CN112695019B - 逆转录酶突变体及其应用 - Google Patents

逆转录酶突变体及其应用 Download PDF

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CN112695019B
CN112695019B CN202110304959.4A CN202110304959A CN112695019B CN 112695019 B CN112695019 B CN 112695019B CN 202110304959 A CN202110304959 A CN 202110304959A CN 112695019 B CN112695019 B CN 112695019B
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沈小波
刘博旭
顾玲
柴常升
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Yisheng Biotechnology (Shanghai) Co.,Ltd.
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Abstract

本发明提供一种MMLV逆转录酶突变体，在野生型MMLV逆转录酶氨基酸序列（如SEQ ID No.1序列所示）中进行七个氨基酸位点的突变，氨基酸突变位点为：R205H；V288T；L304K；G525D；S526D；E531G；E574G。该突变体可以降低MMLV逆转录酶对Taq DNA聚合酶的抑制作用，大大提高了一步法RT‑qPCR的灵敏度。

Description

逆转录酶突变体及其应用
技术领域
本发明专利涉及逆转录酶突变体及其应用，属于生物技术领域。
背景技术
野生型的MMLV逆转录酶会和Taq DNA聚合酶相互作用，抑制Taq DNA聚合酶活性，从而大大降低了一步法RT-qPCR扩增效率。亟需一种可以提升检测灵敏度的MMLV逆转录酶，以满足RNA病毒快速检测的临床需求。
发明内容
本发明的目的是提供一种MMLV逆转录酶突变体，在野生型MMLV逆转录酶氨基酸序列（如SEQ ID No.1序列所示）中进行七个氨基酸位点的突变，氨基酸突变位点为：R205H、V288T、L304K、G525D、S526D、E531G和E574G。该突变体可以降低MMLV逆转录酶对Taq DNA聚合酶的抑制作用，大大提高了一步法RT-qPCR的灵敏度。
本发明的另一目的是提供上述的逆转录酶突变体在进行逆转录反应中的应用，以及在提高一步法RT-qPCR扩增效率中的应用。
其中在提高一步法RT-qPCR扩增效率中的应用的步骤为：
（1）合成待测RNA病毒的特异性引物和荧光探针；
（2）在待测样本中加入合成的引物和探针，和逆转录酶突变体和Taq DNA聚合酶，进行逆转录反应，反应过程中检测荧光强度。
优选的，步骤（2）中在逆转录体系中还加入RNA酶抑制剂。
优选的，步骤（2）中逆转录反应体系为：
组分 体积（μL）
2×基础Buffer 12.5
Taq DNA聚合酶 (5U/μL) 0.2-0.6
MMLV逆转录酶(10U/μL) 1-4
RNasin Inhibitor(10U/μL) 0.5-2
Primer/Probe mix 1
RNA模板 5
RNAse Free H2O Up to 25μL
其中2×基础Buffer的配比为：
组分 浓度
Tris-HCL pH8.5-9.0 40-200mM
MgCl<sub>2</sub> 4-16mM
KCl 100-200mM
优选的，步骤（2）中逆转录反应的过程为：反应体系先在50℃保温10min，然后温度升高到95℃保温5min，再进行95℃ 15sec、60℃30sec的循环，循环数为45，循环时在60℃下采集荧光。
本发明的MMLV逆转录酶突变体，是在野生型MMLV逆转录酶的氨基酸序列中取代了7个氨基酸，可以极大降低MMLV逆转录酶对Taq DNA聚合酶的抑制作用，提高了一步法RT-qPCR的灵敏度。使用本发明的突变型MMLV逆转录酶，一步法RT-qPCR灵敏度可提升到1000Copies/ml。
附图说明
图1为野生型逆转录酶和MMLV逆转录酶突变体A一步法RT-qPCR体系ORf1ab基因扩增性能对比。
图2为野生型逆转录酶和MMLV逆转录酶突变体A一步法RT-qPCR体系N基因扩增性能对比。
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。
MMLV逆转录酶突变体，R205H；V288T；L304K；G525D；S526D；E531G；E574G 7个位点都进行了突变。
针对野生型MMLV逆转录酶和MMLV逆转录酶突变体进行一步法RT-qPCR扩增性能检测，采用CDC新型冠状病毒COVID-19引物和探针Orf1ab/N进行新型冠状病毒COVID-19假病毒RNA检测。具体实验过程如下：
一、所用 CDC新型冠状病毒引物和探针如表1所示。
表1、引物和探针序列
引物名称 引物序列 引物标记
Orf1ab-F CCC TGT GGG TTTTAC ACTTAA
Orf1ab-R ACG ATT GTG CATCAG CTGA
Orf1ab-P CCGTCTGCGGTATGTGGAAAGGTTATGG 5端6-FAM，3端BHQ1
N-F GGG GAACTT CTCCTG CTA GAAT
N-R CAG ACATTTTGCTCT CAA GCTG
N-P TTG CTG CTG CTT GAC AGA TT 5端VIC，3端BHQ1
二、一步法RT-qPCR反应
引物探针mix配置如表2所示。
表2、引物探针mix体系
引物母液 体积（μl）
Orf1ab-F(100μM) 10
Orf1ab-R(100μM) 10
Orf1ab-P(100μM) 7
N-F(100μM) 10
N-R(100μM) 10
N-P(100μM) 7
Rnase Free H2O 46
一步法RT-qPCR体系如表3所示，反应程序如表5所示。对比野生型MMLV逆转录酶和MMLV逆转录酶突变体的一步法RT-qPCR体系的扩增性能，①新型冠状病毒COVID-19假病毒（yeasen Cat.no 11900）RNA 106、105、104copies/ml模板测试扩增性能，3个复孔；②灵敏度对比，新型冠状病毒COVID-19假病毒RNA 105、104、1000copies/ml，20个复孔。
表3、一步法RT-qPCR体系
组分 体积（μL）
2×基础Buffer 12.5
Taq DNA聚合酶 (5U/μL) 0.2-0.6
MMLV逆转录酶(10U/μL) 1-4
RNasin Inhibitor(10U/μL) 0.5-2
Primer/Probe mix 1
RNA模板 5
RNAse Free H2O Up to 25μL
2X基础Buffer配置如表4所示。
表4、2X基础Buffer配比
组分 浓度
Tris-HCL pH8.5-9.0 40-200mM
MgCl<sub>2</sub> 4-16mM
KCl 100-200mM
表5、一步法RT-qPCR反应程序
Figure 100502DEST_PATH_IMAGE001
三、反应结果
如图1-2所示，可以看出：MMLV逆转录酶的一步法RT-qPCR体系扩增新型冠状病毒假病毒RNA 106、105、104copies/mL，野生型MMLV逆转录酶反应体系扩增104copies/mL模板未起峰，扩增106、105 copies/mL模板时，Orf1ab和N基因Ct值均明显大于突变型MMLV逆转录酶扩增体系，表明后者扩增性能更佳。
灵敏度对比，每个模板梯度设置20个重复，统计结果如表6所示。
表6、MMLV逆转录酶突变体与野生型MMLV逆转录酶的灵敏度对比结果
野生型MMLV逆转录酶 MMLV逆转录酶突变体A
Orf1ab 10<sup>5</sup>Copies/ml 17/20 20/20
N 10<sup>5</sup>Copies/ml 16/20 20/20
Orf1ab 10<sup>4</sup>Copies/ml 4/20 20/20
N 10<sup>4</sup>Copies/ml 5/20 20/20
Orf1ab 1000Copies/ml 0/20 19/20
N 1000Copies/ml 0/20 20/20
可以看出，MMLV逆转录酶突变体的一步法RT-qPCR体系灵敏度明显高于野生型MMLV逆转录酶体系。
序列表
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Claims (7)

1.一种逆转录酶突变体，其特征在于：在SEQ ID No.1序列的基础上进行七个氨基酸位点的突变，氨基酸突变位点为：R205H、V288T、L304K、G525D、S526D、E531G和E574G。
2.权利要求1所述的逆转录酶突变体在进行逆转录反应中的应用。
3.权利要求1所述的逆转录酶突变体在提高一步法RT-qPCR扩增效率中的应用。
4.根据权利要求3所述的应用，其特征在于其步骤包括：
（1）合成待测RNA病毒的特异性引物和荧光探针；
（2）在待测样本中加入合成的引物和探针，和权利要求1所述的逆转录酶突变体和TaqDNA聚合酶，进行逆转录反应，反应过程中检测荧光强度。
5.根据权利要求4所述的应用，其特征在于：步骤（2）中在逆转录体系中还加入RNA酶抑制剂。
6.根据权利要求5所述的应用，其特征在于：步骤（2）中逆转录反应体系为：
组分 体积：μL 2×基础Buffer 12.5 5U/μL 的Taq DNA聚合酶 0.2-0.6 10U/μL 的逆转录酶突变体 1-4 10U/μL 的RNasin Inhibitor 0.5-2 Primer/Probe mix 1 RNA模板 5 RNAse Free H<sub>2</sub>O Up to 25μL
其中2×基础Buffer的配比为：
组分 浓度 Tris-HCL pH8.5-9.0 40-200mM MgCl<sub>2</sub> 4-16mM KCl 100-200mM
。
7.根据权利要求6所述的应用，其特征在于：步骤（2）中逆转录反应的过程为：反应体系先在50℃保温10min，然后温度升高到95℃保温5min，再进行95℃ 15sec、60℃30sec的循环，循环数为45，循环时在60℃下采集荧光。
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CN113817707B (zh) * 2021-11-23 2022-02-22 中国医学科学院北京协和医院 一种突变重组逆转录酶及其制备方法和应用
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CN118345060A (zh) * 2023-06-30 2024-07-16 翌圣生物科技(上海)股份有限公司 一种鼠白血病病毒逆转录酶突变体及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002022869A2 (en) * 2000-09-13 2002-03-21 Medical Research Council Directed evolution method
WO2003033680A2 (en) * 2001-10-19 2003-04-24 Incyte Genomics, Inc. Kinases and phosphatases
CN101613680A (zh) * 2009-01-23 2009-12-30 广州华银医药科技有限公司 莫洛尼氏鼠白血病病毒逆转录酶突变体及其表达方法和应用
CN103348004A (zh) * 2011-02-09 2013-10-09 株式会社百奥尼 具有改善的热稳定性的逆转录酶
CN108949937A (zh) * 2018-07-16 2018-12-07 翌圣生物科技（上海）有限公司 一步法rt-pcr预混试剂
CN109073634A (zh) * 2016-11-25 2018-12-21 山映生物制药公司 用于诱导神经干细胞的分化和保护的组合物及使用该组合物诱导神经再生的方法
CN110291196A (zh) * 2016-12-14 2019-09-27 宝生物工程株式会社 耐热逆转录酶突变体

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6607898B1 (en) * 1996-03-26 2003-08-19 Oncomedx, Inc. Method for detection of hTR and hTERT telomerase-associated RNA in plasma or serum

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002022869A2 (en) * 2000-09-13 2002-03-21 Medical Research Council Directed evolution method
WO2003033680A2 (en) * 2001-10-19 2003-04-24 Incyte Genomics, Inc. Kinases and phosphatases
CN101613680A (zh) * 2009-01-23 2009-12-30 广州华银医药科技有限公司 莫洛尼氏鼠白血病病毒逆转录酶突变体及其表达方法和应用
CN103348004A (zh) * 2011-02-09 2013-10-09 株式会社百奥尼 具有改善的热稳定性的逆转录酶
CN109073634A (zh) * 2016-11-25 2018-12-21 山映生物制药公司 用于诱导神经干细胞的分化和保护的组合物及使用该组合物诱导神经再生的方法
CN110291196A (zh) * 2016-12-14 2019-09-27 宝生物工程株式会社 耐热逆转录酶突变体
CN108949937A (zh) * 2018-07-16 2018-12-07 翌圣生物科技（上海）有限公司 一步法rt-pcr预混试剂

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
gag-pro-pol polyprotein [Murine leukemia virus]，GenBank:ABU54793.1;Vu,H.et al.;《GenBank》;20070827;第1-2页 *
HBV逆转录酶区M204位点单独突变患者与M204+A181T联合突变患者临床特征的比较;王春亚等;《实用肝脏病杂志》;20100831;第13卷(第4期);第260-262页 *
Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors;Bahram Arezi et al.;《Analytical Biochemistry》;20100125;第400卷;第301-303页 *
莫洛尼鼠白血病病毒逆转录酶的原核表达纯化及其生物学活性;王贤松等;《生物工程学报》;20080525;第24卷(第5期);第903-906页 *

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