具体实施方式
下面结合实施例对本发明的内容作进一步阐述,但本发明保护范围并不仅局限于此。实施例中未作详细说明的操作方法均为本领域技术人员公知的常规操作方法。
本发明中所用的试剂及药品均为普通市售产品。
实施例1
海杆菌(Marinobacter aquaeolei)的烷烃羟化酶CYP153A与巨大芽孢杆菌(Bacillus megaterium)P450NADH还原酶的融合酶基因,即CYP153A-CPRBM3融合酶基因、CYP153A M228L-CPRBM3融合酶基因,脂酰CoA脱氢酶基因PpFadE、脂酰CoA合成酶基因MaMACS、酯酰辅酶A硫酯酶基因ctydiI的PCR扩增。
根据海杆菌(Marinobacter aquaeolei)的烷烃羟化酶CYP153A与巨大芽孢杆菌(Bacillus megaterium)P450NADH还原酶的融合酶基因,即CYP153A基因、CPRBM3基因、CYP153A M228L-CPRBM3融合酶基因、脂酰CoA脱氢酶基因PpFadE、脂酰CoA合成酶基因MaMACS、酯酰辅酶A硫酯酶基因ctydiI设计PCR扩增引物,上游引物的核苷酸序列分别如SEQID NO.1、3、5、7、9、11所示,下游引物的核苷酸序列如SEQ ID NO.2、4、6、8、10、12所示;
其中,海杆菌(Marinobacter aquaeolei)的烷烃羟化酶CYP153A与巨大芽孢杆菌(Bacillus megaterium)P450NADH还原酶的融合酶基因,即CYP153A-CPRBM3、CYP153AM228L-CPRBM3融合酶基因,脂酰CoA脱氢酶基因PpFadE、脂酰CoA合成酶基因PpFadE、酯酰辅酶A硫酯酶基因ctYdiI的核苷酸序列分别如SEQ ID NO.13、14、15、16、17所示,对应表达的氨基酸序列如SEQ ID NO.18、19、20、21、22所示,CYP153A M228L的核苷酸序列如SEQ IDNO.23所示,CYP153A M228L的氨基酸序列如SEQ ID NO.24所示。
所述构建重组质粒pET21b-CYP153A-CPRBM3,包括如下步骤:
以经过密码子优化的海杆菌(Marinobacter aquaeolei)的烷烃羟化酶CYP153A与巨大芽孢杆菌(Bacillus megaterium)P450NADH还原酶的融合酶基因为模板进行PCR扩增,CYP153A的上游引物的核苷酸序列如SEQ ID NO.1所示,下游引物的核苷酸序列如SEQ IDNO.2所示,CPRBM3的上游引物的核苷酸序列如SEQ ID NO.3所示,下游引物的核苷酸序列如SEQ ID NO.4所示,然后将pET21b质粒用Nde I和Xho I进行双酶切,经多片段无缝克隆试剂盒连接,制得重组质粒pET21b-CYP153A-CPRBM3;
PCR扩增体系如下,总体系25μL:
100μM上游引物1.0μL,100μM下游引物1.0μL,模板1.0μL,5U/μL phanta酶(一种高保真酶)12.5μL,ddH2O 9.5μL;
PCR扩增条件如下:
95℃预变性3min;95℃变性15s,60℃退火15s,72℃延伸CYP153A45s、CPRBM355s,循环30次;72℃延伸5min。
PCR产物回收:
PCR扩增结束后通过1%的琼脂糖凝胶电泳分析片段长短,结果如图2所示,根据片段大小切下目的条带,使用上海生工生物工程股份有限公司的DNA胶回收试剂盒回收PCR产物。
所述构建重组质粒pET21b-CYP153AM228L-CPRBM3,包括如下步骤:
以构建好的pET21b-CYP153A-CPRBM3融合酶基因为模板进行PCR扩增,CYP153AM228L-CPRBM3的上游引物的核苷酸序列如SEQ ID NO.5所示,下游引物的核苷酸序列如SEQID NO.6所示;然后利用Dpn I酶去除原始模板后,转化到感受态细胞Escherichia coliBL21中使其环化,得到重组质粒pET21b-CYP153A M228L-CPRBM3。
PCR扩增体系如下,总体系25μL:
100μM上游引物1.0μL,100μM下游引物1.0μL,模板1.0μL,5U/μL phanta酶(一种高保真酶)12.5μL,ddH2O 9.5μL;
PCR扩增条件如下:
95℃预变性3min;95℃变性15s,60℃退火15s,72℃延伸100s,循环30次;72℃延伸5min。
PCR产物回收:
PCR扩增结束后通过1%的琼脂糖凝胶电泳分析片段长短,结果如图3所示,得到pET21b-CYP153A M228L-CPRBM3的质粒见图1中A,根据片段大小切下目的条带,使用上海生工生物工程股份有限公司的DNA胶回收试剂盒回收PCR产物。
DpnI酶切模板
DpnI酶切体系:
PCR产物 44μL
DpnI 1μL
10X QuickCut Buffer 5μL
反应条件:37℃反应2h。
反应完成后70℃15min失活DpnI。
所述构建重组质粒pCDFDuet-1-MaMACS,包括如此步骤:
以大肠杆菌DH5a基因组为模板,扩增脂酰CoA合成酶基因MaMACS,上游引物的核苷酸序列如SEQ ID NO.7所示,下游引物的核苷酸序列如SEQ ID NO.8所示,然后将pCDFDuet-1质粒用Hind III、BamH I进行双酶切,经连接酶连接,制得重组质粒pCDFDuet-1-MaMACS;
PCR扩增体系如下,总体系25μL:
100μM上游引物1.0μL,100μM下游引物1.0μL,模板1.0μL,5U/μL phanta酶(一种高保真酶)12.5μL,ddH2O 9.5μL;
PCR扩增条件如下:
95℃预变性3min;95℃变性15s,60℃退火15s,72℃延伸50s,循环30次;72℃延伸5min。
PCR产物回收:
PCR扩增结束后通过1%的琼脂糖凝胶电泳分析片段长短,根据片段大小切下目的条带,使用上海生工生物工程股份有限公司的DNA胶回收试剂盒回收PCR产物。
所述构建重组质粒pCDFDuet-1-MaMACS-PpFadE,包括如下步骤:
以大肠杆菌DH5a基因组为模板,扩增脂酰CoA脱氢酶基因PpFadE,上游引物的核苷酸序列如SEQ ID NO.9所示,下游引物的核苷酸序列如SEQ ID NO.10所示,然后将pCDFDuet-1-MaMACS质粒用Nde I、Ava I进行双酶切,经连接酶连接,制得重组质粒pCDFDuet-1-MaMACS-PpFadE;
PCR扩增体系如下,总体系25μL:
100μM上游引物1.0μL,100μM下游引物1.0μL,模板1.0μL,5U/μL phanta酶12.5μL,ddH2O 9.5μL;
PCR扩增条件如下:
95℃预变性3min;95℃变性15s,60℃退火15s,72℃延伸40s,循环30次;72℃延伸5min。
PCR产物回收:
PCR扩增结束后通过1%的琼脂糖凝胶电泳分析片段长短,根据片段大小切下目的条带,使用上海生工生物工程股份有限公司的DNA胶回收试剂盒回收PCR产物。
所述构建重组质粒pET28a-SUMO-ctYdiI,包括如下步骤:
扩增酯酰辅酶A硫酯酶基因ctydiI,上游引物的核苷酸序列如SEQ ID NO.11所示,下游引物的核苷酸序列如SEQ ID NO.12所示,然后将pET28a-SUMO质粒和酯酰辅酶A硫酯酶基因ctydiI分别用BamH I和Xho I分别进行双酶切,经连接酶连接,制得重组质粒pET28a-SUMO-ctYdiI;
PCR扩增体系如下,总体系25μL:
100μM上游引物1.0μL,100μM下游引物1.0μL,模板1.0μL,5U/μL phanta酶12.5μL,ddH2O 9.5μL;
PCR扩增条件如下:
95℃预变性3min;95℃变性15s,60℃退火15s,72℃延伸15s,循环30次;72℃延伸5min。
PCR产物回收:
PCR扩增结束后通过1%的琼脂糖凝胶电泳分析片段长短,结果如图4所示,根据片段大小切下目的条带,使用上海生工生物工程股份有限公司的DNA胶回收试剂盒回收PCR产物。
实施例2
重组质粒载体pET21b、pCDFDuet-1、pCDFDuet-1-MaMACS、pET28a-SUMO及基因ctYdiI的酶切
提取pET21b质粒并对其进行双酶切反应,反应体系如下:
反应条件:37℃反应6h。
pET21b质粒载体双酶切后经1%琼脂糖凝胶电泳纯化图5,并使用DNA胶回收试剂盒进行目的片段回收。
提取pCDFDuet-1质粒并对其进行双酶切反应,反应体系如下:
反应条件:37℃反应6h。
pCDFDuet-1质粒载体双酶切后经1%琼脂糖凝胶电泳纯化,并使用DNA胶回收试剂盒进行目的片段回收。
提取pCDFDuet-1-MaMACS质粒并对其进行双酶切反应,反应体系如下:
反应条件:37℃反应6h。
pCDFDuet-1-MaMACS质粒载体双酶切后经1%琼脂糖凝胶电泳纯化,并使用DNA胶回收试剂盒进行目的片段回收。
提取pET28a-SUMO质粒及酯酰CoA硫酯酶ctYdiI PCR产物进行双酶切反应,反应体系如下:
反应条件:37℃反应6h。
pET28a-SUMO质粒及酯酰CoA硫酯酶ctYdiI PCR产物经双酶切后经1%琼脂糖凝胶电泳纯化图6,并使用DNA胶回收试剂盒进行目的片段回收。
实施例3
重组质粒pET21b-CYP153A-CPRBM3多片段无缝克隆、重组质粒pET28a-SUMO-ctYdiI连接
将经过双酶切的pET21b质粒与CYP153A、CPRBM3 PCR产物连接,连接反应体系如下:
将上述连接反应体系充分混匀后离心数秒,将管壁液滴收到管底,37℃连接30min,得重组质粒pET21b-CYP153A-CPRBM3。
将经过酶切的pCDFDuet-1质粒与MaMACS PCR产物连接,连接反应体系如下:
将上述连接反应体系充分混匀后离心数秒,将管壁液滴收到管底,37℃反应37min,得重组质粒pCDFDuet-1-MaMACS。
将经过酶切的pCDFDuet-1-MaMACS质粒与PpFadE PCR产物连接,连接反应体系如下:
将上述连接反应体系充分混匀后离心数秒,将管壁液滴收到管底,37℃反应37min,得重组质粒pCDFDuet-1-MaMACS-PpFadE见图1中B。
将经过双酶切的pET28a-SUMO质粒与ctYdiI PCR产物连接,连接反应体系如下:
将上述连接反应体系充分混匀后离心数秒,将管壁液滴收到管底,22℃连接2小时,得重组质粒pET28a-SUMO-ctYdiI见图1中C。
实施例4
重组质粒pET21b-CYP153A-CPRBM3、重组质粒pET21b-CYP153A M228L-CPRBM3、重组质粒pCDFDuet-1-MaMACS-PpFadE、重组质粒pET28a-SUMO-ctYdiI的转化:
(1)感受态细胞的制备
①挑取大肠杆菌BL21(DE3)单菌落(或挑取保存菌种)接种至10ml液体LB培养基,37℃、210rpm过夜培养;
②取5ml菌液接种于500ml LB培养基中,37℃、210rpm培养至菌液OD600为0.375左右;
③将菌液放置于冰水混合物上10min,同时预冷50ml离心管;
④将菌液转移到离心管中,4℃,3700rpm离心10min收集菌体;
⑤每个离心管中加入10mL预冷的0.1M CaCl2溶液,重悬菌体,再加入30mL预冷的0.1M CaCl2溶液,颠倒混匀,冰上静置20min;
⑥4℃,3700rpm离心10min收集菌体,按照与步骤④中菌液的体积比为3:125的比例加入预冷的含有15%甘油的0.1M的CaCl2溶液,重悬菌体,得感受态细胞;
⑦将感受态细胞分装,并于-80℃冻存。
同理制备大肠杆菌基因缺失菌BL21(ΔFadB、R、J)的感受态细胞。
大肠杆菌基因缺失菌BL21ΔFadB、R、J的构建方法,包括如下步骤:
Ⅰ利用RED重组法敲除基因,构建基因缺失ΔFadRB菌种,以下称为ΔFadRΔFadB菌种;该菌种的构建方法参见中国专利文献CN110684794A(申请号:201911038194.3)。
Ⅱ构建FadJ敲除框,具体步骤如下:
以大肠杆菌BL21基因组为模板,扩增3-羟酰辅酶A脱氢酶基因的上游同源臂FadJ1,上游引物的核苷酸序列如SEQ ID NO.25所示,下游引物的核苷酸序列如SEQ IDNO.26所示;以大肠杆菌BL21基因组为模板,扩增3-羟酰辅酶A脱氢酶基因的下游同源臂FadJ2,上游引物的核苷酸序列如SEQ ID NO.27所示,下游引物的核苷酸序列如SEQ IDNO.28所示;以pkd3质粒为模板,扩增FRT-RKan—FRT基因片段,上游引物的核苷酸序列如SEQ ID NO.29所示,下游引物的核苷酸序列如SEQ ID NO.30所示;然后将FadJl、FRT-RKan-FRT与FadJ2基因片段多片段无缝克隆,扩增FadJ1-Kan-FadJ2的敲除框片段,上游引物的核苷酸序列如SEQ ID NO.31所示,下游引物的核苷酸序列如SEQ ID NO.32所示,纯化胶回收获得FadJ敲除框(以下将FadJ敲除框缩写为Jk);
PCR扩增体系如下,总体系50μL:
100μM上游引物2.0μL,100μM下游引物2.0μL,模板2.0μL,5U/μL phanta酶25μL,ddH2O19μL;
PCR扩增条件如下:
95℃预变性3min;95℃变性15s,60℃退火15s,72℃延伸1min15s,循环30次;72℃延伸5min。
Ⅲ将步骤Ⅱ制备的FadJ敲除框转化入pkd46-ΔFadRΔFadB重组菌,最终获得大肠杆菌基因缺失菌BL21ΔFadB、R、J,具体步骤如下:
a、将质粒pkd46转化进ΔFadRΔFadB感受态细胞中,获得的pkd46-ΔFadRΔFadB重组菌,制备pkd46-ΔFadRΔFadB感受态细胞,再用质量浓度为10%的甘油保存制备的pkd46-ΔFadRΔFadB感受态细胞;
b、将FadJ敲除框Jk转化进pkd46-ΔFadRΔFadB感受态细胞中,验证pkd46-Jk-BL21重组菌确认敲除框转入后,42℃消除pkd46,筛选后得到Jk-ΔFadRΔFadB重组菌;
c、将Jk-ΔFadRΔFadB重组菌制备感受态转化pcp20质粒,42℃消除Jk抗性以及pcp20质粒,获得ΔFadRBJ重组菌,即大肠杆菌基因缺失菌BL21ΔFadB、R、J。
图11为菌落PCR验证ΔFadRΔFadB敲除FadJ产物琼脂糖凝胶电泳图;
图中,泳道M为Marker;泳道1-2为FadJ基因条带,泳道3-8为FadJ敲除框融合片段基因条带。
(2)重组质粒的转化
①将10μL重组质粒pET21b-CYP153A M228L-CPRBM3加入到100μL新鲜制备的大肠杆菌BL21感受态细胞中,轻轻混匀,冰浴30min;10μL重组质粒pET21b-CYP153A-CPRBM3加入到100μL新鲜制备的大肠杆菌BL21感受态细胞中,轻轻混匀,冰浴30min;将10μL重组质粒pCDFDuet-1-MaMACS-PpFadE、重组质粒pET28a-SUMO-ctYdiI同时加入到100μL新鲜制备的大肠杆菌基因缺失菌BL21ΔFadB、R、J的感受态细胞中。
②42℃热激90s,然后迅速置于冰浴中冷却3min;
③将上述感受态细胞接入到500μL LB培养基,37℃,200rpm振荡培养60min;
④取上述菌液200μL,涂布于带有100mg/mL、50mg/mL、40mg/mL氨苄青霉素、卡娜霉素、链霉素的LB固体培养基;
⑤37℃培养箱中正置30min,待菌液被吸干后,倒置平板于37℃培养12-16h。
(3)阳性克隆的鉴定:
①蛋白表达及可溶性鉴定
取上述菌液900μL,并加入终浓度为0.32mM的IPTG,诱导表达4h,12000rpm离心1min,收集菌体,加入2倍上样缓冲液,重悬菌体,100℃水浴变性10min,用SDS-PAGE检测蛋白表达,结果如图7所示,显示为阳性克隆。
②菌样测序
将用以上两种方法鉴定后的阳性克隆,送至测序公司进行测序,进一步证明构建的阳性克隆的正确性,获得大肠杆菌BL21ΔFadB、R、J,pCDFDuet-1-MaMACS-PpFadE、pET28a-SUMO-ctYdiI;大肠杆菌BL21 pET21b-CYP153A M228L-CPRBM3;大肠杆菌BL21pET21b-CYP153A-CPRBM3;
实施例5
大肠杆菌BL21ΔFadB、R、J,pCDFDuet-1-MaMACS-PpFadE、pET28a-SUMO-ctYdiI及大肠杆菌BL21 pET21b-CYP153A M228L-CPRBM3融合酶工程菌发酵
(1)菌种活化:将实施例4中的大肠杆菌BL21ΔFadB、R、J,pCDFDuet-1-MaMACS-PpFadE、pET28a-SUMO-ctYdiI以1%的接种量接种至50mL的含有卡那霉素和链霉素的液体LB培养基中,在37℃、200rpm振荡培养12h;
(2)菌体转接:取上述活化菌株3mL接入300mL含有卡那霉素和链霉素的液体培养基中,37℃、200rpm振荡培养至菌液OD600为1.0时,降温至20℃适应1小时后,加入IPTG使培养基中IPTG浓度为0.5mM、加入油酸使培养基中油酸质量百分比浓度为0.6%、加入吐温80使培养基中吐温80的质量百分比浓度为0.3%,过夜诱导培养;
(3)收集菌体:取上述菌液300mL,5000rpm,4℃离心15min,收集菌体;
(4)用0.85%生理盐水洗涤沉淀三次,并用转化培养基重悬菌体制备菌悬液。转化培养基包含100mM磷酸钾缓冲液(pH7.4),质量浓度1%的甘油,质量浓度0.4%的葡萄糖,50μg/mL的卡那抗生素、40μg/mL的链霉素,总体积为30mL,分别加入0.5g/L的癸酸进行反应。30℃反应20h并在9h取样。
大肠杆菌BL21 pET21b-CYP153A M228L-CPRBM3融合酶工程菌发酵
(1)菌种活化:将实施例4中的大肠杆菌BL21 pET21b-CYP153A M228L-CPRBM3以1%的接种量接种至50mL的含有氨苄青霉素的液体LB培养基中,在37℃、200rpm振荡培养12h;
(2)菌体转接:取上述活化菌株3mL接入300mL含有氨苄青霉素的液体培养基中,37℃、200rpm振荡培养至菌液OD600为1.0时,降温至20℃适应1小时后,加入加入IPTG使培养基中IPTG浓度为0.5mM、加入终浓度0.5mM的FeCl3、0.5mM终浓度5-ALA(5-氨基乙酰丙酸盐酸盐)继续诱导培养18小时,分离细胞,制得诱导细胞;
(3)收集菌体:取上述菌液300mL,5000rpm,4℃离心15min,收集菌体;
(4)用0.85%生理盐水洗涤沉淀三次,并用转化培养基重悬菌体制备菌悬液。转化培养基包含100mM磷酸钾缓冲液(pH7.4),菌体重悬后体积为3mL,将其加入到上一步反应完成的反应体系中,30℃反应20h取样。反应终止后加入1mL 0.4mol/L HCl终止反应,加入0.1g/L月桂酸作为内标。
发酵液硅烷化处理:取1mL发酵液于1.5mL离心管中,每个样品用1.5mL乙酸乙酯萃取样品溶液中的脂肪酸(每次750μL),旋流混合器混合60s,室温下4000r/m离心10min。取提取液蒸发干燥,干燥后的样品重新溶解在0.5mL乙酸乙酯(色谱纯)、0.5mL正己烷(色谱纯)中,100μL BSTFA-TMCS(99:1,v/v)衍生化试剂加入后室温放置5min,在70℃的烤箱中孵育50min。
气相质谱检测生成产物:气相色谱以氦气为载气,恒流模式,进样体积1μL,分流进样,分流比为1:5,进样温度250℃,50℃保持1min,以15℃/min升至250℃,保持10min。产物色谱图如图8、图9、图10所示。
通过对产物的色谱图、质谱图计算分析,得出癸酸经过两步法生产高附加值10-羟基-2-癸烯酸的过程0.5g/L癸酸经摇瓶发酵,产生0.273g/L的10-羟基-2-癸烯酸,转化率为54.6%
对比例1
与实施例5重组菌发酵的不同之处在于,用实施例4获得的大肠杆菌BL21pET21b-CYP153A-CPRBM3代替大肠杆菌BL21 pET21b-CYP153A M228L-CPRBM3,其他均相同,实验结果得出0.5g/L癸酸经摇瓶发酵,产生0.077g/L的10-羟基-2-癸烯酸,转化率为15.4%对比例2
重组质粒pET21b-CYP153A M228L-CPRBM3、重组质粒pCDFDuet-1-MaMACS-PpFadE、重组质粒pET28a-SUMO-ctYdiI的转化与实施例4的不同之处在于,同理制备大肠杆菌基因缺失菌BL21(ΔFadB、R)的感受态细胞,将重组质粒pCDFDuet-1-MaMACS-PpFadE、重组质粒pET28a-SUMO-ctYdiI转化入新鲜制备的大肠杆菌基因缺失菌BL21ΔFadB、R的感受态细胞中,获得大肠杆菌BL21ΔFadB、R,pCDFDuet-1-MaMACS-PpFadE、pET28a-SUMO-ctYdiI;其他均相同。
将获得的大肠杆菌BL21ΔFadB、R,pCDFDuet-1-MaMACS-PpFadE、pET28a-SUMO-ctYdiI及大肠杆菌BL21 pET21b-CYP153A M228L-CPRBM3融合酶工程菌发酵,发酵方法同实施例5相同,实验结果得出大肠杆菌BL21ΔFadB、R,pCDFDuet-1-MaMACS-PpFadE、pET28a-SUMO-ctYdiI催化癸酸并不能产生反式-2-癸烯酸,所以也就无法产生10-羟基-2癸烯酸。
本实验以癸酸为底物,利用两步法生物级联催化生成10-HDA,前期实验室以癸酸为底物一步法合成10-HAD,本申请通过优化获得的脂酰CoA脱氢酶基因PpFadE、脂酰CoA合成酶基因MaMACS、酰辅酶A硫酯酶基因ctydiI构建的合成途径与专利文献CN109897870A中脂酰CoA脱氢酶基因MCAD、脂酰CoA合成酶基因FadK、酰辅酶A硫酯酶基因ydiI并非相同基因,本文中的P450融合酶是将228位的氨基酸由M变为L,通过催化元件的替换以及生物合成方式的变化,可以显著提高10-HDA产量。
由实施例5与对比例2的实验结果可以看出本发明使用大肠杆菌基因缺失菌BL21ΔFadB、R、J作为重组质粒pCDFDuet-1-MaMACS-PpFadE、重组质粒pET28a-SUMO-ctYdiI的转化宿主菌,催化癸酸产生反式-2-癸烯酸具有特异性,与大肠杆菌BL21 pET21b-CYP153AM228L-CPRBM3融合酶工程菌结合两步法合成10-羟基-2癸烯酸,最终10-羟基-2-癸烯酸的转化率达54.6%显著优于大肠杆菌基因缺失菌BL21ΔFadB、R作为宿主菌的作用效果。
SEQUENCE LISTING
<110> 齐鲁工业大学
<120> 一种突变酶CYP153A M228L及其在合成10-羟基-2-癸烯酸中的应用
<160> 32
<170> PatentIn version 3.5
<210> 1
<211> 42
<212> DNA
<213> 人工序列
<400> 1
taagaaggag atatacatat gatgccgacg ttaccacgta cc 42
<210> 2
<211> 39
<212> DNA
<213> 人工序列
<400> 2
tgccgcccat actattaggt gtcagtttaa ccattaagc 39
<210> 3
<211> 28
<212> DNA
<213> 人工序列
<400> 3
acctaatagt atgggcggca ttccttca 28
<210> 4
<211> 41
<212> DNA
<213> 人工序列
<400> 4
gtggtggtgg tggtgctcga gttacccagc ccacacgtct t 41
<210> 5
<211> 23
<212> DNA
<213> 人工序列
<400> 5
cgcaccggcc aagcgatcgc tcc 23
<210> 6
<211> 23
<212> DNA
<213> 人工序列
<400> 6
ggagcgatcg cttggccggt gcg 23
<210> 7
<211> 30
<212> DNA
<213> 人工序列
<400> 7
atgtcagata ccaccaccgc atttaccgtt 30
<210> 8
<211> 25
<212> DNA
<213> 人工序列
<400> 8
atgtcagata ccaccaccgc attta 25
<210> 9
<211> 32
<212> DNA
<213> 人工序列
<400> 9
atggattttg cctatagtcc gaaagttcag gc 32
<210> 10
<211> 32
<212> DNA
<213> 人工序列
<400> 10
ttccggttga aatgctgcgc tcaggtcgtt aa 32
<210> 11
<211> 41
<212> DNA
<213> 人工序列
<400> 11
agagaacaga ttggtggatc cggatccatg atttggcagc g 41
<210> 12
<211> 41
<212> DNA
<213> 人工序列
<400> 12
gtggtggtgg tggtgctcga gctcgagtta cacaacggcg g 41
<210> 13
<211> 3195
<212> DNA
<213> 人工序列
<400> 13
atgccgacgt taccacgtac ctttgatgac attcagtctc gcttaatcaa tgctacaagt 60
cgtgtggttc caatgcagcg tcagattcag ggtctgaaat ttctgatgag tgccaaacgc 120
aaaacctttg gtccacgtcg cccaatgccg gaatttgtgg aaacacctat cccggatgtt 180
aatacattag ccttagagga cattgatgtg agtaatccgt ttctgtatcg ccagggccag 240
tggcgcgcat attttaaacg cttacgcgat gaagctccag ttcattatca gaaaaatagc 300
ccatttggtc cgttttggag cgtgacccgc tttgaggaca ttctgtttgt ggataaatca 360
catgatctgt ttagcgccga accacagatc atcttaggtg atcctccgga aggcctgtca 420
gtggaaatgt ttattgcgat ggaccctcct aaacatgatg tgcagcgctc tagtgttcag 480
ggtgtggttg cccctaaaaa tctgaaagaa atggaaggcc tgattcgtag tcgtacgggc 540
gatgtgttag attcattacc gacggataaa ccgtttaatt gggttcctgc ggtgagcaaa 600
gaactgacgg gtagaatgct ggctacctta ctggattttc cgtatgaaga acgtcataaa 660
ctggttgaat ggagcgatcg catggccggt gcggcaagtg ctacgggcgg cgaatttgcg 720
gatgaaaatg ctatgtttga tgatgcggca gatatggcac gctctttttc tcgcctgtgg 780
cgcgataaag aagcccgccg tgcagcaggc gaagaaccgg gctttgattt aatctcactg 840
ctacagtcta ataaagaaac caaggatctg atcaatcgtc ctatggaatt tattggcaat 900
ctgaccctgc tgattgtggg cggtaatgat acgacccgca atagcatgtc aggcggctta 960
gttgccatga atgaatttcc tcgtgaattt gaaaaactga aagccaaacc ggaactgatt 1020
ccgaatatgg tgagcgaaat tattcgttgg cagacaccac tggcctatat gcgccgcatt 1080
gccaaacagg atgttgaact gggcggtcag accatcaaaa aaggtgatcg cgttgttatg 1140
tggtatgcct caggtaatcg cgatgaacgt aaatttgata atccggatca gtttattatc 1200
gatcgtaaag atgcacgcaa tcacatgtct tttggctatg gtgttcatcg ctgtatgggt 1260
aatcgtctgg ccgaattaca gctgcgtatt ctgtgggaag aaatcttaaa acgctttgat 1320
aatatcgaag ttgtggaaga accagaacgt gtgcagagca attttgttcg cggctatagc 1380
cgcttaatgg ttaaactgac acctaatagt atgggcggca ttccttcacc aagccgagag 1440
cagtcagcta aaaaagagcg caaaaccgta gaaaacgctc ataatacgcc gcttcttgtg 1500
ctatacggtt caaatatggg aacagccgaa ggaacggcgc gtgatttagc ggatattgcg 1560
atgagcaaag gattcgcacc gcaagtcgca acgcttgatt cccacgcagg aaaccttccg 1620
cgtgaaggag ctgttttaat tgtaacggct tcttataacg gtcatcctcc tgataacgca 1680
aaggaatttg ttgactggtt agaccaagcg tctgctgatg aagtaaaagg cgtgcgctac 1740
tccgtatttg gatgcggtga taaaaactgg gcgacaacgt atcaaaaagt gcctgctttt 1800
attgatgaaa ctcttgccgc taaaggggca gaaaacatag ctgaacgcgg tgaagcagat 1860
gcaagcgacg actttgaagg cacatacgaa gaatggcgtg aacacatgtg gagtgactta 1920
gcagcctact ttaacttaga cattgaaaac agcgaagaaa atgcgtctac gctttcactt 1980
caatttgtcg acagcgctgc ggacatgccg cttgcgaaaa tgcaccgtgc gttttcagca 2040
aacgtcgtag caagcaaaga gcttcaaaag ccaggcagtg cacgaagcac gcgtcatctt 2100
gaaattgaac ttccaaaaga agcttcttat caagaaggag atcatttagg tgttattcct 2160
cgcaactatg aaggaatagt aaatcgtgta gcaacaagat ttggtctaga tgcatcacag 2220
caaatccgtt tggaagctga agaagaaaaa ttagctcatt tgccactcgg taaaacagta 2280
tcagtagaag agcttctgca atacgtggag cttcaagatc ctgttacgcg cacgcagctt 2340
cgcgcaatgg ctgctaaaac agtctgcccg ccgcataaag tagagcttga agtcttgctt 2400
gaaaagcagg cgtacaaaga acaagtgctg gcaaaacgtt taacaatgct tgaactgctt 2460
gaaaaatatc cggcgtgtga aatggaattc agcgaattta tcgcacttct tccaagcatg 2520
cgtccgcgct attactcaat ttcttcatca cctcgtgtcg atgaaaaaca agcaagcatc 2580
acggtcagcg ttgtttcagg agaagcgtgg agcggatacg gagaatacaa aggaattgca 2640
tcgaactatc ttgccaatct gcaagaagga gatacgatta cgtgctttgt ttccacaccg 2700
cagtcaggat ttacgctgcc aaaaggccct gaaacaccac ttatcatggt aggaccggga 2760
acaggcgtcg cgccgtttag aggctttgtg caggctcgca agcagttaaa agaacaagga 2820
cagtcgcttg gagaagcgca tttatacttt ggctgccgtt cacctcatga agattatctg 2880
tatcaaaaag agcttgaaaa cgcccaaaat gaaggcatca ttacgcttca taccgctttt 2940
tctcgcgtac caaatcagcc gaaaacatac gttcaacacg tgatggaaca agacggcaag 3000
aaattgattg aacttcttga ccaaggagcg cacttctata tttgcggaga cggaagccaa 3060
atggcacctg acgttgaagc aacgcttatg aaaagctatg ctgaagttca ccaagtgagt 3120
gaagcagacg ctcgcttatg gctgcagcag ctagaagaaa agggccgata cgcaaaagac 3180
gtgtgggctg ggtaa 3195
<210> 14
<211> 3195
<212> DNA
<213> 人工序列
<400> 14
atgccgacgt taccacgtac ctttgatgac attcagtctc gcttaatcaa tgctacaagt 60
cgtgtggttc caatgcagcg tcagattcag ggtctgaaat ttctgatgag tgccaaacgc 120
aaaacctttg gtccacgtcg cccaatgccg gaatttgtgg aaacacctat cccggatgtt 180
aatacattag ccttagagga cattgatgtg agtaatccgt ttctgtatcg ccagggccag 240
tggcgcgcat attttaaacg cttacgcgat gaagctccag ttcattatca gaaaaatagc 300
ccatttggtc cgttttggag cgtgacccgc tttgaggaca ttctgtttgt ggataaatca 360
catgatctgt ttagcgccga accacagatc atcttaggtg atcctccgga aggcctgtca 420
gtggaaatgt ttattgcgat ggaccctcct aaacatgatg tgcagcgctc tagtgttcag 480
ggtgtggttg cccctaaaaa tctgaaagaa atggaaggcc tgattcgtag tcgtacgggc 540
gatgtgttag attcattacc gacggataaa ccgtttaatt gggttcctgc ggtgagcaaa 600
gaactgacgg gtagaatgct ggctacctta ctggattttc cgtatgaaga acgtcataaa 660
ctggttgaat ggagcgatcg cttggccggt gcggcaagtg ctacgggcgg cgaatttgcg 720
gatgaaaatg ctatgtttga tgatgcggca gatatggcac gctctttttc tcgcctgtgg 780
cgcgataaag aagcccgccg tgcagcaggc gaagaaccgg gctttgattt aatctcactg 840
ctacagtcta ataaagaaac caaggatctg atcaatcgtc ctatggaatt tattggcaat 900
ctgaccctgc tgattgtggg cggtaatgat acgacccgca atagcatgtc aggcggctta 960
gttgccatga atgaatttcc tcgtgaattt gaaaaactga aagccaaacc ggaactgatt 1020
ccgaatatgg tgagcgaaat tattcgttgg cagacaccac tggcctatat gcgccgcatt 1080
gccaaacagg atgttgaact gggcggtcag accatcaaaa aaggtgatcg cgttgttatg 1140
tggtatgcct caggtaatcg cgatgaacgt aaatttgata atccggatca gtttattatc 1200
gatcgtaaag atgcacgcaa tcacatgtct tttggctatg gtgttcatcg ctgtatgggt 1260
aatcgtctgg ccgaattaca gctgcgtatt ctgtgggaag aaatcttaaa acgctttgat 1320
aatatcgaag ttgtggaaga accagaacgt gtgcagagca attttgttcg cggctatagc 1380
cgcttaatgg ttaaactgac acctaatagt atgggcggca ttccttcacc aagccgagag 1440
cagtcagcta aaaaagagcg caaaaccgta gaaaacgctc ataatacgcc gcttcttgtg 1500
ctatacggtt caaatatggg aacagccgaa ggaacggcgc gtgatttagc ggatattgcg 1560
atgagcaaag gattcgcacc gcaagtcgca acgcttgatt cccacgcagg aaaccttccg 1620
cgtgaaggag ctgttttaat tgtaacggct tcttataacg gtcatcctcc tgataacgca 1680
aaggaatttg ttgactggtt agaccaagcg tctgctgatg aagtaaaagg cgtgcgctac 1740
tccgtatttg gatgcggtga taaaaactgg gcgacaacgt atcaaaaagt gcctgctttt 1800
attgatgaaa ctcttgccgc taaaggggca gaaaacatag ctgaacgcgg tgaagcagat 1860
gcaagcgacg actttgaagg cacatacgaa gaatggcgtg aacacatgtg gagtgactta 1920
gcagcctact ttaacttaga cattgaaaac agcgaagaaa atgcgtctac gctttcactt 1980
caatttgtcg acagcgctgc ggacatgccg cttgcgaaaa tgcaccgtgc gttttcagca 2040
aacgtcgtag caagcaaaga gcttcaaaag ccaggcagtg cacgaagcac gcgtcatctt 2100
gaaattgaac ttccaaaaga agcttcttat caagaaggag atcatttagg tgttattcct 2160
cgcaactatg aaggaatagt aaatcgtgta gcaacaagat ttggtctaga tgcatcacag 2220
caaatccgtt tggaagctga agaagaaaaa ttagctcatt tgccactcgg taaaacagta 2280
tcagtagaag agcttctgca atacgtggag cttcaagatc ctgttacgcg cacgcagctt 2340
cgcgcaatgg ctgctaaaac agtctgcccg ccgcataaag tagagcttga agtcttgctt 2400
gaaaagcagg cgtacaaaga acaagtgctg gcaaaacgtt taacaatgct tgaactgctt 2460
gaaaaatatc cggcgtgtga aatggaattc agcgaattta tcgcacttct tccaagcatg 2520
cgtccgcgct attactcaat ttcttcatca cctcgtgtcg atgaaaaaca agcaagcatc 2580
acggtcagcg ttgtttcagg agaagcgtgg agcggatacg gagaatacaa aggaattgca 2640
tcgaactatc ttgccaatct gcaagaagga gatacgatta cgtgctttgt ttccacaccg 2700
cagtcaggat ttacgctgcc aaaaggccct gaaacaccac ttatcatggt aggaccggga 2760
acaggcgtcg cgccgtttag aggctttgtg caggctcgca agcagttaaa agaacaagga 2820
cagtcgcttg gagaagcgca tttatacttt ggctgccgtt cacctcatga agattatctg 2880
tatcaaaaag agcttgaaaa cgcccaaaat gaaggcatca ttacgcttca taccgctttt 2940
tctcgcgtac caaatcagcc gaaaacatac gttcaacacg tgatggaaca agacggcaag 3000
aaattgattg aacttcttga ccaaggagcg cacttctata tttgcggaga cggaagccaa 3060
atggcacctg acgttgaagc aacgcttatg aaaagctatg ctgaagttca ccaagtgagt 3120
gaagcagacg ctcgcttatg gctgcagcag ctagaagaaa agggccgata cgcaaaagac 3180
gtgtgggctg ggtaa 3195
<210> 15
<211> 1653
<212> DNA
<213> 人工序列
<400> 15
atgtcagata ccaccaccgc atttaccgtt ccagcggttg ccaaagcagt tgccgcagcc 60
attccggatc gcgaactgat tattcagggt gatcgtcgct atacctatcg tcaggttatt 120
gaacgctcaa atagattagc tgcatattta cattcacagg gcttaggctg tcataccgaa 180
cgcgaagcct tagcaggtca tgaagttggc caggatctgc tgggcctgta tgcatataat 240
ggcaatgaat ttgtggaagc cttactgggt gcctttgcag ctagggtggc cccgtttaat 300
gtgaattttc gctatgttaa atcagaatta cattatctgt tagccgatag tgaagcaacc 360
gccctgattt atcatgcagc ctttgccccg cgtgtggcag aaattctgcc ggaactgccg 420
cgtctgcgcg tgctgattca gattgcagat gaatcaggta atgaattact ggatggtgca 480
gttgattatg aagatgcctt agcaagcgtg agcgcccagc cgccgccggt tcgtcattgt 540
ccggatgatc tgtatgttct gtataccggc ggtaccaccg gtatgccgaa aggcgtttta 600
tggcgtcagc atgatatttt tatgacctct tttggcggtc gtaatctgat gaccggcgaa 660
cctagctcta gtattgatga aattgttcag cgcgcagcct caggtccggg taccaaatta 720
atgattctgc cgccgttaat tcatggtgcc gcacagtgga gtgttatgac cgcaattacc 780
accggccaga ccgtggtgtt tccgaccgtt gtggatcatc tggatgcaga agatgtggtt 840
cgtaccattg aacgtgaaaa agttatggtt gtgaccgtgg tgggcgatgc aatggcacgt 900
ccgttagttg ccgcaattga aaaaggtatt gccgatgtgt cttcactagc cgtggtagcc 960
aatggcggcg ccctgctgac cccgtttgtt aaacagcgtc tgattgaagt gctgccgaat 1020
gcagtggttg tggatggtgt tggctcaagc gaaaccggtg cacagatgca tcacatgtct 1080
acccccggcg ccgtggcaac cggtaccttt aatgcaggtc cggatacctt tgttgccgca 1140
gaggacctga gcgccatttt accgccgggc catgaaggca tgggctggtt agcccagcgc 1200
ggctatgttc cgttaggcta taaaggcgat gcagccaaaa ccgccaaaac ctttccggtt 1260
attgatggcg tgcgctatgc agttccgggt gatcgcgcac gtcatcatgc agatggtcat 1320
attgaactgc tgggtcgcga tagtgtttgt attaatagcg gcggcgaaaa aatttttgtg 1380
gaagaagtgg aaaccgcaat tgcctcacat ccggcagttg ccgatgtggt tgtggcaggt 1440
cgtccgagtg aacgctgggg tcaggaagtt gttgcagttg tggccctgag cgatggtgca 1500
gcagtggatg caggcgaact gattgcacat gcctctaatt ccctggctcg ctataaactg 1560
ccgaaagcca ttgtgtttcg cccggttatt gaacgctctc cgagcggtaa agccgattat 1620
cgttgggcac gcgaacaggc agttaatggt taa 1653
<210> 16
<211> 1227
<212> DNA
<213> 人工序列
<400> 16
gattttgcct atagtccgaa agttcaggca ttacgtgaac gtgtgaccgc ctttatggat 60
gcacatgtgt atccagccga agcagtgttt gaacgccagg ttgccgaagg tgatcgctgg 120
cagccgaccg ccattatgga agaactgaaa gccaaagcac gcgccgaagg cctgtggaat 180
ctgtttctgc cggaatcaga atatggtgca ggtctgtcta atctggaata tgcaccgtta 240
gcagaaatta tgggccgtag cttactgggc ccggaaccgt ttaattgtag tgccccggat 300
accggtaata tggaagtttt agttcgctat ggtagcgaag cccagaaacg tcagtggtta 360
gaaccgttac tgcgcggtga aattcgtagt gcctttgcaa tgaccgaacc ggatgttgca 420
tctagcgatg ccaccaatat ggcagcaacc gcaattcgcg atggcgatca gtgggttatt 480
aatggtcgca aatggtggac ctcaggcgcc tgtgatccgc gttgtaaagt gatgattttt 540
atgggcttaa gcgatccgga aggcccgcgt catcagcagc attcaatggt gctggttccg 600
accgatacgc caggtgttaa aattgtgcgt ccgctgccgg tgtttggcta tgatgatgcc 660
ccgcatggtc atgccgaagt gctgtttgaa aatgttcgtg ttccgtatga aaatgttatt 720
ttaggcgaag gtcgcggctt tgaaattgca cagggtcgtc tggggcctgg tcgtattcat 780
cattgtatgc gctctattgg catggcagaa cgcgcattag aactgatgtg taaacgctca 840
gttgaacgta ccgcctttgg tcgtccgtta gcacgtctgg gcggtaatgt ggataaaatt 900
gcagattctc gcatggaaat tgatatggca cgcttactga ccttaaaagc cgcctatatg 960
atggataccg tgggtaataa agttgcacgc tctgaaattg cacagattaa agttgtggcc 1020
ccgaatgtgg ccctgaatgt tattgatcgt gccattcaga ttcatggcgg cgcaggcgtg 1080
agcggcgatt ttccgttagc ctatatgtat gccatgcagc gtaccctgcg tttagcagat 1140
ggtccggatg aagttcatcg cgcagccatt ggcaaatatg aaattggtaa atatgttccg 1200
gttgaaatgc tgcgctcagg tcgttaa 1227
<210> 17
<211> 423
<212> DNA
<213> 人工序列
<400> 17
ggatccatga tttggcagcg caccgccacc ctggatgcac tgaatgcaat gggtgcgaat 60
aatatggtgg gcctgctgga tattcgcttt acccgtctgg atgataatga aattgaagca 120
accatgccgg ttgatcatcg tacccatcaa cctttcggtt tactgcatgg cggcgcaagc 180
gtggtgttag ccgaaacctt aggtagtgtt gcaggctatc tgtgtaccga aggcgaacag 240
aatattgtgg gcttagaagt taatgccaat catttacgct cagtgcgtag cggtcgcgtg 300
cgcggcgtgt gtcgcgcagt tcatgtgggt cgtcgtcatc aggtttggca gattgaaatt 360
tttgatgaac aggatcgctt atgttgtagc tctcgtctga ccaccgccgt tgtgtaactc 420
gag 423
<210> 18
<211> 1064
<212> PRT
<213> 人工序列
<400> 18
Met Pro Thr Leu Pro Arg Thr Phe Asp Asp Ile Gln Ser Arg Leu Ile
1 5 10 15
Asn Ala Thr Ser Arg Val Val Pro Met Gln Arg Gln Ile Gln Gly Leu
20 25 30
Lys Phe Leu Met Ser Ala Lys Arg Lys Thr Phe Gly Pro Arg Arg Pro
35 40 45
Met Pro Glu Phe Val Glu Thr Pro Ile Pro Asp Val Asn Thr Leu Ala
50 55 60
Leu Glu Asp Ile Asp Val Ser Asn Pro Phe Leu Tyr Arg Gln Gly Gln
65 70 75 80
Trp Arg Ala Tyr Phe Lys Arg Leu Arg Asp Glu Ala Pro Val His Tyr
85 90 95
Gln Lys Asn Ser Pro Phe Gly Pro Phe Trp Ser Val Thr Arg Phe Glu
100 105 110
Asp Ile Leu Phe Val Asp Lys Ser His Asp Leu Phe Ser Ala Glu Pro
115 120 125
Gln Ile Ile Leu Gly Asp Pro Pro Glu Gly Leu Ser Val Glu Met Phe
130 135 140
Ile Ala Met Asp Pro Pro Lys His Asp Val Gln Arg Ser Ser Val Gln
145 150 155 160
Gly Val Val Ala Pro Lys Asn Leu Lys Glu Met Glu Gly Leu Ile Arg
165 170 175
Ser Arg Thr Gly Asp Val Leu Asp Ser Leu Pro Thr Asp Lys Pro Phe
180 185 190
Asn Trp Val Pro Ala Val Ser Lys Glu Leu Thr Gly Arg Met Leu Ala
195 200 205
Thr Leu Leu Asp Phe Pro Tyr Glu Glu Arg His Lys Leu Val Glu Trp
210 215 220
Ser Asp Arg Met Ala Gly Ala Ala Ser Ala Thr Gly Gly Glu Phe Ala
225 230 235 240
Asp Glu Asn Ala Met Phe Asp Asp Ala Ala Asp Met Ala Arg Ser Phe
245 250 255
Ser Arg Leu Trp Arg Asp Lys Glu Ala Arg Arg Ala Ala Gly Glu Glu
260 265 270
Pro Gly Phe Asp Leu Ile Ser Leu Leu Gln Ser Asn Lys Glu Thr Lys
275 280 285
Asp Leu Ile Asn Arg Pro Met Glu Phe Ile Gly Asn Leu Thr Leu Leu
290 295 300
Ile Val Gly Gly Asn Asp Thr Thr Arg Asn Ser Met Ser Gly Gly Leu
305 310 315 320
Val Ala Met Asn Glu Phe Pro Arg Glu Phe Glu Lys Leu Lys Ala Lys
325 330 335
Pro Glu Leu Ile Pro Asn Met Val Ser Glu Ile Ile Arg Trp Gln Thr
340 345 350
Pro Leu Ala Tyr Met Arg Arg Ile Ala Lys Gln Asp Val Glu Leu Gly
355 360 365
Gly Gln Thr Ile Lys Lys Gly Asp Arg Val Val Met Trp Tyr Ala Ser
370 375 380
Gly Asn Arg Asp Glu Arg Lys Phe Asp Asn Pro Asp Gln Phe Ile Ile
385 390 395 400
Asp Arg Lys Asp Ala Arg Asn His Met Ser Phe Gly Tyr Gly Val His
405 410 415
Arg Cys Met Gly Asn Arg Leu Ala Glu Leu Gln Leu Arg Ile Leu Trp
420 425 430
Glu Glu Ile Leu Lys Arg Phe Asp Asn Ile Glu Val Val Glu Glu Pro
435 440 445
Glu Arg Val Gln Ser Asn Phe Val Arg Gly Tyr Ser Arg Leu Met Val
450 455 460
Lys Leu Thr Pro Asn Ser Met Gly Gly Ile Pro Ser Pro Ser Arg Glu
465 470 475 480
Gln Ser Ala Lys Lys Glu Arg Lys Thr Val Glu Asn Ala His Asn Thr
485 490 495
Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr
500 505 510
Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln
515 520 525
Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala
530 535 540
Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala
545 550 555 560
Lys Glu Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys
565 570 575
Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr
580 585 590
Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys
595 600 605
Gly Ala Glu Asn Ile Ala Glu Arg Gly Glu Ala Asp Ala Ser Asp Asp
610 615 620
Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Leu
625 630 635 640
Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Glu Asn Ala Ser
645 650 655
Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala
660 665 670
Lys Met His Arg Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu
675 680 685
Gln Lys Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu
690 695 700
Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro
705 710 715 720
Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Ala Thr Arg Phe Gly Leu
725 730 735
Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala
740 745 750
His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr
755 760 765
Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala
770 775 780
Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Val Leu Leu
785 790 795 800
Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met
805 810 815
Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu
820 825 830
Phe Ile Ala Leu Leu Pro Ser Met Arg Pro Arg Tyr Tyr Ser Ile Ser
835 840 845
Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val
850 855 860
Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala
865 870 875 880
Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe
885 890 895
Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Gly Pro Glu Thr
900 905 910
Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly
915 920 925
Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly
930 935 940
Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu
945 950 955 960
Tyr Gln Lys Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu
965 970 975
His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln
980 985 990
His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln
995 1000 1005
Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro
1010 1015 1020
Asp Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Glu Val His Gln
1025 1030 1035
Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu
1040 1045 1050
Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly
1055 1060
<210> 19
<211> 1064
<212> PRT
<213> 人工序列
<400> 19
Met Pro Thr Leu Pro Arg Thr Phe Asp Asp Ile Gln Ser Arg Leu Ile
1 5 10 15
Asn Ala Thr Ser Arg Val Val Pro Met Gln Arg Gln Ile Gln Gly Leu
20 25 30
Lys Phe Leu Met Ser Ala Lys Arg Lys Thr Phe Gly Pro Arg Arg Pro
35 40 45
Met Pro Glu Phe Val Glu Thr Pro Ile Pro Asp Val Asn Thr Leu Ala
50 55 60
Leu Glu Asp Ile Asp Val Ser Asn Pro Phe Leu Tyr Arg Gln Gly Gln
65 70 75 80
Trp Arg Ala Tyr Phe Lys Arg Leu Arg Asp Glu Ala Pro Val His Tyr
85 90 95
Gln Lys Asn Ser Pro Phe Gly Pro Phe Trp Ser Val Thr Arg Phe Glu
100 105 110
Asp Ile Leu Phe Val Asp Lys Ser His Asp Leu Phe Ser Ala Glu Pro
115 120 125
Gln Ile Ile Leu Gly Asp Pro Pro Glu Gly Leu Ser Val Glu Met Phe
130 135 140
Ile Ala Met Asp Pro Pro Lys His Asp Val Gln Arg Ser Ser Val Gln
145 150 155 160
Gly Val Val Ala Pro Lys Asn Leu Lys Glu Met Glu Gly Leu Ile Arg
165 170 175
Ser Arg Thr Gly Asp Val Leu Asp Ser Leu Pro Thr Asp Lys Pro Phe
180 185 190
Asn Trp Val Pro Ala Val Ser Lys Glu Leu Thr Gly Arg Met Leu Ala
195 200 205
Thr Leu Leu Asp Phe Pro Tyr Glu Glu Arg His Lys Leu Val Glu Trp
210 215 220
Ser Asp Arg Leu Ala Gly Ala Ala Ser Ala Thr Gly Gly Glu Phe Ala
225 230 235 240
Asp Glu Asn Ala Met Phe Asp Asp Ala Ala Asp Met Ala Arg Ser Phe
245 250 255
Ser Arg Leu Trp Arg Asp Lys Glu Ala Arg Arg Ala Ala Gly Glu Glu
260 265 270
Pro Gly Phe Asp Leu Ile Ser Leu Leu Gln Ser Asn Lys Glu Thr Lys
275 280 285
Asp Leu Ile Asn Arg Pro Met Glu Phe Ile Gly Asn Leu Thr Leu Leu
290 295 300
Ile Val Gly Gly Asn Asp Thr Thr Arg Asn Ser Met Ser Gly Gly Leu
305 310 315 320
Val Ala Met Asn Glu Phe Pro Arg Glu Phe Glu Lys Leu Lys Ala Lys
325 330 335
Pro Glu Leu Ile Pro Asn Met Val Ser Glu Ile Ile Arg Trp Gln Thr
340 345 350
Pro Leu Ala Tyr Met Arg Arg Ile Ala Lys Gln Asp Val Glu Leu Gly
355 360 365
Gly Gln Thr Ile Lys Lys Gly Asp Arg Val Val Met Trp Tyr Ala Ser
370 375 380
Gly Asn Arg Asp Glu Arg Lys Phe Asp Asn Pro Asp Gln Phe Ile Ile
385 390 395 400
Asp Arg Lys Asp Ala Arg Asn His Met Ser Phe Gly Tyr Gly Val His
405 410 415
Arg Cys Met Gly Asn Arg Leu Ala Glu Leu Gln Leu Arg Ile Leu Trp
420 425 430
Glu Glu Ile Leu Lys Arg Phe Asp Asn Ile Glu Val Val Glu Glu Pro
435 440 445
Glu Arg Val Gln Ser Asn Phe Val Arg Gly Tyr Ser Arg Leu Met Val
450 455 460
Lys Leu Thr Pro Asn Ser Met Gly Gly Ile Pro Ser Pro Ser Arg Glu
465 470 475 480
Gln Ser Ala Lys Lys Glu Arg Lys Thr Val Glu Asn Ala His Asn Thr
485 490 495
Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr
500 505 510
Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln
515 520 525
Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala
530 535 540
Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala
545 550 555 560
Lys Glu Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys
565 570 575
Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr
580 585 590
Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys
595 600 605
Gly Ala Glu Asn Ile Ala Glu Arg Gly Glu Ala Asp Ala Ser Asp Asp
610 615 620
Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Leu
625 630 635 640
Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Glu Asn Ala Ser
645 650 655
Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala
660 665 670
Lys Met His Arg Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu
675 680 685
Gln Lys Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu
690 695 700
Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro
705 710 715 720
Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Ala Thr Arg Phe Gly Leu
725 730 735
Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala
740 745 750
His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr
755 760 765
Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala
770 775 780
Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Val Leu Leu
785 790 795 800
Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met
805 810 815
Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu
820 825 830
Phe Ile Ala Leu Leu Pro Ser Met Arg Pro Arg Tyr Tyr Ser Ile Ser
835 840 845
Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val
850 855 860
Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala
865 870 875 880
Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe
885 890 895
Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Gly Pro Glu Thr
900 905 910
Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly
915 920 925
Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly
930 935 940
Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu
945 950 955 960
Tyr Gln Lys Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu
965 970 975
His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln
980 985 990
His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln
995 1000 1005
Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro
1010 1015 1020
Asp Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Glu Val His Gln
1025 1030 1035
Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu
1040 1045 1050
Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly
1055 1060
<210> 20
<211> 550
<212> PRT
<213> 人工序列
<400> 20
Met Ser Asp Thr Thr Thr Ala Phe Thr Val Pro Ala Val Ala Lys Ala
1 5 10 15
Val Ala Ala Ala Ile Pro Asp Arg Glu Leu Ile Ile Gln Gly Asp Arg
20 25 30
Arg Tyr Thr Tyr Arg Gln Val Ile Glu Arg Ser Asn Arg Leu Ala Ala
35 40 45
Tyr Leu His Ser Gln Gly Leu Gly Cys His Thr Glu Arg Glu Ala Leu
50 55 60
Ala Gly His Glu Val Gly Gln Asp Leu Leu Gly Leu Tyr Ala Tyr Asn
65 70 75 80
Gly Asn Glu Phe Val Glu Ala Leu Leu Gly Ala Phe Ala Ala Arg Val
85 90 95
Ala Pro Phe Asn Val Asn Phe Arg Tyr Val Lys Ser Glu Leu His Tyr
100 105 110
Leu Leu Ala Asp Ser Glu Ala Thr Ala Leu Ile Tyr His Ala Ala Phe
115 120 125
Ala Pro Arg Val Ala Glu Ile Leu Pro Glu Leu Pro Arg Leu Arg Val
130 135 140
Leu Ile Gln Ile Ala Asp Glu Ser Gly Asn Glu Leu Leu Asp Gly Ala
145 150 155 160
Val Asp Tyr Glu Asp Ala Leu Ala Ser Val Ser Ala Gln Pro Pro Pro
165 170 175
Val Arg His Cys Pro Asp Asp Leu Tyr Val Leu Tyr Thr Gly Gly Thr
180 185 190
Thr Gly Met Pro Lys Gly Val Leu Trp Arg Gln His Asp Ile Phe Met
195 200 205
Thr Ser Phe Gly Gly Arg Asn Leu Met Thr Gly Glu Pro Ser Ser Ser
210 215 220
Ile Asp Glu Ile Val Gln Arg Ala Ala Ser Gly Pro Gly Thr Lys Leu
225 230 235 240
Met Ile Leu Pro Pro Leu Ile His Gly Ala Ala Gln Trp Ser Val Met
245 250 255
Thr Ala Ile Thr Thr Gly Gln Thr Val Val Phe Pro Thr Val Val Asp
260 265 270
His Leu Asp Ala Glu Asp Val Val Arg Thr Ile Glu Arg Glu Lys Val
275 280 285
Met Val Val Thr Val Val Gly Asp Ala Met Ala Arg Pro Leu Val Ala
290 295 300
Ala Ile Glu Lys Gly Ile Ala Asp Val Ser Ser Leu Ala Val Val Ala
305 310 315 320
Asn Gly Gly Ala Leu Leu Thr Pro Phe Val Lys Gln Arg Leu Ile Glu
325 330 335
Val Leu Pro Asn Ala Val Val Val Asp Gly Val Gly Ser Ser Glu Thr
340 345 350
Gly Ala Gln Met His His Met Ser Thr Pro Gly Ala Val Ala Thr Gly
355 360 365
Thr Phe Asn Ala Gly Pro Asp Thr Phe Val Ala Ala Glu Asp Leu Ser
370 375 380
Ala Ile Leu Pro Pro Gly His Glu Gly Met Gly Trp Leu Ala Gln Arg
385 390 395 400
Gly Tyr Val Pro Leu Gly Tyr Lys Gly Asp Ala Ala Lys Thr Ala Lys
405 410 415
Thr Phe Pro Val Ile Asp Gly Val Arg Tyr Ala Val Pro Gly Asp Arg
420 425 430
Ala Arg His His Asp Ala Gly His Ile Glu Leu Leu Gly Arg Asp Ser
435 440 445
Val Cys Ile Asn Ser Gly Gly Glu Lys Ile Phe Val Glu Glu Val Glu
450 455 460
Thr Ala Ile Ala Ser His Pro Ala Val Ala Asp Val Val Val Ala Gly
465 470 475 480
Arg Pro Ser Glu Arg Trp Gly Gln Glu Val Val Ala Val Val Ala Leu
485 490 495
Ser Asp Gly Ala Ala Val Asp Ala Gly Glu Leu Ile Ala His Ala Ser
500 505 510
Asn Ser Leu Ala Arg Tyr Lys Leu Pro Lys Ala Ile Val Phe Arg Pro
515 520 525
Val Ile Glu Arg Ser Pro Ser Gly Lys Ala Asp Tyr Arg Trp Ala Arg
530 535 540
Glu Gln Ala Val Asn Gly
545 550
<210> 21
<211> 408
<212> PRT
<213> 人工序列
<400> 21
Asp Phe Ala Tyr Ser Pro Lys Val Gln Ala Leu Arg Glu Arg Val Thr
1 5 10 15
Ala Phe Met Asp Ala His Val Tyr Pro Ala Glu Ala Val Phe Glu Arg
20 25 30
Gln Val Ala Glu Gly Asp Arg Trp Gln Pro Thr Ala Ile Met Glu Glu
35 40 45
Leu Lys Ala Lys Ala Arg Ala Glu Gly Leu Trp Asn Leu Phe Leu Pro
50 55 60
Glu Ser Glu Tyr Gly Ala Gly Leu Ser Asn Leu Glu Tyr Ala Pro Leu
65 70 75 80
Ala Glu Ile Met Gly Arg Ser Leu Leu Gly Pro Glu Pro Phe Asn Cys
85 90 95
Ser Ala Pro Asp Thr Gly Asn Met Glu Val Leu Val Arg Tyr Gly Ser
100 105 110
Glu Ala Gln Lys Arg Gln Trp Leu Glu Pro Leu Leu Arg Gly Glu Ile
115 120 125
Arg Ser Ala Phe Ala Met Thr Glu Pro Asp Val Ala Ser Ser Asp Ala
130 135 140
Thr Asn Met Ala Ala Thr Ala Ile Arg Asp Gly Asp Gln Trp Val Ile
145 150 155 160
Asn Gly Arg Lys Trp Trp Thr Ser Gly Ala Cys Asp Pro Arg Cys Lys
165 170 175
Val Met Ile Phe Met Gly Leu Ser Asp Pro Glu Gly Pro Arg His Gln
180 185 190
Gln His Ser Met Val Leu Val Pro Thr Asp Thr Pro Gly Val Lys Ile
195 200 205
Val Arg Pro Leu Pro Val Phe Gly Tyr Asp Asp Ala Pro His Gly His
210 215 220
Ala Glu Val Leu Phe Glu Asn Val Arg Val Pro Tyr Glu Asn Val Ile
225 230 235 240
Leu Gly Glu Gly Arg Gly Phe Glu Ile Ala Gln Gly Arg Leu Gly Pro
245 250 255
Gly Arg Ile His His Cys Met Arg Ser Ile Gly Met Ala Glu Arg Ala
260 265 270
Leu Glu Leu Met Cys Lys Arg Ser Val Glu Arg Thr Ala Phe Gly Arg
275 280 285
Pro Leu Ala Arg Leu Gly Gly Asn Val Asp Lys Ile Ala Asp Ser Arg
290 295 300
Met Glu Ile Asp Met Ala Arg Leu Leu Thr Leu Lys Ala Ala Tyr Met
305 310 315 320
Met Asp Thr Val Gly Asn Lys Val Ala Arg Ser Glu Ile Ala Gln Ile
325 330 335
Lys Val Val Ala Pro Asn Val Ala Leu Asn Val Ile Asp Arg Ala Ile
340 345 350
Gln Ile His Gly Gly Ala Gly Val Ser Gly Asp Phe Pro Leu Ala Tyr
355 360 365
Met Tyr Ala Met Gln Arg Thr Leu Arg Leu Ala Asp Gly Pro Asp Glu
370 375 380
Val His Arg Ala Ala Ile Gly Lys Tyr Glu Ile Gly Lys Tyr Val Pro
385 390 395 400
Val Glu Met Leu Arg Ser Gly Arg
405
<210> 22
<211> 237
<212> PRT
<213> 人工序列
<400> 22
Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro
1 5 10 15
Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser
20 25 30
Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu
35 40 45
Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg
50 55 60
Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp
65 70 75 80
Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile
85 90 95
Gly Gly Ser Gly Ser Met Ile Trp Gln Arg Thr Ala Thr Leu Asp Ala
100 105 110
Leu Asn Ala Met Gly Ala Asn Asn Met Val Gly Leu Leu Asp Ile Arg
115 120 125
Phe Thr Arg Leu Asp Asp Asn Glu Ile Glu Ala Thr Met Pro Val Asp
130 135 140
His Arg Thr His Gln Pro Phe Gly Leu Leu His Gly Gly Ala Ser Val
145 150 155 160
Val Leu Ala Glu Thr Leu Gly Ser Val Ala Gly Tyr Leu Cys Thr Glu
165 170 175
Gly Glu Gln Asn Ile Val Gly Leu Glu Val Asn Ala Asn His Leu Arg
180 185 190
Ser Val Arg Ser Gly Arg Val Arg Gly Val Cys Arg Ala Val His Val
195 200 205
Gly Arg Arg His Gln Val Trp Gln Ile Glu Ile Phe Asp Glu Gln Asp
210 215 220
Arg Leu Cys Cys Ser Ser Arg Leu Thr Thr Ala Val Val
225 230 235
<210> 23
<211> 1410
<212> DNA
<213> 人工序列
<400> 23
atgccgacgt taccacgtac ctttgatgac attcagtctc gcttaatcaa tgctacaagt 60
cgtgtggttc caatgcagcg tcagattcag ggtctgaaat ttctgatgag tgccaaacgc 120
aaaacctttg gtccacgtcg cccaatgccg gaatttgtgg aaacacctat cccggatgtt 180
aatacattag ccttagagga cattgatgtg agtaatccgt ttctgtatcg ccagggccag 240
tggcgcgcat attttaaacg cttacgcgat gaagctccag ttcattatca gaaaaatagc 300
ccatttggtc cgttttggag cgtgacccgc tttgaggaca ttctgtttgt ggataaatca 360
catgatctgt ttagcgccga accacagatc atcttaggtg atcctccgga aggcctgtca 420
gtggaaatgt ttattgcgat ggaccctcct aaacatgatg tgcagcgctc tagtgttcag 480
ggtgtggttg cccctaaaaa tctgaaagaa atggaaggcc tgattcgtag tcgtacgggc 540
gatgtgttag attcattacc gacggataaa ccgtttaatt gggttcctgc ggtgagcaaa 600
gaactgacgg gtagaatgct ggctacctta ctggattttc cgtatgaaga acgtcataaa 660
ctggttgaat ggagcgatcg cttggccggt gcggcaagtg ctacgggcgg cgaatttgcg 720
gatgaaaatg ctatgtttga tgatgcggca gatatggcac gctctttttc tcgcctgtgg 780
cgcgataaag aagcccgccg tgcagcaggc gaagaaccgg gctttgattt aatctcactg 840
ctacagtcta ataaagaaac caaggatctg atcaatcgtc ctatggaatt tattggcaat 900
ctgaccctgc tgattgtggg cggtaatgat acgacccgca atagcatgtc aggcggctta 960
gttgccatga atgaatttcc tcgtgaattt gaaaaactga aagccaaacc ggaactgatt 1020
ccgaatatgg tgagcgaaat tattcgttgg cagacaccac tggcctatat gcgccgcatt 1080
gccaaacagg atgttgaact gggcggtcag accatcaaaa aaggtgatcg cgttgttatg 1140
tggtatgcct caggtaatcg cgatgaacgt aaatttgata atccggatca gtttattatc 1200
gatcgtaaag atgcacgcaa tcacatgtct tttggctatg gtgttcatcg ctgtatgggt 1260
aatcgtctgg ccgaattaca gctgcgtatt ctgtgggaag aaatcttaaa acgctttgat 1320
aatatcgaag ttgtggaaga accagaacgt gtgcagagca attttgttcg cggctatagc 1380
cgcttaatgg ttaaactgac acctaatagt 1410
<210> 24
<211> 470
<212> PRT
<213> 人工序列
<400> 24
Met Pro Thr Leu Pro Arg Thr Phe Asp Asp Ile Gln Ser Arg Leu Ile
1 5 10 15
Asn Ala Thr Ser Arg Val Val Pro Met Gln Arg Gln Ile Gln Gly Leu
20 25 30
Lys Phe Leu Met Ser Ala Lys Arg Lys Thr Phe Gly Pro Arg Arg Pro
35 40 45
Met Pro Glu Phe Val Glu Thr Pro Ile Pro Asp Val Asn Thr Leu Ala
50 55 60
Leu Glu Asp Ile Asp Val Ser Asn Pro Phe Leu Tyr Arg Gln Gly Gln
65 70 75 80
Trp Arg Ala Tyr Phe Lys Arg Leu Arg Asp Glu Ala Pro Val His Tyr
85 90 95
Gln Lys Asn Ser Pro Phe Gly Pro Phe Trp Ser Val Thr Arg Phe Glu
100 105 110
Asp Ile Leu Phe Val Asp Lys Ser His Asp Leu Phe Ser Ala Glu Pro
115 120 125
Gln Ile Ile Leu Gly Asp Pro Pro Glu Gly Leu Ser Val Glu Met Phe
130 135 140
Ile Ala Met Asp Pro Pro Lys His Asp Val Gln Arg Ser Ser Val Gln
145 150 155 160
Gly Val Val Ala Pro Lys Asn Leu Lys Glu Met Glu Gly Leu Ile Arg
165 170 175
Ser Arg Thr Gly Asp Val Leu Asp Ser Leu Pro Thr Asp Lys Pro Phe
180 185 190
Asn Trp Val Pro Ala Val Ser Lys Glu Leu Thr Gly Arg Met Leu Ala
195 200 205
Thr Leu Leu Asp Phe Pro Tyr Glu Glu Arg His Lys Leu Val Glu Trp
210 215 220
Ser Asp Arg Leu Ala Gly Ala Ala Ser Ala Thr Gly Gly Glu Phe Ala
225 230 235 240
Asp Glu Asn Ala Met Phe Asp Asp Ala Ala Asp Met Ala Arg Ser Phe
245 250 255
Ser Arg Leu Trp Arg Asp Lys Glu Ala Arg Arg Ala Ala Gly Glu Glu
260 265 270
Pro Gly Phe Asp Leu Ile Ser Leu Leu Gln Ser Asn Lys Glu Thr Lys
275 280 285
Asp Leu Ile Asn Arg Pro Met Glu Phe Ile Gly Asn Leu Thr Leu Leu
290 295 300
Ile Val Gly Gly Asn Asp Thr Thr Arg Asn Ser Met Ser Gly Gly Leu
305 310 315 320
Val Ala Met Asn Glu Phe Pro Arg Glu Phe Glu Lys Leu Lys Ala Lys
325 330 335
Pro Glu Leu Ile Pro Asn Met Val Ser Glu Ile Ile Arg Trp Gln Thr
340 345 350
Pro Leu Ala Tyr Met Arg Arg Ile Ala Lys Gln Asp Val Glu Leu Gly
355 360 365
Gly Gln Thr Ile Lys Lys Gly Asp Arg Val Val Met Trp Tyr Ala Ser
370 375 380
Gly Asn Arg Asp Glu Arg Lys Phe Asp Asn Pro Asp Gln Phe Ile Ile
385 390 395 400
Asp Arg Lys Asp Ala Arg Asn His Met Ser Phe Gly Tyr Gly Val His
405 410 415
Arg Cys Met Gly Asn Arg Leu Ala Glu Leu Gln Leu Arg Ile Leu Trp
420 425 430
Glu Glu Ile Leu Lys Arg Phe Asp Asn Ile Glu Val Val Glu Glu Pro
435 440 445
Glu Arg Val Gln Ser Asn Phe Val Arg Gly Tyr Ser Arg Leu Met Val
450 455 460
Lys Leu Thr Pro Asn Ser
465 470
<210> 25
<211> 45
<212> DNA
<213> 人工序列
<400> 25
tcgagctccg tcgacaagct tatggaaatg acatcagcgt ttacc 45
<210> 26
<211> 38
<212> DNA
<213> 人工序列
<400> 26
gtccacggag aattcatctc taatgctgtg ctgacgcc 38
<210> 27
<211> 22
<212> DNA
<213> 人工序列
<400> 27
ctgtactgga agccgcttat gg 22
<210> 28
<211> 44
<212> DNA
<213> 人工序列
<400> 28
gtggtggtgg tggtgctcga gttattgcag gtcagttgca gttg 44
<210> 29
<211> 25
<212> DNA
<213> 人工序列
<400> 29
gagatgaatt ctccgtggac ctgca 25
<210> 30
<211> 39
<212> DNA
<213> 人工序列
<400> 30
ataagcggct tccagtacag ggtaccgagc tcggatccg 39
<210> 31
<211> 24
<212> DNA
<213> 人工序列
<400> 31
atggaaatga catcagcgtt tacc 24
<210> 32
<211> 23
<212> DNA
<213> 人工序列
<400> 32
ttattgcagg tcagttgcag ttg 23