CN113174381B - 增强型mmlv逆转录酶突变体及其应用 - Google Patents

增强型mmlv逆转录酶突变体及其应用 Download PDF

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CN113174381B
CN113174381B CN202110638834.5A CN202110638834A CN113174381B CN 113174381 B CN113174381 B CN 113174381B CN 202110638834 A CN202110638834 A CN 202110638834A CN 113174381 B CN113174381 B CN 113174381B
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罗秉轮
戴广伟
曹振
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Abstract

本发明提供一种增强型MMLV逆转录酶突变体，在野生型在MMLV逆转录酶氨基酸序列（如SEQ ID No.1序列所示）进行16个氨基酸位点的突变，氨基酸突变位点如下：A70K；S142P；E201N；D206R；G248D；L300K；A307K；G331P；I434R；S453K；Y522G；E531G；E574G；E596G；E610G；E645G。本发明的MMLV增强型MMLV逆转录酶突变体具有更强的逆转录能力，可用于微量RNA的有效逆转录，可应用在单细胞转录组建库及微量RNA病毒检测等领域。

Description

增强型MMLV逆转录酶突变体及其应用
技术领域
本发明专利涉及增强型MMLV逆转录酶突变体及其应用，属于生物技术领域。
背景技术
在分子检测中经常遇到极低浓度的RNA样本，普通的逆转录酶对这种极低浓度的RNA无法进行有效逆转录，进而影响到了样本中RNA分子的检测，严重限制了微量RNA病毒检测、单细胞RNA分析等应用方面的技术开发。
发明内容
本发明的目的是提供一种增强型MMLV逆转录酶突变体，对野生型MMLV逆转录酶氨基酸序列(SEQ ID No.1)的以下全部16个位点进行突变：A70K；S142P；E201N；D206R；G248D；L300K；A307K；G331P；I434R；S453K；Y522G；E531G；E574G；E596G；E610G；E645G。
本发明的另一目的是提供上述增强型MMLV逆转录酶突变体在进行逆转录反应中的应用。
本发明的增强型MMLV逆转录酶突变体具有更强的逆转录能力，可有效逆转录低至10pg Input Total RNA。本发明的增强型MMLV逆转录酶突变体用于微量RNA的有效逆转录，可应用在单细胞转录组建库及微量RNA病毒检测等领域。
附图说明
图1为阴性对照组扩增产物。
图2为10pg Input RNA使用MMLV逆转录酶突变体扩增产物。
图3为100pg Input RNA使用MMLV逆转录酶突变体扩增产物。
图4为10pg Input RNA使用MMLV逆转录酶野生型扩增产物。
图5为100pg Input RNA使用MMLV逆转录酶野生型扩增产物。
下面结合附图对本发明的具体实施方式做进一步说明。
具体实施方式
实施例1
MMLV逆转录酶突变体，对野生型MMLV以下位点进行突变：A70K；S142P；E201N；D206R；G248D；L300K；A307K；G331P；I434R；S453K；Y522G；E531G；E574G；E596G；E610G；E645G。
参考Smart-seq2单细胞mRNA全长扩增方案，使用该突变的逆转录酶进行微量RNA的逆转录并使用Yeasen高保真酶Super
Figure BDA0003106324200000023
Ⅱ进行逆转录产物扩增，扩增产物使用Hieff
Figure BDA0003106324200000024
DNA Selection Beads进行纯化，纯化产物使用Agilent High SensitivityDNA Kit分析产物大小。实验操作在超净工作台内完成以避免污染。
表1：单细胞建库所用引物
TSO 5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′
Oligo-dT30VN 5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′
ISPCR oligo 5′-AAGCAGTGGTATCAACGCAGAGT-3′
以上合成引物使用1×TE Buffer(nuclease-free)稀释至100μM，分装备用。实验中取适量稀释至所需浓度。
实验方案
以Universal Human Reference RNA(Agilent 740000)为模板，分别使用MMLV逆转录酶突变体和MMLV逆转录酶野生型(简写为WT)对10pg和100pg投入量进行mRNA全长cDNA扩增，同时设置阴性对照组NTC。
1)RNA变性
按照表2的体系于200μL PCR管中加入对应样本及试剂，枪头轻轻吹打混匀，瞬离收集管壁上的液体。将PCR管至于PCR仪中，设置72℃3min进行RNA变性，结束后立即将PCR管至于冰上>1min。
表2：RNA预变性反应体系
Figure BDA0003106324200000021
2)逆转录
按照表3的体系于上一步的PCR管中加入相应试剂，枪头轻轻吹打混匀，瞬离收集管壁上的液体。将PCR管至于PCR仪中，设置42℃90min；70℃15min；4℃hold进行mRNA的逆转录。
表3：逆转录反应体系
Figure BDA0003106324200000022
Figure BDA0003106324200000031
3)扩增
按照表4的体系于上一步的PCR管中加入相应试剂，枪头轻轻吹打混匀，瞬离收集管壁上的液体。
表4：全长cDNA扩增反应体系
Figure BDA0003106324200000032
在PCR仪中运行表5所示的程序，设置NTC组和10pg Input RNA组为18cycles；100pg Input RNA组为16cycles。
表5：全长cDNA扩增反应程序
Figure BDA0003106324200000033
4)扩增产物纯化
PCR产物使用Hieff
Figure BDA0003106324200000041
DNA Selection Beads进行1.0×纯化，纯化产物取1.0μL使用Agilent High Sensitivity DNA Kit分析大小。结果如图1-5所示：从图中可知：10pg和100pg Input Total RNA使用MMLV逆转录酶突变体的扩增产物大小符合mRNA全长扩增结果，比MMLV逆转录酶野生型逆转录效率高，MMLV逆转录酶突变体可有效逆转录低至10pgInput Total RNA，达到单细胞检测级别。
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<110> 翌圣生物科技（上海）股份有限公司
<120> 增强型MMLV逆转录酶突变体及其应用
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Claims (2)

1.一种增强型MMLV逆转录酶突变体，其特征在于：在SEQ ID No.1所示的序列基础上进行16个氨基酸位点的突变，氨基酸突变位点如下：A70K； S142P； E201N；D206R；G248D；L300K；A307K；G331P；I434R；S453K；Y522G；E531G；E574G；E596G；E610G；E645G。
2.权利要求1所述的增强型MMLV逆转录酶突变体在进行逆转录反应中的应用。
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CN114277192B (zh) * 2021-12-30 2022-11-18 南京巨匠生物科技有限公司 用于rt-lamp反应的扩增体系组合物、rna病毒的rt-lamp方法及应用
CN114591930B (zh) * 2022-05-10 2022-08-12 翌圣生物科技(上海)股份有限公司 增强型逆转录酶、编码dna、试剂盒及其在rna文库构建中的应用
WO2024092712A1 (zh) * 2022-11-04 2024-05-10 深圳华大生命科学研究院 Mmlv逆转录酶突变体
WO2024092768A1 (zh) * 2022-11-04 2024-05-10 深圳华大生命科学研究院 一种热稳定逆转录酶及其应用
WO2024092713A1 (zh) * 2022-11-04 2024-05-10 深圳华大生命科学研究院 Mmlv逆转录酶突变体
CN116622669B (zh) * 2023-06-30 2024-06-07 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

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