CN113355303A - 一种m-mlv逆转录酶突变体及其应用 - Google Patents

一种m-mlv逆转录酶突变体及其应用 Download PDF

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CN113355303A
CN113355303A CN202110634550.9A CN202110634550A CN113355303A CN 113355303 A CN113355303 A CN 113355303A CN 202110634550 A CN202110634550 A CN 202110634550A CN 113355303 A CN113355303 A CN 113355303A
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宋丹凤
赵伟
夏娟
严子成
汪瑶
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Abstract

本发明公开了一种M‑MLV逆转录酶突变体及其应用，所述M‑MLV逆转录酶突变体的氨基酸序列如序列表中的SEQ ID NO.2所示。本发明提供的逆转录酶，在野生型M‑MLV逆转录酶的基础上，结合已有对逆转录酶结构研究进展，融合多个突变体的结构特点，通过定点诱变理性设计筛选得到，该逆转录酶突变体具有很强的抑制物耐受性，降低了抑制物对cDNA合成的干扰，在添加全血以及痰液的样本反应过程中，本发明所述的突变型逆转录酶活性不受影响或影响很小。与此同时，本发明提供的逆转录酶突变体能够耐受70℃的高温，在具有较好热稳定性的同时，也具有较高的反应灵敏度和准确性，具有极强的应用前景。

Description

一种M-MLV逆转录酶突变体及其应用
技术领域
本发明涉及生物技术领域，具体涉及一种M-MLV逆转录酶突变体及其应用。
背景技术
逆转录酶(Reverse transcriptase)通常具有三种活性：以RNA为模板的DNA聚合酶活性、以DNA为模板的DNA聚合酶活性以及降解RNA-DNA杂合链中RNA的RNase H活性。因此，逆转录酶常用于cDNA文库构建、mRNA测序、RT-PCR定量等，被广泛应用于研究及医学分子诊断领域。莫洛泥鼠白血病病毒(Moloney Murine LeukemiaVirus,MMLV)或禽类成髓细胞白血病病毒(Avian Myeloblastosis Virus,AMV)pol基因产生的逆转录酶是两种最常用的逆转录酶，AMV逆转录酶的RNase H活性较高且热稳定性高，但产物的产量低；MMLV逆转录酶产量较高，但热稳定性差，因其单体结构特征，易于进行改造，在应用更为广泛。
逆转录酶的合成能力是其应用中十分重要的参数，具有高合成能力的逆转录酶通常具有高的底物亲和力或可高效催化合成cDNA。逆转录酶的RNase H活性可降解RNA-DNA杂合体中的RNA，可能会造成RNA模板在完成全长逆转录前被降解，降低逆转录效率，因此，商业化逆转录酶的RNase H活性通常会被改造以弱化其降解RNA-DNA杂合体中的RNA的功能，提高长链cDNA产量。模板RNA的形成二级结构也是影响逆转录酶催化效率的重要因素，通常可以通过提高反应温度来打开其二级结构，这就要求逆转录酶的反应温度相应提高。此外，生物学样本常含有会抑制逆转录酶活性的物质，如土壤和植物中的腐殖酸、血液中的血红素、粪便中的胆汁盐等，这些物质在RNA提取过程中并不能被完全去除，因此，提高逆转录酶对抑制物的耐受性是亟待解决的重要方面。
在提高逆转录酶的性能的研究中，主要集中于提高其热稳定性和催化效率上，其中Thermo Fisher使用体外区室化核糖体展示技术(Compartmentalized RibosomeDisplay,CRD)进行高通量筛选，获得热稳定性提高的不同的突变株。这些热稳定性提高的突变株，突变位点集中于酶的活性中心及RNase H结构域。但是热稳定性提高的逆转录酶可能会伴随催化活性降低以及抑制物耐受性降低的缺点。具有高温耐受性的逆转录酶可降低反应中引物二聚体的形成，对于检测低浓度模板具有优势，能够耐受血液或痰液等样本的逆转录酶可作为体外诊断试剂中分子诊断用酶，对于简化样本处理流程、减少污染有重要意义，因此临床上亟需一种具有较好热稳定以及催化效率，同时能够耐受抑制物的逆转录酶。
发明内容
基于上述技术问题，发明人根据蛋白结构特征及关键氨基酸位点进行理性设计，将具有不同优良性能的突变株性能结合，在野生型M-MLV逆转录酶的基础上，采用DNA改组技术，融合多个突变体的结构特点，结合高通量筛选方法得到一系列具有更强抑制物耐受性、更高热稳定性以及催化活性的逆转录酶。
本发明提供一种M-MLV逆转录酶突变体，采用如下技术手段：一种M-MLV逆转录酶突变体，其特征在于，所述M-MLV逆转录酶突变体的氨基酸序列如序列表中的SEQ ID NO.2所示。
一种编码权利要求1所述的M-MLV逆转录酶突变体的核苷酸序列。
优选的，根据上述的的核苷酸序列，其特征在于，所述核苷酸序列如SEQ ID NO.1所示。
一种重组表达载体，其特征在于，是将编码上述的M-MLV逆转录酶突变体的DNA分子克隆入表达载体得到。
一种重组工程细胞株，其特征在于，是将上述的重组表达载体转化入工程细胞得到。
一种逆转录反应试剂盒，其特征在于，包括上述的M-MLV逆转录酶突变体。
优选地，所述逆转录反应试剂盒，其特征在于，还包括PCR用水、逆转录反应缓冲液、dNTPs和逆转录反应引物中的至少一种。
一种如上述M-MLV逆转录酶突变体在逆转录反应中的应用。
本发明的有益效果在于：
(1)本发明在野生型M-MLV逆转录酶的基础上，结合已有对逆转录酶结构研究进展，融合多个突变体的结构特点，通过定点诱变理性设计筛选得到新的逆转录酶。该逆转录酶具有很强的抑制物耐受性，降低了抑制物对cDNA合成的额外干扰，在添加全血以及痰液的样本反应过程中，本发明所述的突变型逆转录酶活性不受影响或影响很小。
(2)本发明提供的M-MLV逆转录酶突变体，能够耐受70℃的高温，在具有较好热稳定性的同时，也具有较高的反应灵敏度和准确性，具有极强的应用前景。
附图说明
图1为本发明实施例2验证逆转录酶灵敏度的PCR扩增曲线图。
具体实施例
本文包括以下的实施例，用于以例证的方式更清楚、明确地阐述本发明的技术方案。本领域技术人员根据本文的公开应当理解，可以在所公开的特定的实施方式中进行许多改变但是仍然获得相似或类似的结果，而不偏离本发明的思想和范围。本发明的具体实施方式仅用于解释本发明，而非意图通过任何方式限制本发明。
实施例1M-MLV逆转录酶突变体的构建
1.逆转录酶突变株质粒构建
根据野生型MMLV逆转录酶蛋白序列(MMLV逆转录酶母本的基因序列见SEQ IDNO.1)，进行适用于大肠杆菌的密码子优化后，插入载体pBAD-hisA中作为野生型逆转录酶的表达质粒。根据逆转录酶结构特点，突变位点主要选择：
(1)参与底物结合和cDNA合成的结构域，突变氨基酸可提高底物结合能力；
(2)连接聚合酶结构域与RNaseH结构域的氨基酸，突变氨基酸提高其稳定性；
(3)RNase H结构域，突变氨基酸以改善提热稳定性或抑制物耐受性及合成长链cDNA能力。
在逆转录酶结构基础上，首先将对野生型MMLV的RNaseH结构域D524及D583定点突变为D524G、D583N，得到突变株1，目的为降低其RNaseH活性。在突变株1的基础上，设计饱和突变引物，将突变株1表达蛋白的聚合酶结构域氨基酸E69、L139、F155、V223、E302、W313、L435进行饱和突变，构建人工DNA文库。文库选用的载体为pBAD-HisA，宿主为DH10B，该系统既可用于基因克隆，也可在用于阿拉伯糖诱导情况下的蛋白表达。
2.蛋白表达及突变体筛选
突变体文库用48孔板进行高通量筛选。从平板中选择克隆接种于含有Amp的TY培养基中，37℃培养过夜后，按照1％转接于含有Amp的TY培养基中，37℃培养至OD600＝1时，添加终浓度为0.8mg/mL L-阿拉伯糖，转移至25℃诱导表达约20h。收集菌体，通过溶菌酶破碎获得逆转录酶粗提液。
(1)筛选热稳定性高的逆转录酶
以1ng鼠总RNA为模板，进行逆转录反应。
反应体系：
试剂(Reagent) 体积(Volume)(μL)
5×RT缓冲液 2.0
10mMdNTPmix(各10mM) 2.0
RNA酶抑制剂(40U/μL) 2.5
Oligo(dT)18引物 1.0
RT(逆转录酶)(40U/μL) 0.1
RNase-freeddH<sub>2</sub>O 补齐至20
反应程序：
Figure BDA0003103680770000041
反应结束后，取1μL反应产物作为qPCR模板，用北京擎科新业生物技术有限公司的2×TSINGKE Master qPCRMaster Mix(SYBR Green I)(货号TSE201)及鼠β-actin基因特异性引物(F：GTGACGTTGACATCCGTAAAGA，R：GCCGGACTCATCGTACTCC)进行PCR扩增，筛选扩增Ct低于野生型MMLV的突变株；
(2)筛选耐受全血样品的逆转录酶
将上述筛选出的具有热稳定性的突变体粗酶液，进行抑制物耐受性筛选。
反应体系：
试剂(Reagent) 体积(Volume)(μL)
5×RT缓冲液 2.0
10mMdNTPmix(各10mM) 2.0
RNA酶抑制剂(40U/μL) 2.5
Oligo(dT)18引物 1.0
RT(逆转录酶)(40U/μL) 0.1
RNase-freeddH2O 补齐至20
如上，向体系中额外添加0.1％的全血样本，逆转录温度仍设置为50℃，筛选扩增Ct低于不添加全血的突变株。
经过两轮筛选后，得到的突变体即为兼具热稳定性高及耐受一定程度全血样本的优良菌株。将得到的突变体质粒进行测序，突变菌株用于扩大培养。
3.蛋白纯化
蛋白表达菌体用高压均质机破碎后，通过Ni-NTA亲和纯化、SP阳离子纯化后，透析浓缩获得纯化后蛋白，蛋白经过透析后储存于储存溶液(50mM Tris-HCl,100mM KCl,1mMDTT,0.1％Triton X-100,50％glycerol,pH 8.0)中。通过BCA法测定蛋白浓度，将储存液浓度调整为0.5mg/mL；酶液经SDS-PAGE电泳显示纯度大于98％。
经过上述筛选过程，最终获得一个逆转录酶，所述逆转录酶的核苷酸序列为：SEQID No.1，对应的氨基酸序列为：SEQ ID No.2。
实施例2M-MLV逆转录酶突变体性能验证
对获得的突变型逆转录酶进行性能评价，选取临床样本进行检测，共设置3组反应体系，所述3组反应体系均按照实施例1所述反应体系进行配置，区别仅在于逆转录酶，其余组分及用量完全相同。
A组：逆转录酶为本发明所述逆转录酶；
B组：逆转录酶为Thermo Fisher的SuperScriptⅢ(SSⅢ)；
C组：逆转录酶为Thermo Fisher的SuperScriptⅣ(SSⅣ)。
(1)M-MLV逆转录酶突变体的热稳定性验证
为检测逆转录酶的热稳定性，将上述3组反应体系分别在50℃、60℃以及70℃下发生逆转录反应，随后利用qPCR技术检测反应结果，结果(Ct值)如下表所示：
表1热稳定性验证
Figure BDA0003103680770000061
注：ΔCt(1)＝Ct(60℃)-Ct(50℃)；ΔCt(2)＝Ct(70℃)-Ct(50℃)。
实验结果显示，本发明所述的M-MLV逆转录酶突变体较市售的两种逆转录酶具有更好的热稳定性，能够耐受70℃的高温，在此温度下，所需逆转录的RNA样本的二级结构基本被打开，且提升了引物与样本的亲和结合能力，降低了体系中的非特异性结合，进而能够提升cDNA的合成效率以及合成cDNA的质量。使用本发明提供的逆转录酶突变体进行逆转录反应时，能够对复杂结构的RNA模板进行高效的逆转录反应，合成更高质量的cDNA。
(2)M-MLV逆转录酶突变体的抑制物耐受性验证
为检测逆转录酶对于痰液抑制物的耐受性，向上述3组反应体系中分别添加0％-20％(v/v)的痰液，随后在60℃下发生逆转录反应，利用qPCR技术检测反应结果，结果如下表所示：
表2痰液抑制物验证
痰液浓度/％ 0 2.0 5.0 10 15 20 ΔCt(1) ΔCt(2) ΔCt(3) ΔCt(4) ΔCt(5)
A 28.32 29.27 29.43 29.75 29.8 29.87 0.95 1.11 1.43 1.48 1.55
B 28.33 31.41 31.56 32.31 34.00 34.15 3.08 3.23 3.98 5.67 5.82
C 28.35 29.55 29.6 30.05 31.25 31.75 1.20 1.25 1.70 2.90 3.40
注：ΔCt(1)＝Ct(2.0％)-Ct(0％)；ΔCt(2)＝Ct(5.0％)-Ct(0％)；ΔCt(3)＝Ct(10％)-Ct(0％)；ΔCt(4)＝Ct(15％)-Ct(0％)；ΔCt(5)＝Ct(20％)-Ct(0％)。
为检测逆转录酶对于全血抑制物的耐受性，向上述3组反应体系中分别添加0％-5％(v/v)的全血，随后在60℃下发生逆转录反应，利用qPCR技术检测反应结果，结果如下表所示：
表3全血抑制物验证
全血浓度/％ 0 0.1 0.5 1.0 2.0 5.0 ΔCt(1) ΔCt(2) ΔCt(3) ΔCt(4) ΔCt(5)
A 21.41 21.02 22.07 24.3 24.71 24.61 -0.39 0.66 2.89 3.30 3.20
B 21.81 22.37 26.05 29.12 30.34 33.05 0.56 4.24 7.31 8.53 11.24
C 21.42 21.21 22.53 26.16 25.79 29.12 -0.21 1.11 4.74 4.37 7.70
注：ΔCt(1)＝Ct(0.1％)-Ct(0％)；ΔCt(2)＝Ct(0.5％)-Ct(0％)；ΔCt(3)＝Ct(1.0％)-Ct(0％)；ΔCt(4)＝Ct(2.0％)-Ct(0％)；ΔCt(5)＝Ct(5.％)-Ct(0％)。
实验结果显示，本发明所述的M-MLV逆转录酶突变体较市售的两种逆转录酶具有更强的耐抑制物能力，在全血及痰液存在的情况下，本发明所述的逆转录酶突变体的活性仍不受影响。在20μL逆转录体系中，能够耐受5.0％(v/v)全血以及20％(v/v)痰液，降低了抑制物组分对cDNA合成的干扰，提高cDNA完整率和得率。使用本发明提供的逆转录酶突变体进行逆转录反应时，能够对复杂结构的RNA模板进行高效的逆转录反应，合成更高质量的cDNA。
(3)M-MLV逆转录酶突变体的反应灵敏度验证
为检测逆转录酶对于低浓度样本的灵敏度，将1μg RNA模板进行10倍梯度稀释，使用上述3组反应体系分别在60℃下发生逆转录反应，利用qPCR技术检测反应结果，结果如下表所示：
表4灵敏度验证
Figure BDA0003103680770000071
Figure BDA0003103680770000081
结果显示，本发明所述的逆转录酶对于低浓度样本的检测灵敏度与C组(ThermoFisher的SuperScriptⅣ(SSⅣ)相当，同时显著优于B组(Thermo Fisher的SuperScriptⅣ(SSII)(如附图1所示)。本发明通过对逆转录酶的理性设计，得到了兼具耐受高温及耐受样本中抑制物的逆转录酶突变体，于此同时，该逆转录突变体具有较优的检查灵敏度，可应用于体外分子诊断试剂中，对于降低对进口产品的依赖具有重要意义。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变形。
Figure BDA0003103680770000091
Figure BDA0003103680770000101
Figure BDA0003103680770000111
Figure BDA0003103680770000121
Figure BDA0003103680770000131
Figure BDA0003103680770000141
Figure BDA0003103680770000151
Figure BDA0003103680770000161
Figure BDA0003103680770000171
Figure BDA0003103680770000181
Figure BDA0003103680770000191
序列表
<110> 重庆中元汇吉生物技术有限公司
<120> 一种M-MLV逆转录酶突变体及其应用
<130> 2021.3.29
<160> 2
<170> SIPOSequenceListing 1.0
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attaaacagt atccgatgag ccagaaagcc cgtctgggta ttaaaccgca tattcagcgt 240
ctgctggatc agggtattct ggttccgtgt cagagcccgt ggaatacacc gctgctgccg 300
gttaaaaaac cgggtacaaa tgattatcgt ccggttcagg atctgcgtga agttaataaa 360
cgcgtggaag atattcatcc gaccgttccg aatccgtata atctgctgag cggtccgcct 420
ccgagccatc agtggtatac cgttctggat ctgaaagatg cctatttttg tctgcgtctg 480
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ggtcaactga cctggacccg tctgccgcag ggttttaaaa atagcccgac cctgtttgat 600
gaggccctgc atcgtgatct ggcagatttt cgtattcagc atccggatct gattctgctg 660
cagtatatgg atgatctgct gctggcagca accagcgaac tggattgtca gcagggcacc 720
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atttgtcaga aacaggtgaa atatctgggc tatctgctga aagaaggtca gcgttggctg 840
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gcagcaccgc tgtatccgct gaccaaaacc ggcaccctgt ttaattgggg tccggatcag 1020
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gacctgacca aaccgtttga actgtttgtg gatgaaaaac agggttatgc aaaaggtgtt 1140
ctgacccaga aactgggtcc gtggcgtcgt ccggttgcat atctgagcaa aaaactggat 1200
ccggttgcag caggttggcc tccgtgtctg cgtatggttg cagcaattgc agttctgacc 1260
aaagatgcag gtaaactgac aatgggtcag ccgctggtta ttggggcacc gcatgcagtt 1320
gaagcactgg ttaaacagcc tccggatcgt tggctgagca atgcccgtat gacccattat 1380
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gcaaccctgc tgccgctgcc ggaagaaggt ctgcagcata attgtctgga tattctggcc 1500
gaagcacatg gcacccgtcc ggatctgaca gatcagccgc tgcctgatgc agatcatacc 1560
tggtataccg gcggtagcag cctgctgcaa gagggccagc gtaaagccgg tgcagcagtt 1620
accaccgaaa ccgaagttat ttgggcaaaa gcactgcctg ctggcaccag cgcacagcgt 1680
gcagagctga ttgcactgac ccaggcactg aaaatggccg aaggtaaaaa actgaatgtg 1740
tataccaaca gccgctatgc atttgcaacc gcacatattc atggcgaaat ttatcgtcgt 1800
cgtggtttgc tgaccagcga aggtaaagaa attaaaaata aagatgaaat tctggccctg 1860
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ggtcatagcg cagaagcacg cggtaatcgt atggcagatc aggcagcacg taaagcagca 1980
attaccgaaa ccccggatac cagcaccctg ctgattgaaa atagcagccc g 2031
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<211> 2031
<212> PRT
<213> MOUSE
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Thr Cys Gly Thr Cys Ala Gly Gly Cys Ala Cys Cys Gly Cys Thr Gly
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Ala Thr Thr Ala Thr Thr Cys Cys Gly Cys Thr Gly Ala Ala Ala Gly
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Cys Ala Ala Cys Cys Ala Gly Cys Ala Cys Ala Cys Cys Gly Gly Thr
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Thr Ala Gly Cys Ala Thr Thr Ala Ala Ala Cys Ala Gly Thr Ala Thr
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Cys Cys Gly Ala Thr Gly Ala Gly Cys Cys Ala Gly Ala Ala Ala Gly
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Cys Cys Cys Gly Thr Cys Thr Gly Gly Gly Thr Ala Thr Thr Ala Ala
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Ala Cys Cys Gly Cys Ala Thr Ala Thr Thr Cys Ala Gly Cys Gly Thr
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Cys Thr Gly Cys Thr Gly Gly Ala Thr Cys Ala Gly Gly Gly Thr Ala
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Thr Thr Cys Thr Gly Gly Thr Thr Cys Cys Gly Thr Gly Thr Cys Ala
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Gly Ala Gly Cys Cys Cys Gly Thr Gly Gly Ala Ala Thr Ala Cys Ala
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Cys Cys Gly Cys Thr Gly Cys Thr Gly Cys Cys Gly Gly Thr Thr Ala
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Thr Gly Ala Thr Thr Ala Thr Cys Gly Thr Cys Cys Gly Gly Thr Thr
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Cys Ala Gly Gly Ala Thr Cys Thr Gly Cys Gly Thr Gly Ala Ala Gly
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Thr Thr Ala Ala Thr Ala Ala Ala Cys Gly Cys Gly Thr Gly Gly Ala
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Ala Gly Ala Thr Ala Thr Thr Cys Ala Thr Cys Cys Gly Ala Cys Cys
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Gly Thr Thr Cys Cys Gly Ala Ala Thr Cys Cys Gly Thr Ala Thr Ala
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Ala Thr Cys Thr Gly Cys Thr Gly Ala Gly Cys Gly Gly Thr Cys Cys
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Gly Cys Cys Thr Cys Cys Gly Ala Gly Cys Cys Ala Thr Cys Ala Gly
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Thr Gly Gly Thr Ala Thr Ala Cys Cys Gly Thr Thr Cys Thr Gly Gly
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Ala Thr Cys Thr Gly Ala Ala Ala Gly Ala Thr Gly Cys Cys Thr Ala
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Cys Ala Thr Cys Cys Gly Ala Cys Cys Ala Gly Cys Cys Ala Gly Cys
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Cys Gly Cys Thr Gly Thr Thr Thr Gly Cys Ala Thr Thr Thr Gly Ala
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Ala Thr Gly Gly Cys Gly Thr Gly Ala Thr Cys Cys Gly Gly Ala Ala
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Ala Thr Gly Gly Gly Thr Ala Thr Thr Ala Gly Cys Gly Gly Thr Cys
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Ala Ala Cys Thr Gly Ala Cys Cys Thr Gly Gly Ala Cys Cys Cys Gly
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Thr Cys Thr Gly Cys Cys Gly Cys Ala Gly Gly Gly Thr Thr Thr Thr
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Ala Ala Ala Ala Ala Thr Ala Gly Cys Cys Cys Gly Ala Cys Cys Cys
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Thr Gly Thr Thr Thr Gly Ala Thr Gly Ala Gly Gly Cys Cys Cys Thr
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Gly Cys Ala Thr Cys Gly Thr Gly Ala Thr Cys Thr Gly Gly Cys Ala
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Gly Ala Thr Thr Thr Thr Cys Gly Thr Ala Thr Thr Cys Ala Gly Cys
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Ala Thr Cys Cys Gly Gly Ala Thr Cys Thr Gly Ala Thr Thr Cys Thr
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Gly Cys Thr Gly Cys Ala Gly Thr Ala Thr Ala Thr Gly Gly Ala Thr
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Gly Ala Thr Cys Thr Gly Cys Thr Gly Cys Thr Gly Gly Cys Ala Gly
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Cys Ala Ala Cys Cys Ala Gly Cys Gly Ala Ala Cys Thr Gly Gly Ala
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Thr Thr Gly Thr Cys Ala Gly Cys Ala Gly Gly Gly Cys Ala Cys Cys
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Cys Gly Thr Gly Cys Ala Cys Thr Gly Cys Thr Gly Cys Ala Gly Ala
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Cys Cys Cys Thr Gly Gly Gly Thr Ala Ala Thr Cys Thr Gly Gly Gly
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Thr Thr Ala Thr Cys Gly Thr Gly Cys Ala Ala Gly Cys Gly Cys Ala
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Ala Ala Ala Ala Ala Ala Gly Cys Ala Cys Ala Gly Ala Thr Thr Thr
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Gly Thr Cys Ala Gly Ala Ala Ala Cys Ala Gly Gly Thr Gly Ala Ala
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Ala Thr Ala Thr Cys Thr Gly Gly Gly Cys Thr Ala Thr Cys Thr Gly
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Cys Thr Gly Ala Ala Ala Gly Ala Ala Gly Gly Thr Cys Ala Gly Cys
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Gly Thr Thr Gly Gly Cys Thr Gly Ala Cys Cys Gly Ala Ala Gly Cys
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Ala Cys Gly Thr Ala Ala Ala Gly Ala Ala Ala Cys Cys Gly Thr Thr
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Ala Thr Gly Gly Gly Thr Cys Ala Gly Cys Cys Gly Ala Cys Cys Cys
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Cys Gly Ala Ala Ala Ala Cys Ala Cys Cys Gly Cys Gly Thr Cys Ala
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Gly Cys Thr Gly Cys Gly Thr Ala Ala Ala Thr Thr Thr Cys Thr Gly
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Gly Gly Thr Ala Cys Ala Gly Cys Ala Gly Gly Thr Thr Thr Cys Thr
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Gly Cys Cys Gly Thr Cys Thr Gly Thr Thr Cys Ala Thr Thr Cys Cys
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Gly Gly Gly Thr Thr Thr Thr Gly Cys Ala Gly Ala Ala Ala Thr Gly
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Gly Cys Ala Gly Cys Ala Cys Cys Gly Cys Thr Gly Thr Ala Thr Cys
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Cys Gly Cys Thr Gly Ala Cys Cys Ala Ala Ala Ala Cys Cys Gly Gly
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Cys Ala Cys Cys Cys Thr Gly Thr Thr Thr Ala Ala Thr Thr Gly Gly
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Gly Gly Thr Cys Cys Gly Gly Ala Thr Cys Ala Gly Cys Ala Gly Ala
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Ala Ala Gly Cys Cys Thr Ala Thr Cys Ala Ala Gly Ala Ala Ala Thr
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Thr Ala Ala Ala Cys Ala Gly Gly Cys Ala Cys Thr Gly Cys Thr Gly
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Ala Cys Cys Gly Cys Ala Cys Cys Gly Gly Cys Ala Cys Thr Gly Gly
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Gly Thr Cys Thr Gly Cys Cys Thr Gly Ala Cys Cys Thr Gly Ala Cys
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Cys Ala Ala Ala Cys Cys Gly Thr Thr Thr Gly Ala Ala Cys Thr Gly
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Thr Thr Thr Gly Thr Gly Gly Ala Thr Gly Ala Ala Ala Ala Ala Cys
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Ala Gly Gly Gly Thr Thr Ala Thr Gly Cys Ala Ala Ala Ala Gly Gly
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Thr Gly Thr Thr Cys Thr Gly Ala Cys Cys Cys Ala Gly Ala Ala Ala
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Cys Thr Gly Gly Gly Thr Cys Cys Gly Thr Gly Gly Cys Gly Thr Cys
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Gly Thr Cys Cys Gly Gly Thr Thr Gly Cys Ala Thr Ala Thr Cys Thr
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Gly Ala Gly Cys Ala Ala Ala Ala Ala Ala Cys Thr Gly Gly Ala Thr
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Cys Cys Gly Gly Thr Thr Gly Cys Ala Gly Cys Ala Gly Gly Thr Thr
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Gly Gly Cys Cys Thr Cys Cys Gly Thr Gly Thr Cys Thr Gly Cys Gly
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Thr Ala Thr Gly Gly Thr Thr Gly Cys Ala Gly Cys Ala Ala Thr Thr
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Gly Cys Ala Gly Thr Thr Cys Thr Gly Ala Cys Cys Ala Ala Ala Gly
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Ala Thr Gly Cys Ala Gly Gly Thr Ala Ala Ala Cys Thr Gly Ala Cys
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Ala Ala Thr Gly Gly Gly Thr Cys Ala Gly Cys Cys Gly Cys Thr Gly
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Gly Thr Thr Ala Thr Thr Gly Gly Gly Gly Cys Ala Cys Cys Gly Cys
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Ala Thr Gly Cys Ala Gly Thr Thr Gly Ala Ala Gly Cys Ala Cys Thr
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Gly Gly Thr Thr Ala Ala Ala Cys Ala Gly Cys Cys Thr Cys Cys Gly
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Gly Ala Thr Cys Gly Thr Thr Gly Gly Cys Thr Gly Ala Gly Cys Ala
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Ala Thr Gly Cys Cys Cys Gly Thr Ala Thr Gly Ala Cys Cys Cys Ala
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Thr Thr Ala Thr Cys Ala Gly Gly Cys Cys Cys Thr Gly Cys Thr Gly
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Cys Thr Gly Gly Ala Thr Ala Cys Cys Gly Ala Thr Cys Gly Thr Gly
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Thr Thr Cys Ala Gly Thr Thr Thr Gly Gly Thr Cys Cys Gly Gly Thr
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Thr Gly Thr Thr Gly Cys Ala Cys Thr Gly Ala Ala Thr Cys Cys Gly
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Gly Cys Ala Ala Cys Cys Cys Thr Gly Cys Thr Gly Cys Cys Gly Cys
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Thr Gly Cys Cys Gly Gly Ala Ala Gly Ala Ala Gly Gly Thr Cys Thr
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Gly Cys Ala Gly Cys Ala Thr Ala Ala Thr Thr Gly Thr Cys Thr Gly
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Gly Ala Thr Ala Thr Thr Cys Thr Gly Gly Cys Cys Gly Ala Ala Gly
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Cys Ala Cys Ala Thr Gly Gly Cys Ala Cys Cys Cys Gly Thr Cys Cys
1505 1510 1515 1520
Gly Gly Ala Thr Cys Thr Gly Ala Cys Ala Gly Ala Thr Cys Ala Gly
1525 1530 1535
Cys Cys Gly Cys Thr Gly Cys Cys Thr Gly Ala Thr Gly Cys Ala Gly
1540 1545 1550
Ala Thr Cys Ala Thr Ala Cys Cys Thr Gly Gly Thr Ala Thr Ala Cys
1555 1560 1565
Cys Gly Gly Cys Gly Gly Thr Ala Gly Cys Ala Gly Cys Cys Thr Gly
1570 1575 1580
Cys Thr Gly Cys Ala Ala Gly Ala Gly Gly Gly Cys Cys Ala Gly Cys
1585 1590 1595 1600
Gly Thr Ala Ala Ala Gly Cys Cys Gly Gly Thr Gly Cys Ala Gly Cys
1605 1610 1615
Ala Gly Thr Thr Ala Cys Cys Ala Cys Cys Gly Ala Ala Ala Cys Cys
1620 1625 1630
Gly Ala Ala Gly Thr Thr Ala Thr Thr Thr Gly Gly Gly Cys Ala Ala
1635 1640 1645
Ala Ala Gly Cys Ala Cys Thr Gly Cys Cys Thr Gly Cys Thr Gly Gly
1650 1655 1660
Cys Ala Cys Cys Ala Gly Cys Gly Cys Ala Cys Ala Gly Cys Gly Thr
1665 1670 1675 1680
Gly Cys Ala Gly Ala Gly Cys Thr Gly Ala Thr Thr Gly Cys Ala Cys
1685 1690 1695
Thr Gly Ala Cys Cys Cys Ala Gly Gly Cys Ala Cys Thr Gly Ala Ala
1700 1705 1710
Ala Ala Thr Gly Gly Cys Cys Gly Ala Ala Gly Gly Thr Ala Ala Ala
1715 1720 1725
Ala Ala Ala Cys Thr Gly Ala Ala Thr Gly Thr Gly Thr Ala Thr Ala
1730 1735 1740
Cys Cys Ala Ala Cys Ala Gly Cys Cys Gly Cys Thr Ala Thr Gly Cys
1745 1750 1755 1760
Ala Thr Thr Thr Gly Cys Ala Ala Cys Cys Gly Cys Ala Cys Ala Thr
1765 1770 1775
Ala Thr Thr Cys Ala Thr Gly Gly Cys Gly Ala Ala Ala Thr Thr Thr
1780 1785 1790
Ala Thr Cys Gly Thr Cys Gly Thr Cys Gly Thr Gly Gly Thr Thr Thr
1795 1800 1805
Gly Cys Thr Gly Ala Cys Cys Ala Gly Cys Gly Ala Ala Gly Gly Thr
1810 1815 1820
Ala Ala Ala Gly Ala Ala Ala Thr Thr Ala Ala Ala Ala Ala Thr Ala
1825 1830 1835 1840
Ala Ala Gly Ala Thr Gly Ala Ala Ala Thr Thr Cys Thr Gly Gly Cys
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Cys Cys Thr Gly Cys Thr Gly Ala Ala Ala Gly Cys Ala Cys Thr Gly
1860 1865 1870
Thr Thr Thr Cys Thr Gly Cys Cys Gly Ala Ala Ala Cys Gly Thr Cys
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Thr Gly Ala Gly Cys Ala Thr Thr Ala Thr Thr Cys Ala Thr Thr Gly
1890 1895 1900
Thr Cys Cys Gly Gly Gly Thr Cys Ala Thr Cys Ala Gly Ala Ala Ala
1905 1910 1915 1920
Gly Gly Thr Cys Ala Thr Ala Gly Cys Gly Cys Ala Gly Ala Ala Gly
1925 1930 1935
Cys Ala Cys Gly Cys Gly Gly Thr Ala Ala Thr Cys Gly Thr Ala Thr
1940 1945 1950
Gly Gly Cys Ala Gly Ala Thr Cys Ala Gly Gly Cys Ala Gly Cys Ala
1955 1960 1965
Cys Gly Thr Ala Ala Ala Gly Cys Ala Gly Cys Ala Ala Thr Thr Ala
1970 1975 1980
Cys Cys Gly Ala Ala Ala Cys Cys Cys Cys Gly Gly Ala Thr Ala Cys
1985 1990 1995 2000
Cys Ala Gly Cys Ala Cys Cys Cys Thr Gly Cys Thr Gly Ala Thr Thr
2005 2010 2015
Gly Ala Ala Ala Ala Thr Ala Gly Cys Ala Gly Cys Cys Cys Gly
2020 2025 2030

Claims (8)

1.一种M-MLV逆转录酶突变体，其特征在于，所述M-MLV逆转录酶突变体的氨基酸序列如序列表中的SEQ ID NO.2所示。
2.编码权利要求1所述的M-MLV逆转录酶突变体的核苷酸序列。
3.根据权利要求2所述的核苷酸序列，其特征在于，所述核苷酸序列如SEQ ID NO.1所示。
4.一种重组表达载体，其特征在于，是将编码权利要求1中所述的M-MLV逆转录酶突变体的DNA分子克隆入表达载体得到。
5.一种重组工程细胞株，其特征在于，是将权利要求4所述的重组表达载体转化入工程细胞得到。
6.一种逆转录反应试剂盒，其特征在于，包括如权利要求1任一项所述的M-MLV逆转录酶突变体。
7.根据权利要求6所述的试剂盒，其特征在于，所述试剂盒还包括PCR用水、逆转录反应缓冲液、dNTPs和逆转录反应引物中的至少一种。
8.一种如权利要求1所述的M-MLV逆转录酶突变体在逆转录反应中的应用。
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CN116515792B (zh) * 2023-04-10 2024-01-26 新镁(上海)生物技术有限公司 Mmlv逆转录酶突变体及其应用

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