CN113583988B - 氨基酸脱氢酶突变体及其应用 - Google Patents

氨基酸脱氢酶突变体及其应用 Download PDF

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CN113583988B
CN113583988B CN202010360282.1A CN202010360282A CN113583988B CN 113583988 B CN113583988 B CN 113583988B CN 202010360282 A CN202010360282 A CN 202010360282A CN 113583988 B CN113583988 B CN 113583988B
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贾娴
欧阳敬平
游松
韩冰洋
刘家岱
范多纳
张飞霆
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Shenyang Pharmaceutical University
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Abstract

本发明属于化学催化与酶催化交叉领域，涉及氨基酸脱氢酶突变体及其应用，具体涉及有机合成、枯草芽孢杆菌亮氨酸脱氢酶(leucine dehydrogenase from B.subtilis,BsLDH)的突变改造、底物α‑酮酸及其衍生物的不对称还原氨化，以及手性非天然氨基酸的化学‑酶法连续反应制备过程。本发明公开的突变体，能够催化多种α‑酮酸类化合物的立体选择性还原氨化，合成结构多样的非天然氨基酸。该突变体基因经过重组、转化，在E.coli Rosseta(DE3)中表达，并将其与葡萄糖脱氢酶(GDH)组成辅酶循环体系用于催化α‑酮酸及其衍生物得到手性非天然氨基酸。同时，本发明以甲基酮类化合物为起始原料，构建了立体选择性制备手性非天然氨基酸的化学酶法反应过程。

Description

氨基酸脱氢酶突变体及其应用
技术领域
本发明属于化学催化与酶催化交叉领域，涉及氨基酸脱氢酶突变体及其应用，具体涉及有机合成、枯草芽孢杆菌亮氨酸脱氢酶(leucine dehydrogenase fromB.subtilis,BsLDH)的突变改造、底物α-酮酸及其衍生物的不对称还原氨化，以及手性非天然氨基酸的化学-酶法连续反应制备过程。
背景技术
氨基酸在现代药物发现的早期扮演着重要角色，它们存在于诸如杆菌肽、万古霉素等抗生素中和以胰岛素为代表的多肽中。由于氨基酸类化合物结构的多样性、多肽和修饰性多肽类药物的接受度越来越高以及带有不同侧链的氨基酸在合成上的简便性，氨基酸类化合物在现代药物化学和新药发现中的地位与日俱增。其中，光学纯(S)-苯甘氨酸及其衍生物是一类潜在的先导化合物和药物合成砌块。常用的合成方法包括：酶合成法、α,β-脱氢氨基酸氢化或环加成法、Strecker型反应、亲电或亲核氨基化法以及亲电或亲核烷基化法等，但这些合成方法都或多或少地带有手性控制、反应条件和反应效益方面的不利因素。由于自身理化性质的特殊性，手性氨基酸(尤其是无修饰的游离氨基酸)的级联合成反应报道相对较少，而其中以化学-酶法从简单结构化合物制备手性氨基酸的研究则更少。
发明内容
有鉴于此，本发明的目的之一在于提供能够催化多种α-酮酸及其衍生物的亮氨酸脱氢酶突变体；目的之二在于提供一种以苯乙酮等甲基酮类化合物为起始原料，将化学氧化与酶催化结合起来的化学-酶法连续一锅法制备手性非天然氨基酸的反应过程。
本发明提供了一系列由枯草芽孢杆菌亮氨酸脱氢酶(leucine dehydrogenasefrom B.subtilis,BsLDH)基因(SEQ ID NO.1)通过突变得到的突变体，并在大肠杆菌工程菌中表达。
相较于野生型BsLDH(SEQ ID No.2)，本发明所述的BsLDH突变体是将BsLDH的40位、65位、291位或294位氨基酸中的一种或多个突变为丙氨酸(Ala，A)、甘氨酸(Gly，G)或丝氨酸(Ser，S)中的一个或多个，涉及的突变体列举如下(表1.)。
表1.BsLDH及其突变体列表
a突变体的表述方式。以SEQ ID No.30为例，“L40A”表示将L40(40位亮氨酸)突变为A(丙氨酸)，“/”表示同时在蛋白序列中引入另一个突变，“V294G”表示将V294(294位缬氨酸)突变为G(甘氨酸)。
进一步地，本发明所述的BsLDH突变体的氨基酸序列如SEQ ID No.4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60所示。
相较于野生型BsLDH(SEQ ID No.2)，所述的BsLDH突变体的氨基酸序列与序列SEQID No.4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60中的任意序列具有至少85％同源性，这些序列通过定点突变及组合突变的方法获得。
本发明包括编码所述BsLDH突变体的核酸，例如编码与本发明所述BsLDH突变体的核酸序列相同或具有至少85％的序列同一性的核酸。
本发明提供一种表达载体，该载体包含编码上述BsLDH突变体的核酸且能在宿主细胞中进行表达。
本发明提供一种宿主细胞，所述宿主细胞包含上述的表达载体，如大肠杆菌、枯草芽孢杆菌、地衣芽孢杆菌、黑曲霉、酿酒酵母和毕赤酵母等。优选地，该宿主细胞为大肠杆菌。
本发明所述的BsLDH突变体对α-酮酸类化合物具有不同活性及良好的立体选择性，能够催化生成多种S型非天然α-氨基酸。因此，所述的BsLDH突变体可以制成生物催化剂(包括细胞、粗酶粉、酶溶液、固定化酶等多种形式)，催化α-酮酸的对映选择性还原氨化从而获得S型非天然α-氨基酸。
本发明还提供了以BsLDH突变体作为催化剂制备S型非天然α-氨基酸的方法，所述方法以α-酮酸类化合物为底物，与本发明中的生物催化剂接触。
优选地，所述方法中的生物催化剂为包含上述BsLDH突变体或葡萄糖脱氢酶(GDH)的大肠杆菌冻干细胞；所述反应是在辅酶存在下，在pH 7-10、温度为25-50℃的NH4Cl-NH3·H2O缓冲溶液中进行的。
其中，所述的辅酶再生系统包括但不限于NAD+与如下物质的组合：葡萄糖和葡萄糖脱氢酶、甲酸和甲酸脱氢酶、葡萄糖-6-磷酸和葡萄糖-6-磷酸脱氢酶、仲醇(如异丙醇)和仲醇脱氢酶、亚磷酸和亚磷酸脱氢酶、分子氢和氢化酶以及电化学再生系统。优选的辅酶再生系统包含NAD+，与葡萄糖和GDH。
上述方法中的α-酮酸类化合物如式(I)所示，
(I)
R可以为单取代或多取代的苯环、萘环、五元或六元芳香杂环和C1-C6直链及支链脂肪族取代基；取代基可以为H、卤素、羟基、氨基、巯基、C1-C5饱和或不饱和烷基、C1-C5烷氧基、C1-C5酯基、C1-C5酰胺基。
优选地，取代基为H、羟基、C1-C5烷氧基、硝基、卤素、C1-C5烷基。
本发明提供一种提高亮氨酸脱氢酶对不同α-酮酸类化合物活性的方法，是通过突变库对α-酮酸类化合物进行活性筛选得到每种α-酮酸对应的改良突变体。
本发明提供一种化学氧化制备α-酮酸类化合物的方法。所述方法以甲基酮类化合物为底物，与氧化剂在有机溶剂中回流得到α-酮酸类化合物。
优选地，所述的氧化剂是二氧化硒(SeO2)；所述反应是在吡啶中，50-110℃回流3-10h后完成的；所述的甲基酮类化合物如式(II)所示，
(II)
R可以为单取代或多取代的苯环、萘环、芳香杂环和C1-C6直链及支链脂肪族取代基；取代基可以为H、卤素、羟基、氨基、巯基、C1-C5饱和或不饱和烷基、C1-C5烷氧基、C1-C5酯基、C1-C5酰胺基。
优选地，取代基为H、羟基、C1-C5烷氧基、硝基、卤素、C1-C5烷基。
本发明提供一种化学-酶法连续反应过程，以甲基酮类化合物为起始原料，一锅法反应制备手性氨基酸。该过程将化学氧化制备α-酮酸类化合物的反应结束后经过静置得到的上清液，直接加入到酶催化体系中，以连续反应的方式制得手性氨基酸。
优选地，所述的氧化反应结束后的上清液与酶催化体系的体积比为1:10-1:200。
本发明的主要反应步骤如下：
1.甲基酮类化合物的氧化：
在本发明的一种实施方式中，甲基酮类化合物在氮气保护下于干燥吡啶中，被SeO2氧化生成α-酮酸类化合物。反应温度50-110℃，回流3-10h。所述甲基酮类化合物包括但不限于如上的化合物1-38。
2.α-酮酸类化合物的立体选择性还原氨化：
在本发明的一种实施方式中，将含有BsLDH突变体与GDH的大肠杆菌细胞在NH4Cl-NH3·H2O缓冲溶液中重新悬浮后，向其中加入NH4Cl、葡萄糖、NAD+及α-酮酸类化合物，使底物浓度为20-400mM。反应在pH 7-10、温度25-50℃，在摇床中200rpm进行8-13h。其中，α-酮酸化合物与NH4Cl的摩尔比为1:1.5-1:8；α-酮酸化合物与葡萄糖的摩尔比为1:1.5-1:8；酶催化剂用量为0.02-40g/L(细胞干重)；NAD+的浓度为0.1-0.5mM。反应结束后，反应液用6N盐酸酸化，乙酸乙酯萃取，水层在12000rpm下离心2min，收集上清液，适当稀释后用于HPLC检测转化率及ee值。产物经阳离子交换树脂纯化后，计算收率。
3.化学酶法连续一锅法反应制备手性氨基酸：
在本发明的一种实施方式中，在图1的步骤1中的氧化反应结束后，静置使反应生成的单质硒沉淀，取出一定体积的吡啶反应液(按总体积的百分比计算底物浓度)直接加入到预制的酶促还原氨化体系中，在摇床中以200rpm在pH 7-10、温度25-50℃的条件下反应8-13h。反应结束后，反应液用6N盐酸酸化，乙酸乙酯萃取，水层在12000rpm下离心2min，收集上清液，适当稀释后用于HPLC检测转化率及ee值。产物经阳离子交换树脂纯化后，计算收率。
本发明的优点在于：本发明设计、制备了一系列BsLDH突变体，对多种α-酮酸类化合物具有较好的活性(转化率最高﹥99％)，能够立体选择性地催化生成手性(S)-苯甘氨酸及其衍生物(ee值最高﹥99.9％)。同时，为了提高反应效能，本发明将甲基酮类化合物的氧化反应与酶促还原氨化组成化学-酶法连续一锅法反应过程，简化操作并缩短了反应过程总体时间。
附图说明
图1为化学-酶法连续反应合成手性非天然α-氨基酸的示意图。
具体实施方式
下述实施例将有助于理解本发明，但不能局限本发明的内容。实施例中采用的实施条件可以根据具体使用的不同要求做进一步调整，未注明的实施条件为常规实验中的条件。
实施例涉及的培养基配方：
a.自诱导培养基：0.5％酵母提取物、1％蛋白质胨、0.33％(NH4)2SO4、0.68％KH2PO4、0.71％Na2HPO4、0.024％MgSO4、0.2％甘油(v/v)、0.05％葡萄糖与0.2％乳糖，115℃
高压灭菌30min。
b.LB培养基：0.5％酵母提取物、1％蛋白质胨与1％氯化钠(如配置固体培养基，在灭菌前加入1.5％琼脂)，115℃高压灭菌30min。
实施例1枯草芽孢杆菌(B.subtilis)基因组DNA的提取
将-80℃冷冻保存的B.subtilis在LB平板上划线活化，挑取单菌落接种于4mLLB液体培养基，在37℃过夜培养后用1.5mL离心管3000rpm离心5min，弃上清收集菌体，可重复几次以得到足够量的细胞；b)按TIANGEN细菌基因组DNA提取试剂盒(TIANamp Bacteria DNAKit)操作说明提取枯草芽孢杆菌基因组。
实施例2野生型BsLDH基因的克隆
以实施例1获得的枯草芽孢杆菌基因组DNA为模板进行PCR反应，反应体系如下：
扩增程序：94℃，10min；34个循环(94℃:30s，45℃:30s，72℃:30s)；72℃，10min。
引物：BsLDH-EcoRI-F CCGGAATTCGATGGAACTTTTTAAATAT
BsLDH-XhoI-R CCGCTCGAGACGTCTGCTTAATAC
其中，限制性内切酶酶切位点用下划线标出。
PCR扩增得到的DNA片段用胶回收试剂盒纯化。含有pET-28b-MBP质粒的E.coliDH5α用LB液体培养基，在37℃、200rpm培养过夜，质粒提取采用参照TIANprep MiniPlasmid Kit。
目的片段与质粒pET-28b-MBP经过限制性内切酶EcoRI、XhoI双酶切，酶切体系如下：
酶切产物用胶回收试剂盒纯化，并通过T4连接酶连接。
实施例3E.coli Rosetta(DE3)感受态细胞的制备及转化
a)按CaCl2制备法制备E.coli Rosetta(DE3)的感受态细胞；b)向150μL感受态细胞的悬浮液中加入15μL T4连接酶的反应液并轻轻旋转混匀，冰浴30min；c)在42℃热休克60s，冰浴2min，加入800μL LB液体培养基。在37℃静置培养1h后，在相同温度下150rpm振荡培养1h；d)分别取150μL转化液涂布于LB抗性平板。
实施例4突变体的构建
以含有BsLDH基因的pET-28b-MBP重组质粒作为模板，以含有突变位点的寡核苷酸片段作为突变引物进行重叠延伸PCR。突变位点包括L40、M65、V291和V294，改变后的氨基酸包括Ala、Ser和Gly。
突变库的构建是利用突变引物和侧翼引物，通过重叠延伸PCR进行的。第一轮PCR的体系构成如下：
扩增程序：94℃，10min；34个循环(94℃:30s，45℃:30s，72℃:30s)；72℃，10min。
所得基因片段经过纯化，按如下体系进行第二轮PCR扩增：
所得基因片段按实施例2中所述的方法，经过酶切并纯化后，连接到pET-28b-MBP质粒上，按实施例3所述方法转化到E.coli Rosetta(DE3)中。
实施例5突变体表达
a)取单菌落接种于4mL卡那霉素抗性LB液体培养基中，在37℃，200rpm培养6h后得到种子液；b)取1mL种子液接种于100mL卡那霉素(50μg/mL)抗性自诱导培养基中，30℃，200rpm培养24h；c)取25mL培养液，4000rpm×15min离心收集菌体，生理盐水洗涤两次后用0.1M NH4Cl-NH3·H2O缓冲液重新悬浮菌体，超声破碎菌体，在4℃、12000rpm离心收集上清，进行活性筛选。最终，本发明提供了包含野生型BsLDH在内的30个酶催化剂(突变体编号及突变位点见表1.)。
实施例6苯乙酮的氧化
向50mL圆底烧瓶中加入1.2g(10mmol)苯乙酮、7.5mL干燥吡啶和2.2g(20mmol)二氧化硒，在N2保护下100℃回流5h。反应结束后，减压除去溶剂，随后用240g/L氢氧化钠溶调节pH＝13，并用10mL乙酸乙酯萃取3次，分出水层。水层用6N盐酸调节pH＝1-2，并用20mL乙酸乙酯萃取3次后合并有机相，无水硫酸钠干燥，减压除去溶剂得到苯甲酰甲酸粗品1.44g，收率96％。
所有甲基酮类化合物均采用上述过程氧化得到α-酮酸类化合物。
实施例7苯甲酰甲酸的酶促立体选择性还原氨化
向30mg苯甲酰甲酸中加入100μL 5M氨水，超声使其部分溶解。另向900μL NH4Cl-NH3·H2O缓冲液(100mM，pH 8.5)中加入56mg(0.7mmol)葡萄糖、32.1mg(0.6mmol)氯化铵、0.4mg NAD+(0.5mM)及含有BsLDH-WT(SEQ ID No.2)和GDH的冻干细胞各40mg(40g/L)。将苯甲酰甲酸的氨水悬浮液加入到酶催化体系中，在37℃摇床中反应8h，并用5M氨水维持反应混合物的pH≈8.5。反应结束后，加入100μL 6N盐酸，用1mL乙酸乙酯萃取3次后，水层在12000rpm下离心2min，收集上清液，适当稀释后用于HPLC检测。产物转化率﹥99％，ee﹥99.9％(S)，并在阳离子交换树脂纯化后得到26.8mg产物，收率为88.7％。
所有α-酮酸类化合物均采用上述过程制备，经对应的优势突变体还原氨化得到光学纯的(S)-α-氨基酸。
实施例8BsLDH突变库对α-酮酸类化合物的活性筛选
按照实施例6的方法制备α-酮酸类化合物，并参照实施例7的方法用突变库对底物进行活性筛选。部分化合物与改良突变体的对应关系如下表所示：
实施例9苯乙酮的化学酶法连续一锅法反应制备(S)-苯甘氨酸
将实施例6中氧化反应结束后的反应液静置或离心，弃去生成的单质硒，取1mL反应上清液加入到按实施例7的方法预制的19mL含有BsLDH-WT(SEQ ID No.2)和GDH的冻干细胞各40mg(40g/L)的酶催化体系中，在37℃摇床中反应8h，并用5M氨水维持反应混合物的pH≈8.5。反应结束后，用6N盐酸调节pH＝1，20mL乙酸乙酯萃取3次后，水层在4000rpm下离心15min，收集上清液，适当稀释后用于HPLC检测。产物转化率﹥99％，ee＝98.2(S)，并在阳离子交换树脂纯化后得到117mg产物，两步反应总收率为62.0％。
实施例10对溴苯乙酮的氧化
向50mL圆底烧瓶中加入1.99g(10mmol)对溴苯乙酮和7.5mL干燥吡啶和2.2g(20mmol)二氧化硒，在N2保护下100℃回流5h。反应结束后，减压除去溶剂，随后用240g/L氢氧化钠溶液调节pH＝13，并用10mL乙酸乙酯萃取3次，分出水层。水层用6N盐酸调节pH＝1-2，并用20mL乙酸乙酯萃取3次后合并有机相，无水硫酸钠干燥，减压除去溶剂得到对溴苯甲酰甲酸粗品1.93g，收率84％。
实施例11对溴苯甲酰甲酸的酶促立体选择性还原氨化
向46mg对溴苯甲酰甲酸中加入100μL 5M氨水，超声使其部分溶解。另向900μLNH4Cl-NH3·H2O缓冲液(100mM，pH 8.5)中加入56mg(0.7mmol)葡萄糖、32.1mg(0.6mmol)氯化铵、0.4mg NAD+(0.5mM)及含有L40A/V294G(SEQ ID No.30)和GDH的冻干细胞各40mg(40g/L)。将苯甲酰甲酸的氨水悬浮液加入到酶催化体系中，在37℃摇床中反应8h，并用5M氨水维持反应混合物的pH≈8.5。反应结束后，加入100μL 6N盐酸，用1mL乙酸乙酯萃取3次后，水层在12000rpm下离心2min，收集上清液，适当稀释后用于HPLC检测。产物转化率﹥99％，ee﹥99.9％(S)，并在阳离子交换树脂纯化后得到38.2mg产物，收率为82.7％。
实施例12对溴苯乙酮的化学酶法连续一锅法反应制备(S)-对溴苯甘氨酸
将实施例10中氧化反应结束后的反应液静置或离心，弃去生成的单质硒，取1mL反应上清液加入到按实施例11的方法预制的19mL含有L40A/V294G(SEQ ID No.30)和GDH的冻干细胞各40mg(40g/L)的酶催化体系中，在37℃摇床中反应8h，并用5M氨水维持反应混合物的pH≈8.5。反应结束后，用6N盐酸调节pH＝1，20mL乙酸乙酯萃取3次后，水层在4000rpm下离心15min，收集上清液，适当稀释后用于HPLC检测。产物转化率﹥99％，ee＝99.1％(S)，并在阳离子交换树脂纯化后得到167.5mg产物，两步反应总收率为58％。
SEQUENCE LISTING
<110> 沈阳药科大学
<120> 氨基酸脱氢酶突变体及其应用
<130> 2019.10.28
<160> 60
<170> PatentIn version 3.5
<210> 1
<211> 1095
<212> DNA
<213> Bacillus subtilis, BsLDH-WT
<400> 1
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 2
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<213> 人工序列, BsLDH-WT
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Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
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Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
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Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
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Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
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Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
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Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
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Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
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Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
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Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
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Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
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Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
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Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
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His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
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Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
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Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
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Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
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Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
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Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
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Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
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Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
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Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
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Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
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Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
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<210> 3
<211> 1095
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<400> 3
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
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ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 4
<211> 364
<212> PRT
<213> 人工序列 L40A
<400> 4
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
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Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
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Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
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Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
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Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
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Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
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Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 5
<211> 1095
<212> DNA
<213> 人工序列 L40S
<400> 5
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgagc 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 6
<211> 364
<212> PRT
<213> 人工序列 L40S
<400> 6
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ser Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
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Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 7
<211> 1095
<212> DNA
<213> 人工序列 M65A
<400> 7
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcgcaaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 8
<211> 364
<212> PRT
<213> 人工序列 M65A
<400> 8
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Ala Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 9
<211> 1095
<212> DNA
<213> 人工序列 M65S
<400> 9
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcagcaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 10
<211> 364
<212> PRT
<213> 人工序列 M65S
<400> 10
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Ser Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 11
<211> 1095
<212> DNA
<213> 人工序列 V291A
<400> 11
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gcaatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 12
<211> 364
<212> PRT
<213> 人工序列 V291A
<400> 12
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ala Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 13
<211> 1095
<212> DNA
<213> 人工序列 V291S
<400> 13
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc agcatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 14
<211> 364
<212> PRT
<213> 人工序列 V291S
<400> 14
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ser Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 15
<211> 1095
<212> DNA
<213> 人工序列 V291G
<400> 15
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc agcatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 16
<211> 364
<212> PRT
<213> 人工序列 V291G
<400> 16
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Gly Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 17
<211> 1095
<212> DNA
<213> 人工序列 V294S
<400> 17
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaaca gcgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 18
<211> 364
<212> PRT
<213> 人工序列 V294S
<400> 18
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Ser Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 19
<211> 1095
<212> DNA
<213> 人工序列 V294G
<400> 19
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg gtgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 20
<211> 364
<212> PRT
<213> 人工序列 V294G
<400> 20
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Gly Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 21
<211> 1095
<212> DNA
<213> 人工序列 L40A/M65A
<400> 21
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcgcaaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 22
<211> 364
<212> PRT
<213> 人工序列 L40A/M65A
<400> 22
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Ala Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 23
<211> 1095
<212> DNA
<213> 人工序列 L40A/M65S
<400> 23
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcagcaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 24
<211> 364
<212> PRT
<213> 人工序列 L40A/M65S
<400> 24
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Ser Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 25
<211> 1095
<212> DNA
<213> 人工序列 L40A/V291A
<400> 25
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gcaatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 26
<211> 364
<212> PRT
<213> 人工序列 L40A/V291A
<400> 26
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ala Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 27
<211> 1095
<212> DNA
<213> 人工序列 L40A/V291G
<400> 27
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc ggtatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 28
<211> 364
<212> PRT
<213> 人工序列 L40A/V291G
<400> 28
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Gly Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 29
<211> 1095
<212> DNA
<213> 人工序列 L40A/V294G
<400> 29
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg gtgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 30
<211> 364
<212> PRT
<213> 人工序列 L40A/V294G
<400> 30
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Gly Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 31
<211> 1095
<212> DNA
<213> 人工序列 L40S/M65A
<400> 31
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgagc 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcgcaaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 32
<211> 364
<212> PRT
<213> 人工序列 L40S/M65A
<400> 32
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ser Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Ala Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 33
<211> 1095
<212> DNA
<213> 人工序列 L40S/M65S
<400> 33
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgagc 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcagcaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 34
<211> 364
<212> PRT
<213> 人工序列 L40S/M65S
<400> 34
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ser Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Ser Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 35
<211> 1095
<212> DNA
<213> 人工序列 V291A/V294A
<400> 35
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gcaatcaacg cagcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 36
<211> 364
<212> PRT
<213> 人工序列 V291A/V294A
<400> 36
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ala Ile Asn Ala Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 37
<211> 1095
<212> DNA
<213> 人工序列 V291G/V294A
<400> 37
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc ggtatcaacg cagcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 38
<211> 364
<212> PRT
<213> 人工序列 V291G/V294A
<400> 38
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Gly Ile Asn Ala Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 39
<211> 1095
<212> DNA
<213> 人工序列 V291S/V294A
<400> 39
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gtgatcaacg tggcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 40
<211> 364
<212> PRT
<213> 人工序列 V291S/V294A
<400> 40
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ser Ile Asn Ala Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 41
<211> 1095
<212> DNA
<213> 人工序列 V291S/V294S
<400> 41
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc agcatcaaca gcgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 42
<211> 364
<212> PRT
<213> 人工序列 V291S/V294S
<400> 42
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ser Ile Asn Ser Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 43
<211> 1095
<212> DNA
<213> 人工序列 V291A/V294S
<400> 43
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gcaatcaaca gcgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 44
<211> 364
<212> PRT
<213> 人工序列 V291A/V294S
<400> 44
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ala Ile Asn Ser Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 45
<211> 1095
<212> DNA
<213> 人工序列 V291G/V294G
<400> 45
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgctt 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc ggtatcaacg gtgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 46
<211> 364
<212> PRT
<213> 人工序列 V291G/V294G
<400> 46
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Gly Ile Asn Gly Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 47
<211> 1095
<212> DNA
<213> 人工序列 L40A/V291A/V294A
<400> 47
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gcaatcaacg cagcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 48
<211> 364
<212> PRT
<213> 人工序列 L40A/V291A/V294A
<400> 48
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ala Ile Asn Ala Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 49
<211> 1095
<212> DNA
<213> 人工序列 L40A/V291G/V294G
<400> 49
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc ggtatcaacg gtgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 50
<211> 364
<212> PRT
<213> 人工序列 L40A/V291G/V294G
<400> 50
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Gly Ile Asn Ala Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 51
<211> 1095
<212> DNA
<213> 人工序列 L40A/V291S/V294A
<400> 51
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc agcatcaacg cagcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 52
<211> 364
<212> PRT
<213> 人工序列 L40A/V291S/V294A
<400> 52
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ser Ile Asn Ala Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 53
<211> 1095
<212> DNA
<213> 人工序列 L40A/V291S/V294S
<400> 53
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc agcatcaaca gcgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 54
<211> 364
<212> PRT
<213> 人工序列 L40A/V291S/V294S
<400> 54
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ser Ile Asn Ser Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 55
<211> 1095
<212> DNA
<213> 人工序列 L40A/V291A/V294S
<400> 55
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gcaatcaaca gcgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 56
<211> 364
<212> PRT
<213> 人工序列 L40A/V291A/V294S
<400> 56
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ala Ile Asn Ser Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 57
<211> 1095
<212> DNA
<213> 人工序列 L40S/V291A/V294A
<400> 57
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcgagc 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcatgaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gcaatcaacg cagcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 58
<211> 364
<212> PRT
<213> 人工序列 L40S/V291A/V294A
<400> 58
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ser Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ala Ile Asn Ala Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360
<210> 59
<211> 1095
<212> DNA
<213> 人工序列 L40A/M65A/V291A/V294G
<400> 59
atggaacttt ttaaatatat ggagaaatac gattatgaac aattagtatt ctgccaagat 60
gaacaatcag gcttaaaagc gattatcgca attcatgata caacgcttgg tccggcggca 120
ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga cgcgctcaga 180
ctggcaagag gcgcaaccta taaaaacgcg gctgcaggcc taaaccttgg cggcggaaaa 240
acagtaataa tcggcgatcc acgcaaagac aaaaatgaag aaatgttccg cgcgtttggc 300
cgctatattc aaggactgaa cggcagatac attacagctg aagatgtggg tacaacggtt 360
gaggatatgg acattattca tgatgaaaca gactatgtca cagggatttc tcctgctttc 420
ggctcttctg gaaatccatc tccagttaca gcgtacgggg tgtacagagg aatgaaagca 480
gccgctaaag ctgctttcgg aaccgactct cttgaaggga aaaccatcgc tgtacagggt 540
gtagggaatg tggcctacaa cctatgccgc cacctgcatg aagaaggggc aaacttaatc 600
gttacggata tcaacaaaca gtcagtacag cgcgcagttg aagattttgg cgcccgtgcg 660
gttgatccgg aagagattta ttcacaagag tgcgatattt atgcaccgtg cgcccttgga 720
gcgacaatca acgacgacac cattaaacag ctgaaggcga aagtcatcgc gggtgcggct 780
aataaccaat taaaagaaac gcgccatggc gatcaaattc acgaaatggg tatcgtttac 840
gcaccggatt atgtcattaa cgcaggcggc gcaatcaacg gtgcagatga gctttacggc 900
tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat tgagcgtgta 960
cttgagattt ctcagcgtga cggcattcca acatatttag cagctgaccg cttggcagag 1020
gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080
ttaagcagac gttaa 1095
<210> 60
<211> 364
<212> PRT
<213> 人工序列 L40A/M65A/V291A/V294G
<400> 60
Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Ala Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser
195 200 205
Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Glu
210 215 220
Glu Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Ala Ile Asn Gly Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu
290 295 300
Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val
305 310 315 320
Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Thr Tyr Leu Ala Ala Asp
325 330 335
Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln
340 345 350
Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg
355 360

Claims (10)

1.氨基酸脱氢酶突变体，其特征在于，其氨基酸序列如SEQ ID No. 30、50所示。
2.一种核酸，其能够编码权利要求1所述的氨基酸脱氢酶突变体。
3.一种表达载体，该载体含有权利要求2所述的核酸且能在宿主细胞中进行表达。
4.一种宿主细胞，其含有权利要求3所述的表达载体。
5.权利要求4所述的宿主细胞，其特征在于，所述的宿主细胞为大肠杆菌、枯草芽孢杆菌、地衣芽孢杆菌、黑曲霉、酿酒酵母或毕赤酵母。
6.生物催化剂，包含权利要求1所述的突变体，所述的生物催化剂包括细胞、粗酶粉、酶溶液、固定化酶形式。
7.权利要求1所述的氨基酸脱氢酶突变体在制备手性氨基酸中的应用。
8.如权利要求7所述的应用，其特征在于，以α-酮酸及其衍生物为底物，与权利要求6所述的生物催化剂接触；
反应是在辅酶存在下，在pH 7 - 10、温度为25 - 50℃的缓冲溶液中进行的，其中，所述的辅酶为NAD+，与葡萄糖与GDH构成辅酶再生系统；所述的缓冲溶液为NH4Cl-NH3·H2O缓冲液；
所述的α-酮酸及其衍生物如式（I）所示，
当氨基酸脱氢酶突变体的氨基酸序列为SEQ ID No. 30时，R为单取代或多取代的苯环或C1 - C6直链及支链脂肪族取代基；
当氨基酸脱氢酶突变体的氨基酸序列为SEQ ID No. 50时，R为单取代或多取代的苯环、萘环或C1 - C6直链及支链脂肪族取代基；
所述的单取代或多取代的取代基为H、卤素、羟基、氨基、巯基、C1 - C5饱和或不饱和烷基、C1 - C5烷氧基、C1 - C5酯基、C1 - C5酰胺基。
9.如权利要求8所述应用，其特征在于，α-酮酸及其衍生物通过如下方法制备：甲基酮类化合物为底物，在惰性气体环境中、氧化剂存在下，在有机溶剂中回流得到α-酮酸类化合物，所述的氧化剂是二氧化硒；化学氧化反应是在吡啶中，50 - 110℃回流3 - 10 h后完成的；所述的甲基酮类化合物如式（II）所示，
当氨基酸脱氢酶突变体的氨基酸序列为SEQ ID No. 30时，R为单取代或多取代的苯环或C1 - C6直链及支链脂肪族取代基；
当氨基酸脱氢酶突变体的氨基酸序列为SEQ ID No. 50时，R为单取代或多取代的苯环、萘环或C1 - C6直链及支链脂肪族取代基；
所述的单取代或多取代的取代基为H、卤素、羟基、氨基、巯基、C1 - C5饱和或不饱和烷基、C1 - C5烷氧基、C1 - C5酯基、C1 - C5酰胺基。
10.一种化学-酶法连续反应过程，以甲基酮类化合物为起始原料，一锅法反应制备手性氨基酸，其特征在于，将权利要求9所述化学氧化反应结束后静置得到的上清液，直接加入到酶催化体系中，以连续反应的方式制得手性氨基酸，所述的化学氧化反应结束后的上清液与酶催化体系的体积比为1:10 - 1:200；所述的酶催化体系由含有权利要求1所述的氨基酸脱氢酶突变体与葡萄糖脱氢酶的大肠杆菌冻干细胞各20 - 200 mg、1.35 g/L -252 g/L 葡萄糖和0 - 0.5 mmol NAD+在pH 7 - 10、温度为25 - 50℃的NH4Cl-NH3·H2O缓冲液中构成。
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