CN116515792A - Mmlv逆转录酶突变体及其应用 - Google Patents
Mmlv逆转录酶突变体及其应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体涉及MMLV逆转录酶突变体及其应用,本发明基于MMLV‑RT母本,选择7个突变位点基于理性设计进行多轮筛选,得到的MMLV逆转录酶突变体热稳定性有显著提高,在75℃的逆转录温度下仍然能够保持较高的逆转录效率,不仅可以满足当下直接RT‑qPCR检测领域对逆转录酶的需求,而且可以在更高的温度下处理复杂样本,可应用于医学、法医刑侦、食品检测、环境检测等多领域。
Description
技术领域
本发明涉及生物技术领域,具体涉及MMLV逆转录酶突变体及其应用。
背景技术
莫洛尼鼠白血病病毒来源逆转录酶(MMLV-RT)是一种具有以下三种活性的多功能逆转录酶:
1)RNA和DNA依赖性的DNA聚合活性
2)催化RNA-DNA杂合链中RNA裂解的RNase H活性
3)RNA为模板的DNA聚合酶
MMLV-RT已经被广泛用于许多研究应用中,包括cDNA克隆,逆转录酶-PCR定量,微阵列分析,RACE等。
在逆转录反应的过程中,由于RNA二级结构的复杂性使得逆转录效率显著降低。为了降低RNA结构复杂性的干扰,打开RNA二级结构最简单的方法是通过高温使二级结构的氢键打开,RNA变为线性单链,这就要求MMLV-RT的反应温度相应提高。但野生型的MMLV-RT酶最适反应温度为37℃,高温下稳定性显著下降。设计具有增加的热稳定性的MMLV逆转录酶突变体的方法已有报道。如Thermo Fisher使用核糖体展示(RD)和体外区室化技术(IVC)进行高通量筛选,获得了热稳定性显著提升的突变体(CN107058258A)。赛默飞也推出了新一代热稳定性显著改善的SuperScript IV,其热稳定性显著提高到50℃,但实际上还远远无法满足直接RT-qPCR检测领域的应用需求。
发明内容
为了克服上述技术缺陷的不足,本发明运用结构生物学技术和定点突变技术,基于MMLV-RT母本,选择7个突变位点基于理性设计进行多轮筛选,得到耐高温的逆转录酶突变体。
本发明提供的MMLV逆转录酶突变体是在SEQ ID NO.1所示的氨基酸序列中插入、取代、或缺失1个或多个氨基酸,或者在SEQ ID NO.1所示的氨基酸序列的一个或两个末端添加或删除1个或多个氨基酸,与SEQ ID NO.1具有80%同一性且MMLV-RT功能和热稳定性增强的蛋白。优选的具有90%同一性,更优选的具有95%同一性,最优选的,具有99%同一性。
MMLV逆转录酶具有的RNase H活性对于逆转录反应的产率和DNA合成长度有较大影响,去除RNase H活性后对稳定性也有益处。本发明通过对MMLV逆转录酶的蛋白质结构进行分析,选择G22R突变、A32I突变、K53M突变、D524N突变、E302R突变、T128F突变和D524N突变这7个相关位点,显著提高热稳定性且未损失活力。
在一些实施例中,本发明所述的MMLV逆转录酶突变体在大于37℃的温度具有最佳活性,在一些实施例中,所述MMLV逆转录酶突变体在至少42℃,优选为至少50℃,更优选为至少60℃具有最佳活性。在一些具体的实施例中,所述MMLV逆转录酶突变体在50~75℃下的活性是37℃下活性的至少120%,优选的是,50~75℃下活性是37℃下活性的至少130%,更优选的是至少150%。
在一些实施例中,本发明所述的MMLV逆转录酶突变体耐热能力不低于50℃,在一些实施例中,本发明所述的MMLV逆转录酶突变体耐热能力不低于60℃。
在一些更为具体的实施中,本发明提供了一种MMLV逆转录酶突变体,氨基酸序列如SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示,或与SEQ ID NO.2、SEQ ID NO.3、SEQ IDNO.4所示序列具有80%同一性的具有MMLV-RT功能且热稳定性增强的氨基酸序列;优选的具有85%同一性,更优选的具有90%同一性,更优选的具有95%同一性,最优选的,具有99%同一性。
本发明还提供了编码本发明所述的MMLV逆转录酶突变体的核苷酸序列,本领域技术人员充分了解的是,由于同一氨基酸可能有多种不同的密码子来决定,所以编码上MMLV逆转录酶突变体的核苷酸序列并不仅仅局限于一种,可以是由SEQ ID NO.1所示MMLV逆转录酶突变体核苷酸序列突变一个或多个核苷酸形成同义突变得到同样可编码本发明所述突变体氨基酸序列的核苷酸序列,也可以根据密码子优化设计能够编码本发明所述突变体氨基酸序列的核苷酸序列。
在一些具体的实施例中,本发明还提供编码MMLV逆转录酶突变体的核苷酸序列,所述序列可以是如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7所示,或者由SEQ ID NO.2、SEQID NO.3、SEQ ID NO.4所示的MMLV逆转录酶突变体核苷酸序列突变一个或多个核苷酸形成同义突变得到同样可编码本发明所MMLV逆转录酶突变体氨基酸序列的核苷酸序列,也可以根据密码子优化设计能够编码本发明所述MMLV逆转录酶突变体氨基酸序列的核苷酸序列。该序列是专为大肠杆菌表达系统进行密码子优化得到的序列,能显著提高异源基因在宿主菌中的表达效率。
本发明还提供一种包含编码本发明所述的MMLV逆转录酶突变体的核苷酸序列的重组载体。用于构建本发明的表达载体的母载体不受限制,可使用用于原核生物或真核生物转化的任何传统载体。优选为原核表达载体;更优选为pET系列载体;更优选为pET-28a。
本发明还包含一种重组细胞,可通过将所述重组载体插入随机的原核细胞或真核细胞中来构建,包含可编码本发明所述MMLV逆转录酶突变体的核苷酸序列,或包含可编码所述MMLV逆转录酶突变体的核苷酸序列的载体。所述的工程细胞优选为大肠杆菌细胞;更优选为BL21(DE3)细胞。该重组工程细胞株可快速、可溶表达上述重组表达载体。
本发明还提供所述的MMLV逆转录酶突变体、或所述的重组载体、或所述的重组细胞在生物技术领域中的应用。
本发明还提供所MMLV逆转录酶突变体、或所述的重组载体、或所述的重组细胞在逆转录反应领域中的应用。
本发明的有益效果:本发明找到了新的突变位点,经过采用分子理性设计与功能筛选相结合,在较小范围内筛选后获得MMLV逆转录酶突变体,热稳定性有显著提高,在75℃的逆转录温度下仍然能够保持较高的逆转录效率,不仅可以满足当下直接RT-qPCR检测领域对逆转录酶的需求,而且可以在更高的温度下处理复杂样本,可应用于医学、法医刑侦、食品检测、环境检测等多领域。
附图说明
图1为pET28a-MMLV-RT-W质粒图;
图2为逆转录酶突变体热活力测定图。
具体实施方式
为使本领域技术人员更好的理解本发明的技术方案,下面结合具体实施方式对本发明作详细说明。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。除非另外说明,否则百分比和份数按重量计算。除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1突变体构建
MMLV-RT母本的基因序列见SEQ ID NO.1,使用基因合成获得,合成至载体pET28a上,产生pET28a-MMLV-RT-W质粒,具体合成序列包括3’端添加的纯化标签6×His序列和TAA终止密码子,将此质粒转化大肠杆菌BL21感受态细胞进行表达宿主构建,该质粒诱导表达后产生C端融合6×His纯化标签的MMLV-RT。MMLV-RT突变体的构建以质粒pET28a-MMLV-RT-W为原始模板,按照PCR介导的定点突变方法对MMLV-RT编码基因进行定点突变。利用QuikChange Primer Design网站设计突变引物,并按照PCR介导的定点突变方法对MMLV-RT编码基因进行定点突变。PCR的反应总体系为50μL。具体的,将表1中的组分在冰上混匀后,进行PCR扩增,程序如下:(1)98℃预变性3min;(2)98℃变性20s,60℃退火30s,68℃延伸6min;(3)步骤2执行30个循环;(4)68℃延伸10min;5)4℃保温。按照此程序反应后,得到扩增产物和模板DNA的混合物。按照DpnⅠ消化酶的使用说明,向10μL的PCR扩增产物中加入1μLDpnⅠ和9μL相应的缓冲液,在37℃的条件下保温过夜,彻底消化模板DNA分子。
表1定点突变PCR反应体系组成
将过夜消化的产物用天根通用型DNA纯化回收试剂盒(DP214)进行初步的纯化处理,具体步骤参照说明书。纯化后的PCR产物转化至E.coli DH5α感受态细胞中,具体步骤为:(1)从-80℃冰箱里取出50μL的E.coli DH5α感受态细胞置于冰上,静置5min使其融化;(2)在感受态细胞中加入10μL纯化后的PCR产物,轻轻混匀后在冰上静置60min;(3)将感受态细胞置于42℃的水浴中保持45s,迅速取出放回冰上静置5min;(4)加入500μL不含抗生素的LB液体培养基,37℃孵育1-2h;(5)将含有感受态细胞的液体在4℃,5000×g离心1min,去除400μL的上清;(6)将160μL含感受态细胞的液体混匀,均匀涂布在含有100μg/mL氨苄青霉素的LB平板上,正置至液体全部被吸收后,37℃倒置过夜培养。
实施例2突变体的筛选
每个LB平板挑取6-10个单克隆接种至含有100μg/mL氨苄青霉素的LB培养基中过夜,利用Plasmid DNA Midiprep Kits试剂盒提取质粒。将质粒送基因测序(取突变体V5氨基酸序列如SEQ ID NO.2所示,取突变体V18氨基酸序列如SEQ ID NO.3所示,取突变体V21氨基酸序列如SEQ ID NO.4所示),测序结果采用SnapGene软件进行比对处理,测序正确的质粒转化至E.coli BL21(DE3)感受态细胞中,得到含有目的突变基因的重组菌。
实施例3突变体的发酵和纯化
将得到含有目的突变基因的重组菌接种到1mL含有50μg/mL氨苄青霉素的LB液体培养基中,37℃、180rpm摇床培养活化至OD值为0.5-0.6,加入终浓度为0.05mM的诱导剂IPTG在28℃下继续培养,诱导酶表达;10小时后离心收集发酵菌体,取发酵后的菌体以Ni亲和层析柱结合缓冲液重悬后,以细胞高压匀浆破碎仪破碎,低温高速离心收集破碎上清液,4℃环境下使用Ni亲和层析纯化柱进行纯化收集,得到RI突变体纯酶。纯酶使用储存缓冲液配方为15mM Tris-HCl,pH 7.5,50mM KCl,0.1mM EDTA,10mM DTT,50%甘油,-20℃保存。
实施例4逆转录酶突变体热活力测定:
在PCR反应管中依次加入50mM Tris-HCl(pH 8.2)、100mM NaCl,5mM MgCl2、1mMDTT,0.01%Tween-20、1%海藻糖,1×SYBR Green染料,1mM dTTP,0.01μg/μLpoly(A),0.1μM oligo(dT)20引物和2ng逆转录酶。在不同的温度下孵育,每15s测量一次荧光;计算MMLV逆转录酶突变体的荧光曲线的初始斜率,以各自50℃的活力为100%,并和野生型逆转录酶和市面上所购买的商品化的逆转录酶突变体(SuperScript III、SuperScript IV)做对比,结果如图2所示。表明竞品MMLV逆转录酶55℃下初始活力为50℃下的70%,60℃下已基本无活力,而V5、V18、V21在温度50-70℃仍有很高的活力,温度适用范围显著高于野生型和竞品,特别是V5、V18、V21在温度65-70℃下的活力可达到初始活力的60%以上,显示其突变位点的改变对于热活力有显著的提高。
实施例5逆转录酶突变体储存稳定性测定
将逆转录突变体稀释到保存浓度,置于37℃烘箱放置10天测试其储存稳定性。测试条件如下:以293F细胞总RNA为模板,进行逆转录反应;向含有1μg RNA的PCR观众中加入以下反应组分:1μL Oligo(dT)20VN引物,2μL 10×RT mix(含有dNTPs),1μL RNaseinhibitor,经过10天,37度放置后的1μL逆转录酶突变体纯化酶、野生型逆转录酶或竞品逆转录酶(SuperScriptIII、SuperScript IV),用RNase-free ddH2O补足20μL,在55℃之间的不同反应温度下反应10分钟;反应结束后于95℃孵育30s以终止反应;使用1μL逆转录产物作为模板,使用一对GAPDH基因的特异性引物进行qPCR扩增,检测扩增Ct值,结果见表2。
表2各逆转录酶突变体的Ct值
名称 | Ct |
WT | N/A |
V5 | 18.6 |
V18 | 19.2 |
V21 | 18.2 |
SuperScriptIII | 22 |
SuperScriptIV | 18.7 |
可以看到,本发明的逆转录酶突变体Ct值显著低于SuperscriptIII,和SuperscriptIV性能可比,则认为其突变位点的改变对于储存稳定性也有显著提升。
最后需要说明,上述描述仅为本发明的优选实施例,本领域的技术人员在本发明的启示下,在不违背本发明宗旨及权利要求的前提下,可以做出多种类似的表示,这样的变换均落入本发明的保护范围之内。
Claims (8)
1.MMLV逆转录酶突变体,其特征在于:包含与SEQ ID NO.1至少80%同源性的氨基酸序列,其中所述氨基酸序列包括一个或多个选自由以下组成的组的突变:相对于SEQ ID NO.1的G22R突变、A32I突变、K53M突变、D524N突变、E302R突变、T128F突变和D524N突变。
2.根据权利要求1所述的MMLV逆转录酶突变体,其特征在于:所述氨基酸序列如SEQ IDNO.2所示。
3.根据权利要求1所述的MMLV逆转录酶突变体,其特征在于:所述氨基酸序列如SEQ IDNO.3所示。
4.根据权利要求1所述的MMLV逆转录酶突变体,其特征在于:所述氨基酸序列如SEQ IDNO.4所示。
5.编码权利要求2-4中任一项所述MMLV逆转录酶突变体的核苷酸序列。
6.一种重组表达载体,其特征在于:包含权利要求2-4中任一项所述的MMLV逆转录酶突变体的核苷酸序列。
7.一种重组细胞,其特征在于:是将权利要求6中所述的重组表达载体转化入工程细胞得到。
8.权利要求1所述的MMLV逆转录酶突变体、权利要求6所述的重组载体或权利要求7所述的重组细胞在逆转录反应领域中的应用。
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