CN107475216B - 重组型耐热dna聚合酶及其应用 - Google Patents

重组型耐热dna聚合酶及其应用 Download PDF

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CN107475216B
CN107475216B CN201710695807.5A CN201710695807A CN107475216B CN 107475216 B CN107475216 B CN 107475216B CN 201710695807 A CN201710695807 A CN 201710695807A CN 107475216 B CN107475216 B CN 107475216B
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史进
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Abstract

本发明涉及一种重组型耐热DNA聚合酶:包括DNA结合结构域、蛋白结构域连接接头以及无3’‑5’核酸外切酶活性的耐热DNA聚合酶,DNA结合结构域通过蛋白结构域连接接头连接到无3’‑5’核酸外切酶活性的耐热DNA聚合酶的N端,其中DNA结合结构域为Sso7d结构域、HMf类蛋白或来自甲烷嗜热菌的DNA拓扑异构酶Ⅴ的HhH结构域;无3’‑5’核酸外切酶活性的耐热DNA聚合酶为Taq、Tth、Tma、Pfu、Deep、Vent或Tgo DNA聚合酶的缺失突变体。本发明还提供了上述重组型耐热DNA聚合酶在DNA测序中的应用。本发明的重组型耐热DNA聚合酶的续进性和延伸速度明显增强,在PCArg反应测序过程中,节约时间,使用本发明中的测序酶,可以获得更长的读长,以及更高的测序成功率。

Description

重组型耐热DNA聚合酶及其应用
技术领域
本发明涉及酶工程领域,尤其涉及一种重组型耐热DNA聚合酶及其应用。
背景技术
在测序应用上需要提高聚合酶的效率。通常DNA测序依靠生成四种不同类型的单链 DNA片段库来实现,单链DNA片段的一端是确定不变的,而另一端终止于不同的核苷酸(鸟嘌呤核苷酸G,腺嘌呤核苷酸A,胸腺嘧啶核苷酸T,胞嘧啶核苷酸C)。四种不同类型的DNA片段基于长度大小而被分离,在高分辨率的聚丙烯酰胺凝胶上呈现不同的条带,每个条带线性地对应DNA序列中的每一个特定的核苷酸,从而鉴定出给定的单核苷酸在序列中的位置。目前DNA测序主要有两种方法:Maxam和Gilbert的化学法和Sanger的双脱氧核苷酸终止法。
天然聚合酶的许多特性不适合DNA测序,测序酶应该具备的特性包括:(1)较高的持续合成能力:持续合成能力是指酶从引物-模板退火复合物上脱离之前链持续延伸的程度。(2) 耐热性:在高温条件下抵抗失活或分解的能力是酶在现代高技术投入循环测序反应中的最重要的因素。(3)核苷酸类似物嵌入染色末端:嵌入类似物的能力对双脱氧链终止法起决定性因素。带每个标记染料末端的终止的效率必须与避免低质量的不均匀峰数据的效率相似。此外,聚合酶经常有3’-核酸外切酶“校正”和/或在DNA复制后切掉引物的5’-核酸外切酶能力。由于这两种能力都不是测序所需的,所以聚合酶的突变体中应将其去除。
自动测序的发展催生了对更好的测序酶的追求,但目前应用于测序反应的酶效率不高,缺失型Taq酶去除了5’-3’外切酶活性,续进性只有2个碱基左右。DNA聚合酶的续进性(Processivity)即每次聚合酶结合引物-模板复合体后催化合成的寡核苷酸的数量,这一性能可通过增强酶-核酸复合体的稳定来实现增强。另外,测序酶过早地脱离模板会导致通过测序反应无法得到可靠的序列信息。
发明内容
为解决上述技术问题,本发明的目的是提供一种重组型耐热DNA聚合酶及其应用,本发明的重组型耐热DNA聚合酶的续进性明显增强,具有极快的延伸速度,在PCR反应测序过程中,节约时间,使用本发明中的测序酶,可以获得更长的读长,以及更高的测序成功率。
本发明提供了一种重组型耐热DNA聚合酶,包括DNA结合结构域、蛋白结构域连接接头(linker)以及无3’-5’核酸外切酶活性的耐热DNA聚合酶,DNA结合结构域通过蛋白结构域连接接头连接到无3’-5’核酸外切酶活性的耐热DNA聚合酶的N端,其中
DNA结合结构域为Sso7d结构域、来自耐热菌的HMf类蛋白或来自甲烷嗜热菌的DNA拓扑异构酶Ⅴ的HhH结构域;
无3’-5’核酸外切酶活性的耐热DNA聚合酶为Taq DNA聚合酶缺失突变体、Tth DNA聚合酶缺失突变体、Tma DNA聚合酶缺失突变体、Pfu DNA聚合酶缺失突变体、Deep VentDNA 聚合酶缺失突变体、Vent DNA聚合酶缺失突变体或Tgo DNA聚合酶缺失突变体。优选地, DNA结合结构域为Sso7d结构域。优选地,无3’-5’核酸外切酶活性的耐热DNA聚合酶为Taq DNA聚合酶缺失突变体。
进一步地,Sso7d结构域的氨基酸序列如SEQ ID NO.1所示。
进一步地,蛋白结构域连接接头的氨基酸序列如SEQ ID NO.2所示。
进一步地,Taq DNA聚合酶缺失突变体的N端缺失289个氨基酸,且含有R660D和F667Y 两个突变点。Taq DNA聚合酶N端缺失289个氨基酸,因此无5`-3`核酸外切酶活性;且R660D 和F667Y两个突变点降低了野生型Taq DNA聚合酶催化dNTP和ddNTP及荧光类似物的选择性。
进一步地,Taq DNA聚合酶缺失突变体的氨基酸序列如SEQ ID NO.3所示。
进一步地,Tth DNA聚合酶缺失突变体的氨基酸序列如SEQ ID NO.4所示。其N端缺失 289个氨基酸,且含R662D/F669Y突变点。
进一步地,Tma DNA聚合酶缺失突变体的氨基酸序列如SEQ ID NO.5所示。其N端缺失284个氨基酸。
进一步地,Pfu DNA聚合酶缺失突变体的氨基酸序列如SEQ ID NO.6所示。其含D141A/E143A突变,且无外切酶活性。
本发明还提供了一种上述重组型耐热DNA聚合酶在DNA测序中的应用。
借由上述方案,本发明至少具有以下优点:
本发明的重组型耐热DNA聚合酶耐热温度可高达98℃,在1h内无明显活性变化;续进性明显增强,具有极快的延伸速度,其聚合酶延伸速度可高达20kb/s;在PCR反应测序过程中,节约时间,使用本发明中的测序酶,可以获得更长的读长,以及更高的测序成功率。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是本发明重组型耐热DNA聚合酶的结构示意图;
图2是本发明本发明实施例1中的测序酶与Amplitaq FS在信号易中断测序表现的比较;
附图标记说明:
1-DNA结合结构域;2-蛋白linker;3-无3’-5’核酸外切酶活性的耐热DNA聚合酶。
具体实施方式
下面结合附图和实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1
如图1所示,一种重组型耐热DNA聚合酶,包括DNA结合结构域1、蛋白结构域连接接头(蛋白linker)2以及无3’-5’核酸外切酶活性的耐热DNA聚合酶3,DNA结合结构域1 通过linker 2连接到无3’-5’核酸外切酶活性的耐热DNA聚合酶3的N端。
在本实施例中,DNA结合结构域1为Sso7d蛋白结构域,其氨基酸序列如SEQ IDNO.1 所示。蛋白linker 2为Gly-Gly-Gly-Thr-Val氨基酸序列,编码linker的核苷酸序列如SEQ ID NO.2所示。蛋白linker 2的作用为连接两个独立的蛋白或蛋白结构域,同时保持两个连接的蛋白或蛋白结构域保持独立的活性与功能。
在本实施例中,无3’-5’核酸外切酶活性的耐热DNA聚合酶3为Taq DNA聚合酶缺失突变体,其N端缺失289个氨基酸,且含有R660D和F667Y两个突变点。Taq DNA聚合酶缺失突变体(以下简称TaqΔ289 DNA聚合酶)的氨基酸序列如SEQ ID NO.3所示。
Sso7d蛋白结构域通过蛋白linker连接到Taq DNA缺失突变体的N端,由此构成一个含有两个相对独立的结构域的融合蛋白(以下简称Sso7d-TaqΔ289聚合酶)。Sso7d-TaqΔ289 聚合酶的DNA序列如SEQ ID NO.4所示。Sso7d蛋白结构域赋予融合蛋白非特异性地结合 DNA双链的能力。在聚合酶反应中,聚合酶活性由TaqΔ289 DNA聚合酶提供。
Sso7d蛋白结构域与古生菌Sulfolobus sulfataricus中的染色体结合蛋白Sso7d具有较高的同源性。Sso7d结构域能非特异性地结合到双链DNA上,当Sso7d蛋白结构域融合到TaqΔ 289 DNA聚合酶上时,产生的融合蛋白Sso7d-TaqΔ289 DNA聚合酶的续进性和延伸速度明显增强。)
由于Taq DNA聚合酶N端缺失289个氨基酸,因此无5`-3`核酸外切酶活性;且R660D和F667Y两个突变点降低了野生型Taq DNA聚合酶催化dNTP和ddNTP及荧光类似物的选择性。F667Y突变点降低了测序酶对ddNTP偏爱,使四种碱基的测序信号峰分布更加均匀。而R660D突变点更进一步增强了这一效果。
本实施例中,Sso7d-TaqΔ289聚合酶的合成方法如下:
(1)表达质粒构建及表达菌株转化:
Sso7d-TaqΔ289聚合酶编码DNA序列由人工合成(SEQ ID No.8),并经限制性内切酶 (Nco I/Nde I)消化,连接进入表达载体pET28a,并转化到大肠杆菌表达菌株BL21(DE3)。
(2)Sso7d-TaqΔ289聚合酶诱导表达与纯化
含pET28a(sso7d-taqΔ289)质粒的大肠杆菌BL21(DE3)菌株经过夜活化,以1%体积比转接含卡那霉素的LB液体培养基,37℃培养3-4小时至OD600达0.6-1.0,加入IPTG(至终浓度1mM)诱导表达。继续培养2-3小时。收集菌体,超声波破碎,并以Ni-NTA亲和纯化蛋白。
纯化后,测定酶的纯度以及活性。对获得的Sso7d-TaqΔ289聚合酶进行续进性、延伸速度和耐热性测试,测试结果如表1所示。
表1 Sso7d-TaqΔ289聚合酶性能
Figure BDA0001379134620000041
一般来说,野生型Taq DNA聚合酶延伸速度约为1kb/min,续进性为13-18个核苷酸。本实施例的重组型耐热DNA聚合酶,其续进性约为130-160个核苷酸,是野生型Taq DNA聚合酶续进性的6-10倍。同时,Sso7d-TaqΔ289 DNA聚合酶的延伸速度达到了3kb/min,是野生型Taq DNA聚合酶速度的3倍。
图2是本发明本发明实施例1中的测序酶与AmpliTaq FS在信号易中断测序表现的比较。其中,其中a1、a2为RNAi样本,b1、b2为甲基化样本,c1、c2为Poly-A和PolyT结构, d1、d2为Poly-A和PolyG结构,其中,a1、b1、c1、d1代表Amplitaq FS的结果,a2、b2、 c2、d2代表本实施例的Sso7d-TaqΔ289聚合酶的结果。从图2中可看出,本发明的测序酶与AmpliTaqFS相比,能对RNAi及甲基化样本有更好的延伸,从而成功获得测序结果;对 AT-Rich样本,本发明的测序酶更不易发生信号中断,能获得更长的读长,因而本发明的测序酶在使用时不必特殊处理就可应用于困难样本的测序,保证了测序的成功率。
本实施例中的Sso7d-TaqΔ289聚合酶能耐受98℃,1h内无明显活性变化,这比普通野生型Taq DNA聚合酶有更优良的耐热性(94℃)。通常Taq DNA聚合酶的缺失体(如Klentaq) 的耐热性要好于野生型聚合酶。本实施例中的重组型Taq DNA聚合酶的耐热性较其他Taq DNA聚合酶更好,因而在测序反应中可以使用更高的变性温度(98℃),酶活性损失也最小。
本实施例中的重组型Taq DNA聚合酶具有极好的延伸能力。Sso7d结构域增强了酶-模板 -引物复合物的稳定性,使酶具备了更强的持续合成能力。在测序反应中,体现在测序结果读长(read length)的长度上,以及复杂结构(AT/GC-Rich,Hairpin)测序成功率。使用本发明中的测序酶,可以获得更好的数据质量,更长的读长,以及高的测序成功率。
实施例2
一种重组型耐热DNA聚合酶,包括HMf类蛋白,HMf类蛋白通过蛋白linker连接到Taq Δ289 DNA聚合酶上,其中,蛋白linker 2为Gly-Gly-Gly-Thr-Val氨基酸序列,TaqΔ289 DNA 聚合酶的N端缺失289个氨基酸,且含有R660D和F667Y两个突变点。
以上重组型耐热DNA聚合酶的制备方法如下:
(1)表达质粒构建及表达菌株转化:
Hmf蛋白编码序列人工合成,氨基酸序列见SEQ ID NO.7。并经限制性内切酶NcoI/Spe I连接入pET28a(sso7d-taqΔ289),构建表达质粒pET28a(hmf-taqΔ289),目的基因序列见 SEQ ID NO.9。并转化到大肠杆菌表达菌株BL21(DE3)。
(2)Hmf-TaqΔ289聚合酶诱导表达与纯化
含pET28a pET28a(hmf-taqΔ289)质粒的大肠杆菌BL21(DE3)菌株经过夜活化,以1%体积比转接含卡那霉素的LB液体培养基,37℃培养3-4小时至OD600达0.6-1.0,加入IPTG (至终浓度1mM)诱导表达。继续培养2-3小时。收集菌体,超声波破碎,并以Ni-NTA亲和纯化蛋白。
实施例3
一种重组型耐热DNA聚合酶,包括Sso7d蛋白结构域,Sso7d蛋白结构域通过蛋白linker 连接到无3`-5`核酸外切酶活性的Tth聚合酶突变体上,其中,蛋白linker 2为Gly-Gly-Gly-Thr- Val氨基酸序列,Tth聚合酶突变体的氨基酸序列如SEQ ID NO.4所示,目的基因序列见SEQ ID NO.10。
以上重组型耐热DNA聚合酶的制备方法如下:
(1).表达质粒构建及表达菌株转化:
TthΔ289聚合酶编码DNA序列克隆自Thermus thermophilus HB8基因组DNA(购自TAKAArgA,3071),基因序列见SEQ ID NO.4。并经同源重组替换TaqΔ289同源序列,构建表达质粒pET28a(sso7d-tthΔ289),并转化到大肠杆菌表达菌株BL21(DE3)。
(2)Sso7d-TthΔ289聚合酶诱导表达与纯化
含pET28a pET28a(sso7d-tthΔ289)质粒的大肠杆菌BL21(DE3)菌株经过夜活化,以1%体积比转接含卡那霉素的LB液体培养基,37℃培养3-4小时至OD600达0.6-1.0,加入IPTG(至终浓度1mM)诱导表达。继续培养2-3小时。收集菌体,超声波破碎,并以Ni-NTA亲和纯化蛋白。
以上所述仅是本发明的优选实施方式,并不用于限制本发明,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变型,这些改进和变型也应视为本发明的保护范围。
序列表
<110> 北京擎科新业生物技术有限公司
<120> 重组型耐热DNA聚合酶及其应用
<160> 4
<170> Patent In version 3.3
<210> 1
<211> 63
<212> 氨基酸序列
<213> Sso7d结构域
<400> 1
Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp
5 10 15
Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser
20 25 30
Phe Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val
35 40 45
Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys
50 55 60
Gln Lys Lys
63
<210> 2
<211> 5
<212> 氨基酸序列
<213> 蛋白结构域连接接头
<400> 2
Gly Gly Gly Thr Val
5
<210> 3
<211> 544
<212> 氨基酸序列
<213> Taq DNA聚合酶缺失突变体
<400> 3
Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly
5 10 15
Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala
20 25 30
Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg
35 40 45
Ala Pro Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg
50 55 60
Gly Leu Leu Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly
65 70 75
Leu Gly Leu Pro Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu
80 85 90
Leu Asp Pro Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr
95 100 105
Gly Gly Glu Trp Thr Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser
110 115 120
Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg Leu Glu Gly Glu Glu
125 130 135
Arg Leu Leu Trp Leu Tyr Arg Glu Val Glu Arg Pro Leu Ser Ala
140 145 150
Val Leu Ala His Met Glu Ala Thr Gly Val Arg Leu Asp Val Ala
155 160 165
Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala Glu Glu Ile Ala Arg
170 175 180
Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His Pro Phe Asn Leu
185 190 195
Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp Glu Leu Gly
200 205 210
Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr
215 220 225
Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val
230 235 240
Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr
245 250 255
Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg
260 265 270
Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu
275 280 285
Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro
290 295 300
Leu Gly Gln Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp
305 310 315
Leu Leu Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu
320 325 330
Ala His Leu Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu
335 340 345
Gly Arg Asp Ile His Thr Glu Thr Ala Ser Trp Met Phe Gly Val
350 355 360
Pro Arg Glu Ala Val Asp Pro Leu Met Arg Asp Ala Ala Lys Thr
365 370 375
Ile Asn Tyr Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu Ser
380 385 390
Gln Glu Leu Ala Ile Pro Tyr Glu Glu Ala Gln Ala Phe Ile Glu
395 400 405
Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg Ala Trp Ile Glu Lys
410 415 420
Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val Glu Thr Leu Phe
425 430 435
Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg Val Lys Ser
440 445 450
Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro Val Gln
455 460 465
Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu Phe
470 475 480
Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His
485 490 495
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val
500 505 510
Ala Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala
515 520 525
Val Pro Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser
530 535 540
Ala Lys Glu Gly
544
<210> 4
<211> 834
<212> 氨基酸序列
<213> Tth DNA聚合酶缺失突变体
<400> 4
Met Glu Ala Met Leu Pro Let Phe Glu Pro Lys Gly Arg Val Leu
5 10 15
Leu Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu
20 25 30
Lys Gly Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr
35 40 45
Gly Phe Ala Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr
50 55 60
Lys Ala Val Phe Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg
65 70 75
His Glu Ala Tyr Glu Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro
80 85 90
Glu Asp Phe Pro Arg Gln Leu Ala Leu Ile Lys Glu Leu Val Asp
95 100 105
Leu Leu Gly Phe Thr Arg Leu Glu Val Pro Gly Tyr Glu Ala Asp
110 115 120
Asp Val Leu Ala Thr Leu Ala Lys Lys Ala Glu Lys Glu Gly Tyr
125 130 135
Glu Val Arg Ile Leu Thr Ala Asp Arg Asp Leu Tyr Gln Leu Val
140 145 150
Ser Asp Arg Val Ala Val Leu His Pro Glu Gly His Leu Ile Thr
155 160 165
Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro Glu Gln Trp
170 175 180
Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp Asn Leu Pro
185 190 195
Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu Leu Lys
200 205 210
Gly Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg Val
215 220 225
Lys Pro Glu Asn Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp
230 235 240
Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp Leu Pro
245 250 255
Leu Glu Val Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly
260 265 270
Leu Arg Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His
275 280 285
Glu Phe Gly Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro
290 295 300
Trp Pro Pro Pro Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg
305 310 315
Pro Glu Pro Met Trp Ala Glu Leu Lys Ala Leu Ala Ala Cys Arg
320 325 330
Asp Gly Arg Val His Arg Ala Ala Asp Pro Leu Ala Gly Leu Lys
335 340 345
Asp Leu Lys Glu Val Arg Gly Leu Leu Ala Lys Asp Leu Ala Val
350 355 360
Leu Ala Ser Arg Glu Gly Leu Asp Leu Val Pro Gly Asp Asp Pro
365 370 375
Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr Pro Glu
380 385 390
Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu Asp Ala Ala
395 400 405
His Arg Ala Leu Leu Ser Glu Arg Leu His Arg Asn Leu Leu Lys
410 415 420
Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr His Glu Val
425 430 435
Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala Thr Gly
440 445 450
Val Arg Arg Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu Leu
455 460 465
Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala
470 475 480
Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val
485 490 495
Leu Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gln Lys
500 505 510
Thr Gly Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg
515 520 525
Glu Ala His Pro Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu
530 535 540
Thr Lys Leu Lys Asn Thr Tyr Val Asp Pro Leu Pro Ser Leu Val
545 550 555
His Pro Arg Thr Gly Arg Leu His Thr Arg Phe Asn Gln Thr Ala
560 565 570
Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn
570 580 585
Ile Pro Val Arg Thr Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe
590 595 600
Val Ala Glu Ala Gly Trp Ala Leu Val Ala Leu Asp Tyr Ser Gln
605 610 615
Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly Asp Glu Asn Leu
620 625 630
Ile Arg Val Phe Gln Glu Gly Lys Asp Ile His Thr Gln Thr Ala
635 640 645
Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val Asp Pro Leu Met
650 655 660
Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu Tyr Gly Met
665 670 675
Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
680 685 690
Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val
695 700 705
Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly
710 715 720
Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu
725 730 735
Asn Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala
740 745 750
Phe Asn Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu
755 760 765
Ala Met Val Lys Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg
770 775 780
Met Leu Leu Gln Val His Asp Glu Leu Leu Leu Glu Ala Pro Gln
785 790 795
Ala Arg Ala Glu Glu Val Ala Ala Leu Ala Lys Glu Ala Met Glu
800 805 810
Lys Ala Tyr Pro Leu Ala Val Pro Leu Glu Val Glu Val Gly Met
815 820 825
Gly Glu Asp Trp Leu Ser Ala Lys Gly
830 834
<210> 5
<211> 610
<212> 氨基酸序列
<213> Tma DNA聚合酶缺失突变体
<400> 5
Met Lys Glu Leu Gln Leu Tyr Glu Glu Ser Glu Pro Val Gly Tyr
5 10 15
Arg Ile Val Lys Asp Leu Val Glu Phe Glu Lys Leu Ile Glu Lys
20 25 30
Leu Arg Glu Ser Pro Ser Phe Ala Ile Asp Leu Glu Thr Ser Ser
35 40 45
Leu Asp Pro Phe Asp Cys Asp Ile Val Gly Ile Ser Val Ser Phe
50 55 50
Lys Pro Lys Glu Ala Tyr Tyr Ile Pro Leu His His Arg Asn Ala
65 70 75
Gln Asn Leu Asp Glu Lys Glu Val Leu Lys Lys Leu Lys Glu Ile
80 85 90
Leu Glu Asp Pro Gly Ala Lys Ile Val Gly Gln Asn Leu Lys Phe
95 100 105
Asp Tyr Lys Val Leu Met Val Lys Gly Val Glu Pro Val Pro Pro
110 115 120
Tyr Phe Asp Thr Met Ile Ala Ala Tyr Leu Leu Glu Pro Asn Glu
125 130 135
Lys Lys Phe Asn Leu Asp Asp Leu Ala Leu Lys Phe Leu Gly Tyr
140 145 150
Lys Met Thr Ser Tyr Gln Glu Leu Met Ser Phe Ser Phe Pro Leu
155 160 165
Phe Gly Phe Ser Phe Ala Asp Val Pro Val Glu Lys Ala Ala Asn
170 175 180
Tyr Ser Cys Glu Asp Ala Asp Ile Thr Tyr Arg Leu Tyr Lys Thr
185 190 195
Leu Ser Leu Lys Leu His Glu Ala Asp Leu Glu Asn Val Phe Tyr
200 205 210
Lys Ile Glu Met Pro Leu Val Asn Val Leu Ala Arg Met Glu Leu
215 220 225
Asn Gly Val Tyr Val Asp Thr Glu Phe Leu Lys Lys Leu Ser Glu
230 235 240
Glu Tyr Gly Lys Lys Leu Glu Glu Leu Ala Glu Glu Ile Tyr Arg
245 250 255
Ile Ala Gly Glu Pro Phe Asn Ile Asn Ser Pro Lys Gln Val Ser
260 265 270
Arg Ile Leu Phe Glu Lys Leu Gly Ile Lys Pro Arg Gly Lys Thr
275 280 285
Thr Lys Thr Gly Asp Tyr Ser Thr Arg Ile Glu Val Leu Glu Glu
290 295 300
Leu Ala Gly Glu His Glu Ile Ile Pro Leu Ile Leu Glu Tyr Arg
305 310 315
Lys Ile Gln Lys Leu Lys Ser Thr Tyr Ile Asp Ala Leu Pro Lys
320 325 330
Met Val Asn Pro Lys Thr Gly Arg Ile His Ala Ser Phe Asn Gln
335 340 345
Thr Gly Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu
350 355 360
Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly Lys Glu Ile Arg Lys
365 370 375
Ala Ile Val Pro Gln Asp Pro Asn Trp Trp Ile Val Ser Ala Asp
380 385 390
Tyr Ser Gln Ile Glu Leu Arg Ile Leu Ala His Leu Ser Gly Asp
395 400 405
Glu Asn Leu Leu Arg Ala Phe Glu Glu Gly Ile Asp Val His Thr
410 415 420
Leu Thr Ala Ser Arg Ile Phe Asn Val Lys Pro Glu Glu Val Thr
425 430 435
Glu Glu Met Arg Arg Ala Gly Lys Met Val Asn Phe Ser Ile Ile
440 445 450
Tyr Gly Val Thr Pro Tyr Gly Leu Ser Val Arg Leu Gly Val Pro
455 460 465
Val Lys Glu Ala Glu Lys Met Ile Val Asn Tyr Phe Val Leu Tyr
470 475 480
Pro Lys Val Arg Asp Tyr Ile Gln Arg Val Val Ser Glu Ala Lys
485 490 495
Glu Lys Gly Tyr Val Arg Thr Leu Phe Gly Arg Lys Arg Asp Ile
500 505 510
Pro Gln Leu Met Ala Arg Asp Arg Asn Thr Gln Ala Glu Gly Glu
515 520 525
Arg Ile Ala Ile Asn Thr Pro Ile Gln Gly Thr Ala Ala Asp Ile
530 535 540
Ile Lys Leu Ala Met Ile Glu Ile Asp Arg Glu Leu Lys Glu Arg
545 550 555
Lys Met Arg Ser Lys Met Ile Ile Gln Val His Asp Glu Leu Val
560 565 570
Phe Glu Val Pro Asn Glu Glu Lys Asp Ala Leu Val Glu Leu Val
575 580 585
Lys Asp Arg Met Thr Asn Val Val Lys Leu Ser Val Pro Leu Glu
590 595 600
Val Asp Val Thr Ile Gly Lys Thr Trp Ser
605 610
<210> 6
<211> 774
<212> 氨基酸序列
<213> Pfu聚合酶缺失突变体
<400> 6
Met Ile Leu Asp Val Asp Tyr Ile Thr Glu Glu Gly Lys Pro Val
5 10 15
Ile Arg Leu Phe Lys Lys Glu Asn Gly Lys Phe Lys Ile Glu His
20 25 30
Asp Arg Thr Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp
35 40 45
Ser Lys Ile Glu Glu Val Lys Lys Ile Thr Gly Glu Arg His Gly
50 55 60
Lys Ile Val Arg Ile Val Asp Val Glu Lys Val Glu Lys Lys Phe
65 70 75
Leu Gly Lys Pro Ile Thr Val Trp Lys Leu Tyr Leu Glu His Pro
80 85 90
Gln Asp Val Pro Thr Ile Arg Glu Lys Val Arg Glu His Pro Ala
95 100 105
Val Val Asp Ile Phe Glu Tyr Asp Ile Pro Phe Ala Lys Arg Tyr
110 115 120
Leu Ile Asp Lys Gly Leu Ile Pro Met Glu Gly Glu Glu Glu Leu
125 130 135
Lys Ile Leu Ala Phe Ala Ile Ala Thr Leu Tyr His Glu Gly Glu
140 145 150
Glu Phe Gly Lys Gly Pro Ile Ile Met Ile Ser Tyr Ala Asp Glu
155 160 165
Asn Glu Ala Lys Val Ile Thr Trp Lys Asn Ile Asp Leu Pro Tyr
170 175 180
Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys Arg Phe Leu
185 190 195
Arg Ile Ile Arg Glu Lys Asp Pro Asp Ile Ile Val Thr Tyr Asn
200 205 210
Gly Asp Ser Phe Asp Phe Pro Tyr Lys Ala Lys Arg Ala Glu Lys
215 220 225
Leu Gly Ile Lys Leu Thr Ile Gly Arg Asp Gly Ser Glu Pro Lys
230 235 240
Met Gln Arg Ile Gly Asp Met Thr Ala Val Glu Val Lys Gly Arg
245 250 255
Ile His Phe Asp Leu Tyr His Val Ile Thr Arg Thr Ile Asn Leu
260 265 270
Pro Thr Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys
275 280 285
Pro Lys Glu Lys Val Tyr Ala Asp Glu Ile Ala Lys Ala Trp Glu
290 295 300
Ser Gly Glu Asn Lys Glu Arg Val Ala Lys Tyr Ser Met Glu Asp
305 310 315
Ala Lys Ala Thr Tyr Glu Leu Gly Lys Glu Phe Lys Pro Met Glu
320 325 330
Ile Gln Lys Ser Arg Leu Val Gly Gln Pro Leu Trp Asp Val Ser
335 340 345
Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe Leu Leu Arg Lys
350 355 360
Ala Tyr Glu Arg Asn Glu Val Ala Pro Asn Lys Pro Ser Glu Glu
365 370 375
Glu Tyr Gln Arg Arg Leu Arg Glu Ser Tyr Thr Gly Gly Phe Val
380 385 390
Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn Ile Val Tyr Leu Asp
395 400 405
Phe Arg Ala Leu Tyr Pro Ser Ile Ile Ile Thr His Asn Val Ser
410 415 420
Pro Asp Thr Leu Asn Lys Glu Gly Cys Lys Asn Tyr Asp Ile Ala
425 430 435
Pro Gln Val Gly His Lys Phe Cys Lys Asp Ile Pro Gly Phe Ile
440 445 450
Pro Ser Leu Leu Gly His Leu Leu Glu Glu Arg Gln Lys Ile Lys
455 460 465
Thr Lys Met Lys Glu Thr Gln Asp Pro Ile Glu Lys Ile Leu Leu
470 475 480
Asp Tyr Arg Gln Lys Ala Ile Lys Leu Leu Ala Asn Ser Phe Tyr
485 490 495
Gly Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys
500 505 510
Ala Glu Ser Val Thr Ala Trp Gly Arg Lys Tyr Ile Glu Leu Val
515 520 525
Trp Lys Glu Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ile
530 535 540
Asp Thr Asp Gly Leu Tyr Ala Thr Ile Pro Gly Gly Glu Ser Glu
545 550 555
Glu Ile Lys Lys Lys Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser
560 565 570
Lys Leu Pro Gly Leu Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Lys
575 580 585
Arg Gly Phe Phe Val Thr Lys Lys Arg Tyr Ala Val Ile Asp Glu
590 595 600
Glu Gly Lys Val Ile Thr Arg Gly Leu Glu Ile Val Arg Arg Asp
605 610 615
Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala Arg Val Leu Glu Thr
620 625 630
Ile Leu Lys His Gly Asp Val Glu Glu Ala Val Arg Ile Val Lys
635 640 645
Glu Val Ile Gln Lys Leu Ala Asn Tyr Glu Ile Pro Pro Glu Lys
650 655 660
Leu Ala Ile Tyr Glu Gln Ile Thr Arg Pro Leu His Glu Tyr Lys
665 670 675
Ala Ile Gly Pro His Val Ala Val Ala Lys Lys Leu Ala Ala Lys
680 685 690
Gly Val Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Ile Val Lys
695 700 705
Arg Gly Asp Gly Pro Ile Ser Asn Arg Ala Ile Leu Ala Glu Glu
710 715 720
Tyr Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu
725 730 735
Asn Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Gly Phe Gly
740 745 750
Tyr Arg Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly
755 760 765
Leu Thr Ser Trp Leu Asn Ile Lys Lys
770 774
<210> 7
<211> 69
<212> 氨基酸序列
<213> Hmf蛋白
<400> 7
Met Gly Glu Leu Pro Ile Ala Pro Ile Gly Arg Ile Ile Lys Asn
5 10 15
Ala Gly Ala Glu Arg Val Ser Asp Asp Ala Arg Ile Ala Leu Ala
20 25 30
Lys Val Leu Glu Glu Met Gly Glu Glu Ile Ala Ser Glu Ala Val
35 40 45
Lys Leu Ala Lys His Ala Gly Arg Lys Thr Ile Lys Ala Glu Asp
50 55 60
Ile Glu Leu Ala Arg Lys Met Phe Lys
65 69
<210> 8
<211> 1875
<212> 核苷酸序列
<213> Serso7d- TaqΔ289聚合酶
<400> 8
ATGGGCAGCA GCGCAACCGT AAAGTTCAAG TACAAAGGCG AAGAAAAAGA GGTAGACATC 60
TCCAAGATCA AGAAAGTATG GCGTGTGGGC AAGATGATCT CCTTCACCTA CGACGAGGGC 120
GGTGGCAAGA CCGGCCGTGG TGCGGTAAGC GAAAAGGACG CGCCGAAGGA GCTGCTGCAG 180
ATGCTGGAGA AGCAGAAAAA GGGCGGCGGT GTCACTAGTC CCAAGGCCCT GGAGGAGGCC 240
CCCTGGCCCC CGCCGGAAGG GGCCTTCGTG GGCTTTGTGC TTTCCCGCAA GGAGCCCATG 300
TGGGCCGATC TTCTGGCCCT GGCCGCCGCC AGGGGGGGCC GGGTCCACCG GGCCCCCGAG 360
CCTTATAAAG CCCTCAGGGA CCTGAAGGAG GCGCGGGGGC TTCTCGCCAA AGACCTGAGC 420
GTTCTGGCCC TGAGGGAAGG CCTTGGCCTC CCGCCCGGCG ACGACCCCAT GCTCCTCGCC 480
TACCTCCTGG ACCCTTCCAA CACCACCCCC GAGGGGGTGG CCCGGCGCTA CGGCGGGGAG 540
TGGACGGAGG AGGCGGGGGA GCGGGCCGCC CTTTCCGAGA GGCTCTTCGC CAACCTGTGG 600
GGGAGGCTTG AGGGGGAGGA GAGGCTCCTT TGGCTTTACC GGGAGGTGGA GAGGCCCCTT 660
TCCGCTGTCC TGGCCCACAT GGAGGCCACG GGGGTGCGCC TGGACGTGGC CTATCTCAGG 720
GCCTTGTCCC TGGAGGTGGC CGAGGAGATC GCCCGCCTCG AGGCCGAGGT CTTCCGCCTG 780
GCCGGCCACC CCTTCAACCT CAACTCCCGG GACCAGCTGG AAAGGGTCCT CTTTGACGAG 840
CTAGGGCTTC CCGCCATCGG CAAGACGGAG AAGACCGGCA AGCGCTCCAC CAGCGCCGCC 900
GTCCTGGAGG CCCTCCGCGA GGCCCACCCC ATCGTGGAGA AGATCCTGCA GTACCGGGAG 960
CTCACCAAGC TGAAGAGCAC CTACATTGAC CCCTTGCCGG ACCTCATCCA CCCCAGGACG 1020
GGCCGCCTCC ACACCCGCTT CAACCAGACG GCCACGGCCA CGGGCAGGCT AAGTAGCTCC 1080
GATCCCAACC TCCAGAACAT CCCCGTCCGC ACCCCGCTTG GGCAGAGGAT CCGCCGGGCC 1140
TTCATCGCCG AGGAGGGGTG GCTATTGGTG GCCCTGGACT ATAGCCAGAT AGAGCTCAGG 1200
GTGCTGGCCC ACCTCTCCGG CGACGAGAAC CTGATCCGGG TCTTCCAGGA GGGGCGGGAC 1260
ATCCACACGG AGACCGCCAG CTGGATGTTC GGCGTCCCCC GGGAGGCCGT GGACCCCCTG 1320
ATGCGCGATG CGGCCAAGAC CATCAACTAC GGGGTCCTCT ACGGCATGTC GGCCCACCGC 1380
CTCTCCCAGG AGCTAGCCAT CCCTTACGAG GAGGCCCAGG CCTTCATTGA GCGCTACTTT 1440
CAGAGCTTCC CCAAGGTGCG GGCCTGGATT GAGAAGACCC TGGAGGAGGG CAGGAGGCGG 1500
GGGTACGTGG AGACCCTCTT CGGCCGCCGC CGCTACGTGC CAGACCTAGA GGCCCGGGTG 1560
AAGAGCGTGC GGGAGGCGGC CGAGCGCATG GCCTTCAACA TGCCCGTCCA GGGCACCGCC 1620
GCCGACCTCA TGAAGCTGGC TATGGTGAAG CTCTTCCCCA GGCTGGAGGA AATGGGGGCC 1680
AGGATGCTCC TTCAGGTCCA CGACGAGCTG GTCCTCGAGG CCCCAAAAGA GAGGGCGGAG 1740
GCCGTGGCCC GGCTGGCCAA GGAGGTCATG GAGGGGGTGT ATCCCCTGGC CGTGCCCCTG 1800
GAGGTGGAGG TGGGGATAGG GGAGGACTGG CTCTCCGCCA AGGAGGGGGG TTCTCATCAT 1860
CATCATCATC ATTGA 1875
<210> 9
<211> 1881
<212> 核苷酸序列
<213> Hmf TaqΔ289聚合酶
<400> 9
ATGGGTGAGT TACCAATTGC CCCAATCGGA AGAATCATAA AAAACGCTGG TGCTGAAAGA 60
GTTAGTGACG ATGCAAGAAT TGCATTAGCA AAAGTTTTAG AAGAAATGGG CGAAGAAATA 120
GCTTCAGAAG CTGTGAAATT AGCAAAACAT GCGGGAAGAA AAACAATAAA GGCAGAAGAC 180
ATTGAATTAG CTCGAAAAAT GTTCAAAGGC GGCGGTGTCA CTAGTCCCAA GGCCCTGGAG 240
GAGGCCCCCT GGCCCCCGCC GGAAGGGGCC TTCGTGGGCT TTGTGCTTTC CCGCAAGGAG 300
CCCATGTGGG CCGATCTTCT GGCCCTGGCC GCCGCCAGGG GGGGCCGGGT CCACCGGGCC 360
CCCGAGCCTT ATAAAGCCCT CAGGGACCTG AAGGAGGCGC GGGGGCTTCT CGCCAAAGAC 420
CTGAGCGTTC TGGCCCTGAG GGAAGGCCTT GGCCTCCCGC CCGGCGACGA CCCCATGCTC 480
CTCGCCTACC TCCTGGACCC TTCCAACACC ACCCCCGAGG GGGTGGCCCG GCGCTACGGC 540
GGGGAGTGGA CGGAGGAGGC GGGGGAGCGG GCCGCCCTTT CCGAGAGGCT CTTCGCCAAC 600
CTGTGGGGGA GGCTTGAGGG GGAGGAGAGG CTCCTTTGGC TTTACCGGGA GGTGGAGAGG 660
CCCCTTTCCG CTGTCCTGGC CCACATGGAG GCCACGGGGG TGCGCCTGGA CGTGGCCTAT 720
CTCAGGGCCT TGTCCCTGGA GGTGGCCGAG GAGATCGCCC GCCTCGAGGC CGAGGTCTTC 780
CGCCTGGCCG GCCACCCCTT CAACCTCAAC TCCCGGGACC AGCTGGAAAG GGTCCTCTTT 840
GACGAGCTAG GGCTTCCCGC CATCGGCAAG ACGGAGAAGA CCGGCAAGCG CTCCACCAGC 900
GCCGCCGTCC TGGAGGCCCT CCGCGAGGCC CACCCCATCG TGGAGAAGAT CCTGCAGTAC 960
CGGGAGCTCA CCAAGCTGAA GAGCACCTAC ATTGACCCCT TGCCGGACCT CATCCACCCC 1020
AGGACGGGCC GCCTCCACAC CCGCTTCAAC CAGACGGCCA CGGCCACGGG CAGGCTAAGT 1080
AGCTCCGATC CCAACCTCCA GAACATCCCC GTCCGCACCC CGCTTGGGCA GAGGATCCGC 1140
CGGGCCTTCA TCGCCGAGGA GGGGTGGCTA TTGGTGGCCC TGGACTATAG CCAGATAGAG 1200
CTCAGGGTGC TGGCCCACCT CTCCGGCGAC GAGAACCTGA TCCGGGTCTT CCAGGAGGGG 1260
CGGGACATCC ACACGGAGAC CGCCAGCTGG ATGTTCGGCG TCCCCCGGGA GGCCGTGGAC 1320
CCCCTGATGC GCGATGCGGC CAAGACCATC AACTACGGGG TCCTCTACGG CATGTCGGCC 1380
CACCGCCTCT CCCAGGAGCT AGCCATCCCT TACGAGGAGG CCCAGGCCTT CATTGAGCGC 1440
TACTTTCAGA GCTTCCCCAA GGTGCGGGCC TGGATTGAGA AGACCCTGGA GGAGGGCAGG 1500
AGGCGGGGGT ACGTGGAGAC CCTCTTCGGC CGCCGCCGCT ACGTGCCAGA CCTAGAGGCC 1560
CGGGTGAAGA GCGTGCGGGA GGCGGCCGAG CGCATGGCCT TCAACATGCC CGTCCAGGGC 1620
ACCGCCGCCG ACCTCATGAA GCTGGCTATG GTGAAGCTCT TCCCCAGGCT GGAGGAAATG 1680
GGGGCCAGGA TGCTCCTTCA GGTCCACGAC GAGCTGGTCC TCGAGGCCCC AAAAGAGAGG 1740
GCGGAGGCCG TGGCCCGGCT GGCCAAGGAG GTCATGGAGG GGGTGTATCC CCTGGCCGTG 1800
CCCCTGGAGG TGGAGGTGGG GATAGGGGAG GACTGGCTCT CCGCCAAGGA GGGGGGTTCT 1860
CATCATCATC ATCATCATTG A 1881
<210> 10
<211> 1890
<212> 核苷酸序列
<213> Sso7d- TthΔ289聚合酶
<400> 10
ATGGGCAGCG CAACCGTAAA GTTCAAGTAC AAAGGCGAAG AAAAAGAGGT AGACATCTCC 60
AAGATCAAGA AAGTATGGCG TGTGGGCAAG ATGATCTCCT TCACCTACGA CGAGGGCGGT 120
GGCAAGACCG GCCGTGGTGC GGTAAGCGAA AAGGACGCGC CGAAGGAGCT GCTGCAGATG 180
CTGGAGAAGC AGAAAAAGGG CGGCGGTGTC ACTAGTGGGG GCCTCCTGGA GGCCCCCGCC 240
CCCCTGGAGG AGGCCCCCTG GCCCCCGCCG GAAGGGGCCT TCGTGGGCTT CGTCCTCTCC 300
CGCCCCGAGC CCATGTGGGC GGAGCTTAAA GCCCTGGCCG CCTGCAGGGA CGGCCGGGTG 360
CACCGGGCAG CAGACCCCTT GGCGGGGCTA AAGGACCTCA AGGAGGTCCG GGGCCTCCTC 420
GCCAAGGACC TCGCCGTCTT GGCCTCGAGG GAGGGGCTAG ACCTCGTGCC CGGGGACGAC 480
CCCATGCTCC TCGCCTACCT CCTGGACCCC TCCAACACCA CCCCCGAGGG GGTGGCGCGG 540
CGCTACGGGG GGGAGTGGAC GGAGGACGCC GCCCACCGGG CCCTCCTCTC GGAGAGGCTC 600
CATCGGAACC TCCTTAAGCG CCTCGAGGGG GAGGAGAAGC TCCTTTGGCT CTACCACGAG 660
GTGGAAAAGC CCCTCTCCCG GGTCCTGGCC CACATGGAGG CCACCGGGGT ACGGCGGGAC 720
GTGGCCTACC TTCAGGCCCT TTCCCTGGAG CTTGCGGAGG AGATCCGCCG CCTCGAGGAG 780
GAGGTCTTCC GCTTGGCGGG CCACCCCTTC AACCTCAACT CCCGGGACCA GCTGGAAAGG 840
GTGCTCTTTG ACGAGCTTAG GCTTCCCGCC TTGGGGAAGA CGCAAAAGAC AGGCAAGCGC 900
TCCACCAGCG CCGCGGTGCT GGAGGCCCTA CGGGAGGCCC ACCCCATCGT GGAGAAGATC 960
CTCCAGCACC GGGAGCTCAC CAAGCTCAAG AACACCTACG TGGACCCCCT CCCAAGCCTC 1020
GTCCACCCGA GGACGGGCCG CCTCCACACC CGCTTCAACC AGACGGCCAC GGCCACGGGG 1080
AGGCTTAGTA GCTCCGACCC CAACCTGCAG AACATCCCCG TCCGCACCCC CTTGGGCCAG 1140
AGGATCCGCC GGGCCTTCGT GGCCGAGGCG GGTTGGGCGT TGGTGGCCCT GGACTATAGC 1200
CAGATAGAGC TCCGCGTCCT CGCCCACCTC TCCGGGGACG AAAACCTGAT CAGGGTCTTC 1260
CAGGAGGGGA AGGACATCCA CACCCAGACC GCAAGCTGGA TGTTCGGCGT CCCCCCGGAG 1320
GCCGTGGACC CCCTGATGCG CCGGGCGGCC AAGACGGTGA ACTTCGGCGT CCTCTACGGC 1380
ATGTCCGCCC ATAGGCTCTC CCAGGAGCTT GCCATCCCCT ACGAGGAGGC GGTGGCCTTT 1440
ATAGAGCGCT ACTTCCAAAG CTTCCCCAAG GTGCGGGCCT GGATAGAAAA GACCCTGGAG 1500
GAGGGGAGGA AGCGGGGCTA CGTGGAAACC CTCTTCGGAA GAAGGCGCTA CGTGCCCGAC 1560
CTCAACGCCC GGGTGAAGAG CGTCAGGGAG GCCGCGGAGC GCATGGCCTT CAACATGCCC 1620
GTCCAGGGCA CCGCCGCCGA CCTCATGAAG CTCGCCATGG TGAAGCTCTT CCCCCGCCTC 1680
CGGGAGATGG GGGCCCGCAT GCTCCTCCAG GTCCACGACG AGCTCCTCCT GGAGGCCCCC 1740
CAAGCGCGGG CCGAGGAGGT GGCGGCTTTG GCCAAGGAGG CCATGGAGAA GGCCTATCCC 1800
CTCGCCGTGC CCCTGGAGGT GGAGGTGGGG ATGGGGGAGG ACTGGCTTTC CGCCAAGGGT 1860
GGGGGTTCTC ATCATCATCA TCATCATTGA 1890

Claims (2)

1.一种重组型耐热DNA聚合酶,其特征在于:包括DNA结合结构域、蛋白结构域连接接头以及无3’-5’核酸外切酶活性的耐热DNA聚合酶,所述DNA结合结构域通过所述蛋白结构域连接接头连接到所述无3’-5’核酸外切酶活性的耐热DNA聚合酶的N端,其中
所述DNA结合结构域为Sso7d结构域;
所述无3’-5’核酸外切酶活性的耐热DNA聚合酶为Taq DNA聚合酶缺失突变体;
所述Sso7d结构域的氨基酸序列如SEQ ID NO .1所示;
所述蛋白结构域连接接头的氨基酸序列如SEQ ID NO .2所示;
所述Taq DNA聚合酶缺失突变体的氨基酸序列如SEQ ID NO .3所示。
2.权利要求1所述的重组型耐热DNA聚合酶在DNA测序中的应用。
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CN108129571A (zh) * 2017-12-25 2018-06-08 上海捷瑞生物工程有限公司 Taq DNA连接酶融合蛋白
CN109266628B (zh) * 2018-10-09 2020-04-14 南京市胸科医院 一种融合的TaqDNA聚合酶及其应用
CN109679932B (zh) * 2018-12-05 2020-03-17 广州奇辉生物科技有限公司 一种dna聚合酶、重组载体及它们的制备方法和应用
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CN114015672B (zh) * 2021-12-06 2022-05-31 江南大学 一种Pfu DNA聚合酶

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