CN102000329A - 一种改造的非致病细菌来源的鞭毛素粘膜佐剂及制备方法 - Google Patents
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Abstract
本发明提供了一种优化改造的非致病细菌来源的鞭毛素粘膜佐剂及其制备方法，将鞭毛素粘膜佐剂去除鞭毛蛋白的主要抗原活性区，将其替换为目的抗原，加入免疫原性低的柔性结构，用这种方法得到的改造的鞭毛素粘膜佐剂的基因序列为SEQ ID NO：1-11，氨基酸序列为SEQ ID NO：22-32。本发明的佐剂降低了其可能具有的潜在的危险性、其自身的抗原性、免疫原性以及由其引发的炎症反应和毒性；提高了抗原递呈效果；通过添加低免疫原性的柔性结构，维持其三维结构与天然的鞭毛素蛋白相类似，从而维持其佐剂活性。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种优化改造的非致病细菌来源的鞭毛素粘膜佐剂及其制备方法。
背景技术
现在已知致病菌来源的鞭毛素蛋白具有免疫佐剂效果，它是通过鞭毛素蛋白与Toll样受体(Toll-like receptors，TLRs)5相结合，引发NF-κB途径引发先天性免疫，进而引发特异性免疫。通过鞭毛素蛋白与目的蛋白混合或融合后免疫，可以显著提高对目的抗原的免疫应答，并且可达到抵抗带有目的抗原的病原微生物的效果。但是因其来源于致病菌，可能具有潜在的危险性，并且它还可以引起炎症反应，产生大量针对鞭毛素自身的免疫反应，导致可能的耐受性以及其它可能的免疫副反应。
发明内容
本发明为克服上述技术缺陷，提供了一种优化改造的非致病细菌来源的鞭毛素粘膜佐剂，该佐剂是通过改造一种来源于正常共生菌株的鞭毛素蛋白并且在保证其佐剂活性的前提下降低它的抗原性、免疫原性以及由其引发的炎症反应。
为实现上述目的，采用如下技术方案：
本发明的一种改造的非致病细菌来源的鞭毛素粘膜佐剂去除了鞭毛蛋白的主要抗原活性区。
所述非致病细菌包括大肠杆菌K12株。
所述抗原活性区包括高变区。
所述鞭毛素粘膜佐剂还含有柔性结构，所述柔性结构免疫原性低。
所述柔性结构包括人IgG3铰链序列。
所述抗原活性区替换为目的抗原。
所述目的抗原包括p24抗原。
所述佐剂包括如下的佐剂，该佐剂的
基因序列如SEQ ID：1或氨基酸序列如SEQ ID：22，
基因序列如SEQ ID：2或氨基酸序列如SEQ ID：23，
基因序列如SEQ ID：3或氨基酸序列如SEQ ID：24，
基因序列如SEQ ID：4或氨基酸序列如SEQ ID：25，
基因序列如SEQ ID：5或氨基酸序列如SEQ ID：26，
基因序列如SEQID：6或氨基酸序列如SEQ ID：27，
基因序列如SEQ ID：7或氨基酸序列如SEQ ID：28，
基因序列如SEQ ID：8或氨基酸序列如SEQ ID：29，
基因序列如SEQ ID：9或氨基酸序列如SEQ ID：30，
基因序列如SEQ ID：10或氨基酸序列如SEQ ID：31，
基因序列如SEQ ID：11或氨基酸序列如SEQ ID：32。
本发明的一种改造的非致病细菌来源的鞭毛素粘膜佐剂的制备方法是去除鞭毛蛋白片段中的主要抗原活性区。
选择的非致病细菌包括大肠杆菌K12株。
所述抗原活性区包括高变区。
在所述鞭毛蛋白片段中增加柔性结构，所述柔性结构免疫原性低。
所述柔性结构包括人IgG3铰链序列。
将所述抗原活性区替换为目的抗原。
所述目的抗原包括p24抗原。
本发明的一个优选实施例为选择人正常共生菌株——大肠杆菌K12菌株MG1655亚株的鞭毛素蛋白(简称KF)为目标，确认其佐剂活性；通过构建不同的克隆，通过ELISA(酶联免疫吸附检测法)确定其主要的抗原性区为其内部的高变区；通过去除其高变区并且添加柔性结构，确认去除高变区免疫原性大大降低，即高变区也是主要的免疫原性区，并且柔性结构没有高的免疫原性；通过将目的抗原替换高变区，并添加低免疫原性的柔性结构，得到保留佐剂活性的目的蛋白。
与现有技术相比，本发明具有如下有益效果：
通过选择来源于正常共生菌株的鞭毛素蛋白，从而降低可能具有的潜在的危险性；并且通过去除其主要的免疫原性区及抗原活性区，降低它的抗原性、免疫原性以及由其引发的炎症反应；通过将目的抗原添加到鞭毛素蛋白内部，提高抗原递呈效果；通过添加低免疫原性的柔性结构，维持其三维结构与天然的鞭毛素蛋白相类似，从而维持其佐剂活性。
附图说明
图1是KF不同区段的克隆的构建及其抗原蛋白的表达，其中，图1-1是构建KF不同区段的克隆的方案，图1-2是KF不同区段的抗原蛋白的聚丙烯酰胺凝胶电泳(PAGE)电泳图；
图2是通过酶联免疫吸附检测法(ELISA)确定主要的抗原性区段为其内部的高变区的结果图，其中，图2-1是柱状图，图2-2是点分布图；
图3是KF结构改造方案及其改造得到的片段的鉴定结果，其中，图3-1是KF片段改造方案示意图，图3-2是KF片段改造后得到的片段的示意图，
图3-3是KF D1、D2、D3的SDS-PAGE及Western blot的结果图；图3-4是KFD-p242A、2B、2C、2D、3A、3B、3C和3D的SDS-PAGE及Westernblot的结果图；
图4是KFD1、D2、D3的佐剂活性及抗原性鉴定的结果图；其中，图4-1是以Caco-2细胞为模式细胞通过IL-8分泌水平，比较KF、KFD与TLR5作用能力强弱，图4-2是KFD+p24的10周免疫实验结果的分析图；
图5是KFD-p24的佐剂活性及抗原性鉴定的结果图，其中，图5-1，图5-2以Caco 2细胞为模式细胞通过IL-8分泌水平，比较KF、KFD-p24与TLR5作用能力强弱，图5-3，图5-4，图5-5是KFD-p24免疫实验(0，4，8周免疫)相关结果图；图5-3，图5-4是改造前后免疫所产生的针对目的抗原的抗体(anti-p24IgG)与针对佐剂自身的抗体(anti-KF IgG)的比例；图5-5是全面比较全长KF与改造克隆的代表KFD-p243D的粘膜免疫效果，即不同部位的IgA抗体水平的比较；
图6是小鼠滴鼻免疫后短期内产生的细胞因子的实验数据分析图；其中，图6-1，图6-2，图6-3分别是小鼠滴鼻免疫后短期内产生的IL-6、TNF-α和KC的实验数据分析图；
图7是小鼠滴鼻免疫短期毒性效应数据分析图。
具体实施方式
为使本发明更加容易理解，下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
下面实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆实验手册中所述的条件。
实施例1：构建KF不同区段的克隆及其抗原蛋白的表达
构建KF不同区段的克隆片段如图1所示，其中，图1-1是构建KF不同区段的克隆的方案，包括KF1、KF2、KF3、KFD1和SF3。其中，KFD1是去掉了高变区并插入NheI酶切位点的片段，KF1是KF的高变区片段，KF2是KF高变区中心片段，KF3是在高变区插入KFD1中NheI酶切位点后形成的KF恢复克隆，SF3是将高变区替换为SF高变区并插入NheI酶切位点的片段。
KF是指大肠杆菌K12株MG1655亚株的鞭毛蛋白(E.coli K12 strainMG1655substrain flagellin)，其基因序列号为ID-949101。SF为沙门氏菌的鞭毛蛋白(Salmonalla flagellin)，其基因序列号为ID-1070204。
以抽提所得细菌基因组为模板，选择pET30a作为载体，KF和SF的引物分别如下，其中划线部位为酶切位点：
KF：上游引物：5’-TATACATATGGCACAAGTCATTAATAC-3’NdeI
下游引物：5’-ATATCTCGAGACCCTGCAGCAGAGACAG-3’XhoI
SF：上游引物：5’-CGCGCATATGGCACAAGTCATTAATACAAACA-3’NdeI
下游引物：5’-CGGTCTCGAGACGCAGTAAAGAGAGGACGTTTTG-3’XhoI
克隆表达后得到的蛋白通过Ni柱纯化，超滤浓缩8-10倍后加PBS再超滤浓缩，反复4次；Triton法去内毒素；考马斯亮蓝法确定蛋白浓度；鲎试剂检测内毒素含量＜0.005Eu/μg蛋白，最后SDS-PAGE鉴定，其结果如图1-2所示，图1-2是KF不同区段的抗原蛋白的聚丙烯酰胺凝胶电泳(PAGE)图。该图的结果显示得到的蛋白与预期蛋白的大小一致。
实施例2：确定KF的主要抗原性区段
图2是通过酶联免疫吸附检测法(ELISA)确定主要的抗原性区段为其内部的高变区的结果图。其中，图2-1比较了不同免疫策略所免疫小鼠血清中针对KF不同区段的抗体的比例有何异同，该图的结果说明不同免疫策略所免疫小鼠血清中针对KF不同区段的抗体的比例基本相同。图2-2比较了免疫策略所免疫小鼠血清中针对KF不同区段的抗体的比例有何特征，该图的结果说明恢复性克隆KF3的抗原性与KF基本相同。高变区KF1的抗原性≈90％，高变区的中心部位(KF2)仅占其总抗原性的约1/3，保守区KFD1的抗原性≈10％。高变区替换为SF的抗原性与其缺失(KFD1)的抗原性相似。KF抗原性主要集中在高变区。由图2可知，KF抗原性主要集中在高变区，即高变区也是主要的免疫原性区。
实施例3：KF结构改造方案及其改造得到的片段的鉴定
先构造保守区KFD片段，即在KF片段的基础上，将KF高变区去掉，将KF的N端(KFN)及C端(KFC端)连接起来，得到pET28a-KFD片段；然后将p24片段插入pET28a-KFD中，得到pET28a-KFD-p24。设计了三组KFN和四组p24，选择了两组KFN和四组p24进行组合改造，得到八个改造片段。具体如下：
图3是KF结构改造方案及其改造得到的片段的鉴定结果，KF片段改造方案如图3-1所示，其中，KFN片段改造所用的引物如下：
KF N1段
上游引物(NcoI)
Up-N：5-GGCGTCCATGGCACAAGTCATTAATAC-3 27bp
下游引物(BamHI)：
Low-N1：
5-GGCAAGGATCCGTCGTCGTAGCGCTAGCATCAAGGCCAAGAGTTTTA
G-3 48bp
产物长度541bp；
KF N2段：
上游引物(NcoI)
Up-N：5-GGCGTCCATGGCACAAGTCATTAATAC-3 27bp
下游引物(BamHI)：
Low-N2：
5-TACTAGGATCCGTCGTTCCAGATGTGTGAGTTGTGTCACCAAGTGGAGTGCTAGCATCAAGGCCAAGAGTTTTAG-3 75bp
产物长度568bp；
KF N3段：
上游引物(NcoI)
U p-N：5-GGCGTCCATGGCACAAGTCATTAATAC-3 27bp
下游引物(BamHI)：
Low-N3：
5-TACTAGGATCCGTCGTGCTAGCTCCAGATGTGTGAGTTGTGTCACCAAGTGGAGTATCAAGGCCAAGAGTTTTAG-3 75bp
产物长度568bp；
KFC段：直接酶切KF，根据BamHI和XhoI酶切位点进行连接。
KFD的三个不同片段，简单来说，如下：
KFD1＝NcoI-N1-BamHI-C-XhoI；
KFD2＝NcoI-N2-BamHI-C-XhoI；
KFD3＝NcoI-N3-BamHI-C-XhoI。
进一步，p24的改造具体如下：
p24扩增的引物如下：
Up p24-1NheI：
5’GCATAGCTAGCCCTATAGTGCAGAACCTCCA-3’31bp；
Low-p24-1BamHI：
5’TATGCGGATCCGTCGTCAAAACTCTTGCTTTATGGC-3’36bp；
Up p24-2NheI：
5’ATCGAGCTAGCACACCTCTTGGTGATACTACACACACATCAGGACCTATAGTGCAGAACCTCCA-3’64bp ；
Low-p24-2BamHI：
5’TATGCGGATCCGTCGTTCCAGATGTGTGAGTTGTGTCACCAAGTGGAGTCAAAACTCTTGCTTTATGGC-3’69bp
简单明了的来说，p24A、p24B、p24C、p24D的结构如下：
p24A：Up p24-1+Low-p24-1；
p24B：Up p24-1+Low-p24-2；
p24C：Up p24-2+Low-p24-2；
p24D：Up p24-2+Low-p24-1。
高变区替换改造为KFD-p24，总共有8个片段：KFD-p242A、KFD-p242B、KFD-p242C、KFD-p242D、KFD-p243A、KFD-p243B、KFD-p243C和KFD-p243D，其结构如下：
KFD-p242A：KFD2+p24A
KFD-p242B：KFD2+p24B
KFD-p242C：KFD2+p24C
KFD-p242D：KFD2+p24D
KFD-p243A：KFD3+p24A
KFD-p243B：KFD3+p24B
KFD-p243C：KFD3+p24C
KFD-p243D：KFD3+p24D
图3-2是KF片段改造后得到的片段的示意图所得改造后片段的序列见序列表。
KF D1、D2、D3的SDS-PAGE及Western blot的结果见图3-3；KFD-p242A、2B、2C、2D、3A、3B、3C和3D的SDS-PAGE及Western blot的结果图见图3-4，标号1-9依次代表KFD-p242A、2B、2C、2D、marker、3A、3B、3C、3D。其中，所用marker为Ferment SM0661。预期片段大小KFD-p242A、2B、2C、2D分别为：55KD、56KD、57KD、56KD；KFD-p243A、3B、3C、3D分别为：56KD、57KD、58KD、57KD。由图可知，所得片段与预期片段大小一致。
实施例4：KFD1、D2、D3的佐剂活性及抗原性鉴定
图4是KFD1、D2、D3的佐剂活性及抗原性鉴定的结果图，其中，图4-1是以Caco-2细胞为模式细胞通过IL-8分泌水平，比较KF、KFD1、D2、D3与TLR5作用能力强弱。即KF、KFD1、D2、D3的佐剂活性在细胞水平的鉴定的结果图；分别用KFD1、KFD2、KFD3和KF刺激Caco-2细胞，测量上清中IL-8含量。具体操作：Caco-2细胞于24孔板中培养6-8天至其细胞间界限分隔明显后，用无血清1640培养基饥饿12h后，加无血清1640培养基稀释样品0.5ml刺激6h后去细胞上清，2000rpm 10min离心取上清，-80C冻存备用。该图表明，KF高变区缺失影响到佐剂活性，在较低浓度(10ng/ml)时，全长的鞭毛素KF对Caco-2细胞刺激作用较强，但在较高浓度下(1000ng/ml)改造克隆的刺激作用已经超过KF。
如图所示，图4-2是KFD+p24的免疫实验(0，4，8周滴鼻免疫)第10周结果；即KFD1、D2、D3的佐剂活性及抗原性鉴定的结果图。实验结果说明在保留了佐剂活性的条件下KF自身抗原性明显下降，且不同改造降低的幅度基本相同，均降低了10倍以上。
实施例5：KFD-p242A，2B，2C，2D，3A，3B，3C，3D的佐剂活性及抗原性鉴定
图5是KFD-p24的佐剂活性及抗原性鉴定的结果图，其中，图5-1，图5-2以Caco-2细胞为模式细胞通过IL-8分泌水平，比较KF、KFD-p24与TLR5作用能力强弱，即KF、KFD-p24的佐剂活性在细胞水平的鉴定的结果图；分别用KF和KF-p24刺激Caco-2细胞，测量上清中IL-8含量。具体操作同实施例4。该结果说明：KF高变区替换影响到佐剂活性，在较低浓度(1ng/ml-100ng/ml)时，全长的鞭毛素KF对Caco-2细胞刺激作用较强，但在较高浓度下(1000ng/ml)改造克隆的刺激作用可以与KF相比较。
图5-3，图5-4，图5-5是KFD-p24免疫实验(0，4，8周滴鼻免疫)第10周结果。
图5-3，图5-4是改造前后免疫所产生的针对目的抗原的抗体(anti-p24IgG)与针对佐剂自身的抗体(anti-KF IgG)的比例。图5-3是观察KFD-p24佐剂效应及其佐剂活性部分的抗原性。由以上结果看出，所有的高变区替换的克隆免疫效果均不弱于利用全长KF与同样量的p24混合免疫。并且有些克隆的免疫活性可以与KF与p24直接融合的克隆KF-p24相比较。通过将KF高变区替换为p24，佐剂活性区域的自身抗原性大大降低。图5-4是具体比较KF+p24，KF-p24以及KFD-p24的免疫原性部位；对于KFD-p24来说，抗原活性部位主要在p24部位，对目的抗原p24的抗体是对佐剂活性区域的抗体滴度的约20-200倍。对于直接融合的蛋白KF-p24来说对目的抗原p24的抗体是对佐剂活性区域的抗体滴度的约0.5-1倍。对于混合免疫KF与p24来说对目的抗原p24的抗体是对佐剂活性区域的抗体滴度的约0.2倍。实验结果说明相对于混合免疫KF与p24来说，免疫KFD-p24产生的抗体中，对于佐剂活性区域的抗体减少了两个数量级，大大提高了免疫反应的特异性。
图5-5是全面比较全长KF与改造克隆的代表KFD-p243D的粘膜免疫效果，即不同部位的IgA抗体水平的比较；以KFD-p243D为代表比较不同免疫策略产生的对于目的抗原p24的IgA抗体水平；实验结果说明：从各部位的IgA水平来看，同样剂量的KFD-p243D均好于KF，而且可以同融合相媲美。
实施例6：小鼠细胞因子实验
小鼠提前12小时开始禁食不禁水，用戊巴比妥那麻醉后滴鼻免疫。取样时，拔眼球取血，脱颈处死后，解剖咽喉部，用PBS从气管处灌洗500μL两次，合并后8000rpm 5min离心取上清，-80C冻存备用。图6是小鼠滴鼻免疫后短期内产生的细胞因子的实验数据分析图；其中，图6-1，图6-2，图6-3分别是小鼠滴鼻免疫后短期内产生的IL-6、TNF-α和KC的实验数据分析图。KF高变区缺失及替换诱导产生TNF-α，IL-6，KC(IL-8)的能力比全长低，并且较快的恢复到静息水平(PBS组)，因此其潜在的炎症效应较弱。
实施例7：小鼠滴鼻免疫短期毒性效应
小鼠用戊巴比妥那麻醉后滴鼻免疫，免疫前及免疫后每天称重。体重指标是反映小鼠状态的敏感指标，通过体重反映了小鼠滴鼻免疫的短期毒性效应。图7是小鼠滴鼻免疫毒性炎症效应数据分析图；由图7可知，高变区缺失及替换与阴性对照组相似，对小鼠无显著影响。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
序列表
<110>中国科学院武汉病毒研究所
<160>32
<210>1
<211>827
<212>KFD1 DNA
<400>1
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatgctag cgctacgacg acggatccgc 540
tgaaagcgct ggacgatgct atcgcatctg tagacaaatt ccgttcttcc ctcggtgcgg 600
tgcaaaaccg tctggattcc gcggttacca acctgaacaa caccactacc aacctgtctg 660
aagcgcagtc ccgtattcag gacgccgact atgcgaccga agtgtccaat atgtcgaaag 720
cgcagatcat ccagcaggcc ggtaactccg tgttggcaaa agctaaccag gtaccgcagc 780
aggttctgtc tctgctgcag ggtctcgagc accaccacca ccaccac 827
<210>1
<211>854
<212>KFD2 DNA
<400>2
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatgctag cactccactt ggtgacacaa 540
ctcacacatc tggaacgacg gatccgctga aagcgctgga cgatgctatc gcatctgtag 600
acaaattccg ttcttccctc ggtgcggtgc aaaaccgtct ggattccgcg gttaccaacc 660
tgaacaacac cactaccaac ctgtctgaag cgcagtcccg tattcaggac gccgactatg 720
cgaccgaagt gtccaatatg tcgaaagcgc agatcatcca gcaggccggt aactccgtgt 780
tggcaaaagc taaccaggta ccgcagcagg ttctgtctct gctgcagggt ctcgagcacc 840
accaccacca ccac 854
<210>1
<211>854
<212>KFD3 DNA
<400>3
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatactcc acttggtgac acaactcaca 540
catctggagc tagcacgacg gatccgctga aagcgctgga cgatgctatc gcatctgtag 600
acaaattccg ttcttccctc ggtgcggtgc aaaaccgtct ggattccgcg gttaccaacc 660
tgaacaacac cactaccaac ctgtctgaag cgcagtcccg tattcaggac gccgactatg 720
cgaccgaagt gtccaatatg tcgaaagcgc agatcatcca gcaggccggt aactccgtgt 780
tggcaaaagc taaccaggta ccgcagcagg ttctgtctct gctgcagggt ctcgagcacc 840
accaccacca ccac 854
<210>1
<211>1514
<212>KFD-p24 2A DNA
<400>4
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatgctag ccctatagtg cagaacctcc 540
aggggcaaat ggtacatcag gccatatcac ctagaacttt aaatgcatgg gtaaaagtag 600
tagaagagaa ggctttcagc ccagaagtaa tacccatgtt ttcagcatta tcagaaggag 660
ccaccccaca agatttaaat accatgctaa acacagtggg gggacatcaa gcagccatgc 720
aaatgttaaa agagaccatc aatgaggaag ctgcagaatg ggatagattg catccagtgc 780
atgcagggcc tattgcacca ggccagatga gagaaccaag gggaagtgac atagcaggaa 840
ctactagtac ccttcaggaa caaataggat ggatgacaca taatccacct atcccagtag 900
gagaaatcta taaaagatgg ataatcctgg gattaaataa aatagtaaga atgtatagcc 960
ctaccagcat tctggacata agacaaggac caaaggaacc ctttagagac tatgtagacc 1020
gattctataa aactctaaga gccgagcaag cttcacaaga ggtaaaaaat tggatgacag 1080
aaaccttgtt ggtccaaaat gcgaacccag attgtaagac tattttaaaa gcattgggac 1140
caggagcgac actagaagaa atgatgacag catgtcaggg agtgggggga cccggccata 1200
aagcaagagt tttgacgacg gatccgctga aagcgctgga cgatgctatc gcatctgtag 1260
acaaattccg ttcttccctc ggtgcggtgc aaaaccgtct ggattccgcg gttaccaacc 1320
tgaacaacac cactaccaac ctgtctgaag cgcagtcccg tattcaggac gccgactatg 1380
cgaccgaagt gtccaatatg tcgaaagcgc agatcatcca gcaggccggt aactccgtgt 1440
tggcaaaagc taaccaggta ccgcagcagg ttctgtctct gctgcagggt ctcgagcacc 1500
accaccacca ccac 1514
<210>1
<211>1547
<212>KFD-p24 2B DNA
<400>5
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatgctag ccctatagtg cagaacctcc 540
aggggcaaat ggtacatcag gccatatcac ctagaacttt aaatgcatgg gtaaaagtag 600
tagaagagaa ggctttcagc ccagaagtaa tacccatgtt ttcagcatta tcagaaggag 660
ccaccccaca agatttaaat accatgctaa acacagtggg gggacatcaa gcagccatgc 720
aaatgttaaa agagaccatc aatgaggaag ctgcagaatg ggatagattg catccagtgc 780
atgcagggcc tattgcacca ggccagatga gagaaccaag gggaagtgac atagcaggaa 840
ctactagtac ccttcaggaa caaataggat ggatgacaca taatccacct atcccagtag 900
gagaaatcta taaaagatgg ataatcctgg gattaaataa aatagtaaga atgtatagcc 960
ctaccagcat tctggacata agacaaggac caaaggaacc ctttagagac tatgtagacc 1020
gattctataa aactctaaga gccgagcaag cttcacaaga ggtaaaaaat tggatgacag 1080
aaaccttgtt ggtccaaaat gcgaacccag attgtaagac tattttaaaa gcattgggac 1140
caggagcgac actagaagaa atgatgacag catgtcaggg agtgggggga cccggccata 1200
aagcaagagt tttgactcca cttggtgaca caactcacac atctggaacg acggatccgc 1260
tgaaagcgct ggacgatgct atcgcatctg tagacaaatt ccgttcttcc ctcggtgcgg 1320
tgcaaaaccg tctggattcc gcggttacca acctgaacaa caccactacc aacctgtctg 1380
aagcgcagtc ccgtattcag gacgccgact atgcgaccga agtgtccaat atgtcgaaag 1440
cgcagatcat ccagcaggcc ggtaactccg tgttggcaaa agctaaccag gtaccgcagc 1500
aggttctgtc tctgctgcag ggtctcgagc accaccacca ccaccac 1547
<210>1
<211>1580
<212>KFD-p24 2C DNA
<400>6
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatgctag cacacctctt ggtgatacta 540
cacacacatc aggacctata gtgcagaacc tccaggggca aatggtacat caggccatat 600
cacctagaac tttaaatgca tgggtaaaag tagtagaaga gaaggctttc agcccagaag 660
taatacccat gttttcagca ttatcagaag gagccacccc acaagattta aataccatgc 720
taaacacagt ggggggacat caagcagcca tgcaaatgtt aaaagagacc atcaatgagg 780
aagctgcaga atgggataga ttgcatccag tgcatgcagg gcctattgca ccaggccaga 840
tgagagaacc aaggggaagt gacatagcag gaactactag tacccttcag gaacaaatag 900
gatggatgac acataatcca cctatcccag taggagaaat ctataaaaga tggataatcc 960
tgggattaaa taaaatagta agaatgtata gccctaccag cattctggac ataagacaag 1020
gaccaaagga accctttaga gactatgtag accgattcta taaaactcta agagccgagc 1080
aagcttcaca agaggtaaaa aattggatga cagaaacctt gttggtccaa aatgcgaacc 1140
cagattgtaa gactatttta aaagcattgg gaccaggagc gacactagaa gaaatgatga 1200
cagcatgtca gggagtgggg ggacccggcc ataaagcaag agttttgact ccacttggtg 1260
acacaactca cacatctgga acgacggatc cgctgaaagc gctggacgat gctatcgcat 1320
ctgtagacaa attccgttct tccctcggtg cggtgcaaaa ccgtctggat tccgcggtta 1380
ccaacctgaa caacaccact accaacctgt ctgaagcgca gtcccgtatt caggacgccg 1440
actatgcgac cgaagtgtcc aatatgtcga aagcgcagat catccagcag gccggtaact 1500
ccgtgttggc aaaagctaac caggtaccgc agcaggttct gtctctgctg cagggtctcg 1560
agcaccacca ccaccaccac 1580
<210>1
<211>1547
<212>KFD-p24 2D DNA
<400>7
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatgctag cacacctctt ggtgatacta 540
cacacacatc aggacctata gtgcagaacc tccaggggca aatggtacat caggccatat 600
cacctagaac tttaaatgca tgggtaaaag tagtagaaga gaaggctttc agcccagaag 660
taatacccat gttttcagca ttatcagaag gagccacccc acaagattta aataccatgc 720
taaacacagt ggggggacat caagcagcca tgcaaatgtt aaaagagacc atcaatgagg 780
aagctgcaga atgggataga ttgcatccag tgcatgcagg gcctattgca ccaggccaga 840
tgagagaacc aaggggaagt gacatagcag gaactactag tacccttcag gaacaaatag 900
gatggatgac acataatcca cctatcccag taggagaaat ctataaaaga tggataatcc 960
tgggattaaa taaaatagta agaatgtata gccctaccag cattctggac ataagacaag 1020
gaccaaagga accctttaga gactatgtag accgattcta taaaactcta agagccgagc 1080
aagcttcaca agaggtaaaa aattggatga cagaaacctt gttggtccaa aatgcgaacc 1140
cagattgtaa gactatttta aaagcattgg gaccaggagc gacactagaa gaaatgatga 1200
cagcatgtca gggagtgggg ggacccggcc ataaagcaag agttttgacg acggatccgc 1260
tgaaagcgct ggacgatgct atcgcatctg tagacaaatt ccgttcttcc ctcggtgcgg 1320
tgcaaaaccg tctggattcc gcggttacca acctgaacaa caccactacc aacctgtctg 1380
aagcgcagtc ccgtattcag gacgccgact atgcgaccga agtgtccaat atgtcgaaag 1440
cgcagatcat ccagcaggcc ggtaactccg tgttggcaaa agctaaccag gtaccgcagc 1500
aggttctgtc tctgctgcag ggtctcgagc accaccacca ccaccac 1547
<210>1
<211>1547
<212>KFD-p24 3A DNA
<400>8
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatactcc acttggtgac acaactcaca 540
catctggagc tagccctata gtgcagaacc tccaggggca aatggtacat caggccatat 600
cacctagaac tttaaatgca tgggtaaaag tagtagaaga gaaggctttc agcccagaag 660
taatacccat gttttcagca ttatcagaag gagccacccc acaagattta aataccatgc 720
taaacacagt ggggggacat caagcagcca tgcaaatgtt aaaagagacc atcaatgagg 780
aagctgcaga atgggataga ttgcatccag tgcatgcagg gcctattgca ccaggccaga 840
tgagagaacc aaggggaagt gacatagcag gaactactag tacccttcag gaacaaatag 900
gatggatgac acataatcca cctatcccag taggagaaat ctataaaaga tggataatcc 960
tgggattaaa taaaatagta agaatgtata gccctaccag cattctggac ataagacaag 1020
gaccaaagga accctttaga gactatgtag accgattcta taaaactcta agagccgagc 1080
aagcttcaca agaggtaaaa aattggatga cagaaacctt gttggtccaa aatgcgaacc 1140
cagattgtaa gactatttta aaagcattgg gaccaggagc gacactagaa gaaatgatga 1200
cagcatgtca gggagtgggg ggacccggcc ataaagcaag agttttgacg acggatccgc 1260
tgaaagcgct ggacgatgct atcgcatctg tagacaaatt ccgttcttcc ctcggtgcgg 1320
tgcaaaaccg tctggattcc gcggttacca acctgaacaa caccactacc aacctgtctg 1380
aagcgcagtc ccgtattcag gacgccgact atgcgaccga agtgtccaat atgtcgaaag 1440
cgcagatcat ccagcaggcc ggtaactccg tgttggcaaa agctaaccag gtaccgcagc 1500
aggttctgtc tctgctgcag ggtctcgagc accaccacca ccaccac 1547
<210>1
<211>1580
<212>KFD-p24 3B DNA
<400>9
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatactcc acttggtgac acaactcaca 540
catctggagc tagccctata gtgcagaacc tccaggggca aatggtacat caggccatat 600
cacctagaac tttaaatgca tgggtaaaag tagtagaaga gaaggctttc agcccagaag 660
taatacccat gttttcagca ttatcagaag gagccacccc acaagattta aataccatgc 720
taaacacagt ggggggacat caagcagcca tgcaaatgtt aaaagagacc atcaatgagg 780
aagctgcaga atgggataga ttgcatccag tgcatgcagg gcctattgca ccaggccaga 840
tgagagaacc aaggggaagt gacatagcag gaactactag tacccttcag gaacaaatag 900
gatggatgac acataatcca cctatcccag taggagaaat ctataaaaga tggataatcc 960
tgggattaaa taaaatagta agaatgtata gccctaccag cattctggac ataagacaag 1020
gaccaaagga accctttaga gactatgtag accgattcta taaaactcta agagccgagc 1080
aagcttcaca agaggtaaaa aattggatga cagaaacctt gttggtccaa aatgcgaacc 1140
cagattgtaa gactatttta aaagcattgg gaccaggagc gacactagaa gaaatgatga 1200
cagcatgtca gggagtgggg ggacccggcc ataaagcaag agttttgact ccacttggtg 1260
acacaactca cacatctgga acgacggatc cgctgaaagc gctggacgat gctatcgcat 1320
ctgtagacaa attccgttct tccctcggtg cggtgcaaaa ccgtctggat tccgcggtta 1380
ccaacctgaa caacaccact accaacctgt ctgaagcgca gtcccgtatt caggacgccg 1440
actatgcgac cgaagtgtcc aatatgtcga aagcgcagat catccagcag gccggtaact 1500
ccgtgttggc aaaagctaac caggtaccgc agcaggttct gtctctgctg cagggtctcg 1560
agcaccacca ccaccaccac 1580
<210>1
<211>1613
<212>KFD-p24 3C DNA
<400>10
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatactcc acttggtgac acaactcaca 540
catctggagc tagcacacct cttggtgata ctacacacac atcaggacct atagtgcaga 600
acctccaggg gcaaatggta catcaggcca tatcacctag aactttaaat gcatgggtaa 660
aagtagtaga agagaaggct ttcagcccag aagtaatacc catgttttca gcattatcag 720
aaggagccac cccacaagat ttaaatacca tgctaaacac agtgggggga catcaagcag 780
ccatgcaaat gttaaaagag accatcaatg aggaagctgc agaatgggat agattgcatc 840
cagtgcatgc agggcctatt gcaccaggcc agatgagaga accaagggga agtgacatag 900
caggaactac tagtaccctt caggaacaaa taggatggat gacacataat ccacctatcc 960
cagtaggaga aatctataaa agatggataa tcctgggatt aaataaaata gtaagaatgt 1020
atagccctac cagcattctg gacataagac aaggaccaaa ggaacccttt agagactatg 1080
tagaccgatt ctataaaact ctaagagccg agcaagcttc acaagaggta aaaaattgga 1140
tgacagaaac cttgttggtc caaaatgcga acccagattg taagactatt ttaaaagcat 1200
tgggaccagg agcgacacta gaagaaatga tgacagcatg tcagggagtg gggggacccg 1260
gccataaagc aagagttttg actccacttg gtgacacaac tcacacatct ggaacgacgg 1320
atccgctgaa agcgctggac gatgctatcg catctgtaga caaattccgt tcttccctcg 1380
gtgcggtgca aaaccgtctg gattccgcgg ttaccaacct gaacaacacc actaccaacc 1440
tgtctgaagc gcagtcccgt attcaggacg ccgactatgc gaccgaagtg tccaatatgt 1500
cgaaagcgca gatcatccag caggccggta actccgtgtt ggcaaaagct aaccaggtac 1560
cgcagcaggt tctgtctctg ctgcagggtc tcgagcacca ccaccaccac cac 1613
<210>1
<211>1580
<212>KFD-p24 3D DNA
<400>11
ccatggcaca agtcattaat accaacagcc tctcgctgat cactcaaaat aatatcaaca 60
agaaccagtc tgcgctgtcg agttctatcg agcgtctgtc ttctggcttg cgtattaaca 120
gcgcgaagga tgacgcagcg ggtcaggcga ttgctaaccg tttcacctct aacattaaag 180
gcctgactca ggcggcccgt aacgccaacg acggtatctc cgttgcgcag accaccgaag 240
gcgcgctgtc cgaaatcaac aacaacttac agcgtgtgcg tgaactgacg gtacaggcca 300
ctaccggtac taactctgag tctgatctgt cttctatcca ggacgaaatt aaatcccgtc 360
tggatgaaat tgaccgcgta tctggtcaga cccagttcaa cggcgtgaac gtgctggcaa 420
aaaatggctc catgaaaatc caggttggcg caaatgataa ccagactatc actatcgatc 480
tgaagcagat tgatgctaaa actcttggcc ttgatactcc acttggtgac acaactcaca 540
catctggagc tagcacacct cttggtgata ctacacacac atcaggacct atagtgcaga 600
acctccaggg gcaaatggta catcaggcca tatcacctag aactttaaat gcatgggtaa 660
aagtagtaga agagaaggct ttcagcccag aagtaatacc catgttttca gcattatcag 720
aaggagccac cccacaagat ttaaatacca tgctaaacac agtgggggga catcaagcag 780
ccatgcaaat gttaaaagag accatcaatg aggaagctgc agaatgggat agattgcatc 840
cagtgcatgc agggcctatt gcaccaggcc agatgagaga accaagggga agtgacatag 900
caggaactac tagtaccctt caggaacaaa taggatggat gacacataat ccacctatcc 960
cagtaggaga aatctataaa agatggataa tcctgggatt aaataaaata gtaagaatgt 1020
atagccctac cagcattctg gacataagac aaggaccaaa ggaacccttt agagactatg 1080
tagaccgatt ctataaaact ctaagagccg agcaagcttc acaagaggta aaaaattgga 1140
tgacagaaac cttgttggtc caaaatgcga acccagattg taagactatt ttaaaagcat 1200
tgggaccagg agcgacacta gaagaaatga tgacagcatg tcagggagtg gggggacccg 1260
gccataaagc aagagttttg acgacggatc cgctgaaagc gctggacgat gctatcgcat 1320
ctgtagacaa attccgttct tccctcggtg cggtgcaaaa ccgtctggat tccgcggtta 1380
ccaacctgaa caacaccact accaacctgt ctgaagcgca gtcccgtatt caggacgccg 1440
actatgcgac cgaagtgtcc aatatgtcga aagcgcagat catccagcag gccggtaact 1500
ccgtgttggc aaaagctaac caggtaccgc agcaggttct gtctctgctg cagggtctcg 1560
agcaccacca ccaccaccac 1580
<210>1
<211>27
<212>KF N1(NcoI)人工序列
<400>12
ggcgtccatg gcacaagtca ttaatac 27
<210>1
<211>48
<212>KF Low-N1(BamHI)人工序列
<400>13
ggcaaggatc cgtcgtcgta gcgctagcat caaggccaag agttttag 48
<210>1
<211>27
<212>KF N2(NcoI)人工序列
<400>14
ggcgtccatg gcacaagtca ttaatac 27
<210>1
<211>75
<212>KF Low-N2(BamHI)人工序列
<400>15
tactaggatc cgtcgttcca gatgtgtgag ttgtgtcacc aagtggagtg ctagcatcaa 60
ggccaagagt tttag 75
<210>1
<211>27
<212>KF N3(NcoI)人工序列
<400>16
ggcgtccatg gcacaagtca ttaatac 27
<210>1
<211>75
<212>KF Low-N3(BamHI)人工序列
<400>17
tactaggatc cgtcgtgcta gctccagatg tgtgagttgt gtcaccaagt ggagtatcaa 60
ggccaagagt tttag 75
<210>1
<211>31
<212>Up p24-1(NheI)人工序列
<400>18
gcatagctag ccctatagtg cagaacctcc a 31
<210>1
<211>36
<212>Low-p24-1(BamHI)人工序列
<400>19
tatgcggatc cgtcgtcaaa actcttgctt tatggc 36
<210>1
<211>64
<212>Up p24-2(NheI)人工序列
<400>20
atcgagctag cacacctctt ggtgatacta cacacacatc aggacctata gtgcagaacc 60
tcca 64
<210>1
<211>69
<212>Low-p24-2(BamHI)人工序列
<400>21
tatgcggatc cgtcgttcca gatgtgtgag ttgtgtcacc aagtggagtc aaaactcttg 60
ctttatggc 69
<210>1
<211>274
<212>PRT KFD1
<400>22
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Ala Ser Ala Thr Thr Thr
165 170 175
Asp Pro Leu Lys Ala Leu Asp Asp Ala Ile Ala Ser Val Asp Lys Phe
180 185 190
Arg Ser Ser Leu Gly Ala Val Gln Asn Arg Leu Asp Ser Ala Val Thr
195 200 205
Asn Leu Asn Asn Thr Thr Thr Asn Leu Ser Glu Ala Gln Ser Arg Ile
210 215 220
Gln Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln
225 230 235 240
Ile Ile Gln Gln Ala Gly Asn Ser Val Leu Ala Lys Ala Asn Gln Val
245 250 255
Pro Gln Gln Val Leu Ser Leu Leu Gln Gly Leu Glu His His His His
260 265 270
His His
<210>1
<211>274
<212>PRT KFD2
<400>23
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Ala Ser Thr Pro Leu Gly
165 170 175
Asp Thr Thr His Thr Ser Gly Thr Thr Asp Pro Leu Lys Ala Leu Asp
180 185 190
Asp Ala Ile Ala Ser Val Asp Lys Phe Arg Ser Ser Leu Gly Ala Val
195 200 205
Gln Asn Arg Leu Asp Ser Ala Val Thr Asn Leu Asn Asn Thr Thr Thr
210 215 220
Asn Leu Ser Glu Ala Gln Ser Arg Ile Gln Asp Ala Asp Tyr Ala Thr
225 230 235 240
Glu Val Ser Asn Met Ser Lys Ala Gln Ile Ile Gln Gln Ala Gly Asn
245 250 255
Ser Val Leu Ala Lys Ala Asn Gln Val Pro Gln Gln Val Leu Ser Leu
260 265 270
Leu Gln Gly Leu Glu His His His His His His
275 280
<210>1
<211>283
<212>PRT KFD3
<400>24
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Thr Pro Leu Gly Asp Thr
165 170 175
Thr His Thr Ser Gly Ala Ser Thr Thr Asp Pro Leu Lys Ala Leu Asp
180 185 190
Asp Ala Ile Ala Ser Val Asp Lys Phe Arg Ser Ser Leu Gly Ala Val
195 200 205
Gln Asn Arg Leu Asp Ser Ala Val Thr Asn Leu Asn Asn Thr Thr Thr
210 215 220
Asn Leu Ser Glu Ala Gln Ser Arg Ile Gln Asp Ala Asp Tyr Ala Thr
225 230 235 240
Glu Val Ser Asn Met Ser Lys Ala Gln Ile Ile Gln Gln Ala Gly Asn
245 250 255
Ser Val Leu Ala Lys Ala Asn Gln Val Pro Gln Gln Val Leu Ser Leu
260 265 270
Leu Gln Gly Leu Glu His His His His His His
275 280
<210>1
<211>503
<212>PRT KFD-p24 2A
<400>25
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Ala Ser Pro Ile Val Gln
165 170 175
Asn Leu Gln Gly Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu
180 185 190
Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val
195 200 205
Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu
210 215 220
Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met
225 230 235 240
Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu His
245 250 255
Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg
260 265 270
Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly
275 280 285
Trp Met Thr His Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg
290 295 300
Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr
305 310 315 320
Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr
325 330 335
Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Ser Gln Glu
340 345 350
Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro
355 360 365
Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Gly Ala Thr Leu Glu
370 375 380
Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala
385 390 395 400
Arg Val Leu Thr Thr Asp Pro Leu Lys Ala Leu Asp Asp Ala Ile Ala
405 410 415
Ser Val Asp Lys Phe Arg Ser Ser Leu Gly Ala Val Gln Asn Arg Leu
420 425 430
Asp Ser Ala Val Thr Asn Leu Asn Asn Thr Thr Thr Asn Leu Ser Glu
435 440 445
Ala Gln Ser Arg Ile Gln Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn
450 455 460
Met Ser Lys Ala Gln Ile Ile Gln Gln Ala Gly Asn Ser Val Leu Ala
465 470 475 480
Lys Ala Asn Gln Val Pro Gln Gln Val Leu Ser Leu Leu Gln Gly Leu
485 490 495
Glu His His His His His His
500
<210>1
<211>514
<212>PRT KFD-p24 2B
<400>26
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Ala Ser Pro Ile Val Gln
165 170 175
Asn Leu Gln Gly Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu
180 185 190
Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val
195 200 205
Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu
210 215 220
Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met
225 230 235 240
Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu His
245 250 255
Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg
260 265 270
Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly
275 280 285
Trp Met Thr His Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg
290 295 300
Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr
305 310 315 320
Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr
325 330 335
Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Ser Gln Glu
340 345 350
Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro
355 360 365
Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Gly Ala Thr Leu Glu
370 375 380
Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala
385 390 395 400
Arg Val Leu Thr Pro Leu Gly Asp Thr Thr His Thr Ser Gly Thr Thr
405 410 415
Asp Pro Leu Lys Ala Leu Asp Asp Ala Ile Ala Ser Val Asp Lys Phe
420 425 430
Arg Ser Ser Leu Gly Ala Val Gln Asn Arg Leu Asp Ser Ala Val Thr
435 440 445
Asn Leu Asn Asn Thr Thr Thr Asn Leu Ser Glu Ala Gln Ser Arg Ile
450 455 460
Gln Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln
465 470 475 480
Ile Ile Gln Gln Ala Gly Asn Ser Val Leu Ala Lys Ala Asn Gln Val
485 490 495
Pro Gln Gln Val Leu Ser Leu Leu Gln Gly Leu Glu His His His His
500 505 510
His His
<210>1
<211>525
<212>PRT KFD-p24 2C
<400>27
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Ala Ser Thr Pro Leu Gly
165 170 175
Asp Thr Thr His Thr Ser Gly Pro Ile Val Gln Asn Leu Gln Gly Gln
180 185 190
Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys
195 200 205
Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser
210 215 220
Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn
225 230 235 240
Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile
245 250 255
Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu His Pro Val His Ala Gly
260 265 270
Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala
275 280 285
Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr His Asn
290 295 300
Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly
305 310 315 320
Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile
325 330 335
Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr
340 345 350
Lys Thr Leu Arg Ala Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met
355 360 365
Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile
370 375 380
Leu Lys Ala Leu Gly Pro Gly Ala Thr Leu Glu Glu Met Met Thr Ala
385 390 395 400
Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala Arg Val Leu Thr Pro
405 410 415
Leu Gly Asp Thr Thr His Thr Ser Gly Thr Thr Asp Pro Leu Lys Ala
420 425 430
Leu Asp Asp Ala Ile Ala Ser Val Asp Lys Phe Arg Ser Ser Leu Gly
435 440 445
Ala Val Gln Asn Arg Leu Asp Ser Ala Val Thr Asn Leu Asn Asn Thr
450 455 460
Thr Thr Asn Leu Ser Glu Ala Gln Ser Arg Ile Gln Asp Ala Asp Tyr
465 470 475 480
Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln Ile Ile Gln Gln Ala
485 490 495
Gly Asn Ser Val Leu Ala Lys Ala Asn Gln Val Pro Gln Gln Val Leu
500 505 510
Ser Leu Leu Gln Gly Leu Glu His His His His His His
515 520 525
<210>1
<211>514
<212>PRT KFD-p24 2D
<400>28
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Ala Ser Thr Pro Leu Gly
165 170 175
Asp Thr Thr His Thr Ser Gly Pro Ile Val Gln Asn Leu Gln Gly Gln
180 185 190
Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys
195 200 205
Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser
210 215 220
Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn
225 230 235 240
Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile
245 250 255
Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu His Pro Val His Ala Gly
260 265 270
Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala
275 280 285
Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr His Asn
290 295 300
Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly
305 310 315 320
Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile
325 330 335
Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr
340 345 350
Lys Thr Leu Arg Ala Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met
355 360 365
Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile
370 375 380
Leu Lys Ala Leu Gly Pro Gly Ala Thr Leu Glu Glu Met Met Thr Ala
385 390 395 400
Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala Arg Val Leu Thr Thr
405 410 415
Asp Pro Leu Lys Ala Leu Asp Asp Ala Ile Ala Ser Val Asp Lys Phe
420 425 430
Arg Ser Ser Leu Gly Ala Val Gln Asn Arg Leu Asp Ser Ala Val Thr
435 440 445
Asn Leu Asn Asn Thr Thr Thr Asn Leu Ser Glu Ala Gln Ser Arg Ile
450 455 460
Gln Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln
465 470 475 480
Ile Ile Gln Gln Ala Gly Asn Ser Val Leu Ala Lys Ala Asn Gln Val
485 490 495
Pro Gln Gln Val Leu Ser Leu Leu Gln Gly Leu Glu His His His His
500 505 510
His His
<210>1
<211>514
<212>PRT KFD-p24 3A
<400>29
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Thr Pro Leu Gly Asp Thr
165 170 175
Thr His Thr Ser Gly Ala Ser Pro Ile Val Gln Asn Leu Gln Gly Gln
180 185 190
Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys
195 200 205
Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser
210 215 220
Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn
225 230 235 240
Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile
245 250 255
Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu His Pro Val His Ala Gly
260 265 270
Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala
275 280 285
Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr His Asn
290 295 300
Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly
305 310 315 320
Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile
325 330 335
Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr
340 345 350
Lys Thr Leu Arg Ala Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met
355 360 365
Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile
370 375 380
Leu Lys Ala Leu Gly Pro Gly Ala Thr Leu Glu Glu Met Met Thr Ala
385 390 395 400
Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala Arg Val Leu Thr Thr
405 410 415
Asp Pro Leu Lys Ala Leu Asp Asp Ala Ile Ala Ser Val Asp Lys Phe
420 425 430
Arg Ser Ser Leu Gly Ala Val Gln Asn Arg Leu Asp Ser Ala Val Thr
435 440 445
Asn Leu Asn Asn Thr Thr Thr Asn Leu Ser Glu Ala Gln Ser Arg Ile
450 455 460
Gln Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln
465 470 475 480
Ile Ile Gln Gln Ala Gly Asn Ser Val Leu Ala Lys Ala Asn Gln Val
485 490 495
Pro Gln Gln Val Leu Ser Leu Leu Gln Gly Leu Glu His His His His
500 505 510
His His
<210>1
<211>525
<212>PRT KFD-p24 3B
<400>30
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Thr Pro Leu Gly Asp Thr
165 170 175
Thr His Thr Ser Gly Ala Ser Pro Ile Val Gln Asn Leu Gln Gly Gln
180 185 190
Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys
195 200 205
Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser
210 215 220
Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn
225 230 235 240
Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile
245 250 255
Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu His Pro Val His Ala Gly
260 265 270
Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala
275 280 285
Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr His Asn
290 295 300
Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly
305 310 315 320
Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile
325 330 335
Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr
340 345 350
Lys Thr Leu Arg Ala Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met
355 360 365
Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile
370 375 380
Leu Lys Ala Leu Gly Pro Gly Ala Thr Leu Glu Glu Met Met Thr Ala
385 390 395 400
Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala Arg Val Leu Thr Pro
405 410 415
Leu Gly Asp Thr Thr His Thr Ser Gly Thr Thr Asp Pro Leu Lys Ala
420 425 430
Leu Asp Asp Ala Ile Ala Ser Val Asp Lys Phe Arg Ser Ser Leu Gly
435 440 445
Ala Val Gln Asn Arg Leu Asp Ser Ala Val Thr Asn Leu Asn Asn Thr
450 455 460
Thr Thr Asn Leu Ser Glu Ala Gln Ser Arg Ile Gln Asp Ala Asp Tyr
465 470 475 480
Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln Ile Ile Gln Gln Ala
485 490 495
Gly Asn Ser Val Leu Ala Lys Ala Asn Gln Val Pro Gln Gln Val Leu
500 505 510
Ser Leu Leu Gln Gly Leu Glu His His His His His His
515 520 525
<210>1
<211>536
<212>PRT KFD-p24 3C
<400>31
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Thr Pro Leu Gly Asp Thr
165 170 175
Thr His Thr Ser Gly Ala Ser Thr Pro Leu Gly Asp Thr Thr His Thr
180 185 190
Ser Gly Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala
195 200 205
Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys
210 215 220
Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly
225 230 235 240
Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His
245 250 255
Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala
260 265 270
Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly
275 280 285
Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr
290 295 300
Leu Gln Glu Gln Ile Gly Trp Met Thr His Asn Pro Pro Ile Pro Val
305 310 315 320
Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val
325 330 335
Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys
340 345 350
Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala
355 360 365
Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu
370 375 380
Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly
385 390 395 400
Pro Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly
405 410 415
Gly Pro Gly His Lys Ala Arg Val Leu Thr Pro Leu Gly Asp Thr Thr
420 425 430
His Thr Ser Gly Thr Thr Asp Pro Leu Lys Ala Leu Asp Asp Ala Ile
435 440 445
Ala Ser Val Asp Lys Phe Arg Ser Ser Leu Gly Ala Val Gln Asn Arg
450 455 460
Leu Asp Ser Ala Val Thr Asn Leu Asn Asn Thr Thr Thr Asn Leu Ser
465 470 475 480
Glu Ala Gln Ser Arg Ile Gln Asp Ala Asp Tyr Ala Thr Glu Val Ser
485 490 495
Asn Met Ser Lys Ala Gln Ile Ile Gln Gln Ala Gly Asn Ser Val Leu
500 505 510
Ala Lys Ala Asn Gln Val Pro Gln Gln Val Leu Ser Leu Leu Gln Gly
515 520 525
Leu Glu His His His His His His
530 535
<210>1
<211>525
<212>PRT KFD-p24 3D
<400>32
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Ile Thr Gln Asn Asn
1 5 10 15
Ile Asn Lys Asn Gln Ser Ala Leu Ser Ser Ser Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ala
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95
Gln Ala Thr Thr Gly Thr Asn Ser Glu Ser Asp Leu Ser Ser Ile Gln
100 105 110
Asp Glu Ile Lys Ser Arg Leu Asp Glu Ile Asp Arg Val Ser Gly Gln
115 120 125
Thr Gln Phe Asn Gly Val Asn Val Leu Ala Lys Asn Gly Ser Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Asn Gln Thr Ile Thr Ile Asp Leu Lys
145 150 155 160
Gln Ile Asp Ala Lys Thr Leu Gly Leu Asp Thr Pro Leu Gly Asp Thr
165 170 175
Thr His Thr Ser Gly Ala Ser Thr Pro Leu Gly Asp Thr Thr His Thr
180 185 190
Ser Gly Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala
195 200 205
Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys
210 215 220
Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly
225 230 235 240
Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His
245 250 255
Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala
260 265 270
Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly
275 280 285
Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr
290 295 300
Leu Gln Glu Gln Ile Gly Trp Met Thr His Asn Pro Pro Ile Pro Val
305 310 315 320
Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val
325 330 335
Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys
340 345 350
Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala
355 360 365
Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu
370 375 380
Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly
385 390 395 400
Pro Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly
405 410 415
Gly Pro Gly His Lys Ala Arg Val Leu Thr Thr Asp Pro Leu Lys Ala
420 425 430
Leu Asp Asp Ala Ile Ala Ser Val Asp Lys Phe Arg Ser Ser Leu Gly
435 440 445
Ala Val Gln Asn Arg Leu Asp Ser Ala Val Thr Asn Leu Asn Asn Thr
450 455 460
Thr Thr Asn Leu Ser Glu Ala Gln Ser Arg Ile Gln Asp Ala Asp Tyr
465 470 475 480
Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln Ile Ile Gln Gln Ala
485 490 495
Gly Asn Ser Val Leu Ala Lys Ala Asn Gln Val Pro Gln Gln Val Leu
500 505 510
Ser Leu Leu Gln Gly Leu Glu His His His His His His
515 520 525
Claims (10)
1.一种改造的非致病细菌来源的鞭毛素粘膜佐剂，其特征在于，所述鞭毛素粘膜佐剂去除了鞭毛蛋白的主要抗原活性区。
2.根据权利要求1所述的鞭毛素粘膜佐剂，其特征在于，所述非致病细菌包括大肠杆菌K12株。
3.根据权利要求1所述的鞭毛素粘膜佐剂，其特征在于，所述抗原活性区包括高变区。
4.根据权利要求1所述的鞭毛素粘膜佐剂，其特征在于，所述鞭毛素粘膜佐剂还含有柔性结构，所述柔性结构免疫原性低。
5.根据权利要求4所述的鞭毛素粘膜佐剂，其特征在于，所述柔性结构包括人IgG3铰链序列。
6.根据权利要求1所述的鞭毛素粘膜佐剂，其特征在于，所述抗原活性区替换为目的抗原。
7.根据权利要求6所述的鞭毛素粘膜佐剂，其特征在于，所述目的抗原包括p24抗原。
8.根据权利要求1所述的鞭毛素粘膜佐剂，其特征在于，所述佐剂包括如下的佐剂，该佐剂的
基因序列如SEQ ID：1或氨基酸序列如SEQ ID：22，
基因序列如SEQ ID：2或氨基酸序列如SEQ ID：23，
基因序列如SEQ ID：3或氨基酸序列如SEQ ID：24，
基因序列如SEQ ID：4或氨基酸序列如SEQ ID：25，
基因序列如SEQ ID：5或氨基酸序列如SEQ ID：26，
基因序列如SEQ ID：6或氨基酸序列如SEQ ID：27，
基因序列如SEQ ID：7或氨基酸序列如SEQ ID：28，
基因序列如SEQ ID：8或氨基酸序列如SEQ ID：29，
基因序列如SEQ ID：9或氨基酸序列如SEQ ID：30，
基因序列如SEQ ID：10或氨基酸序列如SEQ ID：31，
基因序列如SEQ ID：11或氨基酸序列如SEQ ID：32。
9.一种改造的非致病细菌来源的鞭毛素粘膜佐剂的制备方法，其特征在于，去除鞭毛蛋白片段中的主要抗原活性区。
10.根据权利要求9所述的制备方法，其特征在于，选择的非致病细菌包括大肠杆菌K12株。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101942856A CN102000329B (zh) | 2009-11-27 | 2009-11-27 | 一种改造的非致病细菌来源的鞭毛素粘膜佐剂及制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101942856A CN102000329B (zh) | 2009-11-27 | 2009-11-27 | 一种改造的非致病细菌来源的鞭毛素粘膜佐剂及制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102000329A true CN102000329A (zh) | 2011-04-06 |
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| CN102816246A (zh) * | 2012-09-04 | 2012-12-12 | 成都蓉生药业有限责任公司 | 一种人巨细胞病毒免疫原融合蛋白及其制备方法和用途 |
| CN103041386A (zh) * | 2012-11-08 | 2013-04-17 | 中国科学院海洋研究所 | 一种鳗弧菌重组蛋白的应用 |
| CN103055311A (zh) * | 2012-12-28 | 2013-04-24 | 中山大学 | 一种传染性喉气管炎粘膜疫苗及其制备方法和应用 |
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