CN114075558B - 一种wh54聚合酶及其在rt-pcr中的应用 - Google Patents
一种wh54聚合酶及其在rt-pcr中的应用 Download PDFInfo
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- CN114075558B CN114075558B CN202010847462.2A CN202010847462A CN114075558B CN 114075558 B CN114075558 B CN 114075558B CN 202010847462 A CN202010847462 A CN 202010847462A CN 114075558 B CN114075558 B CN 114075558B
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Abstract
本发明公开了一种WH54聚合酶及其在RT‑PCR中的应用。本发明提供了蛋白质，为如下1)或2)或3)：1)氨基酸序列是序列表中序列2所示的蛋白质；2)在序列表中序列2所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；3)将1)或2)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到具有相同功能的蛋白质。本发明的DNA聚合酶，同时具有逆转录酶活性以及聚合酶活性，可以被应用于one‑step RT‑PCR中，即只需要WH54聚合酶即可完成逆转录及PCR的步骤，简化实验步骤，降低试剂成本。
Description
技术领域
本发明属于生物技术领域，尤其涉及一种WH54聚合酶及其在RT-PCR中的应用。
背景技术
热稳定性的DNA聚合酶已经在很多嗜热微生物中被广泛发现，例如从Thermusaquaticus中分离出了Taq DNA聚合酶，从Pyrococcus furiosus中分离出了Pfu DNA聚合酶，从Pyrococcus sp.KOD1中分离出了KOD DNA聚合酶。这些聚合酶由于其热稳定性，在核酸高温变性的条件下不会失活，是目前核酸扩增的常用酶，在高温扩增反应如PCR、测序反应中都得到广泛应用。
目前RT-PCR分为2种，two-step RT-PCR，即为常规RT-PCR方案，先用逆转录酶和逆转录酶反应体系进行cDNA合成，再以合成的cDNA为模板利用聚合酶和PCR反应体系进行反应；one-step RT-PCR，市面上常见的方案为将逆转录酶和PCR酶混在一起，通过反应buffer的优化来实现一步RT-PCR。需要两个酶进行反应，试剂成本高，而且需要考虑两个酶的反应buffer的兼容性问题，可能因为需要相互妥协无法达到两个酶的最佳活性条件。
发明内容
本发明一个目的是提供一种蛋白质。
本发明提供的蛋白质，为如下1)或2)或3)：
1)氨基酸序列是序列表中序列2所示的蛋白质；
2)在序列表中序列2所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
3)将1)或2)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到具有相同功能的蛋白质。
上述经过一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述蛋白质具有逆转录酶和/或DNA聚合酶活性。
编码上述蛋白质的核酸分子也是本发明保护的范围。
上述核酸分子为如下(a1)或(a2)或(a3)或(a4)所示的DNA分子：
(a1)编码区包括序列表中序列1的DNA分子；
(a2)核苷酸序列是序列表中序列1的DNA分子；
(a3)与(a1)或(a2)限定的核苷酸序列具有75％或75％以上同一性，且编码上述蛋白质的DNA分子；
(a4)在严格条件下与(a1)或(a2)限定的核苷酸序列杂交，且编码上述蛋白质的DNA分子。
含有上述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系也是本发明保护的范围。
上述蛋白质，或，上述核酸分子，或，上述表达盒、重组载体、重组微生物或转基因细胞系，在制备逆转录酶中的应用也是本发明保护的范围；
或，上述蛋白质，或，上述核酸分子，或，上述表达盒、重组载体、重组微生物或转基因细胞系，在制备DNA聚合酶中的应用也是本发明保护的范围；
或上述蛋白质，或，上述核酸分子，或，上述表达盒、重组载体、重组微生物或转基因细胞系，在制备具有逆转录和DNA聚合双重功能的酶中的应用也是本发明保护的范围。
上述蛋白质在作为逆转录酶或DNA聚合酶或具有逆转录和DNA聚合双重功能的酶中的应用也是本发明保护的范围；
或，上述蛋白质在逆转录反应或PCR反应或one-step RT-PCR中的应用也是本发明保护的范围；
或，含有上述蛋白质的逆转录扩增体系或PCR扩增体系或one-step RT-PCR反应体系也是本发明保护的范围。
本发明另一个目的是提供如下方法:
本发明提供了一种对待测核酸进行逆转录反应的方法，包括如下步骤：将上述蛋白质作为逆转录酶对待测核酸进行逆转录反应。
本发明还提供了一种对待测DNA进行PCR反应，包括如下步骤：将上述蛋白质作为逆转录酶对待测DNA进行PCR反应。
本发明还提供了一种对待测RNA进行one-step RT-PCR反应，包括如下步骤：用上述蛋白质待测RNA进行one-step RT-PCR反应。
本发明的实验证明，本发明的DNA聚合酶，同时具有逆转录酶活性以及聚合酶活性，可以被应用于one-step RT-PCR中，即只需要WH54聚合酶即可完成逆转录及PCR的步骤，简化实验步骤，降低试剂成本。
附图说明
图1为重组质粒EcoRI酶切胶图。
图2为逆转录活性测试。
图3为纯化后蛋白电泳测试。
图4为PCR活性对比测试图。
图5为One-step RT-PCR测试结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中20xBSA为2mgBSA溶在1ml水里。
实施例1、WH54聚合酶基因表达纯化
一、WH54聚合酶基因的获得
人工合成序列1，即为WH54基因，该基因编码的蛋白命名为WH54蛋白，该蛋白的氨基酸序列为序列表中序列2。
二、WH54聚合酶表达纯化
1、重组载体的构建
将上述序列1所示的WH54基因替换pET32a表达载体(Novagen,69015)的EcoRI和NotI酶切位点间的片段，得到重组质粒；转入DH5a中，质粒小提获得重组质粒，再用EcoRI酶切验证。
将重组质粒用EcoRI酶切验证结果如图1所示，从左到右依次为，1：hindIIIdigest marker、2-11:重组质粒，可以看到，得到大小为8.6kb左右片段(箭头所指)为目标重组质粒，泳道3-7、泳道10、泳道11的质粒都为目标重组质粒，即为重组载体。
重组载体为将序列表中序列1所示的WH54基因替换pET32a表达载体的EcoRI和NotI酶切位点间的片段得到的载体，表达序列2所示的WH54蛋白。
2、诱导表达和纯化
将上述1制备的重组载体转入BL21感受态细胞中，涂平板，从平板上挑取单菌落，37℃过夜培养，后转接至30ml新培养基，1:100稀释，培养至OD值为0.5-0.6，加入0.1mMIPTG，30℃过夜诱导，得到诱导培养产物。
诱导培养产物12000rpm离心10min时间，获得0.1g菌体沉淀，向其中加入2.7ml结合缓冲液(50mM NaH2PO4，pH 8.0，300mM NaCl,10mM咪唑，余量为水)、300ul溶菌酶(BBI，A610308-0005)，冰上裂解30min，超声(2son 3soff，60％功率)破碎10min，4℃12000rpm离心30min分离上清和沉淀。
在手工柱(生工，F506605)中加入750ul HisTrap FF填料(GE，17531805)，用3ml结合缓冲液冲洗平衡填料；再加入3ml上清液；再用漂洗液(50mM NaH2PO4，pH 8.0，300mMNaCl,30mM咪唑，余量为水)冲洗10次(3ml/次)，然后用洗脱液(50mM NaH2PO4，pH 8.0，300mMNaCl,250mM咪唑，余量为水)500ul洗脱蛋白4-5次，收集Ni柱洗脱后的蛋白。
将上述洗脱后蛋白取5ul进行SDS-PAGE检测，蛋白预制胶(金斯瑞，M01010C)，电压120V，电泳30min。然后使用金斯瑞的蛋白胶自动染色脱色仪进行染色，拍照。
结果如图3所示，第一个泳道为Ni柱洗脱下蛋白，第二个泳道为蛋白marker(10-180KDa,Thermo,26617)；可以看出，得到大小为94KD的目的蛋白。
将空载体pET32a表达载体转入BL21感受态细胞中，按照上述方法诱导培养和纯化，经过电泳检测，未得到目的大小蛋白。
将Ni柱洗脱后的蛋白用透析缓冲液(20mM KH2PO4-K2HPO4，pH 7.4,50mM KCl,0.1mM DTT,5％(体积百分含量)Glycerol，余量为水)4℃过夜透析，收集透析后产物；再将透析产物用储存buffer溶解，得到蛋白WH54溶液，浓度为0.25mg/ml。
上述储存buffer按照如下方法制备：10mM KH2PO4-K2HPO4、300mM KCl、0.1mMEDTA、1mM DTT、1％(体积百分含量)Tween 20,500ug/ml BSA,50％(体积百分含量)甘油，余量为水。
实施例2、WH54蛋白活性验证
一、反应buffer配制
逆转录反应：
50mM MnSO4水溶液；
逆转录反应10xRT buffer：100mM Tris-HCl，pH8.3，900mM KCl，余量为水。
PCR反应：
PCR反应10xPCR buffer：120mM Tris-HCl，pH8.3,1.18M KCl，7.5mM EGTA，0.5％(体积百分含量)Tween20，余量为水。
二、逆转录活性测试
1、逆转录活性测试1
使用健康人组织的total RNA(Thermo，4307281)作为模板，用N6引物(Random N6primer(Thermo,SO142)，6个nt的随机引物)按照表1所示2组进行RT反应，得到RT产物：
RT反应体系及反应程序为如下表1：
表1为RT反应体系及反应程序
将上述各组RT产物中取1ul作为模板，用人的看家基因的PCR primer mix(biomol,货号HHK-1)进行PCR(rTaq酶)，得到看家基因PCR扩增产物。
30ul的PCR体系如下：
RT产物1ul
PCR反应10xPCR buffer 3ul
2.5mM dNTP mix 3.2ul
10uM primer mix 3ul
20xBSA(2mg/ml)1ul
rTaq(Takara)0.4ul
ddH2O 18.4ul
将看家基因PCR扩增产物电泳，结果如图2所示，1：hindIII digest marker，2:对照组，3:实验组；可以看出，对照组合成了7条带(最上边一条缺失)，实验组合成了6条带，合成6条以上条带即为合格。
上述结果表明，与对照Transcriptor Reverse Transcriptase相比，本发明制备的WH54蛋白也具有很好的逆转录活性。
2、逆转录活性测试2
选取人Hela细胞的总RNA(CLONTECH，636543)作为模板，按照如下各组进行RT反应：
实验组RT反应：
Random primer(Thermo,SO142)1ul
人Hela细胞总RNA(1ug/ul)1ul
RNase free water 13ul
先上述组分65℃加热5min，迅速置于冰水浴降温，并在冰上静置2min，得到RNAmiX；再与如下组分进行如下反应：
RNA mix 15ul
逆转录反应10xRT buffer 2ul
10mM dNTP mix(Fermentas,R0192)1ul
RNase inhibitor(ENZYMATICS，Y9240L)1ul
实施例1制备的0.25mg/ml蛋白WH54溶液1ul
反应条件：25℃5min，60℃反应30min；得到实验组RT反应产物。
对照组1RT反应(商品Tth DNA polymerase)：
Random primer(Thermo,SO142)1ul
人Hela细胞总RNA(1ug/ul)1ul
RNase free water 13ul
先上述组分65℃加热5min，迅速置于冰水浴降温，并在冰上静置2min，得到RNAmix；再与如下组分进行如下反应：
RNA mix 15ul
逆转录反应10xRT buffer 2ul
10mM dNTPmix(Fermentas,R0192)1ul
RNase inhibitor(Enzymatics,Y9240L)1ul
Tth DNA polymerase(Fapon MD012)1ul
反应条件：25℃5min，60℃反应30min；得到对照组1RT反应产物。
对照组2RT反应(MMLV Reverse Transcriptase)：
Random primer(Thermo,SO142)1ul
人Hela细胞总RNA(1ug/ul)1ul
Rnase free water补足至13ul
先上述组分65℃加热5min，迅速置于冰水浴降温，并在冰上静置2min，得到RNAmiX；再与如下组分进行如下反应：
RNA mix 13ul
5x Firststrand buffer 4ul
10mM dNTPmix(Fermentas,R0192)1ul
Rnase inhibitor(Enzymatics,Y9240L)1ul
MMLV Reverse Transcriptase(Thermo,Superscript II)1ul
反应条件：25℃5min，60℃反应30min；得到对照组2RT反应产物。
阴性对照组RT反应：将实验组RT反应体系中不加入酶，其余组分和反应程序与实验组相同，得到阴性照组RT反应产物。
上述各组反应完成后，用1ul 0.5M EDTA终止反应，并使用Qubit dsDNA HS kit(Thermo)进行产物浓度测量。
结果如下：
实验组的RT反应产物浓度为5.98ng/ul；
对照组1的RT反应产物浓度为4.04ng/ul；
对照组2的RT反应产物浓度为5.04ng/ul；
阴性照组的RT反应产物浓度为3.56ng/ul。
上述结果表明，与其他聚合酶相比，实施例1制备的WH54聚合酶具有较好的逆转录酶活性，得到产物量高。
三、聚合酶活性测试
1、聚合酶活性对比测试
以lambda DNA(NEB，N3011S)为模板，进行长度为2kb、4kb、6kb、8kb、12kb、15kb的目的片段PCR扩增，实验条件为：
表2为所有体系中引物
对照组1(rTaq DNA polymerase)的PCR扩增体系：
10x buffer 2.5ul，
10mM dNTPmix 0.5ul,
10uM FW primer 1ul,
10uM不同的RE primer 1ul,
lambda DNA 10ng,
rTaq DNA polymerase(Takara)0.2ul,
Nuclease free water补足至25ul
上述PCR反应条件为：
95℃3min，30cycle x(95℃30s，55℃30s，72℃1kb/min)，72℃7min，12℃forever；
对照组2(商品Tth DNA polymerase)的PCR扩增体系：
2x Tth RT-PCR buffer 12.5ul，
10mM dNTP mix 0.5ul,
10uM FW primer 1ul,
10uM不同的RE primer 1ul,
Lambda DNA 10ng,
商品Tth DNA polymerase(Fapon MD012)0.5ul,
Nuclease free water补足至25ul
反应条件为：
95℃2min，30cycle x(94℃30s，55℃30s，72℃1kb/min)，72℃7min，12℃forever；
实验组(WH54 polymerase)的PCR扩增体系：
2xTth RT-PCR buffer12.5ul，
10mM dNTP mix 0.5ul,
10uM FW primer 1ul,
10uM不同的RE primer 1ul,
Lambda DNA 10ng,
实施例1获得浓度为0.25mg/ml的蛋白WH54溶液0.5ul,
Nuclease free water补足至25ul
上述PCR反应条件均为：95℃2min，30cycle x(94℃30s，55℃30s，72℃1kb/min)，72℃7min，12℃forever。
结果如图4所示，左侧：rTaq DNA pol PCR结果，1，1kb DNA marker，2，2kb扩增，3，4kb扩增，4，6kb扩增，5，8kb扩增，6，12kb扩增，7，15kb扩增。右侧：1-6，实验组WH54 Tth DNApol PCR结果(2kb到15kb)，7，1kb DNA marker，8-13，商品Tth DNA polymerase PCR结果；可以看出，实验组WH54 pol和rTaq DNA聚合酶均可以成功扩增出6kb的目的条带，而商品Tth DNA polymerase则只能扩增出2kb的目的条带，证明实验组WH54有很好的扩增能力。
实施例3、WH54在one-step RT-PCR中的应用
1、模板获得
使用人Hela细胞总RNA，浓度为1ug/ul。
用水稀释上述总RNA，得到不同浓度的RNA 1ug/ul,100ng/ul,10ng/ul以及1ng/ul。
2、one-step RT-PCR扩增目标基因B2M基因
以上述人Hela细胞总RNA为模板，用如下反应体系进行one-step RT-PCR扩增：
实验组：one-step RT-PCR扩增体系：
2XRT-PCR buffer 25ul，
10Mm dNTPmix 1.5ul,
10uM FW primer 2.25ul,
10uM RE primer 2.25ul,
不同浓度的RNA 1ul,
50x MnCl2水溶液1ul,
实施例1的浓度为0.25mg/ml的蛋白WH54溶液1ul，
Nuclease free water补足至50ul
上述体系中，FW引物和RE引物为扩增基因B2M的引物对，其中FW primer为actgaattcacccccactga；RE primer为cctccatgatgctgcttaca。
上述体系中，1ug/ul,100ng/ul,10ng/ul以及1ng/ul的RNA在扩增体系中的质量分别为1ug,100ng，10ng,1ng。
对照组：与实验组相同，不同的仅为实施例1的浓度为0.25mg/ml的蛋白WH54溶液替换为商品Tth DNA polymerase(Fapon MD012)，其他扩增体系中各物质与实验组相同。
上述one-step RT-PCR扩增的反应程序为：
65℃30min，
94℃2min，
30cycles x(94℃30s,55℃30s,70℃1min)，
72℃7min，
12℃forever。
结果如图5所示，Lane 1：100bp marker；Lane2-4：商品Tth DNA polymerase，Lane2，1ug total RNA，Lane3，100ng total RNA，Lane4,10ng total RNA,Lane5,1ngtotal RNA；Lane 6-9：本实施例1的蛋白WH54溶液实验组扩增结果，Lane6，1ug total RNA，Lane7，100ng total RNA，Lane8,10ng total RNA,Lane9,1ng total RNA；相比商品TthDNA polymerase，蛋白WH54在扩增灵敏性上有更优异的表现，可以实现低至1ng RNA模板的RT-PCR扩增。
SEQUENCE LISTING
<110> 深圳华大生命科学研究院
<120> 一种WH54聚合酶及其在RT-PCR中的应用
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gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctgaagga ggacgggtac 180
aaggccgtct tcgtggtctt tgacgccaag gccccctcct tccgccacga ggcctacgag 240
gcctacaagg cggggagggc cccgaccccc gaggacttcc cccggcagct cgccctcatc 300
aaggagctgg tggacctcct ggggtttact cgcctcgagg tccaaggcta cgaggcagac 360
gacgtcctcg ccaccctggc caagaaggcg gaaaaagaag ggtacgaggt gcgcatcctc 420
accgccgacc gggacctcta ccagctcgtc tctgaccgcg tcgccgtcct ccaccccgag 480
ggccacctca tcaccccgga gtggctttgg gagaagtacg gcctcaggcc ggagcagtgg 540
gtggacttcc gcgccctcgt aggggacccc tccgacaacc tccccggggt caagggcatc 600
ggggagaaga ccgccctcaa gctcctcaag gagtggggaa gcctggaaaa cctcctcaag 660
aacctggacc gggtgaagcc ggaaagcgtc cgggagaaga tcaaggccca cctggaagac 720
ctcaggctct ccttggagct ctcccgggtg cgcaccgacc tccccctgga ggtggacctc 780
gcccaggggc gggagcccga ccgggagggg cttagggcct tcctggagag gctggagttc 840
ggcagcctcc tccacgagtt cggcctcctg gaggcccccg cccccctgga ggaggccccc 900
tggcccccgc cggaaggggc cttcgtgggc ttcgtcctct cccgccccga gcccatgtgg 960
gcggagctta aagccctggc cgcctgcagg gacggccggg tgcaccgggc agcggacccc 1020
ttggcggggc ttggggacct caaggaggtc cggggcctcc tcgccaagga cctcgccgtc 1080
ttggcctcga gggaggggct agacctcgtg cccggggacg accccatgct cctcgcctac 1140
ctcctggacc cctccaacac cacccccgag ggggtggcgc ggcgctacgg gggggagtgg 1200
acggaggacg ccgcccaccg ggcccttctc tcggagaggc tccagcagaa cctccttaag 1260
cgcctccagg gggaggagaa gctcctttgg ctctaccacg aggtggaaaa gcccctctcc 1320
cgggtcctgg cccacatgga ggccaccggg gtacggctgg acgtggccta ccttcaggcc 1380
ctttccctgg agcttgcgga ggagatccgc cgcctcgagg aggaggtctt ccgcttggcg 1440
ggccacccct tcaacctcaa ctcccgggac cagctggaaa gggtgctctt tgacgagctt 1500
aggcttcccg ccttggggaa gacgcaaaag acgggcaagc gctccaccag cgccgcggtg 1560
ctggaggccc tacgggaggc ccaccccatc gtggagaaga tcctccagca ccgggagctc 1620
accaagctca agaacaccta cgtggacccc ctcccaagcc tcgtccaccc gaggacgggc 1680
cgcctccaca cccgcttcaa ccagacggcc acggccacgg ggaggcttag tagctccgac 1740
cccaacctgc agaacatccc cgtccgcacc cccttgggcc agaggatccg ccgggccttc 1800
gtggccgagg cgggatgggc gttggtggcc ctggactata gccagataga gctccgcgtc 1860
ctcgcccacc tctccgggga cgagaacctg atcagggtct tccaggaggg gaaggacatc 1920
cacacccaga ccgcaagctg gatgttcggc gtccccccgg aggccgtgga ccccctgatg 1980
cgccgggcag ccaagacggt gaacttcggc gtcctctacg gcatgtccgc ccaccggctc 2040
tcccaggagc tctccatccc ctacgaggag gcctcggcct tcattgagcg ctacttccaa 2100
agcttcccca aggtacgggc ctggatagaa aagaccctgg aggaggggag gaagcggggc 2160
tacgtggaaa ccctcttcgg aagaaggcgc tacgtgcccg acctcaacgc ccgggtgaag 2220
agcgtcaggg aggccgcgga gcgcatggcc ttcaacatgc ccgtccaggg caccgccgcc 2280
gacctcatga aactcgccat ggtgaagctc ttcccccgcc tccgggagat gggggcccgc 2340
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Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly
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Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
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Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe
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Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu
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Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln
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Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu
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Glu Val Gln Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys
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Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg
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Asp Leu Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu
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Gly His Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg
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Pro Glu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp
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Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu
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Leu Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg
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Val Lys Pro Glu Ser Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp
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Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp Leu Pro Leu
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Glu Val Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg
260 265 270
Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly
275 280 285
Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro
290 295 300
Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp
305 310 315 320
Ala Glu Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg
325 330 335
Ala Ala Asp Pro Leu Ala Gly Leu Gly Asp Leu Lys Glu Val Arg Gly
340 345 350
Leu Leu Ala Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp
355 360 365
Leu Val Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro
370 375 380
Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp
385 390 395 400
Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu Gln Gln
405 410 415
Asn Leu Leu Lys Arg Leu Gln Gly Glu Glu Lys Leu Leu Trp Leu Tyr
420 425 430
His Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala
435 440 445
Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu
450 455 460
Leu Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala
465 470 475 480
Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu
485 490 495
Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gln Lys Thr Gly
500 505 510
Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His
515 520 525
Pro Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys
530 535 540
Asn Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly
545 550 555 560
Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu
565 570 575
Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu
580 585 590
Gly Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu
595 600 605
Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu
610 615 620
Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Lys Asp Ile
625 630 635 640
His Thr Gln Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val
645 650 655
Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu
660 665 670
Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ser Ile Pro Tyr
675 680 685
Glu Glu Ala Ser Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys
690 695 700
Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly
705 710 715 720
Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn
725 730 735
Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn
740 745 750
Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val
755 760 765
Lys Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln
770 775 780
Val His Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu
785 790 795 800
Val Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala
805 810 815
Val Pro Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala
820 825 830
Lys Gly
Claims (10)
1.蛋白质，为如下1）或2）：
1）氨基酸序列是序列表中序列2所示的蛋白质；
2）在序列表中序列2所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质。
2.根据权利要求1所述的蛋白质，其特征在于：所述蛋白质具有逆转录酶和/或DNA聚合酶活性。
3.编码权利要求1或2所述蛋白质的核酸分子。
4.如权利要求3所述的核酸分子，其特征在于：所述核酸分子为如下（a1）或（a2）所示：
（a1）编码区为序列表中序列1的DNA分子；
（a2）核苷酸序列是序列表中序列1的 DNA分子。
5.含有权利要求3或4所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系。
6.权利要求1或2所述蛋白质或权利要求3或4所述核酸分子或权利要求5所述表达盒、重组载体、重组微生物或转基因细胞系在制备逆转录酶中的应用；
或，权利要求1或2所述蛋白质或权利要求3或4所述核酸分子或权利要求5所述表达盒、重组载体、重组微生物或转基因细胞系在制备DNA聚合酶中的应用；
或权利要求1或2所述蛋白质或权利要求3或4所述核酸分子或权利要求5所述表达盒、重组载体、重组微生物或转基因细胞系在制备具有逆转录和DNA聚合双重功能的酶中的应用。
7.权利要求1或2所述蛋白质在作为逆转录酶或DNA聚合酶或具有逆转录和DNA聚合双重功能的酶中的应用；
或，权利要求1或2所述蛋白质在逆转录反应或PCR反应或one-step RT-PCR中的应用；
或，含有权利要求1或2所述蛋白质的逆转录扩增体系或PCR扩增体系或one-step RT-PCR反应体系。
8.一种对待测核酸进行逆转录反应的方法，包括如下步骤：将权利要求1或2所述蛋白质作为逆转录酶对待测核酸进行逆转录反应。
9.一种对待测DNA进行PCR反应的方法，包括如下步骤：将权利要求1或2所述蛋白质作为逆转录酶对待测DNA进行PCR反应。
10.一种对待测RNA进行one-step RT-PCR反应的方法，包括如下步骤：用权利要求1或2所述蛋白质待测RNA进行one-step RT-PCR反应。
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