CN114214299A - 一种m-mlv逆转录酶及其制备方法 - Google Patents
一种m-mlv逆转录酶及其制备方法 Download PDFInfo
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- CN114214299A CN114214299A CN202210096427.0A CN202210096427A CN114214299A CN 114214299 A CN114214299 A CN 114214299A CN 202210096427 A CN202210096427 A CN 202210096427A CN 114214299 A CN114214299 A CN 114214299A
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- leu
- ala
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- arg
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Abstract
本发明公开了一种M‑MLV逆转录酶及其制备方法。通过构建包含M‑MLV逆转录酶基因序列，将其连接到载体后导入大肠杆菌表达，基因突变体位点包含D524G、E562Q、D538N、G638H其中的一或多种。通过突变关键位点的氨基酸带电荷或疏水性的改变，从而改变M‑MLV逆转录酶的空间结构，减弱M‑MLV RNase H Minus，提高M‑MLV逆转录酶的活性和稳定性。
Description
技术领域
本发明涉及生物酶技术领域，具体为一种逆转录酶，尤其是一种M-MLV逆转录酶及其制备方法。
背景技术
M-MLV转录酶具有极低RNase H的活性，在提高延伸能力的同时，保证了产物的稳定性，有利于下游实验的展开。M-MLV逆转录酶突变体，具有较高的延伸性、反应效率，同时支持高温反应。对于GC含量高或者具有二级结构的RNA模板也具有较好的兼容性。获得的cDNA若需保存，需进行分装后保存于-20℃或-80℃，避免反复冻融，且尽量在半年内使用。根据需要，可以选用Oligo dT、随机引物或者特异性引物作为逆转录引物，得到的cDNA可以用于后续的PCR、qPCR等实验。因此高活性和高稳定性的M-MLV逆转录酶具有重要的技术价值和商业价值。
但是，目前行业内的M-MLV逆转录酶在进行逆转录体系反应的时候活性不高、识别RNA模板的能力较弱，稳定性也不高，进行逆转录反应中的热稳定性差，逆转录反应的得到cDNA的产量也不高。所以如何构建和制备一种新型的M-MLV逆转录酶，使M-MLV逆转录酶的稳定性和活性提高，是本行业研究的热点和难点。
发明内容
本发明的目的在于提供一种M-MLV转录酶及其制备方法，以解决上述背景技术中提出的问题。
本发明内容提供了一种高稳定性的M-MLV逆转录酶的突变体，并给出了检测方法证实该突变体具体更高的活性和稳定性，可以用于ddPCR，达到代替商品酶的目的。
具体的说：
本发明通过提供一种以大肠杆菌重组的M-MLV逆转录酶野生型氨基酸序列(如SEQID NO.1)的基础上，设计突变体位置包含D524G、E562Q、D538N、G638H不局限于其中的一种或多种突变，通过突变关键位点的氨基酸改变氨基酸的带电荷或疏水性，从而改变M-MLV逆转录酶的空间结构，从而减弱M-MLV RNase H Minus，提高M-MLV逆转录酶的活性和稳定性。
通过PCR以及分子克隆技术改变碱基序列，实现氨基酸序列的改变。
另一方面，实现氨基酸突变的突变体M-MLV逆转录酶，与野生型的M-MLV逆转录酶相比，在37℃金属浴上保存65小时，加速实验发现，突变体的的活性和稳定性与商品酶保持一致。
本发明的包括以下步骤：
1)根据突变位点设计引物，在M-MLV逆转录酶野生型DNA序列(如SEQ ID NO.2)的基础上，通过PCR扩增将突变位点的DNA序列改变，并拼接处全长基因序列。
2)将拼接的全长基因序列XhoI和XbaI双酶切，通过T4连接酶克隆在pET-28a载体上。
3)连接产物转化DH5α感受态细胞中通过涂布卡那霉素抗性的平板培养。
4)通过PCR筛选阳性克隆。并通过测序验证突变体。
5)将筛选好的阳性克隆转化为NiCo21(DE3)E.coli感受态细胞(NEB#C2529)或SHuffle T7表达E.coli感受态细胞(NEB#C3029)感受态细胞，诱导表达。
6)大肠杆菌破碎诱导后的细胞，通过疏水层析色谱、亲和层析色谱和凝胶过滤层析色谱分离纯化目的蛋白。
7)对突变体活性进行检测。
8)通过37℃加热，测试突变体的稳定性。
本发明通过改变M-MLV逆转录酶的D524G、E562Q、D538N、G638H四个位点的氨基酸改变，增加了M-MLV逆转录酶的活性和热稳定性。
与现有技术相比，本发明所达到的有益效果是：
本发明的M-MLV逆转录酶具有很高的纯度和很高的酶活，是一种高活性和高稳定性的M-MLV突变体，搭配相应的Buffer具备以下能力：
1.有较强的识别RNA的能力，可将RNA更高效的逆转录为cDNA，转化率达99％以上；
2.M-MLV逆转录酶纯度高，纯度可达98％以上；
3.M-MLV逆转录酶高活性，与商品酶的活性保持基本一致；
4.热稳定性好，4℃放置10天活性基本无变化，25℃放置4天活性剩余约89％；
5.M-MLV逆转录酶合成cDNA的能力：生成的cDNA产量高，可用于Template Switch实验；
6.可以检测到极低浓度的模板，低至1pg的微量模板；
7.通过ddPCR精准定量；
8.实现一步法RT-PCR。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：
图1是实施例4的疏水层析色谱图1；
图2是实施例4的疏水层析色谱图2；
图3是实施例4的疏水层析电泳图1；
图4是实施例4的疏水层析电泳图2；
图5是实施例4的疏水层析电泳图5；
图6是实施例4的疏水层析电泳图6；
图7是实施例5的亲和层析色谱图1；
图8是实施例5的亲和层析色谱图2；
图9是实施例5的亲和层析电泳图1；
图10是实施例5的亲和层析电泳图2；
图11是实施例5的亲和层析电泳图3；
图12是实施例6的凝胶过滤色谱图1；
图13是实施例6的凝胶过滤色谱图2；
图14是实施例6的凝胶过滤电泳图1；
图15是实施例6的凝胶过滤电泳图2；
图16是实施例6的凝胶过滤电泳图3；
图17是实施例8的SLC34A2-ROS1 E4-E32扩增曲线图；
图18是实施例8的CCDC6-RET E1-E12扩增曲线图；
图19是实施例8的SLC34A2-ROS1 E4-E32不同线性稀释梯度扩增效率图；
图20是实施例8的CCDC6-RET E1-E12不同线性稀释梯度扩增效率图；
图21是实施例9的mRT逆转录酶两组样品的蛋白电泳图；
图22是实施例10的mRT和SSⅢ两种酶的信号变化及活性剩余％变化显示图；
图23是实施例11的mRT的不同温度和放置天数活性显示图；
图24是实施例12中的诺唯赞反应后的液滴状态图；
图25是实施例12中的本发明mRT反应后的液滴状态图；
图26是实施例13中的ACTB基因扩增曲线图；
图27是实施例13中的GAPDH基因扩增曲线图；
图28是实施例13中的CD74-ROS1 E6-E34融合扩增曲线图；
图29是实施例13中的SLC34A2-ROS1 E4-E32融合扩增曲线图；
图30是实施例13中的EML4-EML4-ALK E20-E20融合扩增曲线图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
具体实施方式中M-MLV逆转录酶简称为mRT。
实施例1
构建载体
1)根据SEQ ID NO:1合成基因序列并将全基因片段插入载体pET-28a(XhoI和XbaI)载体中。本实施例中使用的是pET-28a表达载体，但不限于此载体。
2)根据突变点D524G、E562Q、D538N、G638H的位置，修改DNA序列使其对应的氨基酸发生改变，可修改为D524G、E562Q、D538N、G638H不局限于其中的一种或多种突变。
3)本领域技术人员充分了解的是，由于同一氨基酸可能有多重不同的密码子来决定，所以构建上述氨基酸位点发生突变对应的合成基因序列并不仅仅局限于一种，可以是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系列的核苷酸序列，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。
实施例2
诱导表达
选取实施例1中四个位点D524G、E562Q、D538N、G638H全部修改后的重组M-MLV质粒。
转化实验:取1支C3029/C2529感受态细胞冰上放置20min，待感受态细胞解冻为液体状态；取1μL 10ng/μL的M-MLV质粒加入到解冻的感受态细胞中(50～100μL)，轻轻弹两下混匀，冰上静止25min；42℃水浴热激90s后迅速冰浴5min；加入1000μL新鲜无抗LB液体培养基，置于摇床200rpm 37℃培养1h；
取200μL均匀涂布于Kan+抗性的LB平板中(0.1％)，37℃恒温培养箱培养16h。
诱导表达:挑取平板中长势正常的单克隆菌落到150mL Kan+抗性的LB液体培养基中(100μg/mL Kan+，原液50mg/mL)，置于摇床200rpm 37℃培养16h。取10mL上述步骤培养好的菌种接种于1L的Kan+抗性的TB液体培养基中(100μg/mL Kan+)，200rpm 37℃培养摇床培养4h左右。使用可见分光光度计监测OD600值，待培养的菌液浓度OD600在0.8～1.0左右时加入IPTG中浓度为0.25mM，等加完IPTG后，温度降到16℃，开始计时诱导表达16h。使用落地离心机收菌，设置6500rpm，4℃，15min，去尽残留培养基收集沉淀，称量所有菌体湿重并记录。
实施例3
菌体处理
高压破碎菌体：使用电子天平称量湿菌体20g，加入30mL M-MLV Ni BindingBuffer(20mM Tris-HCl，500mM NaCl，pH＝8.0@25℃)，使用涡旋混匀仪快速涡旋均匀直至肉眼观察无明显菌块，继续加入M-MLV Ni Binding Buffer涡旋混匀至肉眼观察无明显菌块，最后使用M-MLV Ni Binding Buffer定容至200mL，置于冰浴中备用。高压细胞破碎仪冷阱提前循环预冷至4℃，排尽管路中75％乙醇，加入500mL工艺用水清洗管路，然后加入200mL M-MLV Ni Binding Buffer润洗管路后排空，倒入重悬菌液，1200bar，流速调至50，破碎6次，细胞破碎液置于冰浴暂存。泄压后加入500mL工艺用水冲洗管路，加入200mL75％乙醇冲洗管路，最后保证整个管路浸泡于75％乙醇。
破碎液处理：离心：将破碎液转移至离心管中，两两配平，当离心管为奇数时，使用空白管子加工艺用水配平，15000rpm、4℃离心30min。过滤：将上清转移至干净容器中，依次用0.45μm、0.22μm滤膜过滤，4℃待用。
PEI沉淀：向离心后得到的上清中使用M-MLV Ni Binding Buffer补足至200mL，边磁力搅拌边加入10％PEI母液，直至加入的PEI终浓度为0.4％为止，共计加入8mL，注意缓慢加入，防止目的蛋白沉淀；室温磁力搅拌平衡10min，然后置于4℃放置20min；使用离心机离心，15000rpm，4℃，离心30min，取上清，使用M-MLV Ni Binding Buffer补体积至200mL。
加入3M(NH4)2SO4补盐：使用电导率仪测试上清，记录电导值。加入3M(NH4)2SO4母液，边磁力搅拌边加入(少量多次加入上清中)，直至电导显示为100ms/cm左右。共计记录加入3M(NH4)2SO4的体积，待完全加入后室温搅拌平衡10min。0.22μm水相微孔滤膜过滤，量取上清液体积，准备上样。
实施例4
疏水柱纯化(疏水层析，XK26/200mm，CV＝50mL)
填料：Capto Butyl
体积：25mL
系统流速：10mL/min
上样量：200mL
缓冲液：
M-MLV HIC Buffer A :20mM Tris-HCl，500mM(NH4)2SO4，pH＝8.0@25℃
M-MLV HIC Buffer B:20mM Tris-HCl，pH＝8.0@25℃
管道清洗
对工艺用水、M-MLV Capto Butyl Buffer A、M-MLV Capto Butyl Buffer B超声脱气5分钟处理。
将AKTA的A1、A2、B1、B2、S1和Buffer泵放入H2O中，设置Pump B2、A2、B1、S1、Buffer和A1 Pump Wash，并执行。
系统泵及Buffer放置
将AKTA的A1、A2、B1、B2、S1和Buffer泵放入下表Buffer中
| Pump | Moving phase |
| System Pump A1 | M-MLV Capto Butyl Buffer A |
| System Pump A2 | 工艺用水 |
| System Pump B1 | M-MLV Capto Butyl Buffer B |
| System Pump B2 | 0.2M NaOH |
| Sample Pump Buffer | M-MLV Capto Butyl Buffer A |
| Sample Pump S1 | M-MLV Capto Butyl Buffer A/Sample |
设置Pump B2、A2、B1、S1、Buffer和A1 Pump Wash，并执行。
设置A2泵流速0.5mL/min，连接层析柱。
上样纯化
样品：补盐过滤后上清液，分别记录体积，pH和电导值。
方法设置：
管道和纯化柱清洗：待实验结束后，将层析柱依次使用20％乙醇清洗，将层析柱及系统管路保存在20％乙醇中，拆下层析柱，连接AKTA管路，将层析柱4℃保存。
将实施例3中制得的样品按照实施例4的方法进行上样疏水层析，获得的疏水层析色谱图详见图1、图2，获得的疏水层析电泳图详见图3、图4、图5、图6。
收集1B1—3E9进行实施例5的Ni亲和层析。
实施例5
亲和柱纯化(亲和层析，XK26/200mm，CV＝50mL)
1.填料：Ni琼脂糖HP
2.体积：25mL
3.系统流速：10mL/min
4.上样量：420mL
5.缓冲液：
M-MLV Binding Buffer:20mM Tris-HCl，500mM NaCl，pH＝8.0@25℃
M-MLV Wash Buffer:20mM Tris-HCl，500mM NaCl，20mM Imidazole，2％TritonX-100，pH＝8.0@25℃
M-MLV Elution Buffer:20mM Tris-HCl，500mM NaCl，150mM Imidazole，pH＝8.0@25℃
管道清洗
对工艺用水、M-MLV Ni Binding Buffer buffer、M-MLV Ni Wash Buffer、M-MLVNi Elution Buffer分别超声脱气5分钟处理。
将AKTA的A1、A2、B1、B2、S1和Buffer泵放入工艺用水中，设置Pump B2、A2、B1、S1、Buffer和A1 Pump Wash，并执行。
系统泵及Buffer放置
将AKTA的A1、A2、B1、B2、S1和Buffer泵放入下表Buffer中
设置Pump B2、A2、B1、S1、Buffer和A1 Pump Wash，并执行。
设置B2泵流速0.5mL/min，连接层析柱。
上样纯化
样品：Capto Butyl目的蛋白洗脱液，记录体积。
方法设置：
管道和纯化柱清洗：待实验结束后，将层析柱依次使用500mM咪唑、工艺用水、20％乙醇清洗，将层析柱及系统管路保存在20％乙醇中，拆下层析柱，连接AKTA管路，将层析柱4℃保存。
实施例4中的1B1—3E9样品经过以上亲和层析，样品处理后获得的亲和层析色谱图见图7、图8，亲和层析电泳图见图9、图10和图11。
收集1B5—2C9进行实施例6的凝胶过滤层析。
实施例6
凝胶过滤层析(16mm×700mm，CV＝120mL)
预装柱：HiLoad Superdex 200pg
体积：120mL
系统流速：1mL/min
上样量：1.2mL
缓冲液：
M-MLV Gel Buffer:100mM Tris-HCl，500mM NaCl，2mM DTT，0.2mM EDTA，pH＝7.6@25℃
管道清洗
对工艺用水、M-MLV Gel Buffer超声脱气处理。
将AKTA的A1、A2、B1、B2、S1和Buffer泵放入工艺用水中，设置Pump B2、B1、A2、S1、Buffer和A1 Pump Wash，并执行。
系统泵及Buffer放置
将AKTA的A1、B1、A2、B2、S1和Buffer泵放入下表Buffer中
| Pump | Moving phase |
| System Pump A1 | M-MLV Gel Buffer |
| Sample Pump Buffer | M-MLV Gel Buffer |
| Sample Pump S1 | M-MLV Gel Buffer/sample |
| System Pump B1 | 工艺用水 |
| System Pump A2 | 0.2M NaOH |
| System Pump B2 | 20％乙醇 |
设置Pump B1、S1、Buffer和A1 Pump Wash，并执行。
设置B1泵流速0.5mL/min，连接层析柱。
上样纯化
样品：Ni柱后目的洗脱液，使用超微量分光光度计测试UV280浓度，消光系数为K＝1.355，记录浓度和体积。加入终浓度为0.05％的Tween 20，使用30KD的超滤管对蛋白洗脱液进行离心(3500rpm，4℃)，使浓度小于10mg/mL，可分批上样，上样量为1～2mL。
方法设置：
管道和纯化柱清洗：待实验结束后，将层析柱及系统管路保存在20％乙醇中，拆下层析柱，连接AKTA管路，将层析柱4℃保存。
备注：如果压力不高，小于0.5Mpa，可以将流速调整为1.6mL/min。
1B5—2C9的凝胶过滤色谱图见图12、图13，凝胶过滤电泳图见图14、图15和图16。
收集1C11—1E9进行实施例7的膜包浓缩。
实施例7
膜包浓缩
使用UV280测试蛋白浓度，消光系数K＝1.355，定量步骤收集的蛋白浓度；
清洗膜包：将膜包连接至蠕动泵上，设置蠕动泵转速300rpm，将进液管放入超纯水中，出液管放入废液桶中，开始清洗膜包，清洗体积约200mL后清空膜包中的液体。
蛋白浓缩：将进液管、出液管同时放入待浓缩酶液中，启动蠕动泵开始浓缩，浓缩至“M-MLV Gel”体积为膜包最小体积。然后清空膜包及管道内酶液。
膜包浓缩至浓度为1.00mg/mL，经0.22μm滤膜过滤后，使用UV280测试蛋白浓度，消光系数K＝1.355，定量过滤的蛋白浓度＝1.00mg/mL；
在使用1×M-MLV Storage Buffer with Glycerol(50mM Tris-HCl，150mM NaCl，1mM DTT，0.1mM EDTA，50％Glycerol，pH＝7.6@25℃)稀释至0.50mg/mL；将高浓度成品M-MLV(0.50mg/mL)，母液分成5mL/tube，-20℃保存。
实施例8
实验操作：
样本稀释：从-80℃取样本1-样本8，放与冰上融化，混匀离心后，
样本信息如下：
| 序号 | 对应样本 | 融合基因 | 融合位置 | 样本货号 | 批号 | 浓度 | 体积 |
| 7 | 样本7 | SLC34A2-ROS1 | E4-E32 | CBP20023R | CGD2020060516 | 60ng/μL | 20μL |
| 8 | 样本8 | CCDC6-RET | E1-E12 | CBP20014R | CGD2020060518 | 60ng/μL | 20μL |
将所有的RNA标准品在冰上融化后混匀离心，分别加入580μL的TE buffer混匀离心后为2ng/μL的工作液，为S1；
分别吸取10μL S1加入0.2mL PCR管中，再分别加入90μL TE buffer，混匀离心后为S2；
分别吸取10μL S2加入0.2mL PCR管中，加入90μL TE buffer，混匀离心后为S3；
分别吸取10μL S3加入0.2mL PCR管中，加入90μL TE buffer，混匀离心后为S4；
所有样本混匀离心后，4℃放置备用
本发明的mRT反应体系配置：
反应程序；
实验结果：
SLC34A2-ROS1 E4-E32扩增曲线如图17所示；
CCDC6-RET E1-E12扩增曲线如图18所示；
SLC34A2-ROS1 E4-E32不同线性稀释梯度的扩增效率见下表，扩增效率如图19所示；
| 线性稀释梯度 | 拷贝数 | SLC34A2-ROS1 E4-E32 |
| 6 | 8125 | 24.9 |
| 5 | 812.5 | 28.2 |
| 4 | 81.25 | 31.6 |
| 3 | 8.125 | 34.9 |
CCDC6-RET E1-E12不同线性稀释梯度的扩增效率见下表，扩增效率如图20所示。
| 线性稀释梯度 | 拷贝数 | CCDC6-RET E1-E12 |
| 6 | 1465.62 | 25.87 |
| 5 | 146.65 | 29.2 |
| 4 | 14.665 | 32.5 |
| 3 | 1.4665 | 36.2 |
实验结论：由本实施例可以得知：mRT有较强的识别RNA的能力，可将RNA更高效的逆转录为cDNA，转化率高达95-100％以上。
实施例9
mRT纯度实验
方法：通过SDS-PAGE电泳，扫描电泳图谱，计算待测样品的纯度。
仪器：SDS-PAGE电泳装置、金属浴、离心机、凝胶成像仪。
试剂：PAGE凝胶快速制备试剂盒、灭菌水、5×蛋白上样缓冲液(含DTT)、彩虹180广谱蛋白Marker、5×Tris-甘氨酸电泳缓冲液、0.05％考马斯亮蓝G250染色液。
操作步骤：
1、样品处理
将待测样品浓缩处理。使用超滤管，加入400μL待测酶，使用离心机，离心2000rpm，10min。量取超滤管上层的酶溶液体积，转移至离心管。根据酶的浓度和浓缩倍数计算超滤后酶溶液的浓度。
使用5×蛋白上样缓冲液(含DTT)稀释待测样品，至终浓度0.40μg/μL和0.05μg/μL。待测样品的稀释量，具体详见下表
| 稀释后的浓度 | 待测样品浓度 | 待测样品体积 | 5×蛋白上样缓冲液(含DTT)体积 |
| 0.40μg/μL | 0.60μg/μL | 50.0μL | 25.0μL |
| 0.40μg/μL | 0.50μg/μL | 50.0μL | 12.5μL |
| 0.05μg/μL | 0.40μg/μL | 10.0μL | 70.0μL |
待测样品的上样量详见下表。上样量为5μg时需做两管重复，其它为1管。
待测样品的上样量电泳、染色及脱色、凝胶成像和记录按照《SDS-PAGE电泳作业指导书》的步骤3至6。
2、分析数据
计算蛋白质纯度
(1)上样量0.3μg和0.4μg均有条带，当上样量为5μg的电泳图谱中无杂带时，则蛋白质纯度为＞94％；当上样量为5μg的电泳图谱中杂带灰度之和小于0.3μg条带灰度时，则蛋白质纯度为＞94％；当上样量为5μg的电泳图谱中杂带灰度之和小于0.4μg条带灰度时，则蛋白质纯度为＞92％。
(2)上样量0.4μg和0.5μg均有条带，当上样量为5μg的电泳图谱中无杂带时，则蛋白质纯度为＞92％；当上样量为5μg的电泳图谱中杂带灰度之和小于0.4μg条带灰度时，则蛋白质纯度为＞92％；当上样量为5μg的电泳图谱中杂带灰度之和小于0.5μg条带灰度时，则蛋白质纯度为＞90％。
(3)上样量0.5μg和0.6μg均有条带，当上样量为5μg的电泳图谱中无杂带时，则蛋白质纯度为＞90％；当上样量为5μg的电泳图谱中杂带灰度之和小于0.5μg条带灰度时，则蛋白质纯度为＞90％；当上样量为5μg的电泳图谱中杂带灰度之和小于0.6μg条带灰度时，则蛋白质纯度为＞88％。
(4)其他纯度对比分析，以此类推。
实验结论：mRT逆转录酶两组样品的蛋白电泳图见图21，mRT逆转录酶大小为78kDa，mRT(20200820#1P1)纯度为＞96％，mRT(20200820#1P2)纯度为＞98％。
实施例10.
mRT逆转录酶的酶活测定
具体实验操作如下：
mRT：200U/μL，SSⅢ：Invitrogen，200U/μL；
MS2序列如SEQ ID NO.3所示；MS2-B序列如SEQ ID NO.4所示。
样品处理方法如下：
Mix1配制(24μL/反应)
| 样品 | 体积/μL | 数量 | 总体积/μL |
| 5×逆转录buffer | 5 | 20 | 100 |
| 10uM MS2-B | 0.5 | 20 | 10 |
| 100mM dNTP | 0.2 | 20 | 4 |
| 100×SG | 0.1 | 20 | 2 |
| 18兆水 | 18.09 | 20 | 361.8 |
| Rnase inhibitor | 0.1 | 20 | 2 |
| mRT 200U/μL | 0.01 | 20 | 0.2 |
5×逆转录buffer
| 序号 | 试剂 | 厂家/来源 | 批号 | 浓度 | 终浓度 | 1mL加入的量μL | 10mL加入的量μL |
| 1 | Tris-HCl | 自配 | 20210311#1 | 1M | 250mM | 250 | 2500 |
| 2 | KCl | 自配 | 20210418#1 | 3M | 375mM | 125 | 1250 |
| 3 | MgCl<sub>2</sub> | Sigma | 20200214 | 1M | 50mM | 50 | 500 |
| 4 | BSA | TAKARA | AI81941A | 20mg/mL | 0.5mg/mL | 25 | 250 |
| 5 | CA-630 | 自配 | 20200302 | 10％ | 1％ | 100 | 1000 |
| 6 | VC-Na | 自配 | 20200302 | 100mM | 5mM | 50 | 500 |
| 7 | 海藻糖 | 自配 | 20200302 | 10mg/mL | 2.5mg/mL | 250 | 2500 |
| 8 | DTT | SIGMA | BCCB2147 | 1M | 2.5mM | 2.5 | 25 |
| 9 | NF水 | Thermo | 20200302 | / | / | 147.5 | 1475 |
将上述Mix混匀后，均分4管每管100μL，一管放置-20℃冰箱保存，其余三管置于PCR加热37℃65小时，加热完成后放置-20℃冰箱保存。
Mix2配制(24μL/反应)
| 样品 | 体积:μL | 数量 | 总体积：μL |
| 5×逆转录buffer | 5 | 20 | 100 |
| 10uM MS2-B | 0.5 | 20 | 10 |
| 100mM dNTP | 0.2 | 20 | 4 |
| 100×SG | 0.1 | 20 | 2 |
| 18兆水 | 18.09 | 20 | 361.8 |
| Ribo lock inhibitor | 0.1 | 20 | 2 |
| SSⅢ200U/μL(Invitrogen) | 0.01 | 20 | 0.2 |
将上述Mix混匀后，均分4管每管100μL，一管放置-20℃冰箱保存，其余三管置于PCR加热37℃65小时，加热完成后放置-20℃冰箱保存。
稀释MS2至250ng/μL MS2 0.8mg/mL，取10μL+21μL 1×TE(10mMTris-HCl1mM EDTAPH＝8.0)+1μL Rnase inhibitor
上述8管样品处理完成后，每管均分3个反应(24μL/反应)，每反应加1μL 250ng/μLMS2，另做不加MS2对照。
反应条件：50℃，30s，35循环。
数据处理：
以加热前信号变化为基础，不同加热时间的信号变化分别除以加热前信号，得出不同加热时间活性剩余百分比。同时需要横向比较每组加热前以及同样加热条件的信号变化值作为参照。
实验结果：
两种酶的信号变化及活性剩余％详见图22。
实验结论：mRT酶活性与商品酶的活性保持基本一致。
实施例11
mRT酶的热稳定性检测
具体实施方式如下：
试剂：5×逆转录buffer，50uM/L MS2-B，0.8mg/mL MS2，75％甘油，100mM dNTP，40U/μLRibiolock，100×SG，15兆水；
材料：96孔透明板、96孔白板、1.5mL EP管、10μL枪头、250μL枪头、1000μL枪头；
仪器：Roche480、10μL移液枪、20μL移液枪、200μL移液枪、1000μL移液枪、离心机、低温PCR冰盒；
样品处理：
取mRT(450U/μL)400μL 20200820#1P2，加入500μL 1×RT storage buffer稀释到200U/μL，分装50μL一管，共10管，一管放在4度冰箱保存，另外各取9个放在Thermo mixer做25度加热。剩余放在-20度冰箱中保存，放置日期为20210105
mRT 200U/μL 20200820#1P2(-20℃放置)
mRT 200U/μL 20200820#1P2(4℃放置)
mRT 200U/μL 20200820#1P2放置于25度Thermo mixer加热20210108取出放置-20℃冰箱保存
mRT 200U/μL 20200820#1P2放置于25度Thermo mixer加热20210109取出放置-20℃冰箱保存
mRT 200U/μL 20200820#1P2放置于25度Thermo mixer加热20210112取出放置-20℃冰箱保存
mRT 200U/μL 20200820#1P2放置于25度Thermo mixer加热20210115取出放置-20℃冰箱保存
样品稀释：(在不高于20℃环境中，在冰盒上进行稀释操作及Mix配制)
20210115将上述6个样品从保存环境取出，分别各做50×稀释：4μL待稀释样品+196μL 1×逆转录buffer 20210110
Mix配制
| 样品 | 体积:μL | 数量 | 总体积：μL |
| 5×逆转录buffer | 5μL | 30μL | 150μL |
| 50uM MS2-B | 0.1μL | 30μL | 3μL |
| 0.8μg/μL MS2(Roche) | 0.3μL | 30μL | 9μL |
| 100mM dNTP | 0.2μL | 30μL | 6μL |
| 100×SG | 0.1μL | 30μL | 3μL |
| 15兆水 | 16.5μL | 30μL | 495μL |
| 75％甘油 | 1.7μL | 30μL | 51μL |
| Ribo lock inhibitor(thermo) | 0.1μL | 30μL | 3μL |
| mRT变量 | 1μL | 30μL | 30μL |
每组样品4个重复
反应条件：46℃、30s、80cycles
数据处理：第80个循环的信号值减去第15个循环的信号值。
实验结果：
mRT的不同温度和放置天数活性见图23所示。
实验结论：热稳定性检测：mRT突变体稳定性好，200U/μL 4℃放置10天活性基本无变化，25℃放置4天活性剩余约89％。
实施例12
验证本发明获得的mRT可以用于ddPCR，达到代替商品酶的目的
实验操作
反应体系配置
Vazyme Mix对照体系(厂家：Vazyme货号：Q222)
配制好的反应体系混匀离心后分装20μL到96孔板中，剩余冰上放置。
本发明的mRT反应体系配置
配制好的反应体系混匀离心后分装到4个0.2mL PCR管中，每管56.5μL(18.8×3)，然后加入相对应的A-Taq酶和A-Fast Taq酶各3.6μL，混匀离心后，各吸取20μL加入96孔板中，剩余反应液用于ddPCR检测。
剩余反应液分别吸取20μL进行液滴制备，每组反应2孔，制备液滴C1000上进行PCR反应，反应程序如下：
反应结束后，镜下观察液滴状态。
实验结果：
对照体系：诺唯赞反应后的液滴状态如图24所示；本发明mRT反应后的液滴状态如图25所示；
实验结论：本发明的mRT亮液滴远远多于诺唯赞。
实施例13.
mRT可以用于一步法RT-PCR，进行融合基因检测，替代Vazyme Q222试剂盒进行一步法荧光定量RT-PCR检测，确定检测灵敏度。
实验操作：
样本稀释：从-80℃取样本1-样本8，放与冰上融化，混匀离心后，
样本信息如下：
| 序号 | 对应样本 | 融合基因 | 融合位置 | 样本货号 | 批号 | 浓度 | 体积 |
| 1 | 样本1 | CD74-ROS1 | E6-E34 | CBP2070R | CBD2020052912 | 60ng/μL | 20μL |
| 3 | 样本3 | EML4-ALK | E20-E20 | CBP20168R | CBD2020082301 | 60ng/μL | 20μL |
| 7 | 样本7 | SLC34A2-ROS1 | E4-E32 | CBP20023R | CGD2020060516 | 60ng/μL | 20μL |
将所有的RNA标准品在冰上融化后混匀离心，分别加入580μL的TE buffer混匀离心后为2ng/μL的工作液，为S1；所有样本混匀离心后，4℃放置备用。
本发明的mRT反应体系配置如下表：
Vazyme Q222 Mix反应体系配置：
反应程序：
实验结果：
ACTB基因扩增曲线如图26所示；
GAPDH基因扩增曲线如图27所示；
CD74-ROS1 E6-E34融合扩增曲线如图28所示；
SLC34A2-ROS1 E4-E32融合扩增曲线如图29所示；
EML4-EML4-ALK E20-E20融合扩增曲线如图30所示。
前面的扩增曲线是本发明的反应体系，后面的是Vazyme反应体系。可以发现本发明的反应体系，无论是Ct还是荧光信号都优于Vazyme反应体系。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 苏州译酶生物科技有限公司
<120> 一种M-MLV逆转录酶及其制备方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 850
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gly Gly Ser Arg Asn Asn Phe Val Leu Glu Gly Asp Leu Asn Met Arg
1 5 10 15
Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Val Asp
20 25 30
Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly Leu Thr
35 40 45
Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala Lys Ser
50 55 60
Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val Val Phe
65 70 75 80
Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly Tyr Lys
85 90 95
Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu Ala Leu
100 105 110
Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu Val Pro
115 120 125
Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys Ala Glu
130 135 140
Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp Leu Tyr
145 150 155 160
Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly Tyr Leu
165 170 175
Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro Asp Gln
180 185 190
Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn Leu Pro
195 200 205
Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu Glu Glu
210 215 220
Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu Lys Pro
225 230 235 240
Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys Leu Ser
245 250 255
Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val Asp Phe
260 265 270
Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe Leu Glu
275 280 285
Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu Glu Ser
290 295 300
Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala Phe
305 310 315 320
Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp Leu Leu
325 330 335
Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro Glu Pro
340 345 350
Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu Ala Lys
355 360 365
Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro Pro Gly
370 375 380
Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr
385 390 395 400
Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu Glu Ala
405 410 415
Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly
420 425 430
Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu Val Glu
435 440 445
Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly Val Arg
450 455 460
Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala Glu Glu
465 470 475 480
Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His Pro Phe
485 490 495
Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp Glu Leu
500 505 510
Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr
515 520 525
Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val Glu
530 535 540
Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr Tyr Ile
545 550 555 560
Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu His Thr
565 570 575
Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp
580 585 590
Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln Arg Ile
595 600 605
Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala Leu Asp
610 615 620
Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly Asp Glu
625 630 635 640
Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr Glu Thr
645 650 655
Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met
660 665 670
Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly Met Ser
675 680 685
Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu Ala Gln
690 695 700
Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg Ala Trp
705 710 715 720
Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val Glu Thr
725 730 735
Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg Val Lys
740 745 750
Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro Val Gln
755 760 765
Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu Phe Pro
770 775 780
Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His Asp Glu
785 790 795 800
Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala Arg Leu
805 810 815
Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro Leu Glu
820 825 830
Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu Leu Glu
835 840 845
Gly Gly
850
<210> 2
<211> 2562
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggtggttcta gaaataattt tgtttaactt taagaaggag atttaaatat gcgtggtatg 60
ttaccattat ttgaaccaaa aggtcgtgtt ttattggttg atggtcatca tctggcctat 120
cgtacctttc atgcactgaa gggattgaca acctcccgtg gcgaacccgt ccaggcagtg 180
tatggattcg caaagtcgct gcttaaagcg cttaaagaag acggagatgc agtcatcgtc 240
gtattcgacg caaaggcgcc gtcctttcgt catgaagcat atggtggcta taaggccggt 300
cgcgctccaa cgccggagga ctttcctcgt cagcttgccc ttattaaaga gttagtcgac 360
ctgctgggtt tggctcgcct tgaagtgcca ggctacgaag cggacgatgt tttagcctcg 420
ttagcgaaaa aggcggaaaa ggaggggtat gaagtgcgta tcctgacagc cgacaaggac 480
ctttaccaac ttttatcaga ccgcattcat gtcttgcacc ctgaaggcta cttgattacg 540
ccagcctggc tttgggaaaa gtatgggttg cgtccagacc aatgggctga ttaccgtgca 600
cttactggcg acgaaagtga taatttgccg ggcgtcaaag gcattggtga aaagacggcc 660
cgcaaattac ttgaggaatg gggtagctta gaggcccttt tgaagaacct ggaccgcctt 720
aaacctgcaa tccgtgaaaa aatcttggct cacatggatg atttaaaatt atcatgggat 780
cttgctaagg ttcgcacaga cttgccactg gaagtcgatt tcgcgaaacg tcgtgaacct 840
gatcgcgagc gccttcgtgc gttcttagaa cgtcttgaat ttggctcgct tctgcatgag 900
tttggactgt tagagtctcc aaaagcgctg gaggaggcgc catggccacc gcctgaggga 960
gcatttgtgg gttttgtttt atcgcgtaaa gaaccaatgt gggcggatct tctggcactg 1020
gctgctgcac gcggtgggcg tgtacaccgc gcgccagagc cctataaagc cttgcgcgat 1080
ttgaaagagg cgcgcggctt gcttgccaag gacctttccg tccttgcatt acgtgagggt 1140
ttgggtttac cgcccggaga cgatccgatg ctgttagcgt atttgttaga cccaagcaac 1200
acgacaccag aaggagtggc acgccgttac gggggagagt ggactgaaga agcaggcgag 1260
cgcgccgcat tatctgaacg tttgtttgcc aatctttggg ggcgcttgga gggtgaggag 1320
cgtcttttat ggctgtatcg tgaagtcgaa cgcccgttga gtgctgtatt agcgcatatg 1380
gaagcgaccg gagtccgcct tgacgtagct tatttgcgcg ccctttcact ggaagtggcc 1440
gaggagatcg cacgtttgga agcggaggtg tttcgtctgg ccggtcaccc cttcaatctg 1500
aatagtcgcg accagttgga acgcgtatta ttcgatgagc ttggtcttcc ggccatcggc 1560
aagactgaga agactggaaa gcgtagcacc agtgccgcgg tgttggaagc cctgcgcgaa 1620
gcacatccta ttgtcgaaaa gattcttcaa tatcgcgaat tgaccaagtt gaagtcgact 1680
tacattgatc cacttcctga ccttattcat cctcgcacag gccgtcttca tactcgcttt 1740
aatcaaaccg caacagcgac cggacgtttg agtagtagcg accctaacct tcaaaatatt 1800
ccggtacgta ctccgttagg tcaacgcatt cgtcgcgcat tcatcgccga agaagggtgg 1860
ttattagtag cattggacta tagccaaatt gagttacgtg tattagctca cttaagtggt 1920
gacgaaaact taattcgcgt atttcaggag ggtcgcgaca tccacactga gaccgcgtcg 1980
tggatgtttg gggttccgcg cgaggccgtg gatcccctta tgcgtcgcgc ggcaaaaacc 2040
atcaattttg gggtattata cggcatgagt gctcaccgct taagccaaga attagctatc 2100
ccttatgagg aggcccaggc cttcattgag cgttatttcc agtcgttccc gaaagtgcgc 2160
gcatggatcg agaaaacatt ggaggaaggt cgccgtcgtg gttacgtcga gaccttattt 2220
gggcgtcgcc gctatgttcc cgatctggaa gcccgtgtta agtcagttcg tgaggcagca 2280
gagcgtatgg cattcaatat gcctgttcaa ggaaccgcag cagatttgat gaaacttgct 2340
atggtcaagc tgttcccgcg cttggaagaa atgggcgcac gtatgctgct tcaggtccat 2400
gacgagttgg tcttggaagc gccaaaggag cgcgccgaag cagtcgcgcg cctggcaaag 2460
gaagtgatgg agggagtcta tcccctggct gtccctttgg aggttgaagt agggattgga 2520
gaggactggt taagcgcaaa agagtaataa ctcgagggtg gt 2562
<210> 3
<211> 3569
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gggtgggacc cctttcgggg tcctgctcaa cttcctgtcg agctaatgcc atttttaatg 60
tctttagcga gacgctacca tggctatcgc tgtaggtagc cggaattcca ttcctaggag 120
gtttgacctg tgcgagcttt tagtaccctt gatagggaga acgagacctt cgtcccctcc 180
gttcgcgttt acgcggacgg tgagactgaa gataactcat tctctttaaa atatcgttcg 240
aactggactc ccggtcgttt taactcgact ggggccaaaa cgaaacagtg gcactacccc 300
tctccgtatt cacggggggc gttaagtgtc acatcgatag atcaaggtgc ctacaagcga 360
agtgggtcat cgtggggtcg cccgtacgag gagaaagccg gtttcggctt ctccctcgac 420
gcacgctcct gctacagcct cttccctgta agccaaaact tgacttacat cgaagtgccg 480
cagaacgttg cgaaccgggc gtcgaccgaa gtcctgcaaa aggtcaccca gggtaatttt 540
aaccttggtg ttgctttagc agaggccagg tcgacagcct cacaactcgc gacgcaaacc 600
attgcgctcg tgaaggcgta cactgccgct cgtcgcggta attggcgcca ggcgctccgc 660
taccttgccc taaacgaaga tcgaaagttt cgatcaaaac acgtggccgg caggtggttg 720
gagttgcagt tcggttggtt accactaatg agtgatatcc agggtgcata tgagatgctt 780
acgaaggttc accttcaaga gtttcttcct atgagagccg tacgtcaggt cggtactaac 840
atcaagttag atggccgtct gtcgtatcca gctgcaaact tccagacaac gtgcaacata 900
tcgcgacgta tcgtgatatg gttttacata aacgatgcac gtttggcatg gttgtcgtct 960
ctaggtatct tgaacccact aggtatagtg tgggaaaagg tgcctttctc attcgttgtc 1020
gactggctcc tacctgtagg taacatgctc gagggcctta cggcccccgt gggatgctcc 1080
tacatgtcag gaacagttac tgacgtaata acgggtgagt ccatcataag cgttgacgct 1140
ccctacgggt ggactgtgga gagacagggc actgctaagg cccaaatctc agccatgcat 1200
cgaggggtac aatccgtatg gccaacaact ggcgcgtacg taaagtctcc tttctcgatg 1260
gtccatacct tagatgcgtt agcattaatc aggcaacggc tctctagata gagccctcaa 1320
ccggagtttg aagcatggct tctaacttta ctcagttcgt tctcgtcgac aatggcggaa 1380
ctggcgacgt gactgtcgcc ccaagcaact tcgctaacgg ggtcgctgaa tggatcagct 1440
ctaactcgcg ttcacaggct tacaaagtaa cctgtagcgt tcgtcagagc tctgcgcaga 1500
atcgcaaata caccatcaaa gtcgaggtgc ctaaagtggc aacccagact gttggtggtg 1560
tagagcttcc tgtagccgca tggcgttcgt acttaaatat ggaactaacc attccaattt 1620
tcgctacgaa ttccgactgc gagcttattg ttaaggcaat gcaaggtctc ctaaaagatg 1680
gaaacccgat tccctcagca atcgcagcaa actccggcat ctactaatag acgccggcca 1740
ttcaaacatg aggattaccc atgtcgaaga caacaaagaa gttcaactct ttatgtattg 1800
atcttcctcg cgatctttct ctcgaaattt accaatcaat tgcttctgtc gctactggaa 1860
gcggtgatcc gcacagtgac gactttacag caattgctta cttaagggac gaattgctca 1920
caaagcatcc gaccttaggt tctggtaatg acgaggcgac ccgtcgtacc ttagctatcg 1980
ctaagctacg ggaggcgaat ggtgatcgcg gtcagataaa tagagaaggt ttcttacatg 2040
acaaatcctt gtcatgggat ccggatgttt tacaaaccag catccgtagc cttattggca 2100
acctcctctc tggctaccga tcgtcgttgt ttgggcaatg cacgttctcc aacggtgctc 2160
ctatggggca caagttgcag gatgcagcgc cttacaagaa gttcgctgaa caagcaaccg 2220
ttaccccccg cgctctgaga gcggctctat tggtccgaga ccaatgtgcg ccgtggatca 2280
gacacgcggt ccgctataac gagtcatatg aatttaggct cgttgtaggg aacggagtgt 2340
ttacagttcc gaagaataat aaaatagatc gggctgcctg taaggagcct gatatgaata 2400
tgtacctcca gaaaggggtc ggtgctttca tcagacgccg gctcaaatcc gttggtatag 2460
acctgaatga tcaatcgatc aaccagcgtc tggctcagca gggcagcgta gatggttcgc 2520
ttgcgacgat agacttatcg tctgcatccg attccatctc cgatcgcctg gtgtggagtt 2580
ttctcccacc agagctatat tcatatctcg atcgtatccg ctcacactac ggaatcgtag 2640
atggcgagac gatacgatgg gaactatttt ccacaatggg aaatgggttc acatttgagc 2700
tagagtccat gatattctgg gcaatagtca aagcgaccca aatccatttt ggtaacgccg 2760
gaaccatagg catctacggg gacgatatta tatgtcccag tgagattgca ccccgtgtgc 2820
tagaggcact tgcctactac ggttttaaac cgaatcttcg taaaacgttc gtgtccgggc 2880
tctttcgcga gagctgcggc gcgcactttt accgtggtgt cgatgtcaaa ccgttttaca 2940
tcaagaaacc tgttgacaat ctcttcgccc tgatgctgat attaaatcgg ctacggggtt 3000
ggggagttgt cggaggtatg tcagatccac gcctctataa ggtgtgggta cggctctcct 3060
cccaggtgcc ttcgatgttc ttcggtggga cggacctcgc tgccgactac tacgtagtca 3120
gcccgcctac ggcagtctcg gtatacacca agactccgta cgggcggctg ctcgcggata 3180
cccgtacctc gggtttccgt cttgctcgta tcgctcgaga acgcaagttc ttcagcgaaa 3240
agcacgacag tggtcgctac atagcgtggt tccatactgg aggtgaaatc accgacagca 3300
tgaagtccgc cggcgtgcgc gttatacgca cttcggagtg gctaacgccg gttcccacat 3360
tccctcagga gtgtgggcca gcgagctctc ctcggtagct gaccgaggga cccccgtaaa 3420
cggggtgggt gtgctcgaaa gagcacgggt gcgaaagcgg tccggctcca ccgaaaggtg 3480
ggcgggcttc ggcccaggga cctcccccta aagagaggac ccgggattct cccgatttgg 3540
taactagctg cttggctagt taccaccca 3569
<210> 4
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cagtatggaa ccacgctat 19
Claims (6)
1.一种M-MLV逆转录酶，其特征在于：在SEQ ID NO.1序列的基础上进行四个氨基酸位点的突变，氨基酸突变位点为：D524G、E562Q、D538N和G638H。
2.编码权利要求1所述的M-MLV逆转录酶的核苷酸序列。
3.权利要求1所述的M-MLV逆转录酶的制备方法。
4.权利要求1所述的M-MLV逆转录酶在逆转录反应领域中的应用。
5.根据权利要求4所述的M-MLV逆转录酶在逆转录反应领域中的应用，其特征在于，所述缓冲液包括：终浓度为50mM Tris-HCl、终浓度为75mM KCl、终浓度为10mM MgCl2、终浓度为0.1mg/mLBSA、终浓度为0.2％CA-630、终浓度为1mM VC-Na、终浓度为0.5mg/mL海藻糖、终浓度为0.5mM DTT和NF水。
6.根据权利要求5所述的M-MLV逆转录酶在逆转录反应领域中的应用，其特征在于，所述反应条件为：50℃，30s，35循环。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020090618A1 (en) * | 2000-05-26 | 2002-07-11 | Invitrogen Corporation | Thermostable reverse transcriptases and uses thereof |
| CN106906237A (zh) * | 2017-04-18 | 2017-06-30 | 淮海工学院 | 一种高性能m‑mlv逆转录酶的制备方法 |
| CN107058258A (zh) * | 2008-04-10 | 2017-08-18 | 赛默飞世尔科技波罗的海Uvb公司 | 一种逆转录酶和编码其的多核苷酸 |
| CN112795546A (zh) * | 2019-11-13 | 2021-05-14 | 中山大学达安基因股份有限公司 | 一种具有高逆转录效率的耐高温逆转录酶突变体及其应用 |
| CN113817707A (zh) * | 2021-11-23 | 2021-12-21 | 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 |
-
2022
- 2022-01-26 CN CN202210096427.0A patent/CN114214299A/zh not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020090618A1 (en) * | 2000-05-26 | 2002-07-11 | Invitrogen Corporation | Thermostable reverse transcriptases and uses thereof |
| CN107058258A (zh) * | 2008-04-10 | 2017-08-18 | 赛默飞世尔科技波罗的海Uvb公司 | 一种逆转录酶和编码其的多核苷酸 |
| CN106906237A (zh) * | 2017-04-18 | 2017-06-30 | 淮海工学院 | 一种高性能m‑mlv逆转录酶的制备方法 |
| CN112795546A (zh) * | 2019-11-13 | 2021-05-14 | 中山大学达安基因股份有限公司 | 一种具有高逆转录效率的耐高温逆转录酶突变体及其应用 |
| CN113817707A (zh) * | 2021-11-23 | 2021-12-21 | 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 |
Non-Patent Citations (2)
| Title |
|---|
| CHAPPEY, C.: "Moloney murine leukemia virus, complete genome", GENBANK: AF033811.1, 16 December 2016 (2016-12-16) * |
| SMITH,M.等: "GenBank:CCA64130.1", GENBANK * |
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