacggtagca tcctgattga atgcgctaaa tgcgaagtac acgctactac gaaagtgaac 1200
gacccggatc gcaatcaccc gacccgtatc tccctggttc tgtaccgcca caaaaacctg 1260
ttcctgccga aacactgcct ggcactgtgg gaagcgaaaa tggccgagaa agcgcgcaaa 1320
gaagaggagt gcggcaaaaa c 1341
<210> 3
<211> 447
<212> PRT
<213> Artificial Sequence
<400> 3
Met Gln Ser Gln Asn Gly Lys Cys Glu Gly Cys Asn Pro Asp Lys Asp
1 5 10 15
Glu Ala Pro Tyr Tyr Thr His Leu Gly Ala Gly Pro Asp Val Ala Ala
20 25 30
Ile Arg Thr Leu Met Glu Glu Arg Tyr Gly Glu Lys Gly Lys Ala Ile
35 40 45
Arg Ile Glu Lys Val Ile Tyr Thr Gly Lys Glu Gly Lys Ser Ser Gln
50 55 60
Gly Cys Pro Ile Ala Lys Trp Val Tyr Arg Arg Ser Ser Glu Glu Glu
65 70 75 80
Lys Leu Leu Cys Leu Val Arg Val Arg Pro Asn His Thr Cys Glu Thr
85 90 95
Ala Val Met Val Ile Ala Ile Met Leu Trp Asp Gly Ile Pro Lys Leu
100 105 110
Leu Ala Ser Glu Leu Tyr Ser Glu Leu Thr Asp Ile Leu Gly Lys Cys
115 120 125
Gly Ile Cys Thr Asn Arg Arg Cys Ser Gln Asn Glu Thr Arg Asn Cys
130 135 140
Cys Cys Gln Gly Glu Asn Pro Glu Thr Cys Gly Ala Ser Phe Ser Phe
145 150 155 160
Gly Cys Ser Trp Ser Met Tyr Tyr Asn Gly Cys Lys Phe Ala Arg Ser
165 170 175
Lys Lys Pro Arg Lys Phe Arg Leu His Gly Ala Glu Pro Lys Glu Glu
180 185 190
Glu Arg Leu Gly Ser His Leu Gln Asn Leu Ala Thr Val Ile Ala Pro
195 200 205
Ile Tyr Lys Lys Leu Ala Pro Asp Ala Tyr Asn Asn Gln Val Glu Phe
210 215 220
Glu His Gln Ala Pro Asp Cys Cys Leu Gly Leu Lys Glu Gly Arg Pro
225 230 235 240
Phe Ser Gly Val Thr Ala Cys Leu Asp Phe Ser Ala His Ser His Arg
245 250 255
Asp Gln Gln Asn Met Pro Asn Gly Ser Thr Val Val Val Thr Leu Asn
260 265 270
Arg Glu Asp Asn Arg Glu Val Gly Ala Lys Pro Glu Asp Glu Gln Phe
275 280 285
His Val Leu Pro Met Tyr Ile Ile Ala Pro Glu Asp Glu Phe Gly Ser
290 295 300
Thr Glu Gly Gln Glu Lys Lys Ile Arg Met Gly Ser Ile Glu Val Leu
305 310 315 320
Gln Ser Phe Arg Arg Arg Arg Val Ile Arg Ile Gly Glu Leu Pro Lys
325 330 335
Ser Cys Lys Lys Gly Gly Gly Gly Ser Val Ser Gly Gln Asp Ala Ala
340 345 350
Ala Val Gln Glu Ile Glu Tyr Trp Ser Asp Ser Glu His Asn Phe Gln
355 360 365
Asp Pro Cys Ile Gly Gly Val Ala Ile Ala Pro Thr His Gly Ser Ile
370 375 380
Leu Ile Glu Cys Ala Lys Cys Glu Val His Ala Thr Thr Lys Val Asn
385 390 395 400
Asp Pro Asp Arg Asn His Pro Thr Arg Ile Ser Leu Val Leu Tyr Arg
405 410 415
His Lys Asn Leu Phe Leu Pro Lys His Cys Leu Ala Leu Trp Glu Ala
420 425 430
Lys Met Ala Glu Lys Ala Arg Lys Glu Glu Glu Cys Gly Lys Asn
435 440 445
<210> 4
<211> 447
<212> PRT
<213> Artificial Sequence
<400> 4
Met Gln Ser Gln Asn Gly Lys Cys Glu Gly Cys Asn Pro Asp Lys Asp
1 5 10 15
Glu Ala Pro Tyr Tyr Thr His Leu Gly Ala Gly Pro Asp Val Ala Ala
20 25 30
Ile Arg Thr Leu Met Glu Glu Arg Tyr Gly Glu Lys Gly Lys Ala Ile
35 40 45
Arg Ile Glu Lys Val Ile Tyr Thr Gly Lys Glu Gly Lys Ser Ser Gln
50 55 60
Gly Cys Pro Ile Ala Lys Trp Val Tyr Arg Arg Ser Ser Glu Glu Glu
65 70 75 80
Lys Leu Leu Cys Leu Val Arg Val Arg Pro Asn His Thr Cys Glu Thr
85 90 95
Ala Val Met Val Ile Ala Ile Met Leu Trp Asp Gly Ile Pro Lys Leu
100 105 110
Leu Ala Ser Glu Leu Tyr Ser Glu Leu Thr Asp Ile Leu Gly Lys Cys
115 120 125
Gly Ile Cys Thr Asn Arg Arg Cys Ser Gln Asn Glu Thr Arg Asn Cys
130 135 140
Cys Cys Gln Gly Glu Asn Pro Glu Thr Cys Gly Ala Ser Phe Ser Phe
145 150 155 160
Gly Cys Ser Trp Ser Met Tyr Tyr Asn Gly Cys Lys Phe Ala Arg Ser
165 170 175
Lys Lys Pro Arg Lys Phe Arg Leu His Gly Ala Glu Pro Lys Glu Glu
180 185 190
Glu Arg Leu Gly Ser His Leu Gln Asn Leu Ala Thr Val Ile Ala Pro
195 200 205
Ile Tyr Lys Lys Leu Ala Pro Asp Ala Tyr Asn Asn Gln Val Glu Phe
210 215 220
Glu His Gln Ala Pro Asp Cys Cys Leu Gly Leu Lys Glu Gly Arg Pro
225 230 235 240
Phe Ser Gly Val Thr Ala Cys Leu Asp Phe Ser Ala His Ser His Arg
245 250 255
Asp Gln Gln Asn Met Pro Asn Gly Ser Thr Leu Val Cys Thr Leu Asn
260 265 270
Arg Glu Asp Asn Arg Glu Val Gly Ala Lys Pro Glu Asp Glu Gln Phe
275 280 285
His Val Leu Pro Met Tyr Ile Ile Ala Pro Glu Asp Glu Phe Gly Ser
290 295 300
Thr Glu Gly Gln Glu Lys Lys Ile Arg Met Gly Ser Ile Glu Val Leu
305 310 315 320
Gln Ser Phe Arg Arg Arg Arg Val Ile Arg Ile Gly Glu Leu Pro Lys
325 330 335
Ser Cys Lys Lys Gly Gly Gly Gly Ser Val Ser Gly Gln Asp Ala Ala
340 345 350
Ala Val Gln Glu Ile Glu Tyr Trp Ser Asp Ser Glu His Asn Phe Gln
355 360 365
Asp Pro Cys Ile Gly Gly Val Ala Ile Ala Pro Thr His Gly Ser Ile
370 375 380
Leu Ile Glu Cys Ala Lys Cys Glu Val His Ala Thr Thr Lys Val Asn
385 390 395 400
Asp Pro Asp Arg Asn His Pro Thr Arg Ile Ser Leu Val Leu Tyr Arg
405 410 415
His Lys Asn Leu Phe Leu Pro Lys His Cys Leu Ala Leu Trp Glu Ala
420 425 430
Lys Met Ala Glu Lys Ala Arg Lys Glu Glu Glu Cys Gly Lys Asn
435 440 445
<210> 5
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 5
attaattatc gttattatat ataataacga taattaat 38
<210> 6
<211> 90
<212> DNA
<213> Artificial Sequence
<400> 6
cccactgctt actggcttat cgaaattaat acgactcact atagggtctg tactgatcga 60
ccgtgcaaca gcagtctcga tcagctgctc 90
<210> 7
<211> 89
<212> DNA
<213> Artificial Sequence
<400> 7
gcagtctcga tcagctgctc cgcatgcaag tagcggtact aacgtacagt acgatgctag 60
ctagtgctta ggatcgagat cgcagaagg 89
<210> 8
<211> 87
<212> DNA
<213> Artificial Sequence
<400> 8
tggacgtacc gtgacgatga tcgatcggat gctagctagt agtgctcgat cgatagctaa 60
tgcttgccct tctgcgatct cgatcct 87