CN107056950A - 一种包含钥孔血蓝蛋白片段的融合蛋白及其应用 - Google Patents

Abstract

本发明公开了一种包含钥孔血蓝蛋白片段的融合蛋白及其应用。所述融合蛋白包含钥孔血蓝蛋白片段及目的蛋白；所述的钥孔血蓝蛋白片段包含钥孔血蓝蛋白第1‑245位氨基酸残基；所述的目的蛋白为植物线粒体膜蛋白或其免疫原性片段。在将钥孔血蓝蛋白片段与目的蛋白融合表达后，钥孔血蓝蛋白片段显著增强了目的蛋白的免疫原性。由于本发明的融合蛋白展示了比单独的目的蛋白更强的免疫原性，因此，通过融合钥孔血蓝蛋白片段可用于制备针对特异性目的蛋白多克隆抗体。

Description

一种包含钥孔血蓝蛋白片段的融合蛋白及其应用

技术领域

本发明涉及生物技术领域，具体涉及一种包含钥孔血蓝蛋白片段的融合蛋白及其应用。

背景技术

抗体是高等动物特异性免疫应答反应所产生的免疫球蛋白，负责特异抗原的识别和清除。抗体不仅是机体抵抗病原体入侵的强大武器，也是基础科学研究中用于特异性分子识别的重要工具。随着基因组学研究深入，人们更加认识到基因组学研究的成果必须在蛋白水平得到进一步的研究和验证。

一直以来，基于亲和(affinity)相互作用的研究手段是研究蛋白质功能的主要手段之一，而基于抗体-抗原亲和相互作用研究手段又是其中最主要的应用方法，在科研与生产应用中有多种衍生技术应用，包括ELISA、免疫印记、免疫荧光、抗体芯片等。因此，制备高质量的抗体成为人们在科研、诊断以及治疗领域中一项核心技术。

在抗体制备过程中，一般都用人工合成的多肽或者细菌表达的重组蛋白质作为抗原。人工合成多肽首先需对该蛋白含有的表位信息进行准确预测，由于合成的片段较短，因此还需要选择合适的免疫原性载体蛋白偶联以形成免疫原，否则用多肽刺激动物产生的抗体对相应的天然蛋白质抗原亲和力低或完全不能识别。而用细菌表达的重组蛋白质经常会遇到一些蛋白难以表达等情况。另外，有些蛋白质免疫原性较低，很难在机体刺激产生特异性抗体。因此，在制备针对各种不同的蛋白质特异性抗体时，首先需要在对抗原制备技术进行革新。

研究发现，将特异性抗原蛋白与某一免疫原性增强蛋白构建成融合蛋白，可明显促进免疫动物对特异性靶蛋白的免疫应答。如Suzue等发现结核分枝杆菌hsp70可加强小鼠对于与之相连的蛋白产生特异性的抗体或细胞免疫应答(Suzue，K.&Young,R.A.:J.Immunol.156:873-9,1996)。专利CN201310437663公开结核分枝杆菌细胞色素C氧化酶亚基Ⅱ蛋白和特异性抗原蛋白组成融合蛋白能显著增强后者的免疫原性。专利CN201210181392公开白喉毒素无毒突变体CRM197或其片段在融合蛋白中作为分子内佐剂增强与其融合的目的蛋白的免疫原性。

钥孔血蓝蛋白(keyhole limpet hemocyanin，KLH)广泛作为载体蛋白交联于半抗原和其他抗原，使它们具有更强的免疫原性以用于抗体的制备。但钥孔血蓝蛋白的氨基酸序列较长，其中的哪个片段与其他抗原连接能够显著增加免疫原性，迄今为止现有技术从未报道过。

发明内容

本发明的目的在于提供一种包含钥孔血蓝蛋白片段的融合蛋白，用以增强与之融合的目的蛋白的免疫原性。

为实现上述目的，本发明通过以下方案予以实现：

一种融合蛋白，包含钥孔血蓝蛋白片段(sKLH片段)及目的蛋白。

优选地，所述的钥孔血蓝蛋白片段包含钥孔血蓝蛋白第1-245位氨基酸残基。

优选地，所述的钥孔血蓝蛋白片段包含SEQ ID NO:1所示的第1-245位氨基酸残基。

我们对KLH蛋白序列研究发现，该蛋白N端含有多段T-细胞和B-细胞表位。动物实验表明，上述融合蛋白sKHL片段能有效刺激针对与之相连蛋白的特异性免疫应答。

优选地，所述的目的蛋白为植物线粒体膜蛋白(NAD4)或其免疫原性片段。

优选地，所述的目的蛋白的序列如SEQ ID NO:5所示。

优选地，所述的钥孔血蓝蛋白片段连接至所述目的蛋白的N端和/或C端。

在本发明中，将两种或更多种蛋白融合表达以形成融合蛋白的技术是本领域公知的(参见例如，J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989)。通常，通过使用重组DNA技术将编码两种或更多种蛋白的DNA片段符合读框地连接在一起，并进行蛋白质表达来获得融合蛋白。

一种编码上述的融合蛋白的核苷酸。

优选地，所述的核苷酸，包含SEQ ID NO:3所述的核苷酸序列。

一种上述融合蛋白在制备针对特异性目的蛋白多克隆抗体中的应用。

一种上述融合蛋白在作为疫苗或药物的应用。

本发明方法具有如下优点：在将sKLH片段与目的蛋白融合表达后，sKLH片段显著增强了目的蛋白的免疫原性。实施例试验数据证明，sKLH片段与目的蛋白NAD4融合后，其作为抗原免疫获得的多克隆抗体特异性显著优于单独使用NAD4蛋白制备的抗体。由于本发明的融合蛋白展示了比单独的目的蛋白更强的免疫原性，因此，通过融合sKLH片段可用于制备针对特异性目的蛋白多克隆抗体。

附图说明

图1为sKLH片段表达载体pE-sKLH质粒图。

图2为NAD4蛋白表达载体pE-NAD4质粒图。

图3为融合蛋白表达载体pE-sKLH-NAD4质粒图。

图4为NAD4蛋白表达、纯化SDS-PAGE图。

图5为sKLH-NAD4融合蛋白表达、纯化SDS-PAGE图。

图6为Western blot鉴定兔抗血清质量效果图。

具体实施方式

以下实施例用于说明本发明，但不用来限制本发明的范围。

实施例1 sKLH片段基因合成及NAD4基因克隆

sKLH片段基因合成根据GenBank数据库中钥孔血蓝蛋白蛋白基因编码序列(Accession Number:AJ698341.2，其核苷酸序列如SEQ ID NO:2所示)，同时依据E.coli遗传密码偏好性将sKLH片段(1-245位氨基酸)基因序列进行了密码子优化，由南京金斯瑞生物科技有限公司合成并构建pUC57-sKLH质粒，经密码子优化并加入酶切位点后的核酸序列如SEQ ID NO:3所示。

NAD4基因克隆拟南芥叶片组织经液氮研磨并经Trizol(Invitrogen)提取获得RNA，再经III反转录酶处理获得cDNA。以cDNA为模板，高保真PCR获得NAD4基因核苷酸序列(如SEQ ID NO:4所示)。扩增引物设计如表1所示：

表1.NAD4基因扩增引物表

SEQ ID NO:	引物名称	引物序列(5’-3’)
			6	NAD4_EcoRI_F	TCCGAATTCGGTGTTTTATATGACC
7	NAD4_HindIII_R	GCAAGCTTATGAAATTTGCCATGTTG

实施例2蛋白表达载体pE-sKLH-NAD4质粒的构建

pE-sKLH质粒的构建质粒pUC57-sKLH(实施例1制备得到)经NcoI和BamHI双酶切(NEB)，胶回收酶切含sKLH片段基因(～740bp)，与同样经NcoI和BamHI双酶切胶回收后的pET-28a质粒连接，转化DH5α感受态细菌，卡拉霉素抗性平板筛选阳性克隆，T7引物测序鉴定pE-sKLH质粒。

pE-NAD4质粒的构建高保真PCR获得的NAD4基因核苷酸序列(实施例1制备得到)经EcoRI和HindIII双酶切(NEB)，胶回收酶切含NAD4核酸片段(～430bp)，与同样经EcoRI和HindIII双酶切胶回收后的pET-28a质粒连接，转化DH5α感受态细菌，卡拉霉素抗性平板筛选阳性克隆，T7引物测序鉴定pE-NAD4质粒。

pE-sKLH-NAD4质粒的构建质粒pE-NAD4经EcoRI和HindIII双酶切(NEB)，胶回收酶切含NAD4核酸片段(～430bp)，与同样经EcoRI和HindIII双酶切胶回收后的pE-sKLH质粒连接，转化DH5α感受态细菌，卡拉霉素抗性平板筛选阳性克隆，T7引物测序鉴定pE-sKLH-NAD4质粒。

实施例3 sKLH-NAD4融合蛋白表达及纯化

从含卡那霉素LB平板上挑取含有pE-NAD4及pE-sKLH-NAD4质粒的单菌落，分别接种于10ml含卡那霉素的液体LB培养基中，在37℃180rpm下振荡培养过夜，取5ml再转接到500mL含卡那霉素的LB培养液中，在37℃180rpm下振荡培养4-5小时。当OD600达0.6左右时，加入IPTG至终浓度0.5mM，在37℃下振荡诱导4个小时。诱导后，以8000g离心10min收集菌体，然后按1g菌体对应20ml裂解液(20mM Tris-HCl，pH7.6，300mM NaCl，5mM EDTA)悬浮，高压均质破碎2次，600bar。12000g离心30min，弃上清保留沉淀(即包涵体)。再用重悬液(20mMTris-HCl pH8.0，300mM NaCl)重悬沉淀，高压均质破碎1次，600bar。离心弃上清，沉淀用含8M尿素上样缓冲液(20mM NaPO₃，pH8.0，300mM NaCl，8M urea)重悬，20000g离心30min后，取上清进行Ni-NTI(qiagen)亲和层析。NAD4蛋白及sKLH-NAD4融合蛋白的表达、纯化情况如图5所示。

实施例4兔多克隆抗血清的制备

用纯化的抗原NAD4蛋白及sKLH-NAD4融合蛋白分别对4月龄的健康新西兰大白兔进行免疫，初免剂量为0.3mg/只，二免、三免剂量为0.2mg/只。初免抗原为蛋白抗原与等体积弗氏完全佐剂混匀乳化，二免、三免、抗原蛋白与等体积弗氏不完全佐剂混匀乳化。每次免疫间隔时间为2周，三免2周后取采血，3000g离心15分钟后得到抗血清。弗氏完全佐剂与弗氏不完全佐剂均购自Sigma。

实施例5 Western blot鉴定兔抗血清质量

拟南芥叶片蛋白裂解提取后，进行SDS-PAGE电泳分离，并转移到PVDF膜上，用于Western blot鉴定。PVDF膜用含5％脱脂牛奶TBST溶液(50mM Tris-HCl，pH7.6，150mMNaCl，0.05％Tween-20)封闭1h，然后加入按1:2500稀释，反应1h；用TBST溶液洗膜3次，每次15min；然后加入羊抗兔lgG偶联(辣根过氧化物酶)二抗孵育1h；继续用TBST溶液洗膜3次，每次15min；然后加入ECL反应液至膜上反应2min，胶片曝光10s-5min。分别用sKLH-NAD4融合蛋白和NAD4蛋白免疫得到的多克隆抗体进行的Western blot检测结果示于图6。结果显示，使用融合蛋白sKLH-NAD融合蛋白作为抗原免疫获得的多克隆抗体特异性显著优于单独使用NAD4蛋白制备的抗体。

虽然，上文中已经用一般性说明及具体实施例对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

SEQUENCE LISTING

<110> 攸归（上海）生物科技有限公司

<120> 一种包含钥孔血蓝蛋白片段的融合蛋白及其应用

<130>

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<170> PatentIn version 3.5

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<212> PRT

<213> 钥孔血蓝蛋白（KLH,keyhole limpet hemocyanin）

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Cys Gln Leu Asn Gly Asn Pro Ile Ser Cys Cys Val His Gly Met

1310 1315 1320

Pro Thr Phe Pro His Trp His Arg Leu Tyr Val Val Val Val Glu

1325 1330 1335

Asn Ala Leu Leu Lys Lys Gly Ser Ser Val Ala Val Pro Tyr Trp

1340 1345 1350

Asp Trp Thr Lys Arg Ile Glu His Leu Pro His Leu Ile Ser Asp

1355 1360 1365

Ala Thr Tyr Tyr Asn Ser Arg Gln His His Tyr Glu Thr Asn Pro

1370 1375 1380

Phe His His Gly Lys Ile Thr His Glu Asn Glu Ile Thr Thr Arg

1385 1390 1395

Asp Pro Lys Asp Ser Leu Phe His Ser Asp Tyr Phe Tyr Glu Gln

1400 1405 1410

Val Leu Tyr Ala Leu Glu Gln Asp Asn Phe Cys Asp Phe Glu Ile

1415 1420 1425

Gln Leu Glu Ile Leu His Asn Ala Leu His Ser Leu Leu Gly Gly

1430 1435 1440

Lys Gly Lys Tyr Ser Met Ser Asn Leu Asp Tyr Ala Ala Phe Asp

1445 1450 1455

Pro Val Phe Phe Leu His His Ala Thr Thr Asp Arg Ile Trp Ala

1460 1465 1470

Ile Trp Gln Asp Leu Gln Arg Phe Arg Lys Arg Pro Tyr Arg Glu

1475 1480 1485

Ala Asn Cys Ala Ile Gln Leu Met His Thr Pro Leu Gln Pro Phe

1490 1495 1500

Asp Lys Ser Asp Asn Asn Asp Glu Ala Thr Lys Thr His Ala Thr

1505 1510 1515

Pro His Asp Gly Phe Glu Tyr Gln Asn Ser Phe Gly Tyr Ala Tyr

1520 1525 1530

Asp Asn Leu Glu Leu Asn His Tyr Ser Ile Pro Gln Leu Asp His

1535 1540 1545

Met Leu Gln Glu Arg Lys Arg His Asp Arg Val Phe Ala Gly Phe

1550 1555 1560

Leu Leu His Asn Ile Gly Thr Ser Ala Asp Gly His Val Phe Val

1565 1570 1575

Cys Leu Pro Thr Gly Glu His Thr Lys Asp Cys Ser His Glu Ala

1580 1585 1590

Gly Met Phe Ser Ile Leu Gly Gly Gln Thr Glu Met Ser Phe Val

1595 1600 1605

Phe Asp Arg Leu Tyr Lys Leu Asp Ile Thr Lys Ala Leu Lys Lys

1610 1615 1620

Asn Gly Val His Leu Gln Gly Asp Phe Asp Leu Glu Ile Glu Ile

1625 1630 1635

Thr Ala Val Asn Gly Ser His Leu Asp Ser His Val Ile His Ser

1640 1645 1650

Pro Thr Ile Leu Phe Glu Ala Gly Thr Asp Ser Ala His Thr Asp

1655 1660 1665

Asp Gly His Thr Glu Pro Val Met Ile Arg Lys Asp Ile Thr Gln

1670 1675 1680

Leu Asp Lys Arg Gln Gln Leu Ser Leu Val Lys Ala Leu Glu Ser

1685 1690 1695

Met Lys Ala Asp His Ser Ser Asp Gly Phe Gln Ala Ile Ala Ser

1700 1705 1710

Phe His Ala Leu Pro Pro Leu Cys Pro Ser Pro Ala Ala Ser Lys

1715 1720 1725

Arg Phe Ala Cys Cys Val His Gly Met Ala Thr Phe Pro Gln Trp

1730 1735 1740

His Arg Leu Tyr Thr Val Gln Phe Gln Asp Ser Leu Arg Lys His

1745 1750 1755

Gly Ala Val Val Gly Leu Pro Tyr Trp Asp Trp Thr Leu Pro Arg

1760 1765 1770

Ser Glu Leu Pro Glu Leu Leu Thr Val Ser Thr Ile His Asp Pro

1775 1780 1785

Glu Thr Gly Arg Asp Ile Pro Asn Pro Phe Ile Gly Ser Lys Ile

1790 1795 1800

Glu Phe Glu Gly Glu Asn Val His Thr Lys Arg Asp Ile Asn Arg

1805 1810 1815

Asp Arg Leu Phe Gln Gly Ser Thr Lys Thr His His Asn Trp Phe

1820 1825 1830

Ile Glu Gln Ala Leu Leu Ala Leu Glu Gln Thr Asn Tyr Cys Asp

1835 1840 1845

Phe Glu Val Gln Phe Glu Ile Met His Asn Gly Val His Thr Trp

1850 1855 1860

Val Gly Gly Lys Glu Pro Tyr Gly Ile Gly His Leu His Tyr Ala

1865 1870 1875

Ser Tyr Asp Pro Leu Phe Tyr Ile His His Ser Gln Thr Asp Arg

1880 1885 1890

Ile Trp Ala Ile Trp Gln Ser Leu Gln Arg Phe Arg Gly Leu Ser

1895 1900 1905

Gly Ser Glu Ala Asn Cys Ala Val Asn Leu Met Lys Thr Pro Leu

1910 1915 1920

Lys Pro Phe Ser Phe Gly Ala Pro Tyr Asn Leu Asn Asp His Thr

1925 1930 1935

His Asp Phe Ser Lys Pro Glu Asp Thr Phe Asp Tyr Gln Lys Phe

1940 1945 1950

Gly Tyr Ile Tyr Asp Thr Leu Glu Phe Ala Gly Trp Ser Ile Arg

1955 1960 1965

Gly Ile Asp His Ile Val Arg Asn Arg Gln Glu His Ser Arg Val

1970 1975 1980

Phe Ala Gly Phe Leu Leu Glu Gly Phe Gly Thr Ser Ala Thr Val

1985 1990 1995

Asp Phe Gln Val Cys Arg Thr Ala Gly Asp Cys Glu Asp Ala Gly

2000 2005 2010

Tyr Phe Thr Val Leu Gly Gly Glu Lys Glu Met Pro Trp Ala Phe

2015 2020 2025

Asp Arg Leu Tyr Lys Tyr Asp Ile Thr Glu Thr Leu Asp Lys Met

2030 2035 2040

Asn Leu Arg His Asp Glu Ile Phe Gln Ile Glu Val Thr Ile Thr

2045 2050 2055

Ser Tyr Asp Gly Thr Val Leu Asp Ser Gly Leu Ile Pro Thr Pro

2060 2065 2070

Ser Ile Ile Tyr Asp Pro Ala His His Asp Ile Ser Ser His His

2075 2080 2085

Leu Ser Leu Asn Lys Val Arg His Asp Leu Ser Thr Leu Ser Glu

2090 2095 2100

Arg Asp Ile Gly Ser Leu Lys Tyr Ala Leu Ser Ser Leu Gln Ala

2105 2110 2115

Asp Thr Ser Ala Asp Gly Phe Ala Ala Ile Ala Ser Phe His Gly

2120 2125 2130

Leu Pro Ala Lys Cys Asn Asp Ser His Asn Asn Glu Val Ala Cys

2135 2140 2145

Cys Ile His Gly Met Pro Thr Phe Pro His Trp His Arg Leu Tyr

2150 2155 2160

Thr Leu Gln Phe Glu Gln Ala Leu Arg Arg His Gly Ser Ser Val

2165 2170 2175

Ala Val Pro Tyr Trp Asp Trp Thr Lys Pro Ile His Asn Ile Pro

2180 2185 2190

His Leu Phe Thr Asp Lys Glu Tyr Tyr Asp Val Trp Arg Asn Lys

2195 2200 2205

Val Met Pro Asn Pro Phe Ala Arg Gly Tyr Val Pro Ser His Asp

2210 2215 2220

Thr Tyr Thr Val Arg Asp Val Gln Glu Gly Leu Phe His Leu Thr

2225 2230 2235

Ser Thr Gly Glu His Ser Ala Leu Leu Asn Gln Ala Leu Leu Ala

2240 2245 2250

Leu Glu Gln His Asp Tyr Cys Asp Phe Ala Val Gln Phe Glu Val

2255 2260 2265

Met His Asn Thr Ile His Tyr Leu Val Gly Gly Pro Gln Val Tyr

2270 2275 2280

Ser Leu Ser Ser Leu His Tyr Ala Ser Tyr Asp Pro Ile Phe Phe

2285 2290 2295

Ile His His Ser Phe Val Asp Lys Val Trp Ala Val Trp Gln Ala

2300 2305 2310

Leu Gln Glu Lys Arg Gly Leu Pro Ser Asp Arg Ala Asp Cys Ala

2315 2320 2325

Val Ser Leu Met Thr Gln Asn Met Arg Pro Phe His Tyr Glu Ile

2330 2335 2340

Asn His Asn Gln Phe Thr Lys Lys His Ala Val Pro Asn Asp Val

2345 2350 2355

Phe Lys Tyr Glu Leu Leu Gly Tyr Arg Tyr Asp Asn Leu Glu Ile

2360 2365 2370

Gly Gly Met Asn Leu His Glu Ile Glu Lys Glu Ile Lys Asp Lys

2375 2380 2385

Gln His His Val Arg Val Phe Ala Gly Phe Leu Leu His Gly Ile

2390 2395 2400

Arg Thr Ser Ala Asp Val Gln Phe Gln Ile Cys Lys Thr Ser Glu

2405 2410 2415

Asp Cys His His Gly Gly Gln Ile Phe Val Leu Gly Gly Thr Lys

2420 2425 2430

Glu Met Ala Trp Ala Tyr Asn Arg Leu Phe Lys Tyr Asp Ile Thr

2435 2440 2445

His Ala Leu His Asp Ala His Ile Thr Pro Glu Asp Val Phe His

2450 2455 2460

Pro Ser Glu Pro Phe Phe Ile Lys Val Ser Val Thr Ala Val Asn

2465 2470 2475

Gly Thr Val Leu Pro Ala Ser Ile Leu His Ala Pro Thr Ile Ile

2480 2485 2490

Tyr Glu Pro Gly Leu Asp His His Glu Asp His His Ser Ser Ser

2495 2500 2505

Met Ala Gly His Gly Val Arg Lys Glu Ile Asn Thr Leu Thr Thr

2510 2515 2520

Ala Glu Val Asp Asn Leu Lys Asp Ala Met Arg Ala Val Met Ala

2525 2530 2535

Asp His Gly Pro Asn Gly Tyr Gln Ala Ile Ala Ala Phe His Gly

2540 2545 2550

Asn Pro Pro Met Cys Pro Met Pro Asp Gly Lys Asn Tyr Ser Cys

2555 2560 2565

Cys Thr His Gly Met Ala Thr Phe Pro His Trp His Arg Leu Tyr

2570 2575 2580

Thr Lys Gln Met Glu Asp Ala Leu Thr Ala His Gly Ala Arg Val

2585 2590 2595

Gly Leu Pro Tyr Trp Asp Gly Thr Thr Ala Phe Thr Ala Leu Pro

2600 2605 2610

Thr Phe Val Thr Asp Glu Glu Asp Asn Pro Phe His His Gly His

2615 2620 2625

Ile Asp Tyr Leu Gly Val Asp Thr Thr Arg Ser Pro Arg Asp Lys

2630 2635 2640

Leu Phe Asn Asp Pro Glu Arg Gly Ser Glu Ser Phe Phe Tyr Arg

2645 2650 2655

Gln Val Leu Leu Ala Leu Glu Gln Thr Asp Phe Cys Gln Phe Glu

2660 2665 2670

Val Gln Phe Glu Ile Thr His Asn Ala Ile His Ser Trp Thr Gly

2675 2680 2685

Gly Leu Thr Pro Tyr Gly Met Ser Thr Leu Glu Tyr Thr Thr Tyr

2690 2695 2700

Asp Pro Leu Phe Trp Leu His His Ala Asn Thr Asp Arg Ile Trp

2705 2710 2715

Ala Ile Trp Gln Ala Leu Gln Glu Tyr Arg Gly Leu Pro Tyr Asp

2720 2725 2730

His Ala Asn Cys Glu Ile Gln Ala Met Lys Arg Pro Leu Arg Pro

2735 2740 2745

Phe Ser Asp Pro Ile Asn His Asn Ala Phe Thr His Ser Asn Ala

2750 2755 2760

Lys Pro Thr Asp Val Phe Glu Tyr Ser Arg Phe Asn Phe Gln Tyr

2765 2770 2775

Asp Asn Leu Arg Phe His Gly Met Thr Ile Lys Lys Leu Glu His

2780 2785 2790

Glu Leu Glu Lys Gln Lys Glu Glu Asp Arg Thr Phe Ala Ala Phe

2795 2800 2805

Leu Leu His Gly Ile Lys Lys Ser Ala Asp Val Ser Phe Asp Val

2810 2815 2820

Cys Asn His Asp Gly Glu Cys His Phe Ala Gly Thr Phe Ala Ile

2825 2830 2835

Leu Gly Gly Glu His Glu Met Pro Trp Ser Phe Asp Arg Leu Phe

2840 2845 2850

Arg Tyr Asp Ile Thr Gln Val Leu Lys Gln Met His Leu Glu Tyr

2855 2860 2865

Asp Ser Asp Phe Thr Phe His Met Arg Ile Ile Asp Thr Ser Gly

2870 2875 2880

Lys Gln Leu Pro Ser Asp Leu Ile Lys Met Pro Thr Val Glu His

2885 2890 2895

Ser Pro Gly Gly Lys His His Glu Lys His His Glu Asp His His

2900 2905 2910

Glu Asp Ile Leu Val Arg Lys Asn Ile His Ser Leu Ser His His

2915 2920 2925

Glu Ala Glu Glu Leu Arg Asp Ala Leu Tyr Lys Leu Gln Asn Asp

2930 2935 2940

Glu Ser His Gly Gly Tyr Glu His Ile Ala Gly Phe His Gly Tyr

2945 2950 2955

Pro Asn Leu Cys Pro Glu Lys Gly Asp Glu Lys Tyr Pro Cys Cys

2960 2965 2970

Val His Gly Met Ser Ile Phe Pro His Trp His Arg Leu His Thr

2975 2980 2985

Ile Gln Phe Glu Arg Ala Leu Lys Lys His Gly Ser His Leu Gly

2990 2995 3000

Ile Pro Tyr Trp Asp Trp Thr Gln Thr Ile Ser Ser Leu Pro Thr

3005 3010 3015

Phe Phe Ala Asp Ser Gly Asn Asn Asn Pro Phe Phe Lys Tyr His

3020 3025 3030

Ile Arg Ser Ile Asn Gln Asp Thr Val Arg Asp Val Asn Glu Ala

3035 3040 3045

Ile Phe Gln Gln Thr Lys Phe Gly Glu Phe Ser Ser Ile Phe Tyr

3050 3055 3060

Leu Ala Leu Gln Ala Leu Glu Glu Asp Asn Tyr Cys Asp Phe Glu

3065 3070 3075

Val Gln Tyr Glu Ile Leu His Asn Glu Val His Ala Leu Ile Gly

3080 3085 3090

Gly Ala Glu Lys Tyr Ser Met Ser Thr Leu Glu Tyr Ser Ala Phe

3095 3100 3105

Asp Pro Tyr Phe Met Ile His His Ala Ser Leu Asp Lys Ile Trp

3110 3115 3120

Ile Ile Trp Gln Glu Leu Gln Lys Arg Arg Val Lys Pro Ala His

3125 3130 3135

Ala Gly Ser Cys Ala Gly Asp Ile Met His Val Pro Leu His Pro

3140 3145 3150

Phe Asn Tyr Glu Ser Val Asn Asn Asp Asp Phe Thr Arg Glu Asn

3155 3160 3165

Ser Leu Pro Asn Ala Val Val Asp Ser His Arg Phe Asn Tyr Lys

3170 3175 3180

Tyr Asp Asn Leu Asn Leu His Gly His Asn Ile Glu Glu Leu Glu

3185 3190 3195

Glu Val Leu Arg Ser Leu Arg Leu Lys Ser Arg Val Phe Ala Gly

3200 3205 3210

Phe Val Leu Ser Gly Ile Arg Thr Thr Ala Val Val Lys Val Tyr

3215 3220 3225

Ile Lys Ser Gly Thr Asp Ser Asp Asp Glu Tyr Ala Gly Ser Phe

3230 3235 3240

Val Ile Leu Gly Gly Ala Lys Glu Met Pro Trp Ala Tyr Glu Arg

3245 3250 3255

Leu Tyr Arg Phe Asp Ile Thr Glu Thr Val His Asn Leu Asn Leu

3260 3265 3270

Thr Asp Asp His Val Lys Phe Arg Phe Asp Leu Lys Lys Tyr Asp

3275 3280 3285

His Thr Glu Leu Asp Ala Ser Val Leu Pro Ala Pro Ile Ile Val

3290 3295 3300

Arg Arg Pro Asn Asn Ala Val Phe Asp Ile Ile Glu Ile Pro Ile

3305 3310 3315

Gly Lys Asp Val Asn Leu Pro Pro Lys Val Val Val Lys Arg Gly

3320 3325 3330

Thr Lys Ile Met Phe Met Ser Val Asp Glu Ala Val Thr Thr Pro

3335 3340 3345

Met Leu Asn Leu Gly Ser Tyr Thr Ala Met Phe Lys Cys Lys Val

3350 3355 3360

Pro Pro Phe Ser Phe His Ala Phe Glu Leu Gly Lys Met Tyr Ser

3365 3370 3375

Val Glu Ser Gly Asp Tyr Phe Met Thr Ala Ser Thr Thr Glu Leu

3380 3385 3390

Cys Asn Asp Asn Asn Leu Arg Ile His Val His Val Asp Asp Glu

3395 3400 3405

<210> 2

<211> 10227

<212> DNA

<213> 钥孔血蓝蛋白（KLH,keyhole limpet hemocyanin）

<400> 2

atgctgtcgg tcaggttgct tatagtcgtg ttggctttgg ccaatgcaga gaacctggtc 60

agaaagagtg tggagcatct gactcaggaa gagaccctgg acctgcaggc tgccctgcgt 120

gagctgcaga tggactcatc cagcattggt ttccagaaaa tagctgctgc tcatggcgcg 180

cccgcatctt gtgtgcataa ggacacttct atagcttgct gtattcacgg catgcctact 240

ttcccgcact ggcacagggc gtacgtagtt cacatggagc gagctctgca gacgaagagg 300

cgtacttctg gcctgcctta ctgggactgg acagaaccca tcacccagtt gccctctctc 360

gctgcggacc ctgtttacat cgattcccag ggcggaaagg ctcacaccaa ctactggtac 420

agagggaaca ttgactttct agacaagaaa accaatcgtg cggtcgacga ccgtcttttt 480

gagaaagtga agccaggcca gcacacccat cttatggaga gtgttttgga cgcactggag 540

caggatgaat tctgtaaatt cgaaatccag ttcgagttag cacataatgc gattcactac 600

ctggtcggtg gaaagcatga ttactctatg gcaaacctcg agtacaccgc ctacgatccg 660

atcttctttc ttcaccactc caacgttgac agaattttcg ccatctggca gaggctccaa 720

gagctccgaa acaaagaccc caaagctatg gattgtgccc aagaattgtt acatcagaaa 780

atggaacctt tcagttggga ggacaatgac ataccactaa caaatgacta tgacacgttg 840

aatcttaatg gtatgactcc agaggaactg aaaacctatt tggatgagcg gtcttcaagg 900

gcaagagcct tcgcaagctt ccgtctgaaa ggatttggag gttcagccaa tgtctttgtg 960

tacgtctgca tccccgacga caacgatcgt aatgatgacc actgtgagaa ggccggtgat 1020

ttcttcgttc tgggcggacc aagcgagatg aaatggcagt tctacaggcc atacttgttt 1080

gacttgtccg acactgttca caagatggga atgaagttgg atggacacta taccgtcaag 1140

gcagagcttt tcagtgtcaa tggtacagct cttcccgatg atttgttacc tcatccagtt 1200

gttgttcatc atccggagaa gggtttcact gatcctccgg tgaagcatca ccaaagcgcc 1260

aatctcctcg ttagaaagaa catcaatgat ctcacccgtg aagaagtgtt gaatctgaga 1320

gaggcgtttc ataaattcca agaagatcgc tccgtcgacg gttatcaagc cacagctgag 1380

tatcatggtc ttccggccag atgccctcgc cctgatgcca aggacagata tgcttgttgt 1440

gtccatggaa tgccaatctt ccctcactgg cacaggcttt ttgtcacaca agttgaagat 1500

gctttagtag gccgtggagc taccattggt atcccgtact ggggcctacc gtactgggac 1560

tggactgaac ccatgacaca cattccgggt ctggcaggaa acaaaactta tgtggattct 1620

catggtgcat cccacacaaa tccttttcat agttcagtga ttgcatttga agaaaatgct 1680

ccccacacca aaagacaaat agatcaaaga ctctttaaac ccgctacctt tggacaccac 1740

acagacctgt tcaaccagat tttgtatgcc tttgaacaag aagattactg tgactttgaa 1800

gtccaatttg agattaccca taacacgatt cacgcttgga caggaggaag cgaacatttc 1860

tcaatgtcgt ccctacatta cacagctttc gatcctttgt tttactttca ccattctaac 1920

gttgatcgtc tttgggccgt ttggcaagcc ttacagatga gacggcataa accctacagg 1980

gcccactgcg ccatatctct ggaacatatg catctgaaac cattcgcctt ttcatctccc 2040

cttaacaata acgaaaagac tcatgccaat gccatgccaa acaagatcta cgactatgaa 2100

aatgtcctcc attacacata cgaagattta acatttggag gcatctctct ggaaaacata 2160

gaaaagatga tccacgaaaa ccagcaagaa gacagaatat atgccggttt tctcctggct 2220

ggcatacgta cttcagcaaa tgttgatatc ttcattaaaa ctaccgattc cgtgcaacat 2280

aaggctggaa catttgcagt gctcggtgga agcaaggaaa tgaagtgggg atttgatcgc 2340

gttttcaagt ttgacatcac gcacgttttg aaagatctcg atctcactgc tgatggcgat 2400

ttcgaagtta ctgttgacat cactgaagtc gatggaacta aacttgcatc cagtcttatt 2460

ccacatgctt ctgtcattcg tgagcatgca cgtggtaagc tgaatagagt taaatttgac 2520

aaagtgccaa ggagtcgtct tattcgaaaa aatgtagacc gtttgagccc cgaggagatg 2580

aatgaacttc gtaaagccct agccttactg aaagaggaca aaagtgccgg tggatttcag 2640

cagcttggtg cattccatgg ggagccaaaa tggtgtccta gtcccgaagc atctaaaaaa 2700

tttgcctgct gtgttcacgg catgtctgtg ttccctcact ggcatcgact gttgacggtt 2760

cagagtgaaa atgctttgag acgacatggc tacgatggag ctttgccgta ctgggattgg 2820

acctctcctc ttaatcacct tcccgaactg gcagatcatg agaagtacgt cgaccctgaa 2880

gatggggtag agaagcataa cccttggttc gatggtcata tagatacagt cgacaaaaca 2940

acaacaagaa gtgttcagaa taaactcttc gaacagcctg agtttggtca ttatacaagc 3000

attgccaaac aagtactgct agcgttggaa caggacaatt tctgtgactt tgaaatccaa 3060

tatgagattg cccataacta catccatgca cttgtaggag gcgctcagcc ttatggtatg 3120

gcatcgcttc gctacactgc ttttgatcca ctattctact tgcatcactc taatacagat 3180

cgtatatggg caatatggca ggctttacag aagtacagag gaaaaccgta caacgttgct 3240

aactgtgctg ttacatcgat gagagaacct ttgcaaccat ttggcctctc tgccaatatc 3300

aacacagacc atgtaaccaa ggagcattca gtgccattca acgtttttga ttacaagacc 3360

aatttcaatt atgaatatga cactttggaa tttaacggtc tctcaatctc tcagttgaat 3420

aaaaagctcg aagcgataaa gagccaagac aggttctttg caggcttcct gttatctggt 3480

ttcaagaaat catctcttgt taaattcaat atttgcaccg atagcagcaa ctgtcacccc 3540

gctggagagt tttaccttct gggtgatgaa aacgagatgc catgggcata cgatagagtc 3600

ttcaaatatg acataaccga aaaactccac gatctaaagc tgcatgcaga agaccacttc 3660

tacattgact atgaagtatt tgaccttaaa ccagcaagcc tgggaaaaga tttgttcaag 3720

cagccttcag tcattcatga accaagaata ggtcaccatg aaggcgaagt atatcaagct 3780

gaagtaactt ctgccaaccg tattcgaaaa aacattgaaa atctgagcct tggtgaactc 3840

gaaagtctga gagctgcctt cctggaaatt gaaaacgatg gaacttacga atcaatagct 3900

aaattccatg gtagccctgg tttgtgccag ttaaatggta accccatctc ttgttgtgtc 3960

catggcatgc caactttccc tcactggcac agactgtacg tggttgtcgt tgagaatgcc 4020

ctcctgaaaa aaggatcatc tgtagctgtt ccctattggg actggacaaa acgaatcgaa 4080

catttacctc acctgatttc agacgccact tactacaatt ccaggcaaca tcactatgag 4140

acaaacccat tccatcatgg caaaatcaca cacgagaatg aaatcactac tagggatccc 4200

aaggacagcc tcttccattc agactacttt tacgagcagg tcctttacgc cttggagcag 4260

gataacttct gtgatttcga gattcagttg gagatattac acaatgcatt gcattcttta 4320

cttggtggca aaggtaaata ttccatgtca aaccttgatt acgctgcttt tgatcctgtg 4380

ttcttccttc atcacgcaac gactgacaga atctgggcaa tctggcaaga ccttcagagg 4440

ttccgaaaac ggccataccg agaagcgaat tgcgctatcc aattgatgca cacgccactc 4500

cagccgtttg ataagagcga caacaatgac gaggcaacga aaacgcatgc cactccacat 4560

gatggttttg aatatcaaaa cagctttggt tatgcttacg ataatctgga actgaatcac 4620

tactcgattc ctcagcttga tcacatgctg caagaaagaa aaaggcatga cagagtattc 4680

gctggcttcc tccttcacaa tattggaaca tctgccgatg gccatgtatt tgtatgtctc 4740

ccaactgggg aacacacgaa ggactgcagt catgaggctg gtatgttctc catcttaggc 4800

ggtcaaacgg agatgtcctt tgtatttgac agactttaca aacttgacat aactaaagcc 4860

ttgaaaaaga acggtgtgca cctgcaaggg gatttcgatc tggaaattga gattacggct 4920

gtgaatggat ctcatctaga cagtcatgtc atccactctc ccactatact gtttgaggcc 4980

ggaacagatt ctgcccacac agatgatgga cacactgaac cagtgatgat tcgcaaagat 5040

atcacacaat tggacaagcg tcaacaactg tcactggtga aagccctcga gtccatgaaa 5100

gccgaccatt catctgatgg gttccaggca atcgcttcct tccatgctct tcctcctctt 5160

tgtccatcac cagctgcttc aaagaggttt gcgtgctgcg tccatggcat ggcaacgttc 5220

ccacaatggc accgtctgta cacagtccaa ttccaagatt ctctcagaaa acatggtgca 5280

gtcgttggac ttccgtactg ggactggacc ctacctcgtt ctgaattacc agagctcctg 5340

accgtctcaa ctattcatga cccggagaca ggcagagata taccaaatcc atttattggt 5400

tctaaaatag agtttgaagg agaaaacgta catactaaaa gagatatcaa tagggatcgt 5460

ctcttccagg gatcaacaaa aacacatcat aactggttta ttgagcaagc actgcttgct 5520

cttgaacaaa ccaactactg cgacttcgag gttcagtttg aaattatgca taatggtgtt 5580

catacctggg ttggaggcaa ggagccctat ggaattggcc atctgcatta tgcttcctat 5640

gatccacttt tctacatcca tcactcccaa actgatcgta tttgggctat atggcaatcg 5700

ttgcagcgtt tcagaggact ttctggatct gaggctaact gtgctgtaaa tctcatgaaa 5760

actcctctga agcctttcag ctttggagca ccatataatc ttaatgatca cacgcatgat 5820

ttctcaaagc ctgaagatac attcgactac caaaagtttg gatacatata tgacactctg 5880

gaatttgcag ggtggtcaat tcgtggcatt gaccatattg tccgtaacag gcaggaacat 5940

tcaagggtct ttgccggatt cttgcttgaa ggatttggca cctctgccac tgtcgatttc 6000

caggtctgtc gcacagcggg agactgtgaa gatgcagggt acttcaccgt gttgggaggt 6060

gaaaaagaaa tgccttgggc ctttgatcgg ctttacaagt acgacataac agaaacctta 6120

gacaagatga accttcgaca tgacgaaatc ttccagattg aagtaaccat tacatcctac 6180

gatggaactg tactcgatag tggccttatt cccacaccgt caatcatcta tgatcctgct 6240

catcatgata ttagttcgca ccacctgtcg ctcaacaagg ttcgtcatga tctgagtaca 6300

ctgagtgagc gagatattgg aagccttaaa tatgctttga gcagcttgca ggcagatacc 6360

tcagcagatg gttttgctgc cattgcatcc ttccatggtc tgcctgccaa atgtaatgac 6420

agccacaata acgaggtggc atgctgtatc catggaatgc ctacattccc ccactggcac 6480

agactctaca ccctccaatt tgagcaagct ctaagaagac atggctctag tgtagcagta 6540

ccctactggg actggacaaa gccaatacat aatattccac atctgttcac agacaaagaa 6600

tactacgatg tctggagaaa taaagtaatg ccaaatccat ttgcccgagg gtatgtcccc 6660

tcacacgata catacacggt aagagacgtc caagaaggcc tgttccacct gacatcaacg 6720

ggtgaacact cagcgcttct gaatcaagct cttttggcgc tggaacagca cgactactgc 6780

gattttgcag tccagtttga agtcatgcac aacacaatcc attacctagt gggaggacct 6840

caagtctatt ctttgtcatc ccttcattat gcttcatatg atccgatctt cttcatacac 6900

cactcctttg tagacaaggt ttgggctgtc tggcaggctc ttcaagaaaa gagaggcctt 6960

ccatcagacc gtgctgactg cgctgttagt ctgatgactc agaacatgag gcctttccat 7020

tacgaaatta accataacca gttcaccaag aaacatgcag ttccaaatga tgttttcaag 7080

tacgaactcc tgggttacag atacgacaat ctggaaatcg gtggcatgaa tttgcatgaa 7140

attgaaaagg aaatcaaaga caaacagcac catgtgagag tgtttgcagg gttcctcctt 7200

cacggaatta gaacctcagc tgatgtccaa ttccagattt gtaaaacatc agaagattgt 7260

caccatggag gccaaatctt cgttcttggg gggactaaag agatggcctg ggcttataac 7320

cgtttattca agtacgatat tacccatgct cttcatgacg cacacatcac tccagaagac 7380

gtattccatc cctctgaacc attcttcatc aaggtgtcag tgacagccgt caacggaaca 7440

gttcttccgg cttcaatcct gcatgcacca accattatct atgaacctgg tctcgaccat 7500

cacgaagatc atcattcttc ttctatggct ggacatggtg tcagaaagga aatcaacaca 7560

cttaccactg cagaggtgga caatctcaaa gatgccatga gagccgtcat ggcagaccac 7620

ggtccaaatg gataccaggc tatagcagcg ttccatggaa acccaccaat gtgccctatg 7680

ccagatggaa agaattactc gtgttgtaca catggcatgg ctactttccc ccactggcac 7740

agactgtaca caaaacagat ggaagatgcc ttgaccgccc atggtgccag agtcggcctt 7800

ccttactggg acgggacaac tgcctttaca gctttgccaa cttttgtcac agatgaagag 7860

gacaatcctt tccatcatgg tcacatagac tatttgggag tggatacaac tcggtcgccc 7920

cgagacaagt tgttcaatga tccagagcga ggatcagaat cgttcttcta caggcaggtt 7980

ctcttggctt tggagcagac agatttttgt cagtttgaag tccagttcga gattacccac 8040

aatgccatcc attcttggac tggtggactg accccctacg gaatgtccac tctggagtac 8100

acgacctatg atcctctttt ctggcttcat catgccaata ctgaccgaat atgggctatt 8160

tggcaagctc ttcaagaata cagaggtctc ccatacgacc atgccaactg cgaaatccag 8220

gcaatgaaga ggcccctacg gccattctct gatcctatta atcacaatgc gttcactcat 8280

tctaacgcaa aaccaacaga cgttttcgag tacagtagat tcaacttcca gtatgataac 8340

ttacggtttc atggtatgac aataaaaaag ttggagcatg agcttgagaa acagaaggaa 8400

gaagacagga cattcgctgc cttcctgctc cacggcatta aaaaaagtgc tgatgtaagc 8460

tttgacgtgt gcaaccatga tggagaatgc cactttgcag gtaccttcgc tattcttgga 8520

ggcgagcacg agatgccttg gtcctttgac agacttttcc gatatgacat cacccaagtt 8580

cttaagcaaa tgcacctgga gtatgattct gatttcacgt tccatatgag aattattgac 8640

acttcgggta aacaactgcc atcagacctg atcaagatgc caacggttga acacagccca 8700

ggaggaaaac atcatgagaa acatcatgaa gatcaccatg aagacatact tgttcgtaaa 8760

aacattcact ctctgtcaca ccatgaagcc gaagaattga gagatgcatt gtacaagctt 8820

caaaacgatg aaagtcacgg tggatatgaa catattgctg gattccatgg ttatcctaac 8880

ctctgtcctg aaaaagggga tgaaaagtat ccttgctgtg ttcatggtat gtctatattc 8940

ccccactggc acaggctcca cacgatacag tttgaaagag ctctgaagaa acatggttcc 9000

catctcggta ttccttactg ggattggact cagacgatat cgtctcttcc taccttcttt 9060

gcagattctg gtaataataa tccattcttc aaataccata tcagaagcat aaatcaagac 9120

actgttcgag acgttaatga agcgatattc caacaaacga agtttggaga attttcgtcc 9180

atattttacc tggccttgca ggctctagaa gaggacaatt attgcgattt tgaggtgcag 9240

tacgagatac ttcacaatga ggtgcatgcc ttgattggtg gagccgagaa gtattccatg 9300

tccacgctgg aatattcagc cttcgatcca tacttcatga tccaccacgc gtctttggac 9360

aagatctgga taatctggca ggagcttcag aaacgccggg ttaagcctgc acatgctggt 9420

tcgtgtgccg gagacattat gcacgttcca cttcatcctt tcaattatga aagcgtcaac 9480

aacgatgact ttaccagaga gaattctctc cccaacgcgg tcgttgacag tcatagattc 9540

aactataaat atgacaatct taatcttcac ggccacaata tagaggagct tgaggaggtt 9600

ctccgcagcc tgagacttaa atctcgtgtt ttcgcaggat ttgttctgtc cggaatccgc 9660

acaactgcag ttgtgaaagt gtatatcaaa agcggaaccg attccgacga cgaatatgcc 9720

ggttcgtttg tcatccttgg aggagccaaa gagatgcctt gggcatatga gaggttgtat 9780

agatttgaca ttacagaaac tgtacacaat cttaacctta cggatgatca tgttaaattc 9840

aggtttgact taaagaaata tgatcatact gaattggatg catcagtact gccagctcct 9900

ataatcgttc gacgtccaaa taatgcggtt tttgatatca ttgagattcc aatagggaaa 9960

gacgtgaatc ttccaccgaa ggtggtcgtc aagagaggca ctaaaatcat gttcatgtct 10020

gttgatgaag ccgttaccac tcccatgctg aacctgggta gttatacagc aatgttcaaa 10080

tgtaaagtgc caccattcag cttccatgct tttgagcttg ggaaaatgta ttctgtcgaa 10140

tctggtgact atttcatgac ggcatcaaca actgaattat gcaacgataa taacttaagg 10200

atacatgtcc atgtggatga cgaatag 10227

<210> 3

<211> 743

<212> DNA

<213> 人工序列

<400> 3

ccatggtttc agtgcgcctg ttgattgtcg tgttggcttt agctaatgca gagaatttgg 60

ttcgcaagtc agttgaacac ttgactcaag aggagactct tgatttacaa gcagcacttc 120

gtgaattgca aatggattcg agatccattg ggtttcagaa aattgcggct gcccacgggg 180

ccccagcttc atgtgtccat aaggatactt cgattgcgtg ttgtatccac ggcatgccta 240

catttccgca ctggcatcgt gcctacgttg ttcatatgga gcgcgccttg cagacaaagc 300

gtcgcacaag cgggcttccg tattgggatt ggacagaacc aattactcaa ttaccaagtc 360

tggcagcgga ccctgtgtat attgattcac agggaggcaa ggcccataca aattactggt 420

atcgcggcaa cattgatttc cttgacaaga aaaccaaccg cgcagttgat gatcgtcttt 480

ttgagaaggt gaagccagga cagcatactc acctgatgga gtcggtattg gacgctcttg 540

aacaagacga gttctgcaaa ttcgaaattc agttcgagct tgcccataac gctattcatt 600

acttggtcgg ggggaaacat gattattcta tggccaacct tgagtacaca gcgtatgacc 660

ctatcttttt tttgcatcat tccaatgtag atcgtatttt tgcgatctgg cagcgtttgc 720

aagagcttcg caacaaagga tcc 743

<210> 4

<211> 1488

<212> DNA

<213> 人工序列

<400> 4

atgttagaac atttctgtga atgctattct aatctaagtg gtcttattct gtgtcctgtg 60

ctaggaagca ttactcttct tttcattcca aattcaagaa tacgaccgat acgattaatt 120

ggtctgtgtg cctctcttat tacttttttg tattctcctg ttcctcggat acaattcgac 180

tcttctacgg ccaaatctca atttgtggaa agccttcgat ggcttcctta tgaaaacatc 240

aatttttatt tgggtataga cggtatctct ttattcttcg tgatattgac cacatttctg 300

atccctattt gcatttcagt gggttggtct ggtatgagaa gttatgggaa agagtatatt 360

acagcatttt taattcgtga atttctaatg atcgccgtgt tccgcatgct agatcttcta 420

ctattctatg tttttcccga aagcgtgcca atccctatgt tcattattat aggagtatgg 480

ggttcgagac aaagaaagat caaggcagca tatcagtttt tcctttatac tttacttgga 540

tctcttttta tgctattagc tattctgttg attcttttcc aaacaggaac caccgattta 600

caaatatcat taaccacaga atttagtgag cggcgccaaa tctttctatg gattgcttct 660

ttcgcctctt tcgccgtcaa agtgcctatg gtaccagttc atatttggtt acctgaagct 720

catgtagagg cacctacggc aggatccgtc atcttggcag gaattccttt aaaatttgga 780

acccacgggt ttttaagatt ttcaataccc atgtttcccg aagcgacact ttgttctact 840

cctttcattt atactttaag cgcgattgct ataatatata cttccttgac cacttcaaga 900

cagatcgatc taaagaagat cattgcttac tcctcagtag cccatatgaa tctggtgact 960

attggtatgt ttagtccgaa catacaggga attggaggta gcattctacc gatgttaagt 1020

catggactgg ttccttcagc cctttttcta tgtgttggtg ttttatatga ccgacataag 1080

actcgacttg ttagatatta cggaggttta gtgagcacca tgccgaatct ctctaccatt 1140

ttcttttctt ttactttggc caatatgagt tcacctggta ctagcagctt tatcggggaa 1200

tttctcatct tagtaggagc tttccaaaga aatagcttag tagccacatt agcagcgctt 1260

gggatgattt taggtgcggc ctattccctt tggctatata atcgtgtggt ttctggaaat 1320

ttaaaacctg atttcctcca taaattctcc gattcaaatg gcagagaagt ttccatattt 1380

ataccttttc ttgttggact cgttcggatg ggtgttcacc ccaaagtgtt cccggactgc 1440

atgcatacat ccgtaagtaa cttagtgcaa catggcaaat ttcattga 1488

<210> 5

<211> 495

<212> PRT

<213> 人工序列

<400> 5

Met Leu Glu His Phe Cys Glu Cys Tyr Ser Asn Leu Ser Gly Leu Ile

1 5 10 15

Leu Cys Pro Val Leu Gly Ser Ile Thr Leu Leu Phe Ile Pro Asn Ser

20 25 30

Arg Ile Arg Pro Ile Arg Leu Ile Gly Leu Cys Ala Ser Leu Ile Thr

35 40 45

Phe Leu Tyr Ser Pro Val Pro Arg Ile Gln Phe Asp Ser Ser Thr Ala

50 55 60

Lys Ser Gln Phe Val Glu Ser Leu Arg Trp Leu Pro Tyr Glu Asn Ile

65 70 75 80

Asn Phe Tyr Leu Gly Ile Asp Gly Ile Ser Leu Phe Phe Val Ile Leu

85 90 95

Thr Thr Phe Leu Ile Pro Ile Cys Ile Ser Val Gly Trp Ser Gly Met

100 105 110

Arg Ser Tyr Gly Lys Glu Tyr Ile Thr Ala Phe Leu Ile Arg Glu Phe

115 120 125

Leu Met Ile Ala Val Phe Arg Met Leu Asp Leu Leu Leu Phe Tyr Val

130 135 140

Phe Pro Glu Ser Val Pro Ile Pro Met Phe Ile Ile Ile Gly Val Trp

145 150 155 160

Gly Ser Arg Gln Arg Lys Ile Lys Ala Ala Tyr Gln Phe Phe Leu Tyr

165 170 175

Thr Leu Leu Gly Ser Leu Phe Met Leu Leu Ala Ile Leu Leu Ile Leu

180 185 190

Phe Gln Thr Gly Thr Thr Asp Leu Gln Ile Ser Leu Thr Thr Glu Phe

195 200 205

Ser Glu Arg Arg Gln Ile Phe Leu Trp Ile Ala Ser Phe Ala Ser Phe

210 215 220

Ala Val Lys Val Pro Met Val Pro Val His Ile Trp Leu Pro Glu Ala

225 230 235 240

His Val Glu Ala Pro Thr Ala Gly Ser Val Ile Leu Ala Gly Ile Pro

245 250 255

Leu Lys Phe Gly Thr His Gly Phe Leu Arg Phe Ser Ile Pro Met Phe

260 265 270

Pro Glu Ala Thr Leu Cys Ser Thr Pro Phe Ile Tyr Thr Leu Ser Ala

275 280 285

Ile Ala Ile Ile Tyr Thr Ser Leu Thr Thr Ser Arg Gln Ile Asp Leu

290 295 300

Lys Lys Ile Ile Ala Tyr Ser Ser Val Ala His Met Asn Leu Val Thr

305 310 315 320

Ile Gly Met Phe Ser Pro Asn Ile Gln Gly Ile Gly Gly Ser Ile Leu

325 330 335

Pro Met Leu Ser His Gly Leu Val Pro Ser Ala Leu Phe Leu Cys Val

340 345 350

Gly Val Leu Tyr Asp Arg His Lys Thr Arg Leu Val Arg Tyr Tyr Gly

355 360 365

Gly Leu Val Ser Thr Met Pro Asn Leu Ser Thr Ile Phe Phe Ser Phe

370 375 380

Thr Leu Ala Asn Met Ser Ser Pro Gly Thr Ser Ser Phe Ile Gly Glu

385 390 395 400

Phe Leu Ile Leu Val Gly Ala Phe Gln Arg Asn Ser Leu Val Ala Thr

405 410 415

Leu Ala Ala Leu Gly Met Ile Leu Gly Ala Ala Tyr Ser Leu Trp Leu

420 425 430

Tyr Asn Arg Val Val Ser Gly Asn Leu Lys Pro Asp Phe Leu His Lys

435 440 445

Phe Ser Asp Ser Asn Gly Arg Glu Val Ser Ile Phe Ile Pro Phe Leu

450 455 460

Val Gly Leu Val Arg Met Gly Val His Pro Lys Val Phe Pro Asp Cys

465 470 475 480

Met His Thr Ser Val Ser Asn Leu Val Gln His Gly Lys Phe His

485 490 495

<210> 6

<211> 25

<212> DNA

<213> 人工序列

<400> 6

tccgaattcg gtgttttata tgacc 25

<210> 7

<211> 26

<212> DNA

<213> 人工序列

<400> 7

gcaagcttat gaaatttgcc atgttg 26

Claims (10)

1.一种融合蛋白，其特征在于，包含钥孔血蓝蛋白片段及目的蛋白。

2.根据权利要求1所述的融合蛋白，其特征在于，所述的钥孔血蓝蛋白片段包含钥孔血蓝蛋白第1-245位氨基酸残基。

3.根据权利要求1所述的融合蛋白，其特征在于，所述的钥孔血蓝蛋白片段包含SEQ IDNO:1所示的第1-245位氨基酸残基。

4.根据权利要求1所述的融合蛋白，其特征在于，所述的目的蛋白为植物线粒体膜蛋白或其免疫原性片段。

5.根据权利要求1所述的融合蛋白，其特征在于，所述的目的蛋白的序列如SEQ ID NO:5所示。

6.根据权利要求1所述的融合蛋白，其特征在于，所述的钥孔血蓝蛋白片段连接至所述目的蛋白的N端和/或C端。

7.编码权利要求1～6任一项所述的融合蛋白的核苷酸。

8.根据权利要求7所述的核苷酸，其特征在于，包含SEQ ID NO:3所述的核苷酸序列。

9.权利要求1～6任一项所述的融合蛋白在制备针对特异性目的蛋白多克隆抗体中的应用。

10.权利要求1～6任一项所述的融合蛋白在作为疫苗或药物的应用。