CN109553689A - 一种含有ApcE2突变体的融合蛋白及其应用 - Google Patents

一种含有ApcE2突变体的融合蛋白及其应用 Download PDF

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CN109553689A
CN109553689A CN201811399622.0A CN201811399622A CN109553689A CN 109553689 A CN109553689 A CN 109553689A CN 201811399622 A CN201811399622 A CN 201811399622A CN 109553689 A CN109553689 A CN 109553689A
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周明
夏坤
付卫雷
吴明
陈彦蓉
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Huazhong Agricultural University
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Abstract

本发明公开了一种融合蛋白，其特征在于：所述融合蛋白的氨基酸序列为：F1多肽—连接保护肽1—F2多肽—连接保护肽2—F1多肽，其中F1多肽为序列为SEQ ID No.1和SEQ ID No.2中的任意一种，F2多肽序列为SEQ ID No.3～SEQ ID No.14中的任意一种。本发明中通过将ApcE2突变体与经过基因工程改造的别藻蓝蛋白亚基BDFP1.1或BDFP1.2进行融合得到一种融合蛋白，提高了ApcE2突变体在哺乳动物细胞中标记的荧光较弱的问题。并且发现在荧光标记过程中体外添加光敏色素PΦB，可有效提高融合蛋白进行细胞标记时的亮度。

Description

一种含有ApcE2突变体的融合蛋白及其应用
技术领域
本发明涉及生物技术领域，特别涉及一种含有ApcE2突变体的融合蛋白及其应用。
背景技术
现代生物学的发展越来越依赖光学技术，例如荧光成像、光学检测及光诱导操纵等等。在活体中对深层组织进行成像定位，最好选用650nm-900nm波段的荧光。因为这个波段的光自发荧光较小、光散射小且对组织损伤较小。近年来，作为荧光探针研究较多的两类蛋白为绿色荧光蛋白家族和细菌光敏色素蛋白(BphP)。
少数蓝细菌为了更适应环境、从环境中捕获更多的能量，慢慢进化出能利用远红光的藻胆体。这些藻胆体中通常存在不同于传统的核膜连接蛋白和一到三个别藻蓝蛋白亚基，其荧光发光机制类似于细菌光敏色素蛋白(BphP)，发色团为非共价结合的藻蓝胆素PCB。
另有研究，对来自蓝细菌Chroococcidiopsisthermalissp.PCC7203的别藻蓝蛋白亚基ApcF2进行基因工程改造，得到一系列远红光和近红外光的荧光蛋白，其被命名为BDFP荧光蛋白。其中的BDFP1.1，共价结合胆绿素BV后，最大吸收波长为682nm，荧光波长707nm；BDFP1.2，最大吸收波长为642nm，荧光波长668nm。BDFP系列荧光蛋白具有较小的分子量，能与色素共价结合，性能稳定，耐受极端环境；可用于对人类细胞系、线虫细胞等活体细胞进行标记，也适用于标记乳酸杆菌、质体、线粒体等。
最新的研究发现，ApcE2(1-273)，来自蓝细菌Synechococcussp.PCC7335，由S7335_3294编码，其序列中保守的半胱氨酸被缬氨酸替代，因此与色素是以非共价方式连接，其光谱均比共价连接色素的ApcE1红移近40nm。它与藻红胆素PEB重组，最大吸收峰在614nm，最大荧光峰在628nm；与藻蓝胆素PCB重组，最大吸收峰在700nm，最大荧光峰在714nm；还可以连接光敏色素PΦB，吸收进一步红移至714nm，荧光在726nm，为目前荧光标记所能达到的最长荧光发射波长。
由于ApcE2是膜蛋白，需要在含高浓度变性剂(3.5mol/L尿素)的条件下溶解；在室温下不够稳定，故而会影响标记。已有文献(缪丹.红移藻胆蛋白和光敏色素的研究及分子进化[D].2017.)报道：对蛋白N端、C端的截短及loop环(77-161)的部分缺失改造，并没有提高蛋白的可溶性。
申请人在研究中以ApcE2(24-245)为模板进行基因改造后，得到的BDFP3等系列蛋白，解决了可溶性问题，结合光敏色素PΦB后，可以对大肠杆菌细胞进行荧光标记，但是在哺乳动物细胞中标记的荧光较弱。
发明内容
本发明的目的在于提供一种含有ApcE2突变体的融合蛋白及其应用，解决ApcE2突变体在哺乳动物细胞中标记的荧光较弱的问题。
本发明所采取的技术方案是：
一种融合蛋白，其氨基酸序列为：F1多肽—连接保护肽1—F2多肽—连接保护肽2—F1多肽，其中F1多肽为序列为SEQ ID No.1和SEQ ID No.2中的任意一种，F2多肽序列为SEQ ID No.3～SEQ ID No.14中的任意一种。
进一步地，F2多肽序列为SEQ ID No.8～SEQ ID No.13中的任意一种。
进一步地，连接保护肽1的长度为10～20个氨基酸。
进一步地，连接保护肽1的序列为：GHGTGSTGSGS(SEQ ID No.15)。
进一步地，连接保护肽2的长度为5～10个氨基酸。
进一步地，连接保护肽2的序列为：GHGTGST(SEQ ID No.16)。
编码上述的融合蛋白的序列。
上述的融合蛋白的制备方法：将上述融合蛋白的编码序列导入微生物进行表达，之后纯化得到。
上述的融合蛋白在对细胞进行荧光标记中的应用，优选地，细胞为哺乳动物细胞。
进一步地，在荧光标记中还包括体外添加光敏色素PΦB。
本发明的有益效果是：本发明中通过将ApcE2突变体与经过基因工程改造的别藻蓝蛋白亚基BDFP1.1或BDFP1.2进行融合得到一种融合蛋白，提高了ApcE2突变体在哺乳动物细胞中标记的荧光较弱的问题。并且发现在荧光标记过程中体外添加光敏色素PΦB，可有效提高融合蛋白进行细胞标记时的亮度。
附图说明
图1为BV-BDFP1.1/1.2的荧光发射光谱与PΦB-BDFP3的吸收光谱重叠图；
图2为色素蛋白在大肠杆菌中的荧光显微镜成像；
图3为BDFP3与融合蛋白有效亮度对比图；
图4为细胞中重组表达生成色素蛋白后的荧光显微镜成像。
具体实施方式
本发明中通过将ApcE2突变体(SEQ ID No.3～SEQ ID No.14所示的任意一种)通过连接保护肽(SEQ ID No.15、SEQ ID No.16)与经过基因工程改造的别藻蓝蛋白亚基BDFP1.1(SEQ ID No.1)或BDFP1.2(SEQ ID No.2)进行融合得到一种融合蛋白，以提高ApcE2突变体在哺乳动物细胞中标记的荧光较弱的问题。
本发明中使用的实验方法如下：
1.克隆
荧光蛋白基因编码序列克隆至载体pET28或者pcDNA3.1(Novagen)，得到pET-fp或者pcDNA3.1-fp:ires:egfp。序列可根据需要，直接进行基因合成。fp序列克隆进入pET28载体时，可采用酶切位点BamHI和HindIII。
为了在大肠杆菌体内产生结合光敏色素PΦB的荧光蛋白，质粒pET-fp通过转化操作进入含有质粒pACYC-ho1-hy2的大肠杆菌BL21细胞中。质粒pACYC-ho1-hy2用于表达生成光敏色素，ho1为血红素氧化还原酶基因，hy2为光敏色素合成酶基因。
2.大肠杆菌体内表达及蛋白纯化
将经转化的BL21细胞在18℃，培养在补充有卡那霉素(20μg/ml)和氯霉素(17μg/ml)LB培养基中。当O.D值达到0.4～0.6时，使用1mM异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达5～16小时，然后在4℃，12,000×g离心3min，收集细胞，经水冲洗2次，短期保存在4℃或者置于-20℃长期保存。
在上述诱导表达的细胞中加入5mL预冷的上样缓冲液充分重悬，利用超声的方式进行破碎，细胞的重悬液必须放置在冰水混合物中。超声功率设置为300W，时长1s，间隔2s，共240次。超声结束后，放置4℃，12000r/min，离心40min后，即可取上清过镍柱提纯。
镍离子金属螯合亲和层析具体步骤如下：首先向Ni柱中加入2倍柱体积的200mM氯化镍，待溶液流尽以后加单蒸水直至留下来的溶液不再含氯化镍(绿色)为止，然后用5倍柱体积的上样缓冲液平衡Ni柱，之后就可以上样。上样后依次用5倍柱体积的上样缓冲液和10倍柱体积的去杂蛋白液清洗柱子，最后用1mL的目的蛋白洗脱液将目的蛋白洗脱下来。Ni柱使用完毕后需要用Elution Buffer洗脱掉Ni离子，最后用5倍柱体积单蒸水处理。层析柱料于4℃用20％乙醇封存。洗脱下来的目的蛋白需要在4℃，不含咪唑的上样缓冲液中过夜透析。
3.哺乳动物细胞体内表达
将HEK 293T HeLa或U-2OS细胞培养在含10％胎牛血清的DMEM培养基(Invitrogen)中，将CHO-K1细胞培养在含10％胎牛血清的F-12K培养基中。使用2000(Invitrogen)进行转染。转染时，2000与DNA以3:1(μl:μg)的比率在无血清培养基Opti-中混合10分钟后，立即加入到要转染的细胞中。5～6h后，更换新鲜的DMEM培养基。
4.广域和超分辨率显微镜成像
成像要求使用individual模式，和配有100×1.49NA油浸物镜的ECLIPSE Ti-E倒置尼康显微镜上的尼康结构化照明系统，来获取广域和结构化照明显微镜(SIM)照片。使用640nm的半导体激光器(100mW,CUBE 640-100C,COHERENT)激发FR荧光。使用受NIS-Elements AR软件(尼康)控制的电子倍增CCD相机(Andor iXon3DU897)进行数据采集。使用NIS-Elements AR对图像进行处理。
5.蛋白定量和结构模拟分析
蛋白浓度检测使用Bradford法，参照物为牛血清白蛋白。
BDFP荧光蛋白结构比对，在SWISS-MODEL远程服务器上完成。采用的模板序列为钝顶螺旋藻(Spirulina platensis)的藻胆蛋白ApcB(pdb code:1ALL)，对比软件为Swiss-PDBViewer，版本4.1。采用PyMOL(http://www.pymol.org/)创建蛋白结构图。
6.光谱分析
通过分光光度计(DU800，Beckman–Coulter)来检测色素蛋白的紫外-可见吸收光谱。共价结合的胆色素BV，在390nm处的吸收系数ε＝39,900M-1cm-1；非共价结合的光敏色素PΦB(溶于尿素溶液，pH2)，在702nm处的吸收系数ε＝38,000M-1cm-1。
通过荧光分光光度计(F320，天津港东科技发展股份有限公司)来检测荧光光谱。
下面将结合实施例进一步阐述本发明，应理解，以下实施例仅用于说明本发明而不用于限制本发明的保护范围。
实施例1
本实施例中使用的ApcE2突变体为BDFP3及BDFP3(Δ88-106)，氨基酸序列如SEQID No.9、SEQ ID No.11所示。图1为BV-BDFP1.1/1.2的荧光发射光谱与PΦB-BDFP3的吸收光谱重叠图，可见用于融合的BDFP1.1、BDFP1.2，其吸收荧光光谱交叉重叠，有利于荧光能量共振转移，能有效地将能量传递到BDFP3系列蛋白。
一种融合蛋白BDFP1.1:3:1.1，其氨基酸序列如SEQ ID No.17所示。
一种融合蛋白BDFP1.2:3:1.2，其氨基酸序列如SEQ ID No.18所示。
一种融合蛋白BDFP1.2:3(Δ88-106):1.2，其氨基酸序列如SEQ ID No.19所示。
将编码上述融合蛋白的基因序列克隆进入大肠杆菌，与光敏色素PΦB进行体内表达重组，生成色素蛋白。图2为色素蛋白在大肠杆菌中的荧光显微镜成像，a为色素蛋白BDFP1.1:3-PΦB:1.1，b为色素蛋白BDFP1.2:3-PΦB:1.2。
提取纯化后得到融合蛋白，进行光谱分析；然后继续体外添加光敏色素PΦB，再次进行光谱分析，结果如下表所示：
荧光发射光谱的激发波长在620nm。色素蛋白的分子亮度，是通过参比IFP2.0(ε·Φfl＝7.5 7.5mM-1cm-1)得到。
a样品通过纯化后，在20mM KPB，0.5M NaCl，pH5.6缓冲环境下进行光谱分析。
b样品通过纯化后，加体外添加光敏色素PΦB。
图3为BDFP3与融合蛋白有效亮度对比图，
A：BDFP1.1/1.2:3:1.1/1.2:IRES:eGFP不添加光敏色素PΦB；
B：BDFP1.1/1.2:3:1.1/1.2:IRES:eGFP添加光敏色素PΦB(5μM)；
C：BDFP1.1/1.2:IRES:eGFP添加光敏色素PΦB；
D：BDFP3:IRES:eGFP添加光敏色素PΦB；
E：iRFP720:IRES:eGFP。
将编码上述融合蛋白的基因序列克隆进入HEK 293T HeLa细胞，重组表达生成色素蛋白；体外添加光敏色素PΦB，荧光显微镜成像如图4所示，(c)BDFP1.1:3:1.1在HEK293T细胞中表达后的荧光成像图；(d)BDFP1.2:3:1.2在HEK293T细胞中表达后的荧光成像图；(e)BDFP1.1:3:1.1在HEK293T细胞中表达并添加光敏色素PΦB后的荧光成像图；(f)BDFP1.2:3:1.2在HEK293T细胞中表达并添加光敏色素PΦB后的荧光成像图。
荧光成像图下角的数字表示亮度，用平均eGFP荧光强度修正表达水平后，再参比IFP2.0得到。BDFP1.1:3:1.1,BDFP1.1:3-PΦB:1.1和iRFP720成像参数为λex＝650/45，λem＝720/40nm；BDFP1.2:3:1.2和BDFP1.2:3-PΦB:1.2成像参数为λex＝630/20，λem＝720/40nm。比例尺50μm。
SEQUENCE LISTING
<110> 华中农业大学
广州天宝颂原生物科技开发有限公司
<120> 一种含有ApcE2突变体的融合蛋白及其应用
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<170> PatentIn version 3.5
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<213> 人工合成
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Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp Gly Glu
1 5 10 15
Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala Glu Val
20 25 30
Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu Leu Val
35 40 45
Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met Cys Val
50 55 60
Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile Val Ala
65 70 75 80
Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Leu Arg Asp
85 90 95
Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg Ser Ile
100 105 110
Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala Gly Met
115 120 125
Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala Arg Glu
130 135 140
Ile Ser Glu Thr Glu Ile
145 150
<210> 2
<211> 150
<212> PRT
<213> 人工合成
<400> 2
Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp Gly Glu
1 5 10 15
Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala Glu Val
20 25 30
Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu Leu Val
35 40 45
Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met Cys Val
50 55 60
Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile Val Ala
65 70 75 80
Gly Gly Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe Arg Asp
85 90 95
Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg Ser Ile
100 105 110
Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala Gly Met
115 120 125
Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala Arg Glu
130 135 140
Ile Ser Glu Thr Glu Ile
145 150
<210> 3
<211> 222
<212> PRT
<213> 人工合成
<400> 3
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Leu Arg Thr Gly Leu Gln Arg Leu Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Pro Glu Glu Leu Val Gly Met Pro Gly Ser Gly Tyr Phe Val Ala Glu
65 70 75 80
Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser
85 90 95
Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Trp Leu Lys
100 105 110
Gly Leu Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala
115 120 125
Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg
130 135 140
Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly
145 150 155 160
Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile
165 170 175
Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln
180 185 190
Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu
195 200 205
Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
210 215 220
<210> 4
<211> 222
<212> PRT
<213> 人工合成
<400> 4
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Leu Arg Thr Gly Asn Gln Arg Leu Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Pro Glu Glu Leu Val Gly Met Pro Gly Ser Gly Tyr Phe Val Ala Glu
65 70 75 80
Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser
85 90 95
Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Trp Leu Lys
100 105 110
Gly Leu Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala
115 120 125
Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg
130 135 140
Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly
145 150 155 160
Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile
165 170 175
Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln
180 185 190
Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu
195 200 205
Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
210 215 220
<210> 5
<211> 222
<212> PRT
<213> 人工合成
<400> 5
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Leu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Pro Glu Glu Leu Val Gly Met Pro Gly Ser Gly Tyr Phe Val Ala Glu
65 70 75 80
Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser
85 90 95
Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Trp Leu Lys
100 105 110
Gly Leu Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala
115 120 125
Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg
130 135 140
Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly
145 150 155 160
Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile
165 170 175
Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln
180 185 190
Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu
195 200 205
Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
210 215 220
<210> 6
<211> 222
<212> PRT
<213> 人工合成
<400> 6
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Pro Glu Glu Leu Val Gly Met Pro Gly Ser Gly Tyr Phe Val Ala Glu
65 70 75 80
Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser
85 90 95
Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Trp Leu Lys
100 105 110
Gly Leu Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala
115 120 125
Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg
130 135 140
Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly
145 150 155 160
Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile
165 170 175
Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln
180 185 190
Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu
195 200 205
Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
210 215 220
<210> 7
<211> 222
<212> PRT
<213> 人工合成
<400> 7
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Pro Glu Glu Thr Ser Gly Met Pro Gly Ser Gly Tyr Phe Val Ala Glu
65 70 75 80
Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser
85 90 95
Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Trp Leu Lys
100 105 110
Gly Leu Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala
115 120 125
Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Arg Ser Met Arg
130 135 140
Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly
145 150 155 160
Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile
165 170 175
Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln
180 185 190
Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu
195 200 205
Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
210 215 220
<210> 8
<211> 222
<212> PRT
<213> 人工合成
<400> 8
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Pro Glu Glu Leu Val Gly Met Pro Gly Ser Gly Tyr Phe Val Ala Glu
65 70 75 80
Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser
85 90 95
Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Trp Glu Lys
100 105 110
Gly Asp Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala
115 120 125
Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg
130 135 140
Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly
145 150 155 160
Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile
165 170 175
Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln
180 185 190
Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu
195 200 205
Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
210 215 220
<210> 9
<211> 222
<212> PRT
<213> 人工合成
<400> 9
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Pro Glu Glu Leu Val Gly Met Pro Gly Ser Gly Tyr Phe Val Ala Glu
65 70 75 80
Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser
85 90 95
Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Tyr Glu Lys
100 105 110
Gly Asp Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala
115 120 125
Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg
130 135 140
Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly
145 150 155 160
Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile
165 170 175
Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln
180 185 190
Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu
195 200 205
Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
210 215 220
<210> 10
<211> 222
<212> PRT
<213> 人工合成
<400> 10
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Pro Glu Glu Leu Val Gly Met Pro Gly Ser Gly Tyr Phe Val Ala Glu
65 70 75 80
Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser
85 90 95
Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Trp Glu Arg
100 105 110
Gly Asp Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala
115 120 125
Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg
130 135 140
Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly
145 150 155 160
Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile
165 170 175
Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln
180 185 190
Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu
195 200 205
Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
210 215 220
<210> 11
<211> 203
<212> PRT
<213> 人工合成
<400> 11
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser Val Gln Asn
65 70 75 80
Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Tyr Glu Lys Gly Asp Phe
85 90 95
Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala Ile Asn Ile
100 105 110
Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg Asp Leu Gly
115 120 125
Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly Asp Thr Ser
130 135 140
Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile Pro Glu Asp
145 150 155 160
Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln Trp Lys Ser
165 170 175
Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu Val Arg Arg
180 185 190
Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
195 200
<210> 12
<211> 195
<212> PRT
<213> 人工合成
<400> 12
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Gly Asn Gly His Ser Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro
65 70 75 80
Val Ala Tyr Glu Lys Gly Asp Phe Phe Ser Gly Lys Pro Ser Val Pro
85 90 95
Ser Arg Phe Gln Ala Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met
100 105 110
Lys Lys Ala Met Arg Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr
115 120 125
Ala Val Val Ala Gly Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly
130 135 140
Leu Arg Gly Ile Ile Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala
145 150 155 160
Leu Gln Glu Met Gln Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser
165 170 175
Ala Ala Ala Ala Leu Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp
180 185 190
Tyr Gln Val
195
<210> 13
<211> 189
<212> PRT
<213> 人工合成
<400> 13
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Tyr Glu Lys Gly
65 70 75 80
Asp Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala Ile
85 90 95
Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg Asp
100 105 110
Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly Asp
115 120 125
Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile Pro
130 135 140
Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln Trp
145 150 155 160
Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu Val
165 170 175
Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val
180 185
<210> 14
<211> 179
<212> PRT
<213> 人工合成
<400> 14
Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser Glu
1 5 10 15
Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr Glu
20 25 30
Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala Gly
35 40 45
Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu Gln
50 55 60
Val Ala Tyr Glu Lys Gly Asp Phe Phe Ser Gly Lys Pro Ser Val Pro
65 70 75 80
Ser Arg Phe Gln Ala Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met
85 90 95
Lys Lys Ala Met Arg Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr
100 105 110
Ala Val Val Ala Gly Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly
115 120 125
Leu Arg Gly Ile Ile Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala
130 135 140
Leu Gln Glu Met Gln Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser
145 150 155 160
Ala Ala Ala Ala Leu Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp
165 170 175
Tyr Gln Val
<210> 15
<211> 11
<212> PRT
<213> 人工合成
<400> 15
Gly His Gly Thr Gly Ser Thr Gly Ser Gly Ser
1 5 10
<210> 16
<211> 7
<212> PRT
<213> 人工合成
<400> 16
Gly His Gly Thr Gly Ser Thr
1 5
<210> 17
<211> 540
<212> PRT
<213> 人工合成
<400> 17
Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp Gly Glu
1 5 10 15
Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala Glu Val
20 25 30
Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu Leu Val
35 40 45
Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met Cys Val
50 55 60
Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile Val Ala
65 70 75 80
Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Leu Arg Asp
85 90 95
Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg Ser Ile
100 105 110
Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala Gly Met
115 120 125
Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala Arg Glu
130 135 140
Ile Ser Glu Thr Glu Ile Gly His Gly Thr Gly Ser Thr Gly Ser Gly
145 150 155 160
Ser Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser
165 170 175
Glu Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr
180 185 190
Glu Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala
195 200 205
Gly Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu
210 215 220
Gln Pro Glu Glu Leu Val Gly Met Pro Gly Ser Gly Tyr Phe Val Ala
225 230 235 240
Glu Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His
245 250 255
Ser Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Tyr Glu
260 265 270
Lys Gly Asp Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln
275 280 285
Ala Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met
290 295 300
Arg Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala
305 310 315 320
Gly Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile
325 330 335
Ile Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met
340 345 350
Gln Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala
355 360 365
Leu Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val Gly
370 375 380
His Gly Thr Gly Ser Thr Asn Arg Glu Val Val Glu Thr Leu Lys Glu
385 390 395 400
Phe Leu Ala Asp Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly
405 410 415
Thr Asn Ala Ala Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln
420 425 430
Glu Tyr Pro Glu Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg
435 440 445
Arg Tyr Asn Met Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys
450 455 460
Ser Tyr Ala Ile Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu
465 470 475 480
Leu Ala Gly Leu Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly
485 490 495
Pro Thr Ala Arg Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys
500 505 510
Leu Val Thr Ala Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe
515 520 525
Asp Tyr Ile Ala Arg Glu Ile Ser Glu Thr Glu Ile
530 535 540
<210> 18
<211> 540
<212> PRT
<213> 人工合成
<400> 18
Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp Gly Glu
1 5 10 15
Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala Glu Val
20 25 30
Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu Leu Val
35 40 45
Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met Cys Val
50 55 60
Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile Val Ala
65 70 75 80
Gly Gly Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe Arg Asp
85 90 95
Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg Ser Ile
100 105 110
Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala Gly Met
115 120 125
Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala Arg Glu
130 135 140
Ile Ser Glu Thr Glu Ile Gly His Gly Thr Gly Ser Thr Gly Ser Gly
145 150 155 160
Ser Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser
165 170 175
Glu Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr
180 185 190
Glu Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala
195 200 205
Gly Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu
210 215 220
Gln Pro Glu Glu Leu Val Gly Met Pro Gly Ser Gly Tyr Phe Val Ala
225 230 235 240
Glu Asp Tyr Leu Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His
245 250 255
Ser Val Gln Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Tyr Glu
260 265 270
Lys Gly Asp Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln
275 280 285
Ala Ile Asn Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met
290 295 300
Arg Asp Leu Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala
305 310 315 320
Gly Asp Thr Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile
325 330 335
Ile Pro Glu Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met
340 345 350
Gln Trp Lys Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala
355 360 365
Leu Val Arg Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val Gly
370 375 380
His Gly Thr Gly Ser Thr Asn Arg Glu Val Val Glu Thr Leu Lys Glu
385 390 395 400
Leu Leu Ala Asp Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly
405 410 415
Thr Asn Ala Ala Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln
420 425 430
Glu Tyr Pro Glu Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg
435 440 445
Arg Tyr Asn Met Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys
450 455 460
Ser Tyr Ala Ile Val Ala Gly Gly Ala Ser Val Leu Asp Glu Arg Leu
465 470 475 480
Leu Ala Gly Phe Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys
485 490 495
Pro Thr Ala Arg Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys
500 505 510
Leu Ala Thr Ala Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe
515 520 525
Asp Tyr Ile Ala Arg Glu Ile Ser Glu Thr Glu Ile
530 535 540
<210> 19
<211> 521
<212> PRT
<213> 人工合成
<400> 19
Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp Gly Glu
1 5 10 15
Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala Glu Val
20 25 30
Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu Leu Val
35 40 45
Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met Cys Val
50 55 60
Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile Val Ala
65 70 75 80
Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Leu Arg Asp
85 90 95
Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg Ser Ile
100 105 110
Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala Gly Met
115 120 125
Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala Arg Glu
130 135 140
Ile Ser Glu Thr Glu Ile Gly His Gly Thr Gly Ser Thr Gly Ser Gly
145 150 155 160
Ser Val Ile Asn Gly Ala His Gln Arg Asp Arg Tyr Pro Asn His Ser
165 170 175
Glu Met Gln Thr Leu Ser Thr Phe Glu Arg Thr Gly Asn Gln Arg Thr
180 185 190
Glu Ile Ala Gln Thr Leu Ala Gln His Ala Asn Glu Ile Val Ala Ala
195 200 205
Gly Gly Lys Arg Ile Phe Val Gly Gly Asn Pro Met Ala Tyr Phe Glu
210 215 220
Gln Ser Pro Lys Ser Arg Arg Gln Thr Gly Asn Gly His Ser Val Gln
225 230 235 240
Asn Ser Ser Ser Ser Ile Thr Asn Pro Val Ala Tyr Glu Lys Gly Asp
245 250 255
Phe Phe Ser Gly Lys Pro Ser Val Pro Ser Arg Phe Gln Ala Ile Asn
260 265 270
Ile Ala Asp Tyr Gly Ala Val Arg Met Lys Lys Ala Met Arg Asp Leu
275 280 285
Gly Trp Phe Leu Arg Tyr Ile Thr Tyr Ala Val Val Ala Gly Asp Thr
290 295 300
Ser Ile Ile Thr Val Asn Thr Arg Gly Leu Arg Gly Ile Ile Pro Glu
305 310 315 320
Asp Val Thr Val Ala Thr Thr Val Ala Leu Gln Glu Met Gln Trp Lys
325 330 335
Ser Leu Ser Phe Phe Pro Val Asp Ser Ala Ala Ala Ala Leu Val Arg
340 345 350
Arg Tyr Phe Asp Val Leu Ile Ala Asp Tyr Gln Val Gly His Gly Thr
355 360 365
Gly Ser Thr Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala
370 375 380
Asp Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala
385 390 395 400
Ala Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro
405 410 415
Glu Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn
420 425 430
Met Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala
435 440 445
Ile Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly
450 455 460
Leu Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala
465 470 475 480
Arg Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr
485 490 495
Ala Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile
500 505 510
Ala Arg Glu Ile Ser Glu Thr Glu Ile
515 520

Claims (10)

1.一种融合蛋白，其特征在于：所述融合蛋白的氨基酸序列为：F1多肽—连接保护肽1—F2多肽—连接保护肽2—F1多肽，其中F1多肽为序列为SEQ ID No.1和SEQ ID No.2中的任意一种，F2多肽序列为SEQ ID No.3～SEQ ID No.14中的任意一种。
2.根据权利要求1所述的融合蛋白，其特征在于：F2多肽序列为SEQ ID No.8～SEQ IDNo.13中的任意一种。
3.根据权利要求1所述的融合蛋白，其特征在于：连接保护肽1的长度为10～20个氨基酸。
4.根据权利要求3所述的融合蛋白，其特征在于：连接保护肽1的序列为：GHGTGSTGSGS(SEQ ID No.15)。
5.根据权利要求1所述的融合蛋白，其特征在于：连接保护肽2的长度为5～10个氨基酸。
6.根据权利要求5所述的融合蛋白，其特征在于：连接保护肽2的序列为：GHGTGST(SEQID No.16)。
7.编码权利要求1～6任一项所述的融合蛋白的序列。
8.权利要求1～6任一项所述的融合蛋白的制备方法：将权利要求1～6任一项所述的融合蛋白的编码序列导入微生物进行表达，之后纯化得到。
9.权利要求1～6任一项所述的融合蛋白在对细胞进行荧光标记中的应用，优选地，细胞为哺乳动物细胞。
10.根据权利要求9所述的应用，其特征在于：在荧光标记中还包括体外添加光敏色素PΦB。
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