CN116004564A - 逆转录突变体及其应用 - Google Patents
逆转录突变体及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，公开了一种MMLV逆转录突变体，其通过将野生型MMLV逆转录酶的RNaseH结构域替换为增强与RNA‑DNA杂交链结合的结构域来获得。本发明还对所述突变体进行了突变设计，其中优选的突变组合为E69K、D200K、P326K、P401K、T330P和T306K。与野生型相比，本发明的MMLV逆转录突变体具有更高的扩增效率。本发明还提供了本发明的MMLV逆转录酶突变体的序列及其在逆转录反应中的应用。
Description
技术领域
本发明属于生物学技术领域，更具体地，涉及逆转录突变体及其应用。
背景技术
逆转录酶是一种以RNA为模板，掺入底物dNTPs合成DNA的聚合酶，在分子生物学领域具有广泛的应用。来源于鼠莫罗尼白血病病毒(Murine-Moloney leukemia virus,MMLV)的逆转录酶(以下简称MMLV逆转录酶)是被广泛应用于基础科研领域的重要工具酶，MMLV逆转录酶在较为广谱的温度范围37℃～60℃中均具有较高活性，其尤其适用于较长片段以及有复杂二级结构的RNA模板的扩增。
野生型MMLV逆转录酶包含逆转录催化结构域和RNaseH结构域，RNaseH结构域本身具有化学小分子的结合位点，这些化学小分子可能包括动物来源的肝素和胆酸盐、植物来源的多糖和多酚和RNA提取过程中用到的醇类物质，这些化学小分子与RNaseH结构域的结合会造成MMLV逆转录酶持续逆转录能力的下降。另外，野生型MMLV催化结构域对模板链RNA的结合能力较弱，这也会造成MMLV逆转录酶持续逆转录能力低下。
因此，本领域需要一种改进的逆转录酶，其具有增强的扩增效率以满足本领域中的逆转录反应应用的需要。
发明内容
鉴于现有技术中存在的问题，本发明将对MMLV逆转录酶进行改进以获得具有增强的扩增效率的MMLV逆转录酶突变体，包括将RNaseH结构域替换为其他增强与RNA-DNA杂交链结合的结构域，并进一步对催化结构域与RNA模板相互作用界面附近的位点进行突变设计，以增强MMLV逆转录酶与RNA模板的相互作用力。
本发明提供了一种MMLV逆转录突变体，与野生型相比，所述突变体具有更高的扩增效率。
本发明提供了一种MMLV逆转录突变体，其是通过将野生型MMLV逆转录酶(SEQ IDNO.1)的RNaseH结构域替换为来自Sulfurisphaera tokodaii的sso7d-like家族蛋白获得的，其中所述sso7d-like家族蛋白的第13位的赖氨酸(K)被突变为亮氨酸(L)，其序列如SEQID NO.2所示。
在一个实施方案中，所述RNaseH结构域替换是在所述野生型MMLV逆转录酶的第474位氨基酸残基以后进行的，优选地，在第474位至第495位进行，更优选地，在第495位进行，替换后的MMLV逆转录酶突变体序列如SEQ ID NO.3所示。
在一个实施方案中，本发明还对替换了RNaseH结构域的MMLV逆转录酶进行了突变设计，所述突变发生的位点包括第69位的谷氨酸(H)、第126位的组氨酸(H)、第131位的天冬酰胺(N)、第200位的天冬氨酸(D)、第204位的组氨酸(H)、第306位的苏氨酸(T)、第326位的脯氨酸(P)、第330位的苏氨酸(T)、第401位的脯氨酸(P)、第374位的谷氨酰胺(Q)、第430位的谷氨酰胺(Q)。
在一个实施方案中，本发明的MMLV逆转录酶突变选自E69K、P326K、P401K、H204R、T306K、H126Y、N131M、D200K、T330P、P326K、P401K、Q374K、Q430K或其组合；优选地，所述MMLV逆转录酶突变体包含选自(1)-(5)的氨基酸置换：
(1)P326K、P401K、H126Y和N131M；
(2)E69K、H126Y、N131M、H204R和T306K；
(3)E69K、D200K和T330P；
(4)E69K、P326K、P401K、Q374K和Q430K；
(5)E69K、D200K、P326K、P401K、T330P和T306K。
本发明还提供了如上所述的MMLV逆转录酶突变体的多核苷酸序列。
本发明还提供了一种表达载体，其包含所述MMLV逆转录酶突变体的多核苷酸序列。
本发明还提供了一种宿主细胞，其包含所述MMLV逆转录酶突变体的多核苷酸序列或如上所述的表达载体。
本发明还提供了本发明的MMLV逆转录酶突变体在逆转录反应中的应用。
本发明还提供了用于进行逆转录反应的试剂盒，其包括如上所述的MMLV逆转录酶突变体。优选地，所述试剂盒还包括逆转录反应缓冲液、dNTP。
附图说明
图1：显示了野生型MMLV逆转录酶和突变体M1至M5的表达纯化的蛋白印迹，其中WT为野生型，M1至M5为5种突变体，Mw为蛋白质分子量marker。
图2：A-C为使用来自鼠的RNA的针对3个不同基因(SCD1、Cyp7a1和GADPH)的荧光定量检测图，其中Y轴指示扩增信号，X轴指示扩增循环数。
图3：A-C为使用来自花生的RNA的针对3个不同基因(60S、E12和FAD8)的荧光定量检测图，其中Y轴指示扩增信号，X轴指示扩增循环数。
具体实施方式
不希望拘囿于具体的理论，本发明人认为，RNaseH结构域具有消化RNA-DNA杂交链中的RNA的活性，不利于RNA的多循环迭代逆转录，因此需要对RNaseH活性进行失活处理。RNaseH结构域本身可以结合RNA-DNA杂交链，对MMLV逆转录酶的持续逆转录能力有重要作用，删除该结构域会造成MMLV逆转录酶持续逆转录能力的下降。因此常规的方法是保留RNaseH结构域，在保证其依旧可以结合RNA-DNA杂交链的条件下，对RNaseH结构域中与消化活性相关的关键位点进行突变。
本发明提供的MMLV逆转录突变体是在野生型MMLV逆转录酶的基础上进行改进而获得的，其中野生型MMLV逆转录酶的序列如SEQ ID NO.1所示。
本发明的具体方案如下：
(1)替换RNaseH结构域。
在本发明的一个实施方案中，使用来自Sulfurisphaera tokodaii的sso7d-like家族蛋白对MMLV逆转录酶的RNaseH结构域进行替换。
由于sso7d-like家族蛋白具有RNase活性，因此在本发明的一个实施方案中，使用来自Sulfurisphaera tokodaii的sso7d-like家族蛋白对MMLV逆转录酶的RNaseH结构域进行替换，其中所述来自Sulfurisphaera tokodaii的sso7d-like家族蛋白中的第13位的赖氨酸(K)被亮氨酸(L)所替代，其序列如SEQ ID NO.2所示。
MMLV逆转录酶的第362～474位的氨基酸残基为连接结构域(connectiondomain)，用于连接本身的位于276～361氨基酸残基区间的拇指结构域(thumb domain)和位于474～672氨基酸残基区间的RNaseH结构域。连接结构域对于MMLV逆转录酶整体活性具有重要调节功能，因此本发明对RNaseH结构域的替换是在MMLV逆转录酶的第474位氨基酸残基以后进行，可以是在第474位至第495位，优选地，选择在第495位对RNaseH结构域的进行替换。
在本发明的一个实施方案中，替换RNaseH结构域后的MMLV逆转录酶序列如SEQ IDNO.3所示。
(2)MMLV逆转录酶突变体设计。
为了进一步提高MMLV逆转录酶的活性，本发明人通过对MMLV的结构进行了分析，获得了可能影响模板结合能力的氨基酸位点，包括第69位的谷氨酸(H)、第126位的组氨酸(H)、第131位的天冬酰胺(N)、第200位的天冬氨酸(D)、第204位的组氨酸(H)、第306位的苏氨酸(T)、第326位的脯氨酸(P)、第330位的苏氨酸(T)、第374位的谷氨酰胺(Q)、第401位的脯氨酸(P)、第430位的谷氨酰胺(Q)。
在本发明中，具体可以对以下位点进行突变设计：
(1)第69位的谷氨酸、第306位的苏氨酸、第326位的脯氨酸、第374位的谷氨酰胺、第401位的脯氨酸和第430位的谷氨酰胺，这些位点位于模板链附近，可以与磷酸骨架结合，因此可以突变为组氨酸、赖氨酸或精氨酸，优选地，突变为赖氨酸(K)；
(2)第126位的组氨酸，其面对模板的磷酸骨架，为了增强其与模板链的相互作用，同时为了保证不影响二级结构，可以将其突变为同属芳香族且仅含有一个苯环侧链的酪氨酸(Y)；
(3)第131位的天冬酰胺，其更靠近碱基，推测可能主要以疏水作用与模板链结合，因而可以将其突变为甲硫氨酸、亮氨酸或异亮氨酸，优选地，突变为甲硫氨酸(M)；
(4)第200位的天冬氨酸和第204位的组氨酸，这两个位点远离模板链，但临近参与催化的β-折叠，间接调节该β-折叠与模板链的相互作用，可以将该两个位点突变为赖氨酸、精氨酸或天冬酰胺，优选地，突变为赖氨酸(K)；
(5)第330位的苏氨酸，临近结合模板的α-螺旋附近，可以影响逆转录酶与模板的相互作用，优选地，突变为脯氨酸(P)。
对于以上突变位点，本发明提供的可选用的具体组合方式如下表1所示：
表1突变位点组合
(3)MMLV逆转录酶突变体的获得。
在设计好MMLV逆转录酶的核酸序列(如SEQ ID NO.10至SEQ ID NO.14所示)后，连入基于T7启动子的商业化表达载体pET系列，使用商业化定点突变试剂盒获取突变体。所用表达载体含有响应IPTG诱导表达的操纵子序列，同时保证了蛋白在大肠杆菌中的高效表达。大肠杆菌的具体培养温度为16℃、25℃、30℃或37℃，培养时间可以是20h、16h、12h或4h。优选地，本发明使用16℃进行诱导表达，诱导时间为20h。
(4)MMLV逆转录酶突变体的分离纯化。
达到设定的诱导表达培养时间后，使用离心方法收集培养的菌体，并用缓冲液进行振荡重悬。缓冲液的pH为7.0～8.0，其含有10～50mM Trima、HEPES或Tricine等作为pH缓冲剂，并含有200mM～500mM NaCl或KCl作为盐。裂解和纯化所用缓冲液含有0.5mM～2mM还原剂，可以防止酶内部错误二硫键折叠。缓冲液使用无核酸酶水进行配制，并进行过滤和高温高压灭菌处理。重悬后的菌体悬浮液，使用超声方式裂解破碎，之后使用亲和层析方法获得较高纯度的MMLV逆转录酶突变体。更进一步的，使用离子交换层析去除更多的杂蛋白，使突变体目的蛋白纯度达到90％以上。突变体目的蛋白最终透析至储存缓冲液中。
(5)MMLV逆转录酶突变体扩增效率的检测。
使用来自2个不同物种的RNA进行逆转录，模板量为500ng。加入逆转录所必需的组分及所得MMLV逆转录酶突变体，设计不同位点的引物，以合成cDNA片段，之后进行qPCR荧光定量检测产物的量。
与现有技术相比较而言，本发明的技术方案带来的有益效果为：本发明所述突变体具有更高的扩增效率。
实施例
实施例1：逆转录酶的表达纯化。
将野生型MMLV逆转录酶及其突变体质粒加进BL21(DE3)感受态，冰浴30min，42℃热激90s，冰浴2min。加入900μL LB，于37℃/220rpm/1h孵育，涂布于含卡那抗性的LB平板，于37℃倒置过夜培养。挑上述单克隆接种于10mL的LB(Kan+)，于37℃，220rpm振荡至浑浊，将菌液接种于1000mL的LB(Kan+)，起始菌量总共为8L，于37℃，220rpm振荡至OD＝0.6，加入0.5mM IPTG 500μL，于16℃/120rpm过夜诱导18h。将诱导后的菌液于4℃，3500rpm离心10min，收集菌体，并重悬于100mL缓冲液中(20mM Tris-Cl 7.6，300mM NaCl，10％甘油)，超声破碎15min，然后于4℃，9500rpm离心30min，收集上清。将上清加入亲和柱，上样后用亲和缓冲液平衡Ni-NTA层析柱5个柱体积，使用洗涤缓冲液(20mM Tris-Cl 7.6，300mM NaCl，50mM咪唑，10％甘油)洗5个柱体积，最后使用洗脱缓冲液(20mM Tris-Cl 7.6，150mM NaCl，300mM咪唑，10％甘油)洗脱目的蛋白。然后用阴离子交换层析进行进一步纯化，使用缓冲液(20mM Tris-Cl 7.6，300mM NaCl，10％甘油)平衡层析柱，之后直接上样进行纯化，收集流传样品，并对其进行SDS-PAGE电泳分析。蛋白样品用透析缓冲液(20mM Tris-Cl 7.6，300mMNaCl，1mM DTT，1mM EDTA，0.2％IPGAL-CA630，50％甘油)过夜透析，收集目的蛋白，并于-20℃保存。
所得蛋白结果如图1所示，野生型MMLV逆转录酶和各突变体均能良好可溶表达。
实施例2：扩增效率检测。
使用1μg的RNA作为模板，RNA模板来源于小鼠和花生。分别选择各物种的三个不同位点的基因(小鼠：SCD1、Cyp7a1和GADPH；花生：60S、E12和FAD8)进行逆转录，具体反应体系见表2。反应条件为：25℃孵育10min，50℃孵育5min，85℃反应5min。
随后使用荧光定量PCR进行检测，将反转录产物稀释3倍后，取1μL进行qRT-PCR反应。反应体系见表3，反应条件见表4，所用的引物序列见表5。
表2逆转录反应体系
表3qRT-PCR反应体系
表4qRT-PCR反应条件
表5引物序列
通过Ct值的比较判断产物量的多数，进而判断扩增效率的高低。简言之，Ct值越小，则达到荧光定量PCR仪阈值所需循环数越低，即初始逆转录产物量越高，逆转录酶效率越高。
结果分别如图2(小鼠基因荧光定量检测)和图3(花生基因荧光定量检测)所示，逆转录酶突变体M1至M5的扩增效率均优于野生型逆转录酶，其中突变体M5表现更佳，其在小鼠基因荧光定量检测中最高可优于野生型4个Ct，在花生基因荧光定量检测中最高可优于野生型2个Ct值。
序列如下：
SEQ ID NO.1(野生型MMLV的序列)：
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
SEQ ID NO.2(突变了的Sulfurisphaera tokodaii序列)：
MVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.3(替换RNaseH结构域后的序列)：
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.4(突变体M1)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIYPTVPMPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYKLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDKVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.5(突变体M2)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIYPTVPMPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALRRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.6(突变体M3)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFKEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.7(突变体M4)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLWIPGFAEMAAPLYKLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKKGYAKGVLTQKLGPWRRPVAYLSKKLDKVAAGWPPCLRMVAAIAVLTKDAGKLTMGKPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGGGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.8(突变体M5)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFKEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLWIPGFAEMAAPLYKLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDKVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.9(替换RNaseH结构域后的核酸序列)
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGATGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAACCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAAGCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGGAAGCGCGTCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCCGTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCAACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGATATTCATCCGACCGTGCCGAACCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGCCATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTGCATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGCATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCCGACCCTGTTTGATGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCATCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAGCGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAATATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAAAGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAATTTCTGGGCACCGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGGCGGCGCCGCTGTATCCGCTGACCAAAACCGGCACCCTGTTTAACTGGGGCCCGGATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGCGCTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACAGGGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGGTGGCGTATCTGAGCAAAAAACTGGATCCGGTGGCGGCGGGCTGGCCGCCGTGCCTGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGACCATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGAAACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCGCTGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCGGCGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGGCGGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGAAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.10（突变体M1核酸序列）ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGATGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAACCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAAGCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGGAAGCGCGTCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCCGTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCAACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGATATTTATCCGACCGTGCCGATGCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGCCATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTGCATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGCATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCCGACCCTGTTTGATGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCATCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAGCGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAATATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAAAGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAATTTCTGGGCACCGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGGCGGCGCCGCTGTATAAACTGACCAAAACCGGCACCCTGTTTAACTGGGGCCCGGATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGCGCTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACAGGGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGGTGGCGTATCTGAGCAAAAAACTGGATAAAGTGGCGGCGGGCTGGCCGCCGTGCCTGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGACCATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGAAACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCGCTGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCGGCGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGGCGGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGAAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.11（突变体M2核酸序列）
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGATGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAACCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAAGCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGAAAGCGCGTCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCCGTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCAACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGATATTTATCCGACCGTGCCGATGCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGCCATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTGCATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGCATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCCGACCCTGTTTGATGAAGCGCTGCGTCGTGATCTGGCGGATTTTCGTATTCAGCATCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAGCGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAATATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAAAGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAATTTCTGGGCAAAGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGGCGGCGCCGCTGTATCCGCTGACCAAAACCGGCACCCTGTTTAACTGGGGCCCGGATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGCGCTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACAGGGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGGTGGCGTATCTGAGCAAAAAACTGGATCCGGTGGCGGCGGGCTGGCCGCCGTGCCTGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGACCATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGAAACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCGCTGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCGGCGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGGCGGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGAAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.12（突变体M3核酸序列）
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGATGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAACCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAAGCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGAAAGCGCGTCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCCGTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCAACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGATATTCATCCGACCGTGCCGAACCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGCCATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTGCATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGCATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCCGACCCTGTTTAAAGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCATCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAGCGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAATATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAAAGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAATTTCTGGGCACCGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGGCGGCGCCGCTGTATCCGCTGACCAAACCGGGCACCCTGTTTAACTGGGGCCCGGATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGCGCTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACAGGGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGGTGGCGTATCTGAGCAAAAAACTGGATCCGGTGGCGGCGGGCTGGCCGCCGTGCCTGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGACCATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGAAACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCGCTGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCGGCGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGGCGGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGAAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.13（突变体M4核酸序列）
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGATGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAACCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAAGCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGAAAGCGCGTCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCCGTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCAACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGATATTCATCCGACCGTGCCGAACCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGCCATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTGCATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGCATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCCGACCCTGTTTGATGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCATCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAGCGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAATATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAAAGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAATTTCTGGGCAAAGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGGCGGCGCCGCTGTATAAACTGACCAAAACCGGCACCCTGTTTAACTGGGGCCCGGATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGCGCTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAAAAAGGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGGTGGCGTATCTGAGCAAAAAACTGGATAAAGTGGCGGCGGGCTGGCCGCCGTGCCTGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGACCATGGGCAAACCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGAAACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCGCTGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCGGCGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCGGCGGCACCGGCGGCGGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGAAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.14（突变体M5核酸序列）
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGATGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAACCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAAGCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGAAAGCGCGTCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCCGTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCAACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGATATTCATCCGACCGTGCCGAACCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGCCATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTGCATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGCATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCCGACCCTGTTTAAAGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCATCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAGCGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAATATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAAAGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAATTTCTGGGCAAAGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGGCGGCGCCGCTGTATAAACTGACCAAACCGGGCACCCTGTTTAACTGGGGCCCGGATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGCGCTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACAGGGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGGTGGCGTATCTGAGCAAAAAACTGGATAAAGTGGCGGCGGGCTGGCCGCCGTGCCTGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGACCATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGAAACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCGCTGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCGGCGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGGCGGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGAAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA 。
序列表
<110> 北京擎科生物科技有限公司
<120> 逆转录突变体及其应用
<140> 2022106214263
<141> 2022-06-01
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 671
<212> PRT
<213> 鼠莫罗尼白血病病毒(Murine-Moloney leukemia virus)
<400> 1
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
<210> 2
<211> 64
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Leu Glu Val Asp
1 5 10 15
Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe
20 25 30
Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys
35 40 45
Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Ser Gly Lys Lys
50 55 60
<210> 3
<211> 564
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Gly Thr
485 490 495
Gly Gly Gly Gly Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu
500 505 510
Leu Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys
515 520 525
Met Ile Ser Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala
530 535 540
Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys
545 550 555 560
Ser Gly Lys Lys
<210> 4
<211> 564
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile Tyr Pro Thr
115 120 125
Val Pro Met Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Lys Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Lys Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Gly Thr
485 490 495
Gly Gly Gly Gly Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu
500 505 510
Leu Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys
515 520 525
Met Ile Ser Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala
530 535 540
Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys
545 550 555 560
Ser Gly Lys Lys
<210> 5
<211> 564
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Lys Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile Tyr Pro Thr
115 120 125
Val Pro Met Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu Arg Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Lys Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Gly Thr
485 490 495
Gly Gly Gly Gly Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu
500 505 510
Leu Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys
515 520 525
Met Ile Ser Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala
530 535 540
Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys
545 550 555 560
Ser Gly Lys Lys
<210> 6
<211> 564
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Lys Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Lys Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Pro Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Gly Thr
485 490 495
Gly Gly Gly Gly Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu
500 505 510
Leu Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys
515 520 525
Met Ile Ser Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala
530 535 540
Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys
545 550 555 560
Ser Gly Lys Lys
<210> 7
<211> 566
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Lys Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Lys Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Lys Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Lys Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Lys Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Lys Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Gly Gly
485 490 495
Gly Thr Gly Gly Gly Gly Met Val Thr Val Lys Phe Lys Tyr Lys Gly
500 505 510
Glu Glu Leu Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val
515 520 525
Gly Lys Met Ile Ser Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg
530 535 540
Gly Ala Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu
545 550 555 560
Glu Lys Ser Gly Lys Lys
565
<210> 8
<211> 564
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Lys Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Lys Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Lys Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Lys Leu Thr Lys Pro Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Lys Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Gly Thr
485 490 495
Gly Gly Gly Gly Met Val Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu
500 505 510
Leu Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys
515 520 525
Met Ile Ser Phe Thr Tyr Asp Asp Asn Gly Lys Thr Gly Arg Gly Ala
530 535 540
Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys
545 550 555 560
Ser Gly Lys Lys
<210> 9
<211> 1695
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
accctgaaca ttgaagatga acatcgtctg catgaaacca gcaaagaacc ggatgtgagc 60
ctgggcagca cctggctgag cgattttccg caggcgtggg cggaaaccgg cggcatgggc 120
ctggcggtgc gtcaggcgcc gctgattatt ccgctgaaag cgaccagcac cccggtgagc 180
attaaacagt atccgatgag ccaggaagcg cgtctgggca ttaaaccgca tattcagcgt 240
ctgctggatc agggcattct ggtgccgtgc cagagcccgt ggaacacccc gctgctgccg 300
gtgaaaaaac cgggcaccaa cgattatcgt ccggtgcagg atctgcgtga agtgaacaaa 360
cgtgtggaag atattcatcc gaccgtgccg aacccgtata acctgctgag cggcctgccg 420
ccgagccatc agtggtatac cgtgctggat ctgaaagatg cgtttttttg cctgcgtctg 480
catccgacca gccagccgct gtttgcgttt gaatggcgtg atccggaaat gggcattagc 540
ggccagctga cctggacccg tctgccgcag ggctttaaaa acagcccgac cctgtttgat 600
gaagcgctgc atcgtgatct ggcggatttt cgtattcagc atccggatct gattctgctg 660
cagtatgtgg atgatctgct gctggcggcg accagcgaac tggattgcca gcagggcacc 720
cgtgcgctgc tgcagaccct gggcaacctg ggctatcgtg cgagcgcgaa aaaagcgcag 780
atttgccaga aacaggtgaa atatctgggc tatctgctga aagaaggcca gcgttggctg 840
accgaagcgc gtaaagaaac cgtgatgggc cagccgaccc cgaaaacccc gcgtcagctg 900
cgtgaatttc tgggcaccgc gggcttttgc cgtctgtgga ttccgggctt tgcggaaatg 960
gcggcgccgc tgtatccgct gaccaaaacc ggcaccctgt ttaactgggg cccggatcag 1020
cagaaagcgt atcaggaaat taaacaggcg ctgctgaccg cgccggcgct gggcctgccg 1080
gatctgacca aaccgtttga actgtttgtg gatgaaaaac agggctatgc gaaaggcgtg 1140
ctgacccaga aactgggccc gtggcgtcgt ccggtggcgt atctgagcaa aaaactggat 1200
ccggtggcgg cgggctggcc gccgtgcctg cgtatggtgg cggcgattgc ggtgctgacc 1260
aaagatgcgg gcaaactgac catgggccag ccgctggtga ttctggcgcc gcatgcggtg 1320
gaagcgctgg tgaaacagcc gccggatcgt tggctgagca acgcgcgtat gacccattat 1380
caggcgctgc tgctggatac cgatcgtgtg cagtttggcc cggtggtggc gctgaacccg 1440
gcgaccctgc tgccgctgcc ggaagaaggc ctgcagcata acggcaccgg cggcggcggc 1500
atggtgaccg tgaaatttaa atataaaggc gaagaactgg aagtggatat tagcaaaatt 1560
aaaaaagtgt ggcgtgtggg caaaatgatt agctttacct atgatgataa cggcaaaacc 1620
ggccgtggcg cggtgagcga aaaagatgcg ccgaaagaac tgctgcagat gctggaaaaa 1680
agcggcaaaa aataa 1695
<210> 10
<211> 1695
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
accctgaaca ttgaagatga acatcgtctg catgaaacca gcaaagaacc ggatgtgagc 60
ctgggcagca cctggctgag cgattttccg caggcgtggg cggaaaccgg cggcatgggc 120
ctggcggtgc gtcaggcgcc gctgattatt ccgctgaaag cgaccagcac cccggtgagc 180
attaaacagt atccgatgag ccaggaagcg cgtctgggca ttaaaccgca tattcagcgt 240
ctgctggatc agggcattct ggtgccgtgc cagagcccgt ggaacacccc gctgctgccg 300
gtgaaaaaac cgggcaccaa cgattatcgt ccggtgcagg atctgcgtga agtgaacaaa 360
cgtgtggaag atatttatcc gaccgtgccg atgccgtata acctgctgag cggcctgccg 420
ccgagccatc agtggtatac cgtgctggat ctgaaagatg cgtttttttg cctgcgtctg 480
catccgacca gccagccgct gtttgcgttt gaatggcgtg atccggaaat gggcattagc 540
ggccagctga cctggacccg tctgccgcag ggctttaaaa acagcccgac cctgtttgat 600
gaagcgctgc atcgtgatct ggcggatttt cgtattcagc atccggatct gattctgctg 660
cagtatgtgg atgatctgct gctggcggcg accagcgaac tggattgcca gcagggcacc 720
cgtgcgctgc tgcagaccct gggcaacctg ggctatcgtg cgagcgcgaa aaaagcgcag 780
atttgccaga aacaggtgaa atatctgggc tatctgctga aagaaggcca gcgttggctg 840
accgaagcgc gtaaagaaac cgtgatgggc cagccgaccc cgaaaacccc gcgtcagctg 900
cgtgaatttc tgggcaccgc gggcttttgc cgtctgtgga ttccgggctt tgcggaaatg 960
gcggcgccgc tgtataaact gaccaaaacc ggcaccctgt ttaactgggg cccggatcag 1020
cagaaagcgt atcaggaaat taaacaggcg ctgctgaccg cgccggcgct gggcctgccg 1080
gatctgacca aaccgtttga actgtttgtg gatgaaaaac agggctatgc gaaaggcgtg 1140
ctgacccaga aactgggccc gtggcgtcgt ccggtggcgt atctgagcaa aaaactggat 1200
aaagtggcgg cgggctggcc gccgtgcctg cgtatggtgg cggcgattgc ggtgctgacc 1260
aaagatgcgg gcaaactgac catgggccag ccgctggtga ttctggcgcc gcatgcggtg 1320
gaagcgctgg tgaaacagcc gccggatcgt tggctgagca acgcgcgtat gacccattat 1380
caggcgctgc tgctggatac cgatcgtgtg cagtttggcc cggtggtggc gctgaacccg 1440
gcgaccctgc tgccgctgcc ggaagaaggc ctgcagcata acggcaccgg cggcggcggc 1500
atggtgaccg tgaaatttaa atataaaggc gaagaactgg aagtggatat tagcaaaatt 1560
aaaaaagtgt ggcgtgtggg caaaatgatt agctttacct atgatgataa cggcaaaacc 1620
ggccgtggcg cggtgagcga aaaagatgcg ccgaaagaac tgctgcagat gctggaaaaa 1680
agcggcaaaa aataa 1695
<210> 11
<211> 1695
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
accctgaaca ttgaagatga acatcgtctg catgaaacca gcaaagaacc ggatgtgagc 60
ctgggcagca cctggctgag cgattttccg caggcgtggg cggaaaccgg cggcatgggc 120
ctggcggtgc gtcaggcgcc gctgattatt ccgctgaaag cgaccagcac cccggtgagc 180
attaaacagt atccgatgag ccagaaagcg cgtctgggca ttaaaccgca tattcagcgt 240
ctgctggatc agggcattct ggtgccgtgc cagagcccgt ggaacacccc gctgctgccg 300
gtgaaaaaac cgggcaccaa cgattatcgt ccggtgcagg atctgcgtga agtgaacaaa 360
cgtgtggaag atatttatcc gaccgtgccg atgccgtata acctgctgag cggcctgccg 420
ccgagccatc agtggtatac cgtgctggat ctgaaagatg cgtttttttg cctgcgtctg 480
catccgacca gccagccgct gtttgcgttt gaatggcgtg atccggaaat gggcattagc 540
ggccagctga cctggacccg tctgccgcag ggctttaaaa acagcccgac cctgtttgat 600
gaagcgctgc gtcgtgatct ggcggatttt cgtattcagc atccggatct gattctgctg 660
cagtatgtgg atgatctgct gctggcggcg accagcgaac tggattgcca gcagggcacc 720
cgtgcgctgc tgcagaccct gggcaacctg ggctatcgtg cgagcgcgaa aaaagcgcag 780
atttgccaga aacaggtgaa atatctgggc tatctgctga aagaaggcca gcgttggctg 840
accgaagcgc gtaaagaaac cgtgatgggc cagccgaccc cgaaaacccc gcgtcagctg 900
cgtgaatttc tgggcaaagc gggcttttgc cgtctgtgga ttccgggctt tgcggaaatg 960
gcggcgccgc tgtatccgct gaccaaaacc ggcaccctgt ttaactgggg cccggatcag 1020
cagaaagcgt atcaggaaat taaacaggcg ctgctgaccg cgccggcgct gggcctgccg 1080
gatctgacca aaccgtttga actgtttgtg gatgaaaaac agggctatgc gaaaggcgtg 1140
ctgacccaga aactgggccc gtggcgtcgt ccggtggcgt atctgagcaa aaaactggat 1200
ccggtggcgg cgggctggcc gccgtgcctg cgtatggtgg cggcgattgc ggtgctgacc 1260
aaagatgcgg gcaaactgac catgggccag ccgctggtga ttctggcgcc gcatgcggtg 1320
gaagcgctgg tgaaacagcc gccggatcgt tggctgagca acgcgcgtat gacccattat 1380
caggcgctgc tgctggatac cgatcgtgtg cagtttggcc cggtggtggc gctgaacccg 1440
gcgaccctgc tgccgctgcc ggaagaaggc ctgcagcata acggcaccgg cggcggcggc 1500
atggtgaccg tgaaatttaa atataaaggc gaagaactgg aagtggatat tagcaaaatt 1560
aaaaaagtgt ggcgtgtggg caaaatgatt agctttacct atgatgataa cggcaaaacc 1620
ggccgtggcg cggtgagcga aaaagatgcg ccgaaagaac tgctgcagat gctggaaaaa 1680
agcggcaaaa aataa 1695
<210> 12
<211> 1695
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
accctgaaca ttgaagatga acatcgtctg catgaaacca gcaaagaacc ggatgtgagc 60
ctgggcagca cctggctgag cgattttccg caggcgtggg cggaaaccgg cggcatgggc 120
ctggcggtgc gtcaggcgcc gctgattatt ccgctgaaag cgaccagcac cccggtgagc 180
attaaacagt atccgatgag ccagaaagcg cgtctgggca ttaaaccgca tattcagcgt 240
ctgctggatc agggcattct ggtgccgtgc cagagcccgt ggaacacccc gctgctgccg 300
gtgaaaaaac cgggcaccaa cgattatcgt ccggtgcagg atctgcgtga agtgaacaaa 360
cgtgtggaag atattcatcc gaccgtgccg aacccgtata acctgctgag cggcctgccg 420
ccgagccatc agtggtatac cgtgctggat ctgaaagatg cgtttttttg cctgcgtctg 480
catccgacca gccagccgct gtttgcgttt gaatggcgtg atccggaaat gggcattagc 540
ggccagctga cctggacccg tctgccgcag ggctttaaaa acagcccgac cctgtttaaa 600
gaagcgctgc atcgtgatct ggcggatttt cgtattcagc atccggatct gattctgctg 660
cagtatgtgg atgatctgct gctggcggcg accagcgaac tggattgcca gcagggcacc 720
cgtgcgctgc tgcagaccct gggcaacctg ggctatcgtg cgagcgcgaa aaaagcgcag 780
atttgccaga aacaggtgaa atatctgggc tatctgctga aagaaggcca gcgttggctg 840
accgaagcgc gtaaagaaac cgtgatgggc cagccgaccc cgaaaacccc gcgtcagctg 900
cgtgaatttc tgggcaccgc gggcttttgc cgtctgtgga ttccgggctt tgcggaaatg 960
gcggcgccgc tgtatccgct gaccaaaccg ggcaccctgt ttaactgggg cccggatcag 1020
cagaaagcgt atcaggaaat taaacaggcg ctgctgaccg cgccggcgct gggcctgccg 1080
gatctgacca aaccgtttga actgtttgtg gatgaaaaac agggctatgc gaaaggcgtg 1140
ctgacccaga aactgggccc gtggcgtcgt ccggtggcgt atctgagcaa aaaactggat 1200
ccggtggcgg cgggctggcc gccgtgcctg cgtatggtgg cggcgattgc ggtgctgacc 1260
aaagatgcgg gcaaactgac catgggccag ccgctggtga ttctggcgcc gcatgcggtg 1320
gaagcgctgg tgaaacagcc gccggatcgt tggctgagca acgcgcgtat gacccattat 1380
caggcgctgc tgctggatac cgatcgtgtg cagtttggcc cggtggtggc gctgaacccg 1440
gcgaccctgc tgccgctgcc ggaagaaggc ctgcagcata acggcaccgg cggcggcggc 1500
atggtgaccg tgaaatttaa atataaaggc gaagaactgg aagtggatat tagcaaaatt 1560
aaaaaagtgt ggcgtgtggg caaaatgatt agctttacct atgatgataa cggcaaaacc 1620
ggccgtggcg cggtgagcga aaaagatgcg ccgaaagaac tgctgcagat gctggaaaaa 1680
agcggcaaaa aataa 1695
<210> 13
<211> 1701
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
accctgaaca ttgaagatga acatcgtctg catgaaacca gcaaagaacc ggatgtgagc 60
ctgggcagca cctggctgag cgattttccg caggcgtggg cggaaaccgg cggcatgggc 120
ctggcggtgc gtcaggcgcc gctgattatt ccgctgaaag cgaccagcac cccggtgagc 180
attaaacagt atccgatgag ccagaaagcg cgtctgggca ttaaaccgca tattcagcgt 240
ctgctggatc agggcattct ggtgccgtgc cagagcccgt ggaacacccc gctgctgccg 300
gtgaaaaaac cgggcaccaa cgattatcgt ccggtgcagg atctgcgtga agtgaacaaa 360
cgtgtggaag atattcatcc gaccgtgccg aacccgtata acctgctgag cggcctgccg 420
ccgagccatc agtggtatac cgtgctggat ctgaaagatg cgtttttttg cctgcgtctg 480
catccgacca gccagccgct gtttgcgttt gaatggcgtg atccggaaat gggcattagc 540
ggccagctga cctggacccg tctgccgcag ggctttaaaa acagcccgac cctgtttgat 600
gaagcgctgc atcgtgatct ggcggatttt cgtattcagc atccggatct gattctgctg 660
cagtatgtgg atgatctgct gctggcggcg accagcgaac tggattgcca gcagggcacc 720
cgtgcgctgc tgcagaccct gggcaacctg ggctatcgtg cgagcgcgaa aaaagcgcag 780
atttgccaga aacaggtgaa atatctgggc tatctgctga aagaaggcca gcgttggctg 840
accgaagcgc gtaaagaaac cgtgatgggc cagccgaccc cgaaaacccc gcgtcagctg 900
cgtgaatttc tgggcaaagc gggcttttgc cgtctgtgga ttccgggctt tgcggaaatg 960
gcggcgccgc tgtataaact gaccaaaacc ggcaccctgt ttaactgggg cccggatcag 1020
cagaaagcgt atcaggaaat taaacaggcg ctgctgaccg cgccggcgct gggcctgccg 1080
gatctgacca aaccgtttga actgtttgtg gatgaaaaaa aaggctatgc gaaaggcgtg 1140
ctgacccaga aactgggccc gtggcgtcgt ccggtggcgt atctgagcaa aaaactggat 1200
aaagtggcgg cgggctggcc gccgtgcctg cgtatggtgg cggcgattgc ggtgctgacc 1260
aaagatgcgg gcaaactgac catgggcaaa ccgctggtga ttctggcgcc gcatgcggtg 1320
gaagcgctgg tgaaacagcc gccggatcgt tggctgagca acgcgcgtat gacccattat 1380
caggcgctgc tgctggatac cgatcgtgtg cagtttggcc cggtggtggc gctgaacccg 1440
gcgaccctgc tgccgctgcc ggaagaaggc ctgcagcata acggcggcgg caccggcggc 1500
ggcggcatgg tgaccgtgaa atttaaatat aaaggcgaag aactggaagt ggatattagc 1560
aaaattaaaa aagtgtggcg tgtgggcaaa atgattagct ttacctatga tgataacggc 1620
aaaaccggcc gtggcgcggt gagcgaaaaa gatgcgccga aagaactgct gcagatgctg 1680
gaaaaaagcg gcaaaaaata a 1701
<210> 14
<211> 1695
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
accctgaaca ttgaagatga acatcgtctg catgaaacca gcaaagaacc ggatgtgagc 60
ctgggcagca cctggctgag cgattttccg caggcgtggg cggaaaccgg cggcatgggc 120
ctggcggtgc gtcaggcgcc gctgattatt ccgctgaaag cgaccagcac cccggtgagc 180
attaaacagt atccgatgag ccagaaagcg cgtctgggca ttaaaccgca tattcagcgt 240
ctgctggatc agggcattct ggtgccgtgc cagagcccgt ggaacacccc gctgctgccg 300
gtgaaaaaac cgggcaccaa cgattatcgt ccggtgcagg atctgcgtga agtgaacaaa 360
cgtgtggaag atattcatcc gaccgtgccg aacccgtata acctgctgag cggcctgccg 420
ccgagccatc agtggtatac cgtgctggat ctgaaagatg cgtttttttg cctgcgtctg 480
catccgacca gccagccgct gtttgcgttt gaatggcgtg atccggaaat gggcattagc 540
ggccagctga cctggacccg tctgccgcag ggctttaaaa acagcccgac cctgtttaaa 600
gaagcgctgc atcgtgatct ggcggatttt cgtattcagc atccggatct gattctgctg 660
cagtatgtgg atgatctgct gctggcggcg accagcgaac tggattgcca gcagggcacc 720
cgtgcgctgc tgcagaccct gggcaacctg ggctatcgtg cgagcgcgaa aaaagcgcag 780
atttgccaga aacaggtgaa atatctgggc tatctgctga aagaaggcca gcgttggctg 840
accgaagcgc gtaaagaaac cgtgatgggc cagccgaccc cgaaaacccc gcgtcagctg 900
cgtgaatttc tgggcaaagc gggcttttgc cgtctgtgga ttccgggctt tgcggaaatg 960
gcggcgccgc tgtataaact gaccaaaccg ggcaccctgt ttaactgggg cccggatcag 1020
cagaaagcgt atcaggaaat taaacaggcg ctgctgaccg cgccggcgct gggcctgccg 1080
gatctgacca aaccgtttga actgtttgtg gatgaaaaac agggctatgc gaaaggcgtg 1140
ctgacccaga aactgggccc gtggcgtcgt ccggtggcgt atctgagcaa aaaactggat 1200
aaagtggcgg cgggctggcc gccgtgcctg cgtatggtgg cggcgattgc ggtgctgacc 1260
aaagatgcgg gcaaactgac catgggccag ccgctggtga ttctggcgcc gcatgcggtg 1320
gaagcgctgg tgaaacagcc gccggatcgt tggctgagca acgcgcgtat gacccattat 1380
caggcgctgc tgctggatac cgatcgtgtg cagtttggcc cggtggtggc gctgaacccg 1440
gcgaccctgc tgccgctgcc ggaagaaggc ctgcagcata acggcaccgg cggcggcggc 1500
atggtgaccg tgaaatttaa atataaaggc gaagaactgg aagtggatat tagcaaaatt 1560
aaaaaagtgt ggcgtgtggg caaaatgatt agctttacct atgatgataa cggcaaaacc 1620
ggccgtggcg cggtgagcga aaaagatgcg ccgaaagaac tgctgcagat gctggaaaaa 1680
agcggcaaaa aataa 1695
Claims (10)
1.一种MMLV逆转录酶突变体，其特征在于，所述突变体是通过将野生型MMLV逆转录酶(SEQ ID NO.1)的RNaseH结构域替换为来自Sulfurisphaera tokodaii的sso7d-like家族蛋白获得的，其中所述sso7d-like家族蛋白的第13位的赖氨酸(K)被突变为亮氨酸(L)，其序列如SEQ ID NO.2所示。
2.如权利要求1所述MMLV逆转录酶突变体，其特征在于，所述RNaseH结构域替换是在所述野生型MMLV逆转录酶的第474位氨基酸残基以后进行的，优选地，在第474位至第495位进行，更优选地，在第495位进行，替换后的MMLV逆转录酶突变体序列如SEQ ID NO.3所示。
3.如权利要求1或2所述MMLV逆转录酶突变体，其特征在于，还对所述突变体进行了突变设计，所述突变发生的位点包括第69位的谷氨酸(H)、第126位的组氨酸(H)、第131位的天冬酰胺(N)、第200位的天冬氨酸(D)、第204位的组氨酸(H)、第306位的苏氨酸(T)、第326位的脯氨酸(P)、第330位的苏氨酸(T)、第401位的脯氨酸(P)、第374位的谷氨酰胺(Q)、第430位的谷氨酰胺(Q)。
4.如权利要求3所述MMLV逆转录酶突变体，其特征在于，所述突变选自E69K、P326K、P401K、H204R、T306K、H126Y、N131M、D200K、T330P、P326K、P401K、Q374K、Q430K或其组合；优选地，所述MMLV逆转录酶突变体包含选自(1)-(5)的氨基酸置换：
(1)P326K、P401K、H126Y和N131M；
(2)E69K、H126Y、N131M、H204R和T306K；
(3)E69K、D200K和T330P；
(4)E69K、P326K、P401K、Q374K和Q430K；
(5)E69K、D200K、P326K、P401K、T330P和T306K。
5.编码如权利要求1至4中任一项所述MMLV逆转录酶突变体的多核苷酸序列。
6.一种表达载体，其包含如权利要求5所述的多核苷酸序列。
7.包含权利要求5所述的多核苷酸序列或权利要求6所述的表达载体的宿主细胞。
8.如权利要求1-4中任一项所述MMLV逆转录酶突变体在逆转录反应中的应用。
9.用于进行逆转录反应的试剂盒，其特征在于，包括权利要求1至4中任一项所述MMLV逆转录酶突变体。
10.根据权利要求9所述的用于进行逆转录反应的试剂盒，其特征在于，还包括：逆转录反应缓冲液、dNTP。
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