一种降低淀粉消化率的方法及其应用
阅读说明：本技术 一种降低淀粉消化率的方法及其应用 (Method for reducing starch digestibility and application thereof ) 是由 吴敬 宿玲恰 李玲玲 李娜 于 2019-10-25 设计创作，主要内容包括：本发明公开了一种降低淀粉消化率的方法及其应用,属于酶工程技术领域。本发明提供了一种降低淀粉消化率的方法,此方法可用于制备抗性淀粉,且生产效率十分高；利用此方法将糖原分支酶ChlGBE加入淀粉溶液中改性6h,即可使淀粉溶液中抗性淀粉的含量高达35.06％,较未经改性的淀粉溶液提高9.75％；利用此方法将糖原分支酶HosGBE加入淀粉溶液中改性8h,即可使淀粉溶液中抗性淀粉的含量高达47.36％,较未经改性的淀粉溶液提高22.05％；利用此方法将糖原分支酶VvGBE加入淀粉溶液中改性10h,即可使淀粉溶液中抗性淀粉的含量高达51.4％,较未经改性的淀粉溶液提高26.09％。(The invention discloses a method for reducing starch digestibility and application thereof, belonging to the technical field of enzyme engineering. The invention provides a method for reducing the digestibility of starch, which can be used for preparing resistant starch and has high production efficiency; the glycogen branching enzyme ChlGBE is added into the starch solution for modification for 6 hours by using the method, so that the content of resistant starch in the starch solution can reach 35.06 percent, and is improved by 9.75 percent compared with the unmodified starch solution; by using the method, glycogen branching enzyme HosGBE is added into the starch solution for modification for 8 hours, so that the content of resistant starch in the starch solution can reach 47.36 percent, which is increased by 22.05 percent compared with the unmodified starch solution; the glycogen branching enzyme VvGBE is added into the starch solution for modification for 10 hours by using the method, so that the content of resistant starch in the starch solution can reach 51.4 percent, and is improved by 26.09 percent compared with the unmodified starch solution.)
技术领域
本发明涉及一种降低淀粉消化率的方法及其应用，属于酶工程技术领域。
背景技术
淀粉是自然界中主要的储碳和储能多糖，它能够为人体提供70～80％的能量，是人体最重要的膳食能量来源。依据消化率不同，可将淀粉分为三类，分别为快消化淀粉(RDS)、慢消化淀粉(SDS)以及抗性淀粉(RS)。其中，快消化淀粉(RDS)是指在小肠内完全消化吸收的淀粉，慢消化淀粉是指20～120min在小肠内完全消化吸收的淀粉，抗性淀粉是指120min后仍不能被小肠消化吸收的淀粉。
研究表明，由于具备消化率低以及可被肠道益生菌特异性吸收等特点，抗性淀粉在调节血糖、降低血脂、促进矿物质吸收、保护肠道等方面均具有重要作用(具体可参考文献“Glycemi c and insulinemic response ofsubjects with type 2diabetes afterconsumption ofthree energy bars[J].Journal oftheAmerican DieteticAssociation,2002,102(8):1139-1142”、“Han K H,Sekikawa M,Shimada K I,et al.Resistant starchfraction prepared from kintoki bean affects gene expression ofgenesassociated with cholesterol metabolism in rats[J].Experimental Biology andMedicine,2004,229(8):787-792.”以及“SchulzAG M,Amelsvoort J M M V,BeynenAC.Dietary native resistant starch not retrograded resistant starchraises magnesium and calcium absorption in rats[J].Journal ofNutrition,1993,123(10):1724-1731.”)。因此，降低淀粉的消化率以制备得到更多的抗性淀粉十分重要。
目前，制备抗性淀粉的方法主要有三种，分别为物理改性、化学改性和酶法改性。其中，物理改性与化学改性均存在副产物多、生产效率低以及安全性差等问题(具体可参考文献“Goesaert H,BijttebierA,Delcour JA.Hydrolysis ofamylopectinby amylolyticenzymes:level ofinner chain attack as an important analytical differentiationcriterion[J].Carbohydrate Research,2010,345(3):397-401.”)，因此，酶法改性在制备抗性淀粉的领域越发受到关注。
酶法改性主要是通过使用麦芽糖淀粉酶、β-淀粉酶、淀粉蔗糖酶和/或葡萄糖基转移酶等酶对淀粉进行处理，将分子量大的淀粉切割成较小的分子或增加淀粉分子内如α-1,6键、α-1,3键等的抗消化的化学键以降低淀粉的消化率。但是，由于现有的麦芽糖淀粉酶、β-淀粉酶、淀粉蔗糖酶和/或葡萄糖基转移酶等酶切割淀粉以及在淀粉内生成抗消化化学键的效率过低，使用酶法改性制备抗性淀粉时具备生产效率低的问题。因此，仍需找到一种可高效降低淀粉消化率的方法，以提高抗性淀粉的生产效率。
发明内容
[技术问题]
本发明要解决的技术问题是提供一种可高效降低淀粉消化率的方法。
[技术方案]
为解决上述技术问题，本发明提供了一种降低淀粉消化率的方法，所述方法为利用氨基酸序列如SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3所示的糖原分支酶(glycogenbranching enzyme)中的一种或多种对淀粉进行改性。所述消化率是指淀粉被人体消化释放葡萄糖的速率。
在本发明的一种实施方式中，所述方法为先将淀粉加入缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，接着将糊化淀粉溶液进行温育，得到温育淀粉溶液，最后将氨基酸序列如SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3所示的糖原分支酶中的一种或多种加入糊化淀粉溶液中进行改性。
在本发明的一种实施方式中，所述糖原分支酶的添加量为100～1000U/g淀粉。
在本发明的一种实施方式中，所述糖原分支酶的添加量为500U/g淀粉。
在本发明的一种实施方式中，所述改性的温度为25～45℃、pH为5.5～8.5、时间为4～12h。
在本发明的一种实施方式中，所述方法为先将淀粉加入缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，接着将糊化淀粉溶液进行温育，得到温育淀粉溶液，最后将氨基酸序列如SEQ ID NO:1所示的糖原分支酶加入糊化淀粉溶液中，于温度为30℃、pH为7.0的条件下改性6h；
或者，所述方法为先将淀粉加入缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，接着将糊化淀粉溶液进行温育，得到温育淀粉溶液，最后将氨基酸序列如SEQ ID NO:2所示的糖原分支酶加入糊化淀粉溶液中，于温度为30℃、pH为7.0的条件下改性8h；
或者，所述方法为先将淀粉加入缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，接着将糊化淀粉溶液进行温育，得到温育淀粉溶液，最后将氨基酸序列如SEQ ID NO:3所示的糖原分支酶加入糊化淀粉溶液中，于温度为35℃、pH为7.5的条件下改性10h。
在本发明的一种实施方式中，所述糊化淀粉溶液以及温育淀粉溶液中淀粉的浓度为10～100g/L。
在本发明的一种实施方式中，所述糊化淀粉溶液以及温育淀粉溶液中淀粉的浓度为25g/L。
在本发明的一种实施方式中，所述氨基酸序列如SEQ ID NO:1所示的糖原分支酶来源于小球藻(Chlorella kessleri)。
在本发明的一种实施方式中，所述氨基酸序列如SEQ ID NO:2所示的糖原分支酶来源于智人(Homo sapiens)。
在本发明的一种实施方式中，所述氨基酸序列如SEQ ID NO:3所示的糖原分支酶来源于创伤弧菌(Vibrio vulnificus)。
在本发明的一种实施方式中，所述缓冲液为磷酸二氢钠-磷酸氢二钠缓冲液、Tris-HCl缓冲液、磷酸氢二钠-柠檬酸缓冲液或柠檬酸缓冲液。
在本发明的一种实施方式中，所述缓冲液为磷酸二氢钠-磷酸氢二钠缓冲液。
在本发明的一种实施方式中，所述缓冲液的浓度为20～100mmol/L。
在本发明的一种实施方式中，所述缓冲液的浓度为50mmol/L
在本发明的一种实施方式中，所述糊化为将淀粉溶液在沸水浴中以100～200r/min的速度搅拌20～40min。
在本发明的一种实施方式中，所述糊化为将淀粉溶液在沸水浴中以150r/min的速度搅拌30min。
在本发明的一种实施方式中，所述温育为将糊化淀粉溶液在25～45℃的条件下以100～200r/min的速度搅拌10～20min。
在本发明的一种实施方式中，所述温育为将糊化淀粉溶液在30℃的条件下以150r/min的速度搅拌10min。
在本发明的一种实施方式中，所述淀粉为玉米淀粉、马铃薯淀粉、木薯淀粉、或甘薯淀粉。
本发明提供了上述降低淀粉消化率的方法在生产抗性淀粉中的应用。所述慢消化淀粉是指20～120min在小肠内完全消化吸收的淀粉。所述抗性淀粉是指120min后仍不能被小肠消化吸收的淀粉。
[有益效果]
本发明提供了一种降低淀粉消化率的方法，此方法可用于制备抗性淀粉，且生产效率十分高；利用此方法将氨基酸序列如SEQ ID NO:1所示的糖原分支酶(ChlGBE)加入淀粉溶液中改性6h，即可使淀粉溶液中抗性淀粉的含量高达35.06％，较未经改性的淀粉溶液提高9.75％；利用此方法将氨基酸序列如SEQ ID NO:2所示的糖原分支酶(HosGBE)加入淀粉溶液中改性8h，即可使淀粉溶液中抗性淀粉的含量高达47.36％，较未经改性的淀粉溶液提高22.05％；利用此方法将氨基酸序列如SEQ ID NO:3所示的糖原分支酶(VvGBE)加入淀粉溶液中改性10h，即可使淀粉溶液中抗性淀粉的含量高达51.4％，较未经改性的淀粉溶液提高26.09％。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述。
下述实施例中涉及的大肠杆菌E.coli BL21(DE3)购自宝生物工程(大连)有限公司；下述实施例中涉及的玉米淀粉购自山东临沂实业有限公司；
下述实施例中涉及的培养基如下：
LB液体培养基：酵母粉5.0g·L-1、胰蛋白胨10.0g·L-1、NaCl 10.0g·L-1、卡那霉素100mg·L-1、氨苄青霉素100mg·L-1。
LB固体培养基：酵母粉5.0g·L-1、胰蛋白胨10.0g·L-1、NaCl 10.0g·L-1、琼脂粉15g·L-1、卡那霉素100mg·L-1、氨苄青霉素100mg·L-1。
TB液体培养基：酵母粉24g·L-1、胰蛋白胨12g·L-1、甘油5g·L-1、K2HPO412.54g·L-1、KH2PO42.31g·L-1、卡那霉素100mg·L-1、氨苄青霉素100mg·L-1。
下述实施例中涉及的检测方法如下：
抗性淀粉含量的测定方法：
将来自猪胰腺的α-淀粉酶(2g，购自Sigma化学试剂有限公司)加入蒸馏水(24mL)中并充分搅拌10min，1500g离心10min，将上清液(20mL)转移至烧杯中，然后与淀粉葡萄糖苷酶(0.4mL，购自Sigma化学试剂有限公司)和蒸馏水(3.6mL)混合，得到消化酶溶液；取反应液，将反应液的一部分于12000rpm离心15min取上清，用GOD-POD试剂盒检测游离葡萄糖的含量，将反应液的另一部分煮沸灭酶调节pH至5.2，在37℃温育10min，然后加入0.75mL消化酶溶液，分别在反应20min和120min时取样，同时加热煮沸灭酶10min，用GOD-POD试剂盒测定葡萄糖的含量，计算反应液中快消化淀粉(RDS)、慢消化淀粉(SDS)和抗性淀粉(RS)的含量。
快消化淀粉(RDS)、慢消化淀粉(SDS)和抗性淀粉(RS)含量的计算：
RDS(％)＝(G20-G0)×0.9×100；
SDS(％)＝(G120-G20)×0.9×100；
RS(％)＝100％-RDS(％)-SDS(％)；
式中，G0为游离葡萄糖含量，单位(mg)；G20为消化20min内释放的葡萄糖量，单位(mg)；G120为消化120min内释放的葡萄糖量，单位(mg)。
分支酶酶活的测定方法：
准确称取马铃薯支链淀粉(购自上海荣盛生物药业有限公司)0.5g(直至±0.001g)加入至盛有缓冲液的烧杯中，将其置于沸水浴中加热煮沸30min，直至底物溶液澄清透明，然后用100mL容量瓶定容，得到底物溶液；称取0.26g碘和2.6g碘化钾加入至盛有蒸馏水的烧杯中，搅拌均匀，然后用10mL棕色容量瓶定容，得到卢戈斯溶液(该溶液应该在使用前至少3天进行配置，以确保所有的碘溶解，并且该溶液应避光放置于阴凉处，可保持半年的有效期)；将100μL卢戈斯溶液、50μL盐酸(2M)、26mL蒸馏水混合，得到终止液(该溶液避光放置，现配现用)；设置对照组和实验组，其中，对照组：200μL底物溶液+200μL蒸馏水；实验组：200μL底物+200μL粗酶液；将对照组和未加酶液的实验组充分混匀后置于分支酶的最适温度(ChlGBE、HosGBE最适温度为30℃，VvGBE最适温度为35℃)下孵育10min；10min后，在实验组中加入稀释好的粗酶液200μL，将其置于糖原分支酶的最适温度下反应15min；15min后，分别从对照组和实验组取200μL产物加入到4mL终止液中，避光放置20min左右，直至颜色稳定；20min后，在分光光度计下测定吸光值(A530)，计算糖原分支酶酶活。
分支酶酶活的定义：
单位时间单位体积内每减少1mg支链淀粉所需要的酶量定义为1个酶活单位(1U)。
分支酶酶活的计算：
式中，A为分支酶酶活，单位(U/mL)；m0为对照组中支链淀粉含量，单位(mg)；m1为实验组中支链淀粉含量，单位(mg)；D为粗酶液稀释倍数；t为粗酶液与底物反应时间，单位(min)；V为粗酶液的体积，单位(mL)。
实施例1：不同分支酶的制备
具体步骤如下：
化学合成编码来源于小球藻(Chlorella kessleri)的糖原分支酶ChlGBE(氨基酸序列如SEQ ID NO:1所示)、来源于智人(Homo sapiens)的糖原分支酶HosGBE(氨基酸序列如SEQ ID NO:2所示)、来源于创伤弧菌(Vibrio vulnificus)的糖原分支酶VvGBE(氨基酸序列如SEQ ID NO:3所示)、来源于蓝藻(Cyanothece sp.ATCC 51142)的淀粉分支酶CySBE(氨基酸序列如SEQ ID NO:4所示)或来源于小浜红嗜热菌(Rhodothermus obamensis)的淀粉分支酶RoSBE(氨基酸序列如SEQ ID NO:5所示)的基因(核苷酸序列分别如SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10所示)，并将合成得到的基因和pET-24a(+)质粒进行连接，得到重组质粒pET-24a-ChlGBE、pET-24a-HosGBE、pET-24a-VvGBE、pET-24a-CySBE、pET-24a-RoSBE(此步骤由上海捷瑞生物工程有限公司完成)；将重组质粒pET-24a-ChlGBE、pET-24a-HosGBE、pET-24a-VvGBE、pET-24a-CySBE、pET-24a-RoSBE分别转化大肠杆菌E.coli BL21(DE3)，转化产物涂布于LB固体培养基，于37℃培养8～10h，在LB固体培养基上挑取转化子，接入LB液体培养基培养，于37℃培养10h，获得菌液；提取菌液中的质粒，进行双酶切验证，并将此质粒进行序列测定，测序正确即获得重组大肠杆菌pET-24a-ChlGBE/E.coli BL21、pET-24a-HosGBE/E.coli BL21、pET-24a-VvGBE/E.coliBL21、pET-24a-CySBE/E.coli BL21、pET-24a-RoSBE/E.coli BL21及其菌液。
将重组大肠杆菌pET-24a-ChlGBE/E.coli BL21、pET-24a-HosGBE/E.coli BL21、pET-24a-VvGBE/E.coli BL21、pET-24a-CySBE/E.coli BL21、pET-24a-RoSBE/E.coli BL21的菌液按照2‰(v/v)的接种量接入LB液体培养基，于37℃培养8～12h，获得种子液；将种子液按照5％(v/v)的接种量接入TB液体培养基，于37℃培养2～3h后，于25℃继续诱导培养24h，得到发酵液；将发酵液于4℃、8000rpm离心15min后，收集细胞并破碎，收集细胞破碎液；将细胞破碎液于4℃、8000rpm离心15min，得到细胞破碎上清液，此细胞破碎上清液即为糖原分支酶ChlGBE、糖原分支酶HosGBE、糖原分支酶VvGBE、淀粉分支酶CySBE或淀粉分支酶RoSBE的粗酶液。
实施例2：不同分支酶降低淀粉消化率的能力
具体步骤如下：
称取2.5g玉米淀粉加入50mM、pH 7.0的磷酸二氢钠-磷酸氢二钠缓冲液中，得到淀粉溶液；将淀粉溶液在沸水浴中加热搅拌30min，加热完毕，用容量瓶定容至100mL，配制成2.5％的糊化淀粉溶液；将糊化淀粉溶液置于30℃、150rpm恒温水浴摇床中温育10min，得到温育淀粉溶液；在温育淀粉溶液分别加入100、200、500、1000U/g淀粉的实施例1获得的糖原分支酶ChlGBE、糖原分支酶HosGBE、糖原分支酶VvGBE、分支酶CySBE或分支酶RoSBE的粗酶液，其中，空白对照组(Control)不添加酶液，用缓冲液补足，在30℃条件下反应12h，加热煮沸灭酶终止反应，得到反应液。检测反应液中慢消化淀粉(SDS)和抗性淀粉(RS)含量，检测结果如见表1。
由表1可知，仅有糖原分支酶ChlGBE、糖原分支酶HosGBE、糖原分支酶VvGBE可高效降低淀粉的消化率，在制备抗性淀粉中极具应用前景。
表1不同分支酶在不同添加量下反应获得的反应液中慢消化淀粉(SDS)和抗性淀粉(RS)的含量
其中，“/”为未测定。
实施例3：反应时间对分支酶降低淀粉消化率能力的影响
称取2.5g玉米淀粉加入50mM、pH 7.0的磷酸二氢钠-磷酸氢二钠缓冲液中，得到淀粉溶液；将淀粉溶液在沸水浴中加热搅拌30min，加热完毕，用容量瓶定容至100mL，配制成2.5％的糊化淀粉溶液；将糊化淀粉溶液置于30℃、150rpm恒温水浴摇床中温育10min，得到温育淀粉溶液；在温育淀粉溶液分别加入500U/g淀粉的实施例1获得的糖原分支酶ChlGBE、糖原分支酶HosGBE或糖原分支酶VvGBE的粗酶液，在30℃条件下分别反应0、4、8、10、12h，加热煮沸灭酶终止反应，得到反应液。检测反应液中抗性淀粉(RS)含量，检测结果如见表2。
由表2可知，反应时间为6h时，ChlGBE反应获得的反应液中抗性淀粉含量最高，为35.06％；反应时间为8h时，HosGBE反应获得的反应液中抗性淀粉含量最高，为47.36％；反应时间为10h时，VvGBE反应获得的反应液中抗性淀粉含量最高，为49.28％。
表2不同糖原分支酶在不同反应时间下反应获得的反应液中抗性淀粉(RS)的含量
实施例4：反应温度对分支酶降低淀粉消化率能力的影响
在实施例3的基础上，将反应温度分别调整为25、30、35、40、45℃，并且，将ChlGBE的反应时间限定为6h，将HosGBE的反应时间限定为8h，将VvGBE的反应时间限定为10h。检测反应液中抗性淀粉(RS)含量，检测结果如见表3。
由表3可知，反应温度为30℃时，ChlGBE、HosGBE反应获得的反应液中抗性淀粉的含量最高，分别为35.06％、47.36％；反应温度为35℃时，VvGBE反应获得的反应液中抗性淀粉的含量最高，为50.4％。
表3不同糖原分支酶在不同反应温度下反应获得的反应液中抗性淀粉(RS)的含量
实施例5：反应pH对分支酶降低淀粉消化率能力的影响
配置50mM、pH为5.5～6.0的柠檬酸缓冲液，50mM、pH为6.5～8.0的磷酸二氢钠-磷酸氢二钠缓冲液以及50mM、pH为8.5的Tris-HCl缓冲液；在实施例4的基础上，将50mM、pH7.0的磷酸二氢钠-磷酸氢二钠缓冲液分别调整为配置得到的50mM、pH为5.5～6.0的柠檬酸缓冲液，50mM、pH为6.5～8.0的磷酸二氢钠-磷酸氢二钠缓冲液以及50mM、pH为8.5的Tris-HCl缓冲液，并且，将ChlGBE的反应时间限定为6h、反应温度限定为30℃，将HosGBE的反应时间限定为8h、反应温度限定为30℃，将VvGBE的反应时间限定为10h、反应温度限定为35℃。检测反应液中抗性淀粉(RS)含量，检测结果如见表4。
由表4可知，反应pH为7.0时，ChlGBE、HosGBE反应获得的反应液中抗性淀粉的含量最高，分别为35.06％、47.36％；反应pH为7.5时，VvGBE反应获得的反应液中抗性淀粉的含量最高，为51.4％。
表4不同糖原分支酶在不同反应pH下反应获得的反应液中抗性淀粉(RS)的含量
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 江南大学
<120> 一种降低淀粉消化率的方法及其应用
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 880
<212> PRT
<213> 小球藻（Chlorella kessleri）
<400> 1
Met Gln Ser Gln Leu Gly Met Leu Gln His Ser Thr Thr Ser Ala Pro
1 5 10 15
Pro Gly Pro Arg Ser Gly Val Ser Gly Arg Leu Asn Arg Phe Val Ala
20 25 30
Arg Pro Thr Gln Gly Lys Arg Leu Gly Arg Arg Leu Ile Ala Asn Val
35 40 45
Arg Ser Asp Ser Pro Ser Asn Glu Thr Leu Ser Pro Leu Glu Ile Leu
50 55 60
Lys Arg Glu Asn Glu Leu Leu Arg Arg Thr Val Glu Ala Thr Glu Arg
65 70 75 80
Ser Val Gly Glu Leu Glu Ala Gly Leu Thr Asp Ala Gly Val Gly Leu
85 90 95
Pro Pro Ala Ala Gly Ala Gly Arg Ala Thr Ala Gly Gly Pro Thr Ala
100 105 110
Ala Ala Leu Glu Ser Asp Thr Pro Glu Asp Ala Trp Ser Pro Ala Val
115 120 125
His Val Pro Glu Gly Gln Ala Phe Glu Glu Val Tyr Gly Leu Ile Ser
130 135 140
Pro Ile Pro Asp His Asp Gly Thr Glu Cys Leu Lys Trp Asp Pro Thr
145 150 155 160
Leu Trp Ser His Ala Asp His Phe Lys Tyr Arg Trp His Val Phe Lys
165 170 175
Ser Ile Arg Ala Ala Ile Asp Gln Asn Glu Gly Gly Leu Glu Lys Phe
180 185 190
Thr Gln Gly Tyr Lys Tyr Tyr Gly Phe Thr Arg Gly Glu His Glu Gly
195 200 205
Lys Lys Gly Ile Trp Tyr Arg Glu Trp Ala Pro Gly Ala Lys Ala Leu
210 215 220
Ala Leu Ile Gly Glu Phe Asn Ala Trp Gln Pro Lys Asp Glu His Trp
225 230 235 240
Ala Ile Lys Asn Asp Phe Gly Val Trp Gln Leu Phe Leu Pro Asp Asn
245 250 255
Pro Asp Gly Thr Ser Ala Ile Thr His Arg Thr Lys Val Lys Leu Arg
260 265 270
Leu Glu Thr Ala Tyr Gly Glu Trp Val Glu Arg Ile Pro Ala Trp Ile
275 280 285
Lys Trp Ala Thr Gln Glu Trp Asn Glu Val Gln Phe Asn Gly Val Tyr
290 295 300
Tyr Gln Pro Pro Gln Val Gly Ala Pro Gly Glu Ile Asp Pro Asn Lys
305 310 315 320
Ser Tyr Thr Phe Lys Tyr Pro Arg Pro Ala Arg Pro Arg Ala Leu Arg
325 330 335
Ile Tyr Glu Cys His Val Gly Met Ser Ser Gln Glu Pro Lys Val Asn
340 345 350
Ser Tyr Leu Glu Phe Lys Glu Glu Val Leu Pro Arg Ile Arg Ser Leu
355 360 365
Gly Tyr Asn Ala Ile Gln Ile Met Ala Ile Gln Glu His Ala Tyr Tyr
370 375 380
Gly Ser Phe Gly Tyr His Val Thr Asn Phe Phe Ala Ala Ser Ser Arg
385 390 395 400
Cys Gly Thr Pro Asp Glu Leu Lys Ala Met Ile Asp Glu Ala His Arg
405 410 415
Leu Gly Met Val Val Leu Met Asp Ile Val His Ser His Ala Ser Lys
420 425 430
Asn Thr Met Asp Gly Ile Asn Met Phe Asp Gly Thr Asp Gly Met Tyr
435 440 445
Phe His Gly Gly Gly Arg Gly Tyr His Trp Met Trp Asp Ser Arg Cys
450 455 460
Phe Asn Tyr Gly Asn Trp Glu Thr Leu Arg Phe Leu Leu Ser Asn Ala
465 470 475 480
Arg Trp Trp Met Asp Glu Tyr Lys Phe Asp Gly Tyr Arg Phe Asp Gly
485 490 495
Val Thr Ser Met Met Tyr His His His Gly Leu Gln Thr Thr Phe Thr
500 505 510
Gly Asn Tyr Asp Glu Tyr Phe Gly Met Ala Thr Asp Val Asp Ala Val
515 520 525
Val Tyr Leu Gln Leu Val Asn His Thr Leu His Asp Leu Phe Pro Thr
530 535 540
Ala Ile Thr Ile Gly Glu Asp Val Ser Gly Met Pro Thr Phe Cys Arg
545 550 555 560
Pro Trp Thr Glu Gly Gly Val Gly Phe Asp Tyr Arg Leu Asn Met Ala
565 570 575
Ile Ala Asp Lys Trp Ile Glu Met Leu Ser Lys Leu Asp Asp Tyr Ser
580 585 590
Trp Asn Met Gly Asn Ile Val His Thr Met Thr Asn Arg Arg Tyr Met
595 600 605
Glu Ala Cys Val Gly Tyr Ala Glu Ser His Asp Gln Ala Leu Val Gly
610 615 620
Asp Lys Thr Ile Ala Phe Trp Leu Met Asp Lys Asp Met Tyr Asp Cys
625 630 635 640
Met Ala Ala Pro Gly Tyr Gly Ser Ser Ser Pro Val Val Asp Arg Gly
645 650 655
Ile Ala Leu His Lys Met Ile Arg Leu Leu Thr Met Ala Leu Gly Gly
660 665 670
Glu Ser Tyr Leu Asn Phe Met Gly Asn Glu Phe Gly His Pro Glu Trp
675 680 685
Ile Asp Phe Pro Arg Asp Asp Ser Tyr Asp Thr Ser Thr Gly Ala Phe
690 695 700
Val Pro Gly Asn Gly Gly Ser Leu Glu Lys Cys Arg Arg Arg Trp Asp
705 710 715 720
Leu Ala Asp Ala Pro Phe Leu Lys Tyr Lys Phe Met Asn Ala Tyr Asp
725 730 735
Arg Ala Ile Met His Leu Asp Lys Ala Phe Gly Phe Ile Ser Ala Pro
740 745 750
His Asn Trp Val Ser Arg Lys Asp Glu Gly Asp Lys Ile Ile Val Ala
755 760 765
Glu Lys Gly Asp Leu Val Met Val Phe Asn Phe His Pro Thr Asn Ser
770 775 780
Tyr Ser Asp Tyr Arg Val Gly Cys Tyr Lys Pro Gly Pro Tyr Lys Val
785 790 795 800
Ala Leu Ser Ser Asp Glu Glu Val Phe Gly Gly Trp Arg Asn Val Thr
805 810 815
Lys Asp Asn Asp Val Glu Phe Tyr Thr Ala Glu Gly Asn Tyr Asp Asn
820 825 830
Arg Pro His Ser Leu Gln Val Tyr Ala Pro Ser Arg Thr Val Val Val
835 840 845
Tyr Ala Pro Thr Glu Phe Cys Asp Lys Asp Ala Asp Arg Thr Pro Val
850 855 860
Gly Ile Pro Gly Leu Ser Val Lys Gly Leu Gly Pro Tyr Tyr Gly Phe
865 870 875 880
<210> 2
<211> 702
<212> PRT
<213> 智人（Homo sapiens）
<400> 2
Met Ala Ala Pro Met Thr Pro Ala Ala Arg Pro Glu Asp Tyr Glu Ala
1 5 10 15
Ala Leu Asn Ala Ala Leu Ala Asp Val Pro Glu Leu Ala Arg Leu Leu
20 25 30
Glu Ile Asp Pro Tyr Leu Lys Pro Tyr Ala Val Asp Phe Gln Arg Arg
35 40 45
Tyr Lys Gln Phe Ser Gln Ile Leu Lys Asn Ile Gly Glu Asn Glu Gly
50 55 60
Gly Ile Asp Lys Phe Ser Arg Gly Tyr Glu Ser Phe Gly Val His Arg
65 70 75 80
Cys Ala Asp Gly Gly Leu Tyr Cys Lys Glu Trp Ala Pro Gly Ala Glu
85 90 95
Gly Val Phe Leu Thr Gly Asp Phe Asn Gly Trp Asn Pro Phe Ser Tyr
100 105 110
Pro Tyr Lys Lys Leu Asp Tyr Gly Lys Trp Glu Leu Tyr Ile Pro Pro
115 120 125
Lys Gln Asn Lys Ser Val Leu Val Pro His Gly Ser Lys Leu Lys Val
130 135 140
Val Ile Thr Ser Lys Ser Gly Glu Ile Leu Tyr Arg Ile Ser Pro Trp
145 150 155 160
Ala Lys Tyr Val Val Arg Glu Gly Asp Asn Val Asn Tyr Asp Trp Ile
165 170 175
His Trp Asp Pro Glu His Ser Tyr Glu Phe Lys His Ser Arg Pro Lys
180 185 190
Lys Pro Arg Ser Leu Arg Ile Tyr Glu Ser His Val Gly Ile Ser Ser
195 200 205
His Glu Gly Lys Val Ala Ser Tyr Lys His Phe Thr Cys Asn Val Leu
210 215 220
Pro Arg Ile Lys Gly Leu Gly Tyr Asn Cys Ile Gln Leu Met Ala Ile
225 230 235 240
Met Glu His Ala Tyr Tyr Ala Ser Phe Gly Tyr Gln Ile Thr Ser Phe
245 250 255
Phe Ala Ala Ser Ser Arg Tyr Gly Thr Pro Glu Glu Leu Gln Glu Leu
260 265 270
Val Asp Thr Ala His Ser Met Gly Ile Ile Val Leu Leu Asp Val Val
275 280 285
His Ser His Ala Ser Lys Asn Ser Ala Asp Gly Leu Asn Met Phe Asp
290 295 300
Gly Thr Asp Ser Cys Tyr Phe His Ser Gly Pro Arg Gly Thr His Asp
305 310 315 320
Leu Trp Asp Ser Arg Leu Phe Ala Tyr Ser Ser Trp Glu Ile Leu Arg
325 330 335
Phe Leu Leu Ser Asn Ile Arg Trp Trp Leu Glu Glu Tyr Arg Phe Asp
340 345 350
Gly Phe Arg Phe Asp Gly Val Thr Ser Met Leu Tyr His His His Gly
355 360 365
Val Gly Gln Gly Phe Ser Gly Asp Tyr Ser Glu Tyr Phe Gly Leu Gln
370 375 380
Val Asp Glu Asp Ala Leu Thr Tyr Leu Met Leu Ala Asn His Leu Val
385 390 395 400
His Thr Leu Cys Pro Asp Ser Ile Thr Ile Ala Glu Asp Val Ser Gly
405 410 415
Met Pro Ala Leu Cys Ser Pro Ile Ser Gln Gly Gly Gly Gly Phe Asp
420 425 430
Tyr Arg Leu Ala Met Ala Ile Pro Asp Lys Trp Ile Gln Leu Leu Lys
435 440 445
Glu Phe Lys Asp Glu Asp Trp Asn Met Gly Asp Ile Val Tyr Thr Leu
450 455 460
Thr Asn Arg Arg Tyr Leu Glu Lys Cys Ile Ala Tyr Ala Glu Ser His
465 470 475 480
Asp Gln Ala Leu Val Gly Asp Lys Ser Leu Ala Phe Trp Leu Met Asp
485 490 495
Ala Glu Met Tyr Thr Asn Met Ser Val Leu Thr Pro Phe Thr Pro Val
500 505 510
Ile Asp Arg Gly Ile Gln Leu His Lys Met Ile Arg Leu Ile Thr His
515 520 525
Gly Leu Gly Gly Glu Gly Tyr Leu Asn Phe Met Gly Asn Glu Phe Gly
530 535 540
His Pro Glu Trp Leu Asp Phe Pro Arg Lys Gly Asn Asn Glu Ser Tyr
545 550 555 560
His Tyr Ala Arg Arg Gln Phe His Leu Thr Asp Asp Asp Leu Leu Arg
565 570 575
Tyr Lys Phe Leu Asn Asn Phe Asp Arg Asp Met Asn Arg Leu Glu Glu
580 585 590
Arg Tyr Gly Trp Leu Ala Ala Pro Gln Ala Tyr Val Ser Glu Lys His
595 600 605
Glu Gly Asn Lys Ile Ile Ala Phe Glu Arg Ala Gly Leu Leu Phe Ile
610 615 620
Phe Asn Phe His Pro Ser Lys Ser Tyr Thr Asp Tyr Arg Val Gly Thr
625 630 635 640
Ala Leu Pro Gly Lys Phe Lys Ile Val Leu Asp Ser Asp Ala Ala Glu
645 650 655
Tyr Gly Gly His Gln Arg Leu Asp His Ser Thr Asp Phe Phe Ser Glu
660 665 670
Ala Phe Glu His Asn Gly Arg Pro Tyr Ser Leu Leu Val Tyr Ile Pro
675 680 685
Ser Arg Val Ala Leu Ile Leu Gln Asn Val Asp Leu Pro Asn
690 695 700
<210> 3
<211> 742
<212> PRT
<213> 创伤弧菌（Vibrio vuLnificus）
<400> 3
Met Arg Ile Val Phe Thr Thr Leu Ala Val Trp Arg Gly Lys Lys Gly
1 5 10 15
Leu Lys Lys Leu Val Lys Ser Lys Val Asn Gln Met Phe Glu Lys Leu
20 25 30
Ser Gln Ala Ala Cys Ser Glu Pro Phe Ala Phe Leu Gly Pro Phe Ile
35 40 45
Asp Pro Thr Gln Gly Ala Leu Arg Val Trp Met Pro Gly Ala Thr Gly
50 55 60
Val Ala Leu Val Leu Glu Gly Gln Pro Arg Ile Ala Leu Glu Arg Glu
65 70 75 80
Lys Glu Ser Ala Phe Ile Leu Lys Ala Asp Leu Asn Leu His Leu Thr
85 90 95
His Tyr Gln Leu Ala Ile Asp Trp Asn Gly Val Glu Gln Leu Ile Asp
100 105 110
Asp Pro Tyr Gln Tyr His Gly Ile Tyr Ala Glu Tyr Asp Asp Leu His
115 120 125
Thr Pro Lys Thr Met Tyr Gln His Met Gly Ser Gln Phe Met Thr Leu
130 135 140
Glu Arg Asp Gly Lys Ser Ile Ser Gly Ile Arg Phe Leu Val Tyr Ala
145 150 155 160
Pro His Ala Thr Ala Val Ser Leu Val Gly Cys Phe Asn Asp Trp Asp
165 170 175
Gly Arg Arg His Pro Met Gln Arg Leu Asp Tyr Gly Ile Trp Gly Leu
180 185 190
Phe Ile Pro Gly Leu Thr Glu Gly Val Ser Tyr Lys Phe Glu Met Lys
195 200 205
Gly Pro Lys Gly Glu Gly Leu Pro His Lys Ala Asp Pro Trp Gly Phe
210 215 220
Tyr Ala Glu Gln Tyr Pro Ser Phe Ala Ser Val Thr Tyr Asp His Ala
225 230 235 240
Arg Tyr Gln Trp Gln Asp Ala Gln Trp Gln Thr Arg Pro Val Thr Glu
245 250 255
Lys Arg Lys Glu Ala Leu Ser Phe Tyr Glu Leu His Ala Gly Ser Trp
260 265 270
Lys Arg Asn Glu Gln Gly Glu Phe Leu Asn Tyr Arg Glu Leu Ala Ala
275 280 285
Glu Leu Val Pro Tyr Leu Val Asp Met Gly Tyr Thr His Val Glu Leu
290 295 300
Met Pro Val Ser Glu His Pro Phe Tyr Gly Ser Trp Gly Tyr Gln Pro
305 310 315 320
Val Gly Leu Phe Ala Pro Thr Ser Arg Tyr Gly Ser Pro Asp Asp Phe
325 330 335
Lys Phe Phe Val Asp Ala Cys His Gln Ala Gly Ile Gly Val Val Leu
340 345 350
Asp Trp Val Pro Ala His Phe Pro Ser Asp Asp His Gly Leu Ala Asn
355 360 365
Phe Asp Gly Thr Pro Leu Phe His Asp Pro Asp Pro Arg Arg Gly Trp
370 375 380
His Gln Asp Trp Asn Ser Phe Ile Tyr Asp Leu Gly Arg Glu Gln Val
385 390 395 400
Arg Arg Phe Leu Val Ser Asn Ala Leu Tyr Trp Phe Glu Gln Phe His
405 410 415
Ile Asp Gly Ile Arg Val Asp Ala Val Ala Ser Met Leu Tyr Leu Asp
420 425 430
Tyr Ser Arg Ser His Gly Gln Trp Ile Pro Asn Met Asp Gly Gly Asn
435 440 445
Glu Asn Tyr Asp Ala Ile Ala Thr Leu Lys Trp Met Asn Glu Glu Val
450 455 460
Tyr Lys Tyr Phe Pro Asn Ala Met Thr Ile Ala Glu Glu Ser Thr Ala
465 470 475 480
Phe Pro Gly Val Ser Ala Pro Thr Phe Met Gly Gly Leu Gly Phe Gly
485 490 495
Phe Lys Trp Asn Met Gly Trp Met His Asp Ser Leu Ser Tyr Ile Lys
500 505 510
Glu Glu Pro Val His Arg Lys Tyr His His Asn Thr Leu Thr Phe Pro
515 520 525
Leu Val Tyr Ala His Ser Glu Asn Tyr Val Leu Ser Leu Ser His Asp
530 535 540
Glu Val Val Tyr Gly Lys Gly Ser Ile His Asn Lys Met Pro Gly Asp
545 550 555 560
Glu Trp Gln Gln Thr Ala Asn Leu Arg Ala Tyr Phe Gly Tyr Met Tyr
565 570 575
Gly Gln Pro Gly Lys Lys Leu Asn Phe Met Gly Ala Glu Ile Gly Gln
580 585 590
Thr Ala Glu Trp Asn His Asp Asp Gln Leu Gln Trp Phe Leu Leu Asp
595 600 605
Phe Pro Arg His Gln Gly Val Gln Ala Leu Thr Arg Asp Leu Asn His
610 615 620
Leu Tyr Arg Asn Glu Ala Ala Leu His Asp Gln Asp Cys Ile Pro Ala
625 630 635 640
Gly Phe Glu Trp Arg Leu Gln Asp Ala Ala Glu Gln Ser Ile Ile Ala
645 650 655
His Glu Arg Ile Ser Glu Ala Gly Glu Arg Ile Leu Val Val Ser Asn
660 665 670
Phe Thr Pro Val Pro Arg Asp Glu Phe Arg Leu Gly Val Pro Asn Lys
675 680 685
Gly Arg Tyr Gln Leu Leu Leu Asn Thr Asp Asp Ser Lys Tyr Ala Gly
690 695 700
Ser Gly Tyr Glu Val Val Val Asp Ala Lys Ser Glu Ala Val Val Ser
705 710 715 720
Glu Asp Leu Ala Gln Ser Ile Val Leu Arg Leu Pro Pro Leu Ser Thr
725 730 735
Leu Phe Tyr Lys Leu Val
740
<210> 4
<211> 773
<212> PRT
<213> 蓝藻（Cyanothece sp.）
<400> 4
Met Thr Thr Thr Ile Ser Ala Asp Gln Val Asn Gln Ile Ile Tyr Asn
1 5 10 15
Leu His His Asp Pro Phe Glu Ile Leu Gly Cys His Leu Leu Glu Glu
20 25 30
Gly Lys Asn Thr Lys Lys Trp Val Val Arg Ala Tyr Leu Pro Lys Ala
35 40 45
Glu Ala Ala Trp Val Ile Arg Pro Thr Glu Arg Lys Glu Asp Pro Met
50 55 60
Asn Ser Val His His Pro Asn Phe Phe Glu Cys Ile Ile Glu Thr Pro
65 70 75 80
Glu Leu Asn His Tyr Gln Leu Lys Val Lys Glu Gly Glu His Glu Lys
85 90 95
Val Ile Tyr Asp Pro Tyr Ala Phe Ser Ser Pro Tyr Leu Thr Asp Glu
100 105 110
Asp Ile Tyr Leu Phe Ser Glu Gly Asn His His Arg Ile Tyr Glu Lys
115 120 125
Leu Gly Ala His Val Gly Glu Ile Asn Gly Val Lys Gly Val Tyr Phe
130 135 140
Ala Val Trp Ala Pro Asn Ala Arg Asn Val Ser Val Ile Gly Asp Phe
145 150 155 160
Asn Asn Trp Asp Gly Arg Glu His Gln Met Arg Lys Arg Asn Tyr Thr
165 170 175
Ile Trp Glu Leu Phe Val Pro Glu Ile Gly Ser Gly Thr Val Tyr Lys
180 185 190
Tyr Glu Ile Lys Asn Ser Glu Gly His Ile Tyr Glu Lys Ser Asp Pro
195 200 205
Tyr Gly Phe Tyr Arg Glu Val Arg Pro Asn Thr Ala Ser Ile Val Val
210 215 220
Asp Ile Asp Asn Ile Tyr Gln Trp His Asp Glu Glu Trp Leu Glu Lys
225 230 235 240
Arg Arg Asn Ser Asp Pro Leu Lys Gln Pro Val Ser Val Tyr Glu Val
245 250 255
His Leu Gly Ser Trp Leu His Gly Ser Ser Ala Glu Lys Met Pro Leu
260 265 270
Leu Asn Gly Glu Ala Asp Pro Val Ile Val Ser Glu Trp Asn Pro Gly
275 280 285
Ala Arg Phe Leu Ser Tyr Tyr Glu Leu Ala Glu Lys Leu Ile Pro Tyr
290 295 300
Val Lys Asp Met Gly Tyr Thr His Ile Glu Leu Leu Pro Ile Ala Glu
305 310 315 320
His Pro Phe Asp Gly Ser Trp Gly Tyr Gln Val Thr Gly Phe Tyr Ser
325 330 335
Pro Thr Ser Arg Phe Gly Arg Pro Glu Asp Phe Met Tyr Phe Val Asp
340 345 350
Lys Cys His Glu Asn Gly Ile Gly Val Ile Leu Asp Trp Val Pro Gly
355 360 365
His Phe Pro Lys Asp Ser His Gly Leu Ala Tyr Phe Asp Gly Thr His
370 375 380
Leu Tyr Glu His Ala Asp Pro Arg Ile Gly Glu His Lys Glu Trp Gly
385 390 395 400
Thr Leu Val Phe Asn Tyr Gly Arg His Glu Val Arg Asn Phe Leu Val
405 410 415
Ala Asn Val Leu Phe Trp Phe Asp Lys Tyr His Val Asp Gly Ile Arg
420 425 430
Val Asp Ala Val Ala Ser Met Leu Tyr Arg Asn Tyr Leu Arg Lys Glu
435 440 445
Gly Glu Trp Ile Ala Asn Glu Tyr Gly Gly Asp Glu His Ile Glu Ala
450 455 460
Val Ser Phe Ile Arg Glu Val Asn Thr Leu Leu Phe Glu Tyr Phe Pro
465 470 475 480
Gly Ile Leu Ser Ile Ala Glu Glu Ser Thr Glu Trp Glu Lys Val Ser
485 490 495
Arg Pro Val Tyr Asp Gly Gly Leu Gly Phe Asn Leu Lys Trp Asp Met
500 505 510
Gly Trp Met His Asp Met Leu Asp Tyr Phe Asn Ile Asp Pro Tyr Phe
515 520 525
Arg Gln Tyr His Gln Asn Asn Val Thr Phe Ser Met Leu Tyr Tyr Tyr
530 535 540
Asn Glu Asn Phe Met Leu Ala Leu Ser His Asp Glu Ile Val His Gly
545 550 555 560
Lys Ser Asn Met Leu Gly Lys Met Pro Gly Asp Glu Trp Gln Lys Tyr
565 570 575
Ala Asn Val Arg Ala Leu Phe Thr Tyr Met Tyr Thr His Pro Gly Lys
580 585 590
Lys Thr Met Phe Met Ser Met Glu Phe Gly Gln Trp Ser Glu Trp Asn
595 600 605
Val Trp Gly Asp Leu Glu Trp His Leu Leu Gln Tyr Glu Pro His Gln
610 615 620
Gln Leu Lys Gln Phe Phe Thr Asp Leu Asn Ala Leu Tyr Gln Gln Glu
625 630 635 640
Pro Ala Leu Tyr Thr His Asp Phe Glu Tyr His Gly Phe Glu Trp Ile
645 650 655
Asp Cys Asn Asp Asn Thr His Ser Val Val Ser Phe Leu Arg Arg Ser
660 665 670
Asp Asp Pro Asn Asp Ser Leu Val Val Val Cys Asn Phe Thr Pro Gln
675 680 685
Pro His Ser His Tyr Arg Ile Gly Val Pro Glu Ala Gly Tyr Tyr Val
690 695 700
Glu Leu Phe Asn Ser Asp Ala Lys Gln Tyr Gly Gly Ser Asn Met Gly
705 710 715 720
Asn Leu Gly Gly Lys Trp Ala Asp Glu Trp Ser Phe His Asn Lys Pro
725 730 735
Tyr Ser Leu Asp Leu Cys Leu Pro Pro Leu Ala Val Leu Ile Leu Lys
740 745 750
Leu Asp Pro Thr Lys Val Pro Glu Gly Thr Thr Ile Lys Glu Ile Ala
755 760 765
Ala Asp Glu Glu Glu
770
<210> 5
<211> 621
<212> PRT
<213> 小浜红嗜热菌（Rhodothermus obamensis）
<400> 5
Met Ser Trp Leu Thr Glu Glu Asp Ile Arg Arg Trp Glu Ser Gly Thr
1 5 10 15
Phe Tyr Asp Ser Tyr Arg Lys Leu Gly Ala His Pro Asp Asp Glu Gly
20 25 30
Thr Trp Phe Cys Val Trp Ala Pro His Ala Asp Gly Val Ser Val Leu
35 40 45
Gly Ala Phe Asn Asp Trp Asn Pro Glu Ala Asn Pro Leu Glu Arg Tyr
50 55 60
Gly Gly Gly Leu Trp Ala Gly Tyr Val Pro Gly Ala Arg Pro Gly His
65 70 75 80
Thr Tyr Lys Tyr Arg Ile Arg His Gly Phe Tyr Gln Ala Asp Lys Thr
85 90 95
Asp Pro Tyr Ala Phe Ala Met Glu Pro Pro Thr Gly Ser Pro Ile Glu
100 105 110
Gly Leu Ala Ser Ile Ile Thr Arg Leu Asp Tyr Thr Trp His Asp Asp
115 120 125
Glu Trp Met Arg Arg Arg Lys Gly Pro Ala Ser Leu Tyr Glu Pro Val
130 135 140
Ser Ile Tyr Glu Val His Leu Gly Ser Trp Arg His Lys Arg Pro Gly
145 150 155 160
Glu Ser Phe Ser Tyr Arg Glu Ile Ala Glu Pro Leu Ala Asp Tyr Val
165 170 175
Gln Glu Met Gly Phe Thr His Val Glu Leu Leu Pro Val Met Glu His
180 185 190
Pro Tyr Tyr Gly Ser Trp Gly Tyr Gln Val Val Gly Tyr Tyr Ala Pro
195 200 205
Thr Phe Arg Tyr Gly Ser Pro Gln Asp Leu Met Tyr Leu Ile Asp Tyr
210 215 220
Leu His Gln Arg Gly Ile Gly Val Ile Leu Asp Trp Val Pro Ser His
225 230 235 240
Phe Ala Ala Asp Pro Gln Gly Leu Val Phe Phe Asp Gly Thr Thr Leu
245 250 255
Phe Glu Tyr Asp Asp Pro Lys Met Arg Tyr His Pro Asp Trp Gly Thr
260 265 270
Tyr Val Phe Asp Tyr Asn Lys Pro Gly Val Arg Asn Phe Leu Ile Ser
275 280 285
Asn Ala Leu Phe Trp Leu Glu Lys Tyr His Val Asp Gly Leu Arg Val
290 295 300
Asp Ala Val Ala Ser Met Leu Tyr Arg Asp Tyr Ser Arg Lys Glu Trp
305 310 315 320
Thr Pro Asn Ile Phe Gly Gly Arg Glu Asn Leu Glu Ala Ile Asp Phe
325 330 335
Ile Lys Lys Phe Asn Glu Thr Val Tyr Leu His Phe Pro Glu Ala Met
340 345 350
Thr Ile Ala Glu Glu Ser Thr Ala Trp Pro Gly Val Ser Ala Pro Thr
355 360 365
Tyr Asn Asn Gly Leu Gly Phe Leu Tyr Lys Trp Asn Met Gly Trp Met
370 375 380
His Asp Thr Leu Asp Tyr Ile Gln Arg Asp Pro Ile Tyr Arg Lys Tyr
385 390 395 400
His His Asp Glu Leu Thr Phe Ser Leu Trp Tyr Ala Phe Ser Glu His
405 410 415
Tyr Val Leu Pro Leu Ser His Asp Glu Val Val His Gly Lys Gly Ser
420 425 430
Leu Trp Gly Lys Met Pro Gly Asp Asp Trp Gln Lys Ala Ala Asn Leu
435 440 445
Arg Leu Leu Phe Gly His Met Trp Gly His Pro Gly Lys Lys Leu Leu
450 455 460
Phe Met Gly Gly Glu Phe Gly Gln His His Glu Trp Asn His Asp Thr
465 470 475 480
Gln Leu Glu Trp His Leu Leu Asp Gln Pro Tyr His Arg Gly Ile Gln
485 490 495
Leu Trp Val Cys Asp Leu Asn His Leu Tyr Arg Thr Asn Pro Ala Leu
500 505 510
Trp His Asp Gly Pro Glu Gly Phe Glu Trp Ile Asp Phe Ser Asp Arg
515 520 525
Asp Gln Ser Val Ile Cys Tyr Leu Arg Lys Asn Ala Gly Arg Met Leu
530 535 540
Leu Phe Val Leu Asn Phe Thr Pro Val Pro Arg Glu His Tyr Arg Val
545 550 555 560
Gly Val Pro Ile Gly Gly Pro Trp His Glu Val Leu Asn Ser Asp Ala
565 570 575
Val Ala Tyr Gly Gly Ser Gly Met Gly Asn Phe Gly Arg Val Glu Ala
580 585 590
Val Pro Glu Ser Trp His Gly Arg Pro Phe His Leu Glu Leu Thr Leu
595 600 605
Pro Pro Leu Ala Ala Leu Ile Leu Glu Pro Glu His Gly
610 615 620
<210> 6
<211> 2649
<212> DNA
<213> 人工序列
<400> 6
catatgcagt cacagttagg catgttacag catagtacaa cgagtgctcc tccgggtcca 60
cgtagcggcg tgagcggtcg tctgaatcgt tttgttgcac gcccgacaca gggcaaacgc 120
ttaggtcgtc gcctgattgc caatgttcgc tcagatagtc ctagtaatga aacactgagt 180
ccattagaaa tcctgaaacg cgaaaatgaa ctgttacgcc gtaccgttga agcaacggaa 240
cgtagcgtgg gcgaactgga agccggtctg acggatgcgg gcgtgggctt acctccagcc 300
gccggcgccg gtcgtgcaac cgcaggcggc cctacagcag cagccttaga aagcgatacc 360
ccggaagatg cctggagccc agcagttcat gttccagaag gtcaggcctt tgaagaagtg 420
tatggcttaa tctctcctat cccagatcat gatggtacgg aatgtctgaa atgggaccca 480
accctgtgga gtcatgcaga tcattttaaa tatcgctggc atgtgtttaa atctattcgt 540
gcagccatcg atcagaatga aggcggttta gaaaaattta cacagggcta taaatattat 600
ggctttaccc gcggcgaaca tgaaggcaaa aaaggcatct ggtatcgcga atgggccccg 660
ggcgccaaag cgttagcact gatcggcgaa tttaatgctt ggcagccaaa agatgaacat 720
tgggccatca aaaatgattt tggtgtgtgg cagctgtttc tgccggataa tccggatggt 780
acgagcgcaa tcacacatcg caccaaagtt aaactgcgct tagaaaccgc ctatggcgaa 840
tgggttgaac gcattcctgc ttggatcaaa tgggccaccc aggaatggaa tgaagttcag 900
tttaatggtg tgtattatca gccacctcag gttggtgcac cgggcgaaat cgatcctaat 960
aaaagctata cgtttaaata tcctcgtcct gcgcgcccac gtgcgttacg catctatgaa 1020
tgtcatgtgg gcatgtcaag tcaggaacct aaagttaata gctatctgga atttaaagaa 1080
gaagttctgc cacgcatccg tagtctgggc tataatgcta ttcagattat ggccattcag 1140
gaacatgcgt attatggttc ttttggttat catgtgacca atttttttgc tgccagttca 1200
cgctgcggta caccagatga actgaaagcc atgatcgatg aagcacatcg cttaggtatg 1260
gttgttctga tggatattgt tcatagccat gcctctaaaa atacaatgga tggcatcaat 1320
atgtttgatg gtacggatgg catgtatttt catggcggtg gccgcggcta tcattggatg 1380
tgggattcac gttgctttaa ttatggtaat tgggaaaccc tgcgctttct gctgagtaat 1440
gctcgctggt ggatggatga atataaattt gatggttatc gctttgatgg tgtgacctct 1500
atgatgtatc atcatcatgg cttacagacg acctttacgg gcaattatga tgaatatttt 1560
ggcatggcaa ccgatgttga tgctgttgtg tatctgcagt tagttaatca taccctgcat 1620
gatctgtttc cgacagccat tacaatcggc gaagatgtga gcggcatgcc tacgttttgt 1680
cgtccttgga cggaaggcgg tgtgggcttt gattatcgtc tgaatatggc cattgccgat 1740
aaatggatcg aaatgctgtc taaactggat gattatagct ggaatatggg taatattgtg 1800
cataccatga ccaatcgtcg ctatatggaa gcgtgcgtgg gctatgccga atctcatgat 1860
caggccttag tgggtgataa aacgattgcg ttttggttaa tggataaaga tatgtatgat 1920
tgtatggccg ccccgggtta tggtagtagc tctccagtgg ttgatcgcgg cattgcctta 1980
cataaaatga ttcgcttact gactatggcc ttaggcggtg aaagctatct gaattttatg 2040
ggtaatgaat ttggtcatcc agaatggatc gattttccac gcgatgattc ttatgatacc 2100
tctaccggtg cctttgttcc aggtaatggc ggctcactgg aaaaatgtcg tcgccgctgg 2160
gatttagcag atgccccgtt tctgaaatat aaatttatga atgcgtatga tcgcgcaatc 2220
atgcatttag ataaagcctt tggctttatt agtgcaccac ataattgggt gagtcgcaaa 2280
gatgaaggtg ataaaatcat tgttgccgaa aaaggcgatc tggttatggt gtttaatttt 2340
catccgacca atagctatag cgattatcgc gtgggttgct ataaaccagg tccgtataaa 2400
gttgcactgt caagcgatga agaagtgttt ggcggctggc gcaatgtgac caaagataat 2460
gatgtggaat tttatacagc agaaggcaat tatgataatc gtccacattc actgcaggtg 2520
tatgcaccgt ctcgtacagt tgttgtgtat gctccgacgg aattttgcga taaagatgcc 2580
gatcgtaccc cagtgggtat cccgggtctg tcagtgaaag gcttaggtcc gtattatggc 2640
tttaagctt 2649
<210> 7
<211> 2115
<212> DNA
<213> 人工序列
<400> 7
catatggccg ccccgatgac cccggcagca cgtccggaag attatgaagc agcactgaat 60
gccgccctgg cagatgttcc ggaactggca cgcctgctgg aaattgatcc gtatctgaaa 120
ccgtatgcag tggattttca gcgtcgttat aaacagtttt ctcagattct gaaaaatatt 180
ggcgaaaatg aaggcggtat tgataaattt tctcgcggct atgaatcttt tggcgtgcat 240
cgttgtgccg atggcggtct gtattgtaaa gaatgggcac cgggtgcaga aggcgtgttt 300
ctgaccggcg attttaatgg ttggaatccg tttagctatc cgtataaaaa actggattat 360
ggcaaatggg aactgtatat tccgccgaaa cagaataaaa gcgtgctggt tccgcatggt 420
agcaaactga aagttgtgat tacctctaaa tcaggcgaaa ttctgtatcg cattagcccg 480
tgggccaaat atgttgtgcg cgaaggcgat aatgttaatt atgattggat tcattgggac 540
ccggaacata gctatgaatt taaacatagt cgtccgaaaa aaccgcgctc actgcgcatt 600
tatgaaagtc atgtgggcat tagtagccat gaaggcaaag ttgcctctta taaacatttt 660
acctgtaatg tgctgccgcg cattaaaggt ttaggctata attgtattca gttaatggcc 720
attatggaac atgcatatta tgcgtcattt ggctatcaga ttaccagctt ttttgcagcc 780
tctagtcgct atggcacccc ggaagaactc caggaactgg tggataccgc ccattcaatg 840
ggcattattg tgctgctgga tgttgttcat agtcatgcaa gcaaaaatag tgcagatggt 900
ctgaatatgt ttgatggtac cgatagttgt tattttcata gtggtccgcg cggtacccat 960
gatctgtggg atagtcgcct gtttgcctat tcaagttggg aagtgctgcg ctttctgctg 1020
tctaatattc gttggtggtt agaagaatat cgctttgatg gctttcgctt tgatggtgtg 1080
acctcaatgc tgtatcatca tcatggcgtg ggccagggct ttagcggcga ttatagcgaa 1140
tattttggct tacaggtgga tgaagatgca ctgacctatc tgatgttagc caatcatctg 1200
gttcataccc tgtgtccgga tagtattacc attgcagaag atgtgagcgg tatgccggcc 1260
ctgtgttctc cgatttcaca gggcggtggc ggctttgatt atcggttagc gatggccatt 1320
ccggataaat ggattcagct gctgaaagaa tttaaagatg aagattggaa tatgggtgat 1380
attgtgtata ccttaaccaa tcgtcgctat ctggaaaaat gtattgccta tgccgaatca 1440
catgatcagg ccttagtggg tgataaatca ctggcctttt ggttaatgga tgccgaaatg 1500
tataccaata tgtcagtgct gaccccgttt accccggtga ttgatcgcgg cattcagtta 1560
cataaaatga ttcgtttaat tacccacggc ttaggcggcg aaggctatct gaattttatg 1620
ggcaatgaat ttggccatcc ggaatggtta gattttccgc gcaaaggtaa taatgaaagc 1680
tatcattatg cacgtcgtca gtttcatctg accgatgatg atctgttacg ctataaattt 1740
ctgaataatt ttgatcgtga tatgaatcgt ctggaagaac gctatggttg gttagccgca 1800
ccgcaggcct atgtgagcga aaaacatgaa ggtaataaaa ttattgcctt tgaacgcgca 1860
ggtctgctgt ttatttttaa ttttcatccg agcaaatctt ataccgatta tcgcgtgggt 1920
accgccctgc cgggtaaatt taaaattgtg ttagatagcg atgcagcaga atatggcggt 1980
catcagcgct tagatcatag taccgatttt tttagcgaag cctttgaaca taatggtcgc 2040
ccgtatagct tactggtgta tattccgtct cgcgttgcct taattttaca gaatgtggat 2100
ctgccgaata agctt 2115
<210> 8
<211> 2238
<212> DNA
<213> 人工序列
<400> 8
catatgcgca ttgtgtttac caccttagcc gtgtggcgcg gcaaaaaagg tctgaaaaaa 60
ctggttaaat ctaaagttaa tcagatgttt gaaaaactgt cacaggcagc ctgtagcgaa 120
ccgtttgcct ttctgggtcc gtttattgat ccgacccagg gcgccctgcg tgtgtggatg 180
ccgggtgcca ccggcgttgc actggtgctg gaaggccagc cgcgcattgc cctggaacgt 240
gaaaaagaaa gcgcctttat tctgaaagcc gatctgaatc tgcatctgac ccattatcag 300
ctggccattg attggaatgg cgtggaacag ttaattgatg atccgtatca gtatcatggc 360
atttatgcag aatatgatga tttacatacc ccgaaaacca tgtatcagca catgggtagt 420
cagtttatga ccttagaacg cgatggcaaa tctattagcg gcattcgctt tctggtgtat 480
gcaccccatg caaccgccgt gtcactggtg ggttgtttta atgattggga tggtcgtcgt 540
catccgatgc agcgcttaga ttatggtatt tggggcctgt ttattccggg cttaaccgaa 600
ggtgtgagct ataaatttga aatgaaaggc ccgaaaggcg aaggcttacc gcataaagca 660
gatccgtggg gcttttatgc agaacagtat ccgagctttg cctcagtgac ctatgatcat 720
gcacgctatc agtggcagga tgcccagtgg cagacccgtc cggtgaccga aaaacgcaaa 780
gaagcactga gcttttatga attacatgca ggctcttgga aacgtaatga acagggtgaa 840
tttctgaatt atcgtgaact ggcagcagaa ctggttccgt atctggtgga tatgggctat 900
acccatgtgg aattaatgcc ggtgagcgaa catccgtttt atggctcttg gggctatcag 960
ccggtgggtc tgtttgcccc gacctctcgc tatggtagtc cggatgattt taaatttttt 1020
gttgatgcct gtcatcaggc cggcattggc gttgtgttag attgggttcc ggcccatttt 1080
ccgagcgatg atcatgggct ggctaatttt gatggtaccc cgctgtttca tgatcccgac 1140
ccgcgtcgcg gttggcatca ggattggaat agctttattt atgatttagg tcgcgaacag 1200
gttcgtcgct ttctggtgtc taatgcactg tattggtttg aacagtttca tattgatggt 1260
attcgcgtgg atgccgttgc aagcatgtta tatttagatt atagtcgctc acatggccag 1320
tggattccga atatggatgg cggtaatgaa aattatgatg ccattgccac cttaaaatgg 1380
atgaatgaag aagtgtataa atattttccg aatgcaatga ccattgcaga agaaagtacc 1440
gcctttccgg gcgtgagtgc cccgaccttt atgggcggct taggttttgg ctttaaatgg 1500
aatatgggtt ggatgcatga tagtctgagc tatattaaag aagaaccggt tcatcgcaaa 1560
tatcatcata ataccctgac ctttccgtta gtgtatgcac atagcgaaaa ttatgtgctg 1620
tcactgtcac atgatgaagt tgtgtatggt aaaggttcaa ttcataataa aatgccgggc 1680
gatgaatggc agcagaccgc caatctgcgc gcctattttg gctatatgta tggccagccg 1740
ggcaaaaaac tgaattttat gggtgcagaa attggccaga ccgcagaatg gaatcatgat 1800
gatcagttac agtggtttct gttagatttt ccgcgtcatc agggcgttca ggccttaacc 1860
cgcgatctga atcatctgta tcgtaatgaa gcagccttac atgatcagga ttgtattccc 1920
gcgggctttg aatggcgctt acaggatgca gcagaacaga gtattattgc acatgaacgc 1980
attagcgaag caggcgaacg cattctggtt gtgtctaatt ttaccccggt gccgcgcgat 2040
gaatttcgtc tgggcgttcc gaataaaggt cgctatcagc tgctgttaaa taccgatgat 2100
agtaaatatg ccggctcagg ctatgaagtt gttgtggatg ccaaaagtga agccgttgtg 2160
agcgaagatt tagcccagtc aattgtgctg cgcctgccgc cgctgagtac cctgttttat 2220
aaactggtgt aaggatcc 2238
<210> 9
<211> 2331
<212> DNA
<213> 人工序列
<400> 9
catatgacca ccaccattag cgcagatcag gttaatcaga ttatttataa tctgcatcat 60
gatccgtttg aaattctggg ttgtcatctg ctggaagaag gtaaaaatac caaaaaatgg 120
gttgtgcgcg catatctgcc gaaagcagaa gcagcatggg tgattcgccc gaccgaacgc 180
aaagaagatc cgatgaatag cgttcatcat ccgaattttt ttgaatgtat tattgaaacc 240
ccggaactga atcattatca gctgaaagtt aaagaaggcg aacatgaaaa agtgatttat 300
gatccgtatg catttagtag cccgtatctg accgatgaag atatttatct gtttagcgaa 360
ggcaatcatc atcgtattta tgaaaaactg ggtgcacatg tgggcgaaat taatggcgtt 420
aaaggcgtgt attttgcagt gtgggcaccg aatgcacgca atgttagcgt gattggtgat 480
tttaataatt gggatggtcg cgaacatcag atgcgcaaac gcaattatac catttgggaa 540
ctgtttgtgc cggaaattgg ttcagggact gtgtataaat atgaaattaa aaattcagaa 600
ggccatattt atgaaaaaag cgatccgtat ggcttttatc gcgaagttcg cccgaatacc 660
gcaagcattg ttgtggatat tgataatatt tatcagtggc atgatgaaga atggctggaa 720
aaacgtcgta atagcgatcc gctgaaacag ccggtgagcg tgtatgaagt tcatctgggc 780
tcttggctgc atggtagctc agcagaaaaa atgccgctgc tgaatggcga agcagatccg 840
gtgattgtta gcgaatggaa tccgggtgca cgctttctgt cttattatga actggcagaa 900
aaactgattc cgtatgttaa agatatgggc tatacccata ttgaactgct gccgattgca 960
gaacatccgt ttgatggtag ttggggctat caggtgaccg gcttttatag tccgacctct 1020
cgctttggtc gtccggaaga ttttatgtat tttgtggata aatgtcatga aaatggcatt 1080
ggcgtgattc tggattgggt tccgggccat tttccgaaag attcacatgg cctggcatat 1140
tttgatggta cgcatctgta tgaacatgca gatccgcgca ttggcgaaca taaagaatgg 1200
ggaactctgg tgtttaatta tggtcgtcat gaagtgcgca attttctggt tgcaaatgtt 1260
ctgttttggt ttgataaata tcatgtggat ggcattcgcg tggatgcagt tgcatcgatg 1320
ctgtatcgta attatctgcg taaagaaggc gaatggattg caaatgaata tggcggcgat 1380
gaacatattg aagcagtgag ctttattcgc gaagttaata ccctgctgtt tgaatatttt 1440
ccgggcattc tgtcaattgc agaagaatct accgaatggg aaaaagtgtc acgcccggtg 1500
tatgatggcg gcctgggctt taatctgaaa tgggatatgg gttggatgca tgatatgctg 1560
gattatttta atattgatcc gtattttcgc cagtatcatc agaataatgt gaccttttct 1620
atgctgtatt attataatga aaattttatg ctggcactga gtcatgatga aattgttcat 1680
ggtaaatcta atatgctggg taaaatgccg ggcgatgaat ggcagaaata tgcaaatgtg 1740
cgcgcactgt ttacctatat gtatacccat ccgggtaaaa aaaccatgtt tatgtctatg 1800
gaatttggcc agtggagcga atggaatgtg tggggcgatc tggaatggca tctgcttcag 1860
tatgaaccgc atcagcagct gaaacagttt tttaccgatc tgaatgcact gtatcagcag 1920
gaaccggcac tgtataccca tgattttgaa tatcatggct ttgaatggat tgattgtaat 1980
gataataccc atagcgttgt gagctttctg cgtcgctcag atgatccgaa tgatagcctg 2040
gttgttgtgt gtaattttac cccgcagccg catagtcatt atcgcattgg tgtgccggaa 2100
gcaggctatt atgtggaact gtttaatagc gatgcaaaac agtatggcgg ctctaatatg 2160
ggcaatctgg gcggtaaatg ggcagatgaa tggagctttc ataataaacc gtatagcctg 2220
gatctgtgtc tgccgccgct ggcagtgctg attctgaaac tggacccgac caaagtgccg 2280
gaaggaacga ccattaaaga aattgcagca gatgaagaag aataaaagct t 2331
<210> 10
<211> 1875
<212> DNA
<213> 人工序列
<400> 10
catatgtctt ggttaaccga agaagatatt cgtcgttggg aaagcggtac gttttatgat 60
agctatcgca aactgggtgc acatccggat gatgaaggga catggttttg tgtgtgggcc 120
ccacatgccg atggcgtgtc agtgttaggt gcctttaatg attggaatcc ggaagccaat 180
ccgttagaac gctatggcgg cggtctgtgg gcaggctatg ttccgggtgc acgtccgggt 240
catacctata aatatcgtat tcgacacggc ttttatcagg cagataaaac cgatccgtat 300
gcctttgcaa tggaaccgcc gaccggctct ccgattgaag gcttagcctc aattattacc 360
cgcttagatt atacctggca tgatgatgaa tggatgcgtc gtcgtaaagg cccggcctct 420
ctgtatgagc ctgtgtcaat ttatgaagtt catttaggct cttggcgcca taaacgtcca 480
ggtgaaagtt tttcttatcg cgaaattgca gaaccgttag cagattatgt tcaggaaatg 540
ggctttaccc atgttgaact gctgccggtg atggaacatc cgtattatgg tagctggggc 600
tatcaggttg tgggctatta tgccccgacc tttcgctatg gtagtccgca ggatctgatg 660
tatttaattg attatttaca tcagcgcggc attggcgtga ttctggattg ggtgccgtca 720
cattttgcag cagatccgca gggcttagtg ttttttgatg gcactaccct gtttgaatat 780
gatgatccga aaatgcgcta tcatccggat tggggcacct atgtgtttga ttataataaa 840
ccgggcgttc gtaattttct gattagtaat gcactgtttt ggttagaaaa atatcatgtg 900
gatggtctgc gcgtggatgc agttgcctca atgctgtatc gcgattatag tcgcaaagaa 960
tggaccccga atatttttgg cggtcgcgaa aatctggaag ccattgattt tattaaaaaa 1020
tttaatgaaa ccgtgtattt acattttccg gaagccatga ccattgcaga agaaagtacc 1080
gcctggccgg gcgtgagtgc accgacctat aataatggct taggctttct gtataaatgg 1140
aatatgggtt ggatgcatga taccttagat tatattcagc gcgatccgat ttatcgcaaa 1200
tatcatcatg atgaactgac cttttcactg tggtatgcct ttagcgaaca ttatgtgctg 1260
ccgctgagtc atgatgaagt tgttcatggc aaaggctcac tgtggggtaa aatgccgggc 1320
gatgattggc agaaagcagc caatctgcgc ttactgtttg gtcacatgtg gggccatccg 1380
ggtaaaaaac tgctgtttat gggcggcgaa tttggccagc atcatgaatg gaatcatgat 1440
acccagttag aatggcatct gctggatcag ccgtatcatc gcggcattca gctgtgggtt 1500
tgtgatctga atcatctgta tcgcaccaat ccggccctgt ggcatgatgg cccggaaggc 1560
tttgaatgga ttgatttttc agatcgcgat cagagcgtta tttgttatct gcgcaaaaat 1620
gcaggtcgta tgttactgtt tgtgctgaat tttaccccgg ttccgcgcga acattatcgc 1680
gttggtgttc cgattggcgg cccgtggcat gaagtgctga atagcgatgc agttgcctat 1740
ggcggtagcg gcatgggtaa ttttggtcgt gttgaagcag tgccggaaag ctggcatggt 1800
cgtccgtttc atctggaact gaccctgccg ccgttagcag ccttaattct ggaaccggaa 1860
catggttaaa agctt 1875
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