專利名稱：一種改造的非致病細(xì)菌來源的鞭毛素粘膜佐劑及制備方法
技術(shù)領(lǐng)域：
本發(fā)明涉及生物技術(shù)領(lǐng)域，具體涉及一種優(yōu)化改造的非致病細(xì)菌來源的鞭毛素粘 膜佐劑及其制備方法。
背景技術(shù)：
現(xiàn)在已知致病菌來源的鞭毛素蛋白具有免疫佐劑效果，它是通過鞭毛素蛋白與 Toll樣受體(Toll-like rec印tors，TLRs) 5相結(jié)合，引發(fā)NF-κ B途徑引發(fā)先天性免疫，進(jìn) 而引發(fā)特異性免疫。通過鞭毛素蛋白與目的蛋白混合或融合后免疫，可以顯著提高對目的 抗原的免疫應(yīng)答，并且可達(dá)到抵抗帶有目的抗原的病原微生物的效果。但是因其來源于致 病菌，可能具有潛在的危險(xiǎn)性，并且它還可以引起炎癥反應(yīng)，產(chǎn)生大量針對鞭毛素自身的免 疫反應(yīng)，導(dǎo)致可能的耐受性以及其它可能的免疫副反應(yīng)。

發(fā)明內(nèi)容
本發(fā)明為克服上述技術(shù)缺陷，提供了一種優(yōu)化改造的非致病細(xì)菌來源的鞭毛素粘 膜佐劑，該佐劑是通過改造一種來源于正常共生菌株的鞭毛素蛋白并且在保證其佐劑活性 的前提下降低它的抗原性、免疫原性以及由其引發(fā)的炎癥反應(yīng)。為實(shí)現(xiàn)上述目的，采用如下技術(shù)方案本發(fā)明的一種改造的非致病細(xì)菌來源的鞭毛素粘膜佐劑去除了鞭毛蛋白的主要 抗原活性區(qū)。所述非致病細(xì)菌包括大腸桿菌Κ12株。所述抗原活性區(qū)包括高變區(qū)。所述鞭毛素粘膜佐劑還含有柔性結(jié)構(gòu)，所述柔性結(jié)構(gòu)免疫原性低。所述柔性結(jié)構(gòu)包括人IgG3鉸鏈序列。所述抗原活性區(qū)替換為目的抗原。所述目的抗原包括p24抗原。所述佐劑包括如下的佐劑，該佐劑的基因序列如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，
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基因序列如SEQ ID 11或氨基酸序列如SEQ ID :32。本發(fā)明的一種改造的非致病細(xì)菌來源的鞭毛素粘膜佐劑的制備方法是去除鞭毛 蛋白片段中的主要抗原活性區(qū)。選擇的非致病細(xì)菌包括大腸桿菌K12株。所述抗原活性區(qū)包括高變區(qū)。在所述鞭毛蛋白片段中增加柔性結(jié)構(gòu)，所述柔性結(jié)構(gòu)免疫原性低。所述柔性結(jié)構(gòu)包括人IgG3鉸鏈序列。將所述抗原活性區(qū)替換為目的抗原。所述目的抗原包括p24抗原。本發(fā)明的一個(gè)優(yōu)選實(shí)施例為選擇人正常共生菌株——大腸桿菌K12菌株MG1655 亞株的鞭毛素蛋白(簡稱KF)為目標(biāo)，確認(rèn)其佐劑活性；通過構(gòu)建不同的克隆，通過 ELISA(酶聯(lián)免疫吸附檢測法)確定其主要的抗原性區(qū)為其內(nèi)部的高變區(qū)；通過去除其高變 區(qū)并且添加柔性結(jié)構(gòu)，確認(rèn)去除高變區(qū)免疫原性大大降低，即高變區(qū)也是主要的免疫原性 區(qū)，并且柔性結(jié)構(gòu)沒有高的免疫原性；通過將目的抗原替換高變區(qū)，并添加低免疫原性的柔 性結(jié)構(gòu)，得到保留佐劑活性的目的蛋白。與現(xiàn)有技術(shù)相比，本發(fā)明具有如下有益效果通過選擇來源于正常共生菌株的鞭毛素蛋白，從而降低可能具有的潛在的危險(xiǎn) 性；并且通過去除其主要的免疫原性區(qū)及抗原活性區(qū)，降低它的抗原性、免疫原性以及由其 引發(fā)的炎癥反應(yīng)；通過將目的抗原添加到鞭毛素蛋白內(nèi)部，提高抗原遞呈效果；通過添加 低免疫原性的柔性結(jié)構(gòu)，維持其三維結(jié)構(gòu)與天然的鞭毛素蛋白相類似，從而維持其佐劑活 性。


圖1是KF不同區(qū)段的克隆的構(gòu)建及其抗原蛋白的表達(dá)，其中，圖1-1是構(gòu)建KF不 同區(qū)段的克隆的方案，圖1-2是KF不同區(qū)段的抗原蛋白的聚丙烯酰胺凝膠電泳(PAGE)電 泳圖；圖2是通過酶聯(lián)免疫吸附檢測法(ELISA)確定主要的抗原性區(qū)段為其內(nèi)部的高變 區(qū)的結(jié)果圖，其中，圖2-1是柱狀圖，圖2-2是點(diǎn)分布圖；圖3是KF結(jié)構(gòu)改造方案及其改造得到的片段的鑒定結(jié)果，其中，圖3-1是KF片段 改造方案示意圖，圖3-2是KF片段改造后得到的片段的示意圖，圖 3-3 是 KF Dl、D2、D3 的 SDS-PAGE 及 Western blot 的結(jié)果圖；圖 3-4 是 KFD-p242A、2B、2C、2D、3A、3B、3C 和 3D 的 SDS-PAGE 及 Westernblot 的結(jié)果圖；圖4是KFD1、D2、D3的佐劑活性及抗原性鑒定的結(jié)果圖；其中，圖4_1是以Caco-2 細(xì)胞為模式細(xì)胞通過IL-8分泌水平，比較KF、KFD與TLR5作用能力強(qiáng)弱，圖4_2是KFD+p24 的10周免疫實(shí)驗(yàn)結(jié)果的分析圖；圖5是KFD-p24的佐劑活性及抗原性鑒定的結(jié)果圖，其中，圖5_1，圖5_2以Caco 2細(xì)胞為模式細(xì)胞通過IL-8分泌水平，比較KF、KFD-p24與TLR5作用能力強(qiáng)弱，圖5_3，圖 5-4，圖5-5是KFD-p24免疫實(shí)驗(yàn)(0，4，8周免疫)相關(guān)結(jié)果圖；圖5_3，圖5_4是改造前后 免疫所產(chǎn)生的針對目的抗原的抗體(anti-p24IgG)與針對佐劑自身的抗體(anti-KF IgG)
4的比例；圖5-5是全面比較全長KF與改造克隆的代表KFD-p243D的粘膜免疫效果，即不同 部位的IgA抗體水平的比較；圖6是小鼠滴鼻免疫后短期內(nèi)產(chǎn)生的細(xì)胞因子的實(shí)驗(yàn)數(shù)據(jù)分析圖；其中，圖6-1， 圖6-2，圖6-3分別是小鼠滴鼻免疫后短期內(nèi)產(chǎn)生的IL-6、TNF-a和KC的實(shí)驗(yàn)數(shù)據(jù)分析圖；圖7是小鼠滴鼻免疫短期毒性效應(yīng)數(shù)據(jù)分析圖。
具體實(shí)施例方式為使本發(fā)明更加容易理解，下面結(jié)合具體實(shí)施例，進(jìn)一步闡述本發(fā)明。應(yīng)理解，這 些實(shí)施例僅用于說明本發(fā)明而不用于限制本發(fā)明的范圍。下面實(shí)施例中未注明具體條件的實(shí)驗(yàn)方法，通常按照常規(guī)條件，例如Sambrook等 分子克隆實(shí)驗(yàn)手冊中所述的條件。實(shí)施例1 構(gòu)建KF不同區(qū)段的克隆及其抗原蛋白的表達(dá)構(gòu)建KF不同區(qū)段的克隆片段如圖1所示，其中，圖1-1是構(gòu)建KF不同區(qū)段的克隆 的方案，包括KFl、KF2、KF3、KFDl和SF3。其中，KFDl是去掉了高變區(qū)并插入NheI酶切位 點(diǎn)的片段，KFl是KF的高變區(qū)片段，KF2是KF高變區(qū)中心片段，KF3是在高變區(qū)插入KFDl 中NheI酶切位點(diǎn)后形成的KF恢復(fù)克隆，SF3是將高變區(qū)替換為SF高變區(qū)并插入NheI酶 切位點(diǎn)的片段。KF是指大腸桿菌K12株MG1655亞株的鞭毛蛋白(E. coli K12 strainMG1655substrain flagellin)，其基因序列號(hào)為ID-949101。SF為沙門氏菌的鞭毛 蛋白(Salmonalla flagellin)，其基因序列號(hào)為 ID-1070204。以抽提所得細(xì)菌基因組為模板，選擇pET30a作為載體，KF和SF的引物分別如下， 其中劃線部位為酶切位點(diǎn)KF 上游引物5，-TATACATATGGCACAAGTCATTAATAC-3，Nde I下游引物5，-ATATCTCGAGACCCTGCAGCAGAGACAG-3，XhoISF 上游引物5，-CGCGCATATGGCACAAGTCATTAATACAAACA-3，Nde I
下游引物5，-CGGTCTCGAGACGCAGTAAAGAGAGGACGTTTTG-3，Xho I克隆表達(dá)后得到的蛋白通過Ni柱純化，超濾濃縮8-10倍后加PBS再超濾濃 縮，反復(fù)4次；Triton法去內(nèi)毒素；考馬斯亮藍(lán)法確定蛋白濃度；鱟試劑檢測內(nèi)毒素含量 < 0. 005Eu/ μ g蛋白，最后SDS-PAGE鑒定，其結(jié)果如圖1_2所示，圖1_2是KF不同區(qū)段的 抗原蛋白的聚丙烯酰胺凝膠電泳(PAGE)圖。該圖的結(jié)果顯示得到的蛋白與預(yù)期蛋白的大 小一致。實(shí)施例2 確定KF的主要抗原性區(qū)段圖2是通過酶聯(lián)免疫吸附檢測法(ELISA)確定主要的抗原性區(qū)段為其內(nèi)部的高變 區(qū)的結(jié)果圖。其中，圖2-1比較了不同免疫策略所免疫小鼠血清中針對KF不同區(qū)段的抗體 的比例有何異同，該圖的結(jié)果說明不同免疫策略所免疫小鼠血清中針對KF不同區(qū)段的抗 體的比例基本相同。圖2-2比較了免疫策略所免疫小鼠血清中針對KF不同區(qū)段的抗體的 比例有何特征，該圖的結(jié)果說明恢復(fù)性克隆KF3的抗原性與KF基本相同。高變區(qū)KFl的 抗原性 90%，高變區(qū)的中心部位(KF2)僅占其總抗原性的約1/3，保守區(qū)KFDl的抗原性 ^ 10%。高變區(qū)替換為SF的抗原性與其缺失(KFDl)的抗原性相似。KF抗原性主要集中
5在高變區(qū)。由圖2可知，KF抗原性主要集中在高變區(qū)，即高變區(qū)也是主要的免疫原性區(qū)。實(shí)施例3 =KF結(jié)構(gòu)改造方案及其改造得到的片段的鑒定先構(gòu)造保守區(qū)KFD片段，即在KF片段的基礎(chǔ)上，將KF高變區(qū)去掉，將KF的N端 (KFN)及C端(KFC端)連接起來，得到pET28a-KFD片段；然后將p24片段插入pET28a_KFD 中，得到pET28a-KFD-p24。設(shè)計(jì)了三組KFN和四組p24，選擇了兩組KFN和四組p24進(jìn)行組 合改造，得到八個(gè)改造片段。具體如下圖3是KF結(jié)構(gòu)改造方案及其改造得到的片段的鑒定結(jié)果，KF片段改造方案如圖 3-1所示，其中，KFN片段改造所用的引物如下KF Nl 段上游引物(NcoI)Up-N 5-GGCGTCCATGGCACAAGTCATTAATAC-3 27bp下游引物(BamHI)Low-Nl 5-GGCAAGGATCCGTCGTCGTAGCGCTAGCATCAAGGCCAAGAGTTTTAG-3 48bp產(chǎn)物長度541bp ；KF N2 段上游引物(NcoI)Up-N 5-GGCGTCCATGGCACAAGTCATTAATAC-3 27bp下游引物(BamHI)Low-N2 5-TACTAGGATCCGTCGTTCCAGATGTGTGAGTTGTGTCACCAAGTGGAGTGCTAGCATCAAGGCCAAG AGTTTTAG-3 75bp產(chǎn)物長度568bp ；KF N3 段上游引物(NcoI)U p-N 5-GGCGTCCATGGCACAAGTCATTAATAC-3 27bp下游引物(BamHI)Low-N3 5-TACTAGGATCCGTCGTGCTAGCTCCAGATGTGTGAGTTGTGTCACCAAGTGGAGTATCAAGGCCAAG AGTTTTAG-3 75bp產(chǎn)物長度568bp ；KFC段直接酶切KF，根據(jù)BamHI和XhoI酶切位點(diǎn)進(jìn)行連接。KFD的三個(gè)不同片段，簡單來說，如下KFDl = NcoI-Nl-BamHI-C-XhoI ；KFD2 = NcoI-N2-BamHI-C-XhoI ；KFD3 = NcoI-N3-BamHI-C-XhoI。進(jìn)一步，p24的改造具體如下p24擴(kuò)增的引物如下
Up p24-lNheI 5，GCATAGCTAGCCCTATAGTGCAGAACCTCCA-3‘ 3 Ibp ；Low-p24-lBamHI 5， TATGCGGATCCGTCGTCAAAACTCTTGCTTTATGGC-3' 36bp ；Up p24-2NheI 5’ ATCGAGCTAGCACACCTCTTGGTGATACTACACACACATCAGGACCTATAGTGCAGAACCTCCA-3’ 64bp ；Low-p24-2BamHI 5 ’ TATGCGGATCCGTCGTTCCAGATGTGTGAGTTGTGTCACCAAGTGGAGTCAAAACTCTTGCTTTATG GC-3，69bp簡單明了的來說，p24A、p24B、p24C、p24D的結(jié)構(gòu)如下p24A :Up p24-l+Low-p24_l ；p24B :Up p24_l+Low-p24_2 ；p24C :Up p24_2+Low-p24_2 ；p24D :Up p24-2+Low-p24_l。高變區(qū)替換改造為KFD-p24，總共有 8 個(gè)片段KFD-p242A、KFD-p242B、KFD_p242C、 KFD-p242D、KFD_p243A、KFD_p243B、KFD_p243C 和 KFD_p243D，其結(jié)構(gòu)如下KFD-P242A :KFD2+p24AKFD-p242B :KFD2+p24BKFD-p242C :KFD2+p24CKFD-p242D :KFD2+p24DKFD-p243A :KFD3+p24AKFD-p243B :KFD3+p24BKFD-p243C :KFD3+p24CKFD-p243D :KFD3+p24D圖3-2是KF片段改造后得到的片段的示意圖所得改造后片段的序列見序列表。KF DU D2、D3 的 SDS-PAGE 及 Western blot 的結(jié)果見圖 3-3 ;KFD_p242A、2B、2C、 2D、3A、3B、3C和3D的SDS-PAGE及Western blot的結(jié)果圖見圖3_4，標(biāo)號(hào)1_9依次代表 KFD-p242A、2B、2C、2D、marker、3A、3B、3C、3D。其中，所用 marker 為 Ferment SM0661。預(yù)期 片段大小 KFD-p242A、2B、2C、2D 分別為55KD、56KD、57KD、56KD ;KFD_p243A、3B、3C、3D 分別 為56KD、57KD、58KD、57KD。由圖可知，所得片段與預(yù)期片段大小一致。實(shí)施例4 :KFD1、D2、D3的佐劑活性及抗原性鑒定圖4是KFD1、D2、D3的佐劑活性及抗原性鑒定的結(jié)果圖，其中，圖4_1是以Caco-2 細(xì)胞為模式細(xì)胞通過IL-8分泌水平，比較KF、KFD1、D2、D3與TLR5作用能力強(qiáng)弱。即KF、 KFDU D2、D3的佐劑活性在細(xì)胞水平的鑒定的結(jié)果圖；分別用KFD1、KFD2、KFD3和KF刺激 Caco-2細(xì)胞，測量上清中IL-8含量。具體操作Cac0-2細(xì)胞于24孔板中培養(yǎng)6_8天至其 細(xì)胞間界限分隔明顯后，用無血清1640培養(yǎng)基饑餓12h后，加無血清1640培養(yǎng)基稀釋樣品 0. 5ml刺激6h后去細(xì)胞上清，2000rpm IOmin離心取上清，-80C凍存?zhèn)溆谩Ｔ搱D表明，KF高 變區(qū)缺失影響到佐劑活性，在較低濃度(lOng/ml)時(shí)，全長的鞭毛素KF對Caco-2細(xì)胞刺激
7作用較強(qiáng)，但在較高濃度下(lOOOng/ml)改造克隆的刺激作用已經(jīng)超過KF。如圖所示，圖4-2是KFD+p24的免疫實(shí)驗(yàn)(0，4，8周滴鼻免疫)第10周結(jié)果；即 KFDU D2、D3的佐劑活性及抗原性鑒定的結(jié)果圖。實(shí)驗(yàn)結(jié)果說明在保留了佐劑活性的條件 下KF自身抗原性明顯下降，且不同改造降低的幅度基本相同，均降低了 10倍以上。實(shí)施例5 :KFD-p242A，2B，2C，2D，3A，3B，3C，3D的佐劑活性及抗原性鑒定圖5是KFD-p24的佐劑活性及抗原性鑒定的結(jié)果圖，其中，圖5_1，圖5_2以Caco-2 細(xì)胞為模式細(xì)胞通過IL-8分泌水平，比較KF、KFD-p24與TLR5作用能力強(qiáng)弱，即KF、 KFD-p24的佐劑活性在細(xì)胞水平的鑒定的結(jié)果圖；分別用KF和KF-p24刺激Caco-2細(xì)胞，測 量上清中IL-8含量。具體操作同實(shí)施例4。該結(jié)果說明KF高變區(qū)替換影響到佐劑活性， 在較低濃度(lng/ml-lOOng/ml)時(shí)，全長的鞭毛素KF對Caco-2細(xì)胞刺激作用較強(qiáng)，但在較 高濃度下(lOOOng/ml)改造克隆的刺激作用可以與KF相比較。圖5-3，圖5-4，圖5-5是KFD-p24免疫實(shí)驗(yàn)(0，4，8周滴鼻免疫)第10周結(jié)果。圖5-3，圖5-4是改造前后免疫所產(chǎn)生的針對目的抗原的抗體(anti-p24IgG)與 針對佐劑自身的抗體(anti-KF IgG)的比例。圖5_3是觀察KFD_p24佐劑效應(yīng)及其佐劑活 性部分的抗原性。由以上結(jié)果看出，所有的高變區(qū)替換的克隆免疫效果均不弱于利用全長 KF與同樣量的p24混合免疫。并且有些克隆的免疫活性可以與KF與p24直接融合的克隆 KF-p24相比較。通過將KF高變區(qū)替換為p24，佐劑活性區(qū)域的自身抗原性大大降低。圖 5-4是具體比較KF+p24，KF-p24以及KFD_p24的免疫原性部位；對于KFD_p24來說，抗原活 性部位主要在P24部位，對目的抗原p24的抗體是對佐劑活性區(qū)域的抗體滴度的約20-200 倍。對于直接融合的蛋白KF-p24來說對目的抗原p24的抗體是對佐劑活性區(qū)域的抗體滴 度的約0. 5-1倍。對于混合免疫KF與p24來說對目的抗原p24的抗體是對佐劑活性區(qū)域 的抗體滴度的約0. 2倍。實(shí)驗(yàn)結(jié)果說明相對于混合免疫KF與p24來說，免疫KFD-p24產(chǎn)生 的抗體中，對于佐劑活性區(qū)域的抗體減少了兩個(gè)數(shù)量級(jí)，大大提高了免疫反應(yīng)的特異性。圖5-5是全面比較全長KF與改造克隆的代表KFD-p243D的粘膜免疫效果，即不同 部位的IgA抗體水平的比較；以KFD-p243D為代表比較不同免疫策略產(chǎn)生的對于目的抗原 P24的IgA抗體水平；實(shí)驗(yàn)結(jié)果說明從各部位的IgA水平來看，同樣劑量的KFD-p243D均 好于KF，而且可以同融合相媲美。實(shí)施例6 小鼠細(xì)胞因子實(shí)驗(yàn)小鼠提前12小時(shí)開始禁食不禁水，用戊巴比妥那麻醉后滴鼻免疫。取樣時(shí)，拔眼 球取血，脫頸處死后，解剖咽喉部，用PBS從氣管處灌洗500 μ L兩次，合并后8000rpm 5min 離心取上清，-80C凍存?zhèn)溆谩D6是小鼠滴鼻免疫后短期內(nèi)產(chǎn)生的細(xì)胞因子的實(shí)驗(yàn)數(shù)據(jù)分 析圖；其中，圖6-1，圖6-2，圖6-3分別是小鼠滴鼻免疫后短期內(nèi)產(chǎn)生的IL-6,TNF-a和KC 的實(shí)驗(yàn)數(shù)據(jù)分析圖。KF高變區(qū)缺失及替換誘導(dǎo)產(chǎn)生TNF-a，IL-6，KC(IL-S)的能力比全長 低，并且較快的恢復(fù)到靜息水平(PBS組)，因此其潛在的炎癥效應(yīng)較弱。實(shí)施例7 小鼠滴鼻免疫短期毒性效應(yīng)小鼠用戊巴比妥那麻醉后滴鼻免疫，免疫前及免疫后每天稱重。體重指標(biāo)是反映 小鼠狀態(tài)的敏感指標(biāo)，通過體重反映了小鼠滴鼻免疫的短期毒性效應(yīng)。圖7是小鼠滴鼻免 疫毒性炎癥效應(yīng)數(shù)據(jù)分析圖；由圖7可知，高變區(qū)缺失及替換與陰性對照組相似，對小鼠無
顯著影響。
8
最后應(yīng)當(dāng)說明的是，以上實(shí)施例僅用以說明本發(fā)明的技術(shù)方案而非對本發(fā)明保護(hù) 范圍的限制，盡管參照較佳實(shí)施例對本發(fā)明作了詳細(xì)說明，本領(lǐng)域的普通技術(shù)人員應(yīng)當(dāng)理 解，可以對本發(fā)明的技術(shù)方案進(jìn)行修改或者等同替換，而不脫離本發(fā)明技術(shù)方案的實(shí)質(zhì)和
范圍。
序列表
〈110〉中國科學(xué)院武漢病毒研究所
<160>32
<210>1
<211>827
<212>KFD1DNA
<400>1
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatgctagcgctacgacgacggatccgc540
tgaaagcgctggacgatgctatcgcatctgtagacaaattccgttcttccctcggtgcgg600
tgcaaaaccgtctggattccgcggttaccaacctgaacaacaccactaccaacctgtctg660
aagcgcagtcccgtattcaggacgccgactatgcgaccgaagtgtccaatatgtcgaaag720
cgcagatcatccagcaggccggtaactccgtgttggcaaaagctaaccaggtaccgcagc780
aggttctgtctctgctgcagggtctcgagcaccaccaccaccaccac827
<210>1
<211>854
<212>KFD2 DNA
<400>2
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatgctagcactccacttggtgacacaa540
ctcacacatctggaacgacggatccgctgaaagcgctggacgatgctatcgcatctgtag600
acaaattccgttcttccctcggtgcggtgcaaaaccgtctggattccgcggttaccaacc660
tgaacaacaccactaccaacctgtctgaagcgcagtcccgtattcaggacgccgactatg720
cgaccgaagtgtccaatatgtcgaaagcgcagatcatccagcaggccggtaactccgtgt780
tggcaaaagctaaccaggtaccgcagcaggttctgtctctgctgcagggtctcgagcacc840
accaccaccaccac854
<210>1
<211>854
<212>KFD3 DNA
<400>3
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatactccacttggtgacacaactcaca540
catctggagctagcacgacggatccgctgaaagcgctggacgatgctatcgcatctgtag600
acaaattccgttcttccctcggtgcggtgcaaaaccgtctggattccgcggttaccaacc660
tgaacaacaccactaccaacctgtctgaagcgcagtcccgtattcaggacgccgactatg720
cgaccgaagtgtccaatatgtcgaaagcgcagatcatccagcaggccggtaactccgtgt780
tggcaaaagctaaccaggtaccgcagcaggttctgtctctgctgcagggtctcgagcacc840
accaccaccaccac854
<210>1
<211>1514
<212>KFD-p24 2A DNA
<400>4
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatgctagccctatagtgcagaacctcc540
aggggcaaatggtacatcaggccatatcacctagaactttaaatgcatgggtaaaagtag600
tagaagagaaggctttcagcccagaagtaatacccatgttttcagcattatcagaaggag660
10
ccaccccacaagatttaaataccatgctaaacacagtggggggacatcaagcagccatgc720
aaatgttaaaagagaccatcaatgaggaagctgcagaatgggatagattgcatccagtgc780
atgcagggcctattgcaccaggccagatgagagaaccaaggggaagtgacatagcaggaa840
ctactagtacccttcaggaacaaataggatggatgacacataatccacctatcccagtag900
gagaaatctataaaagatggataatcctgggattaaataaaatagtaagaatgtatagcc960
ctaccagcattctggacataagacaaggaccaaaggaaccctttagagactatgtagacc1020
gattctataaaactctaagagccgagcaagcttcacaagaggtaaaaaattggatgacag1080
aaaccttgttggtccaaaatgcgaacccagattgtaagac 3.3.3.3.3.gcattgggac1140
caggagcgacactagaagaaatgatgacagcatgtcagggagtggggggacccggccata1200
aagcaagagttttgacgacggatccgctgaaagcgctggacgatgctatcgcatctgtag1260
acaaattccgttcttccctcggtgcggtgcaaaaccgtctggattccgcggttaccaacc1320
tgaacaacaccactaccaacctgtctgaagcgcagtcccgtattcaggacgccgactatg1380
cgaccgaagtgtccaatatgtcgaaagcgcagatcatccagcaggccggtaactccgtgt1440
tggcaaaagctaaccaggtaccgcagcaggttctgtctctgctgcagggtctcgagcacc1500
accaccaccaccac1514
<210>1
<211>1547
<212>KFD-p24 2B DNA
〈400>5
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaat￡1￡Ι ￡Ι 0￡1￡10￡160
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatgctagccctatagtgcagaacctcc540
aggggcaaatggtacatcaggccatatcacctagaactttaaatgcatgggtaaaagtag600
tagaagagaaggctttcagcccagaagtaatacccatgttttcagcattatcagaaggag660
ccaccccacaagatttaaataccatgctaaacacagtggggggacatcaagcagccatgc720
aaatgttaaaagagaccatcaatgaggaagctgcagaatgggatagattgcatccagtgc780
atgcagggcctattgcaccaggccagatgagagaaccaaggggaagtgacatagcaggaa840
ctactagtacccttcaggaacaaataggatggatgacacataatccacctatcccagtag900
gagaaatctataaaagatggataatcctgggattaaataaaatagtaagaatgtatagcc960
ctaccagcattctggacataagacaaggaccaaaggaaccctttagagactatgtagacc1020
gattctataaaactctaagagccgagcaagcttcacaagaggtaaaaaattggatgacag1080
aaaccttgttggtccaaaatgcgaacccagattgtaagac 3.3.3.3.3.gcattgggac1140
caggagcgacactagaagaaatgatgacagcatgtcagggagtggggggacccggccata1200
11
aagcaagagttttgactccacttggtgacacaactcacacatctggaacgacggatccgc1260
tgaaagcgctggacgatgctatcgcatctgtagacaaattccgttcttccctcggtgcgg1320
tgcaaaaccgtctggattccgcggttaccaacctgaacaacaccactaccaacctgtctg1380
aagcgcagtcccgtattcaggacgccgactatgcgaccgaagtgtccaatatgtcgaaag1440
cgcagatcatccagcaggccggtaactccgtgttggcaaaagctaaccaggtaccgcagc1500
aggttctgtctctgctgcagggtctcgagcaccaccaccaccaccac1547
<210>1
<211>1580
<212>KFD-p24 2C DNA
<400>6
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatgctagcacacctcttggtgatacta540
cacacacatcaggacctatagtgcagaacctccaggggcaaatggtacatcaggccatat600
cacctagaactttaaatgcatgggtaaaagtagtagaagagaaggctttcagcccagaag660
taatacccatgttttcagcattatcagaaggagccaccccacaagatttaaataccatgc720
taaacacagtggggggacatcaagcagccatgcaaatgttaaaagagaccatcaatgagg780
aagctgcagaatgggatagattgcatccagtgcatgcagggcctattgcaccaggccaga840
tgagagaaccaaggggaagtgacatagcaggaactactagtacccttcaggaacaaatag900
gatggatgacacataatccacctatcccagtaggagaaatctataaaagatggataatcc960
tgggattaaataaaatagta agaatgtata gccctaccag cattctggac ataagacaag1020
gaccaaaggaaccctttaga gactatgtag accgattcta taaaactcta agagccgagc1080
aagcttcacaagaggtaaaa aattggatga cagaaaccttgttggtccaa aatgcgaacc1140
cagattgtaagactatttta aaagcattgg gaccaggagcgacactagaa gaaatgatga1200
cagcatgtcagggagtgggg ggacccggcc ataaagcaag agttttgactccacttggtg1260
￡lC￡lC￡l￡lCtC￡lcacatctgga acgacggatccgctgaaagcgctggacgatgctatcgcat1320
ctgtagacaaattccgttcttccctcggtg cggtgcaaaa ccgtctggattccgcggtta1380
ccaacctgaacaacaccact accaacctgtctgaagcgca gtcccgtattcaggacgccg1440
actatgcgaccgaagtgtccaatatgtcga aagcgcagatcatccagcag gccggtaact1500
CCgtgttggCaaaagctaaccaggtaccgc agcaggttctgtctctgctg cagggtctcg1560
agcaccaccaccaccaccac1580
<210>1
<211>1547
12
<212>KFD-p24 2D DNA
<400>7
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatgctagcacacctcttggtgatacta540
cacacacatcaggacctatagtgcagaacctccaggggcaaatggtacatcaggccatat600
cacctagaactttaaatgcatgggtaaaagtagtagaagagaaggctttcagcccagaag660
taatacccatgttttcagcattatcagaaggagccaccccacaagatttaaataccatgc720
taaacacagtggggggacatcaagcagccatgcaaatgttaaaagagaccatcaatgagg780
aagctgcagaatgggatagattgcatccagtgcatgcagggcctattgcaccaggccaga840
tgagagaaccaaggggaagtgacatagcaggaactactagtacccttcaggaacaaatag900
gatggatgacacataatccacctatcccagtaggagaaatctataaaagatggataatcc960
tgggattaaataaaatagtaagaatgtatagccctaccagcattctggacataagacaag1020
gaccaaaggaaccctttagagactatgtagaccgattctataaaactctaagagccgagc1080
aagcttcacaagaggtaaaaaattggatgacagaaaccttgttggtccaaaatgcgaacc1140
cagattgtaagactattttaaaagcattgggaccaggagcgacactagaagaaatgatga1200
cagcatgtcagggagtggggggacccggccataaagcaagagttttgacgacggatccgc1260
tgaaagcgctggacgatgctatcgcatctgtagacaaattccgttcttccctcggtgcgg1320
tgcaaaaccgtctggattccgcggttaccaacctgaacaacaccactaccaacctgtctg1380
aagcgcagtcccgtattcaggacgccgactatgcgaccgaagtgtccaatatgtcgaaag1440
cgcagatcatccagcaggccggtaactccgtgttggcaaaagctaaccaggtaccgcagc1500
aggttctgtctctgctgcagggtctcgagcaccaccaccaccaccac1547
<210>1
<211>1547
<212>KFD-p24 3A DNA
<400>8
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
13
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatactccacttggtgacacaactcaca540
catctggagctagccctatagtgcagaacctccaggggcaaatggtacatcaggccatat600
cacctagaactttaaatgcatgggtaaaagtagtagaagagaaggctttcagcccagaag660
taatacccatgttttcagcattatcagaaggagccaccccacaagatttaaataccatgc720
taaacacagtggggggacatcaagcagccatgcaaatgttaaaagagaccatcaatgagg780
aagctgcagaatgggatagattgcatccagtgcatgcagggcctattgcaccaggccaga840
tgagagaaccaaggggaagtgacatagcaggaactactagtacccttcaggaacaaatag900
gatggatgacacataatccacctatcccagtaggagaaatctataaaagatggataatcc960
tgggattaaataaaatagtaagaatgtatagccctaccagcattctggacataagacaag1020
gaccaaaggaaccctttagagactatgtagaccgattctataaaactctaagagccgagc1080
aagcttcacaagaggtaaaaaattggatgacagaaaccttgttggtccaaaatgcgaacc1140
cagattgtaagactattttaaaagcattgggaccaggagcgacactagaagaaatgatga1200
cagcatgtcagggagtggggggacccggccataaagcaagagttttgacgacggatccgc1260
tgaaagcgctggacgatgctatcgcatctgtagacaaattccgttcttccctcggtgcgg1320
tgcaaaaccgtctggattccgcggttaccaacctgaacaacaccactaccaacctgtctg1380
aagcgcagtcccgtattcaggacgccgactatgcgaccgaagtgtccaatatgtcgaaag1440
cgcagatcatccagcaggccggtaactccgtgttggcaaaagctaaccaggtaccgcagc1500
aggttctgtctctgctgcagggtctcgagcaccaccaccaccaccac1547
<210>1
<211>1580
<212>KFD-p24 3B DNA
<400>9
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatactccacttggtgacacaactcaca540
catctggagctagccctatagtgcagaacctccaggggcaaatggtacatcaggccatat600
cacctagaactttaaatgcatgggtaaaagtagtagaagagaaggctttcagcccagaag660
taatacccatgttttcagcattatcagaaggagccaccccacaagatttaaataccatgc720
taaacacagtggggggacatcaagcagccatgcaaatgttaaaagagaccatcaatgagg780
aagctgcagaatgggatagattgcatccagtgcatgcagggcctattgcaccaggccaga840
tgagagaaccaaggggaagtgacatagcaggaactactagtacccttcaggaacaaatag900
gatggatgacacataatccacctatcccagtaggagaaatctataaaagatggataatcc960
tgggattaaataaaatagtaagaatgtata gccctaccag cattctggacataagacaag1020
gaccaaaggaaccctttagagactatgtag accgattcta taaaactcta agagccgagc1080
aagcttcacaagaggtaaaaaattggatga cagaaaccttgttggtccaa aatgcgaacc1140
cagattgtaagactattttaaaagcattgg gaccaggagcgacactagaa gaaatgatga1200
cagcatgtcagggagtggggggacccggcc ataaagcaag agttttgactccacttggtg1260
acacaactcacacatctggaacgacggatccgctgaaagcgctggacgatgctatcgcat1320
ctgtagacaaattccgttcttccctcggtg cggtgcaaaa ccgtctggattccgcggtta1380
ccaacctgaacaacaccactaccaacctgtctgaagcgca gtcccgtattcaggacgccg1440
actatgcgaccgaagtgtccaatatgtcga aagcgcagatcatccagcag gccggtaact1500
CCgtgttggCaaaagctaaccaggtaccgc agcaggttctgtctctgctg cagggtctcg1560
agcaccaccaccaccaccac1580
<210>1
<211>1613
<212>KFD-p24 3C DNA
<400>10
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatactccacttggtgacacaactcaca540
catctggagctagcacacctcttggtgatactacacacacatcaggacctatagtgcaga600
acctccaggggcaaatggtacatcaggccatatcacctagaactttaaatgcatgggtaa660
aagtagtagaagagaaggctttcagcccagaagtaatacccatgttttcagcattatcag720
aaggagccaccccacaagatttaaataccatgctaaacacagtggggggacatcaagcag780
ccatgcaaatgttaaaagagaccatcaatgaggaagctgcagaatgggatagattgcatc840
cagtgcatgcagggcctattgcaccaggccagatgagagaaccaaggggaagtgacatag900
caggaactactagtacccttcaggaacaaataggatggatgacacataatccacctatcc960
cagtaggagaaatctataaaagatggataatcctgggatt3.3.3. 3.3.3.3. 3.gtaagaatgt1020
atagccctaccagcattctggacataagacaaggaccaaaggaaccctttagagactatg1080
tagaccgattctataaaactctaagagccgagcaagcttcacaagaggtaaaaaattgga1140
tgacagaaaccttgttggtccaaaatgcgaacccagattgtaagactattttaaaagcat1200
tgggaccaggagcgacactagaagaaatgatgacagcatgtcagggagtggggggacccg1260
gccataaagcaagagttttgactccacttggtgacacaactcacacatctggaacgacgg1320
atccgctgaaagcgctggacgatgctatcgcatctgtagacaaattccgttcttccctcg1380
gtgcggtgcaaaaccgtctggattccgcggttaccaacctgaacaacaccactaccaacc1440
tgtctgaagcgcagtcccgtattcaggacgccgactatgcgaccgaagtgtccaatatgt1500
cgaaagcgcagatcatccagcaggccggtaactccgtgttggcaaaagctaaccaggtac1560
cgcagcaggttctgtctctgctgcagggtctcgagcaccaccaccaccaccac1613
<210>1
〈211>1580
<212>KFD-p24 3D DNA
〈400〉11
ccatggcacaagtcattaataccaacagcctctcgctgatcactcaaaataatatcaaca60
agaaccagtctgcgctgtcgagttctatcgagcgtctgtcttctggcttgcgtattaaca120
gcgcgaaggatgacgcagcgggtcaggcgattgctaaccgtttcacctctaacattaaag180
gcctgactcaggcggcccgtaacgccaacgacggtatctccgttgcgcagaccaccgaag240
gcgcgctgtccgaaatcaacaacaacttacagcgtgtgcgtgaactgacggtacaggcca300
ctaccggtactaactctgagtctgatctgtcttctatccaggacgaaattaaatcccgtc360
tggatgaaattgaccgcgtatctggtcagacccagttcaacggcgtgaacgtgctggcaa420
aaaatggctccatgaaaatccaggttggcgcaaatgataaccagactatcactatcgatc480
tgaagcagattgatgctaaaactcttggccttgatactccacttggtgacacaactcaca540
catctggagctagcacacctcttggtgatactacacacacatcaggacctatagtgcaga600
acctccaggggcaaatggtacatcaggccatatcacctagaactttaaatgcatgggtaa660
aagtagtagaagagaaggctttcagcccagaagtaatacccatgttttcagcattatcag720
aaggagccaccccacaagatttaaataccatgctaaacacagtggggggacatcaagcag780
ccatgcaaatgttaaaagagaccatcaatgaggaagctgcagaatgggatagattgcatc840
cagtgcatgcagggcctattgcaccaggccagatgagagaaccaaggggaagtgacatag900
caggaactactagtacccttcaggaacaaataggatggatgacacataatccacctatcc960
cagtaggagaaatctataaaagatggataatcctgggatt3.3.3. 3.3.3.3. 3.gtaagaatgt1020
atagccctaccagcattctggacataagacaaggaccaaaggaaccctttagagactatg1080
tagaccgattctataaaactctaagagccgagcaagcttcacaagaggtaaaaaattgga1140
tgacagaaaccttgttggtccaaaatgcgaacccagattgtaagactattttaaaagcat1200
tgggaccaggagcgacactagaagaaatgatgacagcatgtcagggagtggggggacccg1260
gccataaagcaagagttttgacgacggatccgctgaaagcgctggacgatgctatcgcat1320
ctgtagacaaattccgttcttccctcggtgcggtgcaaaaccgtctggattccgcggtta1380
ccaacctgaacaacaccactaccaacctgtctgaagcgcagtcccgtattcaggacgccg1440
actatgcgaccgaagtgtccaatatgtcgaaagcgcagatcatccagcaggccggtaact1500
CCgtgttggCaaaagctaaccaggtaccgcagcaggttctgtctctgctgcagggtctcg1560
agcaccaccaccaccaccac1580
<210>1
〈211>27
<212>KF Nl (NcoI)人工序列
<400>12
ggcgtccatg gcacaagtca ttaatac27
16
<210>1<211>48<212>KF Low-Nl(BamHI)人工序列<400>13ggcaaggatc cgtcgtcgta gcgctagcat caaggccaag agttttag48<210>1<211>27<212>KF N2 (NcoI)人工序列<400>14ggcgtccatg gcacaagtca ttaatac27<210>1<211>75<212>KF Low-N2 (BamHI)人工序列<400>15tactaggatc cgtcgttcca gatgtgtgag ttgtgtcacc aagtggagtg ctagcatcaa 60ggccaagagt tttag75<210>1<211>27<212>KF N3 (NcoI)人工序列<400>16ggcgtccatg gcacaagtca ttaatac27<210>1<211>75<212>KF Low-N3 (BamHI)人工序列<400>17tactaggatc cgtcgtgcta gctccagatg tgtgagttgt gtcaccaagt ggagtatcaa 60ggccaagagt tttag75<210>1<211>31<212>Up p24-l (NheI)人工序列<400>18gcatagctag ccctatagtg cagaacctcc a31<210>1<211>36<212>Low-p24-l (BamHI)人工序列<400>19tatgcggatc cgtcgtcaaa actcttgctt tatggc36<210>1<211>64
<212>Up p24-2 (NheI)人工序列
<400>20
atcgagctag cacacctctt ggtgatacta cacacacatc aggacctata gtgcagaacc
tcca
<210>1
<211>69
<212>Low-p24-2 (BamHI)人工序列
<400>21
tatgcggatc cgtcgttcca gatgtgtgag ttgtgtcacc aagtggagtcaaaactcttg
ctttatggc
<210>1
<211>274
<212>PRT KFDl
<400>22
Ala Gln ValIleAsnThrAsnSerLeuSerLeuIleThrGlnAsnAsn
151015
lie Asn LysAsnGlnSerAlaLeuSerSerSerIleGluArgLeuSer
202530
Ser Gly LeuArgIleAsnSerAlaLysAspAspAlaAlaGlyGlnAla
354045
lie Ala AsnArgPheThrSerAsnIleLysGlyLeuThrGlnAlaAla
505560
Arg Asn AlaAsnAspGlyIleSerValAlaGlnThrThrGluGlyAla
65707580
Leu Ser GluIleAsnAsnAsnLeuGlnArgValArgGluLeuThrVal
859095
Gln Ala ThrThrGlyThrAsnSerGluSerAspLeuSerSerIleGln
100105110
Asp Glu IleLysSerArgLeuAspGluIleAspArgValSerGlyGln
115120125
Thr Gln PheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys
130135140
lie Gln ValGlyAlaAsnAspAsnGlnThrIleThrIleAspLeuLys
145150155160
Gln lie AspAlaLysThrLeuGlyLeuAspAlaSerAlaThrThrThr
165170175
Asp Pro LeuLysAlaLeuAspAspAlaIleAlaSerValAspLysPhe
180185190
Arg Ser SerLeuGlyAlaValGlnAsnArgLeuAspSerAlaValThr
195200205
AsnLeuAsnAsnThrThrThrAsnLeuSerGluAlaGlnSerArglie
210215220
GlnAspAlaAspTyrAlaThrGluValSerAsnMetSerLysAlaGln
225230235240
IleIleGlnGlnAlaGlyAsnSerValLeuAlaLysAlaAsnGlnVal
245250255
ProGlnGlnValLeuSerLeuLeuGlnGlyLeuGluHisHisHisHis
260265270
HisHis
<210>1
<211>274
<212>PRT KFD2
<400>23
AlaGlnValIleAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn
151015
IleAsnLysAsnGlnSerAlaLeuSerSerSerlieGluArgLeuSer
202530
SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla
354045
IleAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla
505560
ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla
65707580
LeuSerGluIleAsnAsnAsnLeuGlnArgValArgGluLeuThrVal
859095
GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln
100105110
AspGluIleLysSerArgLeuAspGlulieAspArgValSerGlyGln
115120125
ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys
130135140
IleGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys
145150155160
GlnIleAspAlaLysThrLeuGlyLeuAspAlaSerThrProLeuGly
165170175
AspThrThrHisThrSerGlyThrThrAspProLeuLysAlaLeuAsp
180185190
AspAlaIleAlaSerValAspLysPheArgSerSerLeuGlyAlaVal
19
195200205
GlnAsnArgLeuAspSerAlaValThrAsnLeuAsnAsnThrThrThr
210215220
AsnLeuSerGluAlaGlnSerArglieGlnAspAlaAspTyrAlaThr
225230235240
GluValSerAsnMetSerLysAlaGlnlielieGlnGlnAlaGlyAsn
245250255
SerValLeuAlaLysAlaAsnGlnValProGlnGlnValLeuSerLeu
260265270
LeuGlnGlyLeuGluHisHisHisHisHisHis
275280
<210>1
<211>283
<212>PRT KFD3
<400>24
AlaGlnValIleAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn
151015
IleAsnLysAsnGlnSerAlaLeuSerSerSerlieGluArgLeuSer
202530
SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla
354045
IleAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla
505560
ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla
65707580
LeuSerGluIleAsnAsnAsnLeuGlnArgValArgGluLeuThrVal
859095
GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln
100105110
AspGluIleLysSerArgLeuAspGlulieAspArgValSerGlyGln
115120125
ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys
130135140
IleGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys
145150155160
GlnIleAspAlaLysThrLeuGlyLeuAspThrProLeuGlyAspThr
165170175
ThrHisThrSerGlyAlaSerThrThrAspProLeuLysAlaLeuAsp
180185190
20
AspAlaIleAlaSerValAspLysPheArgSerSerLeuGlyAlaVal
195200205
GlnAsnArgLeuAspSerAlaValThrAsnLeuAsnAsnThrThrThr
210215220
AsnLeuSerGluAlaGlnSerArglieGlnAspAlaAspTyrAlaThr
225230235240
GluValSerAsnMetSerLysAlaGlnlielieGlnGlnAlaGlyAsn
245250255
SerValLeuAlaLysAlaAsnGlnValProGlnGlnValLeuSerLeu
260265270
LeuGlnGlyLeuGluHisHisHisHisHisHis
275280
<210>1
<211>503
<212>PRT KFD-p24 2A
<400>25
AlaGlnValIleAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn
151015
IleAsnLysAsnGlnSerAlaLeuSerSerSerlieGluArgLeuSer
202530
SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla
354045
IleAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla
505560
ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla
65707580
LeuSerGluIleAsnAsnAsnLeuGlnArgValArgGluLeuThrVal
859095
GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln
100105110
AspGluIleLysSerArgLeuAspGlulieAspArgValSerGlyGln
115120125
ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys
130135140
IleGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys
145150155160
GlnIleAspAlaLysThrLeuGlyLeuAspAlaSerProlieValGln
165170175
AsnLeuGlnGlyGlnMetValHisGlnAlalieSerProArgThrLeu
21
180185190
AsnAlaTrpValLysValValGluGluLysAlaPheSerProGluVal
195200205
IleProMetPheSerAlaLeuSerGluGlyAlaThrProGlnAspLeu
210215220
AsnThrMetLeuAsnThrValGlyGlyHisGlnAlaAlaMetGlnMet
225230235240
LeuLysGluThrlieAsnGluGluAlaAlaGluTrpAspArgLeuHis
245250255
ProValHisAlaGlyProlieAlaProGlyGlnMetArgGluProArg
260265270
GlySerAsplieAlaGlyThrThrSerThrLeuGlnGluGlnlieGly
275280285
TrpMetThrHisAsnProProlieProValGlyGlulieTyrLysArg
290295300
TrplielieLeuGlyLeuAsnLyslieValArgMetTyrSerProThr
305310315320
SerlieLeuAsplieArgGlnGlyProLysGluProPheArgAspTyr
325330335
ValAspArgPheTyrLysThrLeuArgAlaGluGlnAlaSerGlnGlu
340345350
ValLysAsnTrpMetThrGluThrLeuLeuValGlnAsnAlaAsnPro
355360365
AspCysLysThrlieLeuLysAlaLeuGlyProGlyAlaThrLeuGlu
370375380
GluMetMetThrAlaCysGlnGlyValGlyGlyProGlyHisLysAla
385390395400
ArgValLeuThrThrAspProLeuLysAlaLeuAspAspAlalieAla
405410415
SerValAspLysPheArgSerSerLeuGlyAlaValGlnAsnArgLeu
420425430
AspSerAlaValThrAsnLeuAsnAsnThrThrThrAsnLeuSerGlu
435440445
AlaGlnSerArglieGlnAspAlaAspTyrAlaThrGluValSerAsn
450455460
MetSerLysAlaGlnlielieGlnGlnAlaGlyAsnSerValLeuAla
465470475480
LysAlaAsnGlnValProGlnGlnValLeuSerLeuLeuGlnGlyLeu
485490495
220666]GluHisHisHisHisHisHis0667]5000668]<210>10669]<211>5140670]<212>PRT KFD-p24 2B0671]<400>260672]AlaGlnVallieAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn0673]1510150674]IleAsnLysAsnGlnSerAlaLeuSerSerSerlieGluArgLeuSer0675]2025300676]SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla0677]3540450678]IleAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla0679]5055600680]ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla0681]657075800682]LeuSerGlulieAsnAsnAsnLeuGlnArgValArgGluLeuThrVal0683]8590950684]GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln0685]1001051100686]AspGlulieLysSerArgLeuAspGlulieAspArgValSerGlyGln0687]1151201250688]ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys0689]1301351400690]IleGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys0691]1451501551600692]GlnlieAspAlaLysThrLeuGlyLeuAspAlaSerProlieValGln0693]1651701750694]AsnLeuGlnGlyGlnMetValHisGlnAlalieSerProArgThrLeu0695]1801851900696]AsnAlaTrpValLysValValGluGluLysAlaPheSerProGluVal0697]1952002050698]IleProMetPheSerAlaLeuSerGluGlyAlaThrProGlnAspLeu0699]2102152200700]AsnThrMetLeuAsnThrValGlyGlyHisGlnAlaAlaMetGlnMet0701]2252302352400702]LeuLysGluThrlieAsnGluGluAlaAlaGluTrpAspArgLeuHis0703]2452502550704]ProValHisAlaGlyProlieAlaProGlyGlnMetArgGluProArg0705]2602652700706]GlySerAsplieAlaGlyThrThrSerThrLeuGlnGluGlnlieGly0707]2752802850708]TrpMetThrHisAsnProProlieProValGlyGlulieTyrLysArg0709]2902953000710]TrplielieLeuGlyLeuAsnLyslieValArgMetTyrSerProThr0711]3053103153200712]SerlieLeuAsplieArgGlnGlyProLysGluProPheArgAspTyr0713]3253303350714]ValAspArgPheTyrLysThrLeuArgAlaGluGlnAlaSerGlnGlu0715]3403453500716]ValLysAsnTrpMetThrGluThrLeuLeuValGlnAsnAlaAsnPro0717]3553603650718]AspCysLysThrlieLeuLysAlaLeuGlyProGlyAlaThrLeuGlu0719]3703753800720]GluMetMetThrAlaCysGlnGlyValGlyGlyProGlyHisLysAla0721]3853903954000722]ArgValLeuThrProLeuGlyAspThrThrHisThrSerGlyThrThr0723]4054104150724]AspProLeuLysAlaLeuAspAspAlalieAlaSerValAspLysPhe0725]4204254300726]ArgSerSerLeuGlyAlaValGlnAsnArgLeuAspSerAlaValThr0727]4354404450728]AsnLeuAsnAsnThrThrThrAsnLeuSerGluAlaGlnSerArglie0729]4504554600730]GlnAspAlaAspTyrAlaThrGluValSerAsnMetSerLysAlaGln0731]4654704754800732]IlelieGlnGlnAlaGlyAsnSerValLeuAlaLysAlaAsnGlnVal0733]4854904950734]ProGlnGlnValLeuSerLeuLeuGlnGlyLeuGluHisHisHisHis0735]5005055100736]HisHis0737]<210>10738]<211>5250739]<212>PRT KFD-p24 2C0740]<400>270741]AlaGlnVallieAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn0742]1510150743]lie Asn LysAsnGlnSerAlaLeuSerSerSerlieGluArg LeuSer
24
202530
SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla
354045
IleAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla
505560
ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla
65707580
LeuSerGlulieAsnAsnAsnLeuGlnArgValArgGluLeuThrVal
859095
GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln
100105110
AspGlulieLysSerArgLeuAspGlulieAspArgValSerGlyGln
115120125
ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys
130135140
IleGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys
145150155160
GlnlieAspAlaLysThrLeuGlyLeuAspAlaSerThrProLeuGly
165170175
AspThrThrHisThrSerGlyProlieValGlnAsnLeuGlnGlyGln
180185190
MetValHisGlnAlalieSerProArgThrLeuAsnAlaTrpValLys
195200205
ValValGluGluLysAlaPheSerProGluVallieProMetPheSer
210215220
AlaLeuSerGluGlyAlaThrProGlnAspLeuAsnThrMetLeuAsn
225230235240
ThrValGlyGlyHisGlnAlaAlaMetGlnMetLeuLysGluThrlie
245250255
AsnGluGluAlaAlaGluTrpAspArgLeuHisProValHisAlaGly
260265270
ProlieAlaProGlyGlnMetArgGluProArgGlySerAsplieAla
275280285
GlyThrThrSerThrLeuGlnGluGlnlieGlyTrpMetThrHisAsn
290295300
ProProlieProValGlyGlulieTyrLysArgTrplielieLeuGly
305310315320
LeuAsnLyslieValArgMetTyrSerProThrSerlieLeuAsplie
325330335
250783]ArgGlnGlyProLysGluProPheArgAspTyrValAspArgPheTyr0784]3403453500785]LysThrLeuArgAlaGluGlnAlaSerGlnGluValLysAsnTrpMet0786]3553603650787]ThrGluThrLeuLeuValGlnAsnAlaAsnProAspCysLysThrlie0788]3703753800789]LeuLysAlaLeuGlyProGlyAlaThrLeuGluGluMetMetThrAla0790]3853903954000791]CysGlnGlyValGlyGlyProGlyHisLysAlaArgValLeuThrPro0792]4054104150793]LeuGlyAspThrThrHisThrSerGlyThrThrAspProLeuLysAla0794]4204254300795]LeuAspAspAlalieAlaSerValAspLysPheArgSerSerLeuGly0796]4354404450797]AlaValGlnAsnArgLeuAspSerAlaValThrAsnLeuAsnAsnThr0798]4504554600799]ThrThrAsnLeuSerGluAlaGlnSerArglieGlnAspAlaAspTyr0800]4654704754800801]AlaThrGluValSerAsnMetSerLysAlaGlnlielieGlnGlnAla0802]4854904950803]GlyAsnSerValLeuAlaLysAlaAsnGlnValProGlnGlnValLeu0804]5005055100805]SerLeuLeuGlnGlyLeuGluHisHisHisHisHisHis0806]5155205250807]<210>10808]<211>5140809]<212>PRT KFD-p24 2D0810]<400>280811]AlaGlnVallieAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn0812]1510150813]IleAsnLysAsnGlnSerAlaLeuSerSerSerlieGluArgLeuSer0814]2025300815]SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla0816]3540450817]IleAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla0818]5055600819]ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla0820]657075800821]LeuSerGlulieAsnAsnAsnLeuGlnArgValArgGluLeuThrVal
26
859095
GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln
100105110
AspGlulieLysSerArgLeuAspGlulieAspArgValSerGlyGln
115120125
ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys
130135140
IleGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys
145150155160
GlnlieAspAlaLysThrLeuGlyLeuAspAlaSerThrProLeuGly
165170175
AspThrThrHisThrSerGlyProlieValGlnAsnLeuGlnGlyGln
180185190
MetValHisGlnAlalieSerProArgThrLeuAsnAlaTrpValLys
195200205
ValValGluGluLysAlaPheSerProGluVallieProMetPheSer
210215220
AlaLeuSerGluGlyAlaThrProGlnAspLeuAsnThrMetLeuAsn
225230235240
ThrValGlyGlyHisGlnAlaAlaMetGlnMetLeuLysGluThrlie
245250255
AsnGluGluAlaAlaGluTrpAspArgLeuHisProValHisAlaGly
260265270
ProlieAlaProGlyGlnMetArgGluProArgGlySerAsplieAla
275280285
GlyThrThrSerThrLeuGlnGluGlnlieGlyTrpMetThrHisAsn
290295300
ProProlieProValGlyGlulieTyrLysArgTrplielieLeuGly
305310315320
LeuAsnLyslieValArgMetTyrSerProThrSerlieLeuAsplie
325330335
ArgGlnGlyProLysGluProPheArgAspTyrValAspArgPheTyr
340345350
LysThrLeuArgAlaGluGlnAlaSerGlnGluValLysAsnTrpMet
355360365
ThrGluThrLeuLeuValGlnAsnAlaAsnProAspCysLysThrlie
370375380
LeuLysAlaLeuGlyProGlyAlaThrLeuGluGluMetMetThrAla
385390395400
27
CysGlnGlyValGlyGlyProGlyHisLysAlaArgValLeuThrThr
405410415
AspProLeuLysAlaLeuAspAspAlalieAlaSerValAspLysPhe
420425430
ArgSerSerLeuGlyAlaValGlnAsnArgLeuAspSerAlaValThr
435440445
AsnLeuAsnAsnThrThrThrAsnLeuSerGluAlaGlnSerArglie
450455460
GlnAspAlaAspTyrAlaThrGluValSerAsnMetSerLysAlaGln
465470475480
IlelieGlnGlnAlaGlyAsnSerValLeuAlaLysAlaAsnGlnVal
485490495
ProGlnGlnValLeuSerLeuLeuGlnGlyLeuGluHisHisHisHis
500505510
HisHis
<210>1
<211>514
<212>PRT KFD-p24 3A
<400>29
AlaGlnVallieAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn
151015
IleAsnLysAsnGlnSerAlaLeuSerSerSerlieGluArgLeuSer
202530
SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla
354045
IleAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla
505560
ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla
65707580
LeuSerGlulieAsnAsnAsnLeuGlnArgValArgGluLeuThrVal
859095
GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln
100105110
AspGlulieLysSerArgLeuAspGlulieAspArgValSerGlyGln
115120125
ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys
130135140
IleGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys
145150155160
GlnlieAspAlaLysThrLeuGlyLeuAspThrProLeuGlyAspThr
165170175
ThrHisThrSerGlyAlaSerProlieValGlnAsnLeuGlnGlyGln
180185190
MetValHisGlnAlalieSerProArgThrLeuAsnAlaTrpValLys
195200205
ValValGluGluLysAlaPheSerProGluVallieProMetPheSer
210215220
AlaLeuSerGluGlyAlaThrProGlnAspLeuAsnThrMetLeuAsn
225230235240
ThrValGlyGlyHisGlnAlaAlaMetGlnMetLeuLysGluThrlie
245250255
AsnGluGluAlaAlaGluTrpAspArgLeuHisProValHisAlaGly
260265270
ProlieAlaProGlyGlnMetArgGluProArgGlySerAsplieAla
275280285
GlyThrThrSerThrLeuGlnGluGlnlieGlyTrpMetThrHisAsn
290295300
ProProlieProValGlyGlulieTyrLysArgTrplielieLeuGly
305310315320
LeuAsnLyslieValArgMetTyrSerProThrSerlieLeuAsplie
325330335
ArgGlnGlyProLysGluProPheArgAspTyrValAspArgPheTyr
340345350
LysThrLeuArgAlaGluGlnAlaSerGlnGluValLys Asn Trp Met
355360365
ThrGluThrLeuLeuValGlnAsnAlaAsnProAspCysLysThrlie
370375380
LeuLysAlaLeuGlyProGlyAlaThrLeuGluGluMetMetThrAla
385390395400
CysGlnGlyValGlyGlyProGlyHisLysAlaArgValLeuThrThr
405410415
AspProLeuLysAlaLeuAspAspAlalieAlaSerValAspLysPhe
420425430
ArgSerSerLeuGlyAlaValGlnAsnArgLeuAspSerAlaValThr
435440445
AsnLeuAsnAsnThrThrThrAsnLeuSerGluAlaGlnSerArglie
450455460
GlnAspAlaAspTyrAlaThrGluValSerAsnMetSerLysAlaGln
29
465470475480
IlelieGlnGlnAlaGlyAsnSerValLeuAlaLysAlaAsnGlnVal
485490495
ProGlnGlnValLeuSerLeuLeuGlnGlyLeuGluHisHisHisHis
500505510
HisHis
<210>1
<211>525
<212>PRT KFD-p24 3B
<400>30
AlaGlnVallieAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn
151015
IleAsnLysAsnGlnSerAlaLeuSerSerSerlieGluArgLeuSer
202530
SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla
354045
IleAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla
505560
ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla
65707580
LeuSerGlulieAsnAsnAsnLeuGlnArgValArgGluLeuThrVal
859095
GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln
100105110
AspGlulieLysSerArgLeuAspGlulieAspArgValSerGlyGln
115120125
ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys
130135140
IleGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys
145150155160
GlnlieAspAlaLysThrLeuGlyLeuAspThrProLeuGlyAspThr
165170175
ThrHisThrSerGlyAlaSerProlieValGlnAsnLeuGlnGlyGln
180185190
MetValHisGlnAlalieSerProArgThrLeuAsnAlaTrpValLys
195200205
ValValGluGluLysAlaPheSerProGluVallieProMetPheSer
210215220
AlaLeuSerGluGlyAlaThrProGlnAspLeuAsnThrMetLeuAsn0978]2252302352400979]ThrValGlyGlyHisGlnAlaAlaMetGlnMetLeuLysGluThrlie0980]2452502550981]AsnGluGluAlaAlaGluTrpAspArgLeuHisProValHisAlaGly0982]2602652700983]ProlieAlaProGlyGlnMetArgGluProArgGlySerAsplieAla0984]2752802850985]GlyThrThrSerThrLeuGlnGluGlnlieGlyTrpMetThrHisAsn0986]2902953000987]ProProlieProValGlyGlulieTyrLysArgTrplielieLeuGly0988]3053103153200989]LeuAsnLyslieValArgMetTyrSerProThrSerlieLeuAsplie0990]3253303350991]ArgGlnGlyProLysGluProPheArgAspTyrValAspArgPheTyr0992]3403453500993]LysThrLeuArgAlaGluGlnAlaSerGlnGluValLysAsnTrpMet0994]3553603650995]ThrGluThrLeuLeuValGlnAsnAlaAsnProAspCysLysThrlie0996]3703753800997]LeuLysAlaLeuGlyProGlyAlaThrLeuGluGluMetMetThrAla0998]3853903954000999]CysGlnGlyValGlyGlyProGlyHisLysAlaArgValLeuThrPro1000]4054104151001]LeuGlyAspThrThrHisThrSerGlyThrThrAspProLeuLysAla1002]4204254301003]LeuAspAspAlalieAlaSerValAspLysPheArgSerSerLeuGly1004]4354404451005]AlaValGlnAsnArgLeuAspSerAlaValThrAsnLeuAsnAsnThr1006]4504554601007]ThrThrAsnLeuSerGluAlaGlnSerArglieGlnAspAlaAspTyr1008]4654704754801009]AlaThrGluValSerAsnMetSerLysAlaGlnlielieGlnGlnAla1010]4854904951011]GlyAsnSerValLeuAlaLysAlaAsnGlnValProGlnGlnValLeu1012]5005055101013]SerLeuLeuGlnGlyLeuGluHisHisHisHisHisHis1014]5155205251015]<210>11016]<211>536
31[1017]<212>PRT KFD-p24 3C[1018]<400>31[1019]AlaGlnVallieAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn[1020]151015[1021]lieAsnLysAsnGlnSerAlaLeuSerSerSerlieGluArgLeuSer[1022]202530[1023]SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla[1024]354045[1025]lieAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla[1026]505560[1027]ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla[1028]65707580[1029]LeuSerGlulieAsnAsnAsnLeuGlnArgValArgGluLeuThrVal[1030]859095[1031]GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln[1032]100105110[1033]AspGlulieLysSerArgLeuAspGlulieAspArgValSerGlyGln[1034]115120125[1035]ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys[1036]130135140[1037]lieGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys[1038]145150155160[1039]GlnlieAspAlaLysThrLeuGlyLeuAspThrProLeuGlyAspThr[1040]165170175[1041]ThrHisThrSerGlyAlaSerThrProLeuGlyAspThrThrHisThr[1042]180185190[1043]SerGlyProlieValGlnAsnLeuGlnGlyGlnMetValHisGlnAla[1044]195200205[1045]lieSerProArgThrLeuAsnAlaTrpValLysValValGluGluLys[1046]210215220[1047]AlaPheSerProGluVallieProMetPheSerAlaLeuSerGluGly[1048]225230235240[1049]AlaThrProGlnAspLeuAsnThrMetLeuAsnThrValGlyGlyHis[1050]245250255[1051]GlnAlaAlaMetGlnMetLeuLysGluThrlieAsnGluGluAlaAla[1052]260265270[1053]GluTrpAspArgLeuHisProValHisAlaGlyProlieAlaProGly[1054]275280285[1055]GlnMetArgGluProArgGlySerAsplieAlaGlyThrThrSerThr1056]2902953001057]LeuGlnGluGlnlieGlyTrpMetThrHisAsnProProlieProVal1058]3053103153201059]GlyGlulieTyrLysArgTrplielieLeuGlyLeuAsnLyslieVal1060]3253303351061]ArgMetTyrSerProThrSerlieLeuAsplieArgGlnGlyProLys1062]3403453501063]GluProPheArgAspTyrValAspArgPheTyrLysThrLeuArgAla1064]3553603651065]GluGlnAlaSerGlnGluValLysAsnTrpMetThrGluThrLeuLeu1066]3703753801067]ValGlnAsnAlaAsnProAspCysLysThrlieLeuLysAlaLeuGly1068]3853903954001069]ProGlyAlaThrLeuGluGluMetMetThrAlaCysGlnGlyValGly1070]4054104151071]GlyProGlyHisLysAlaArgValLeuThrProLeuGlyAspThrThr1072]4204254301073]HisThrSerGlyThrThrAspProLeuLysAlaLeuAspAspAlalie1074]4354404451075]AlaSerValAspLysPheArgSerSerLeuGlyAlaValGlnAsnArg1076]4504554601077]LeuAspSerAlaValThrAsnLeuAsnAsnThrThrThrAsnLeuSer1078]4654704754801079]GluAlaGlnSerArglieGlnAspAlaAspTyrAlaThrGluValSer1080]4854904951081]AsnMetSerLysAlaGlnlielieGlnGlnAlaGlyAsnSerValLeu1082]5005055101083]AlaLysAlaAsnGlnValProGlnGlnValLeuSerLeuLeuGlnGly1084]5155205251085]LeuGluHisHisHisHisHisHis1086]5305351087]<210>11088]<211>5251089]<212>PRT KFD-p24 3D1090]<400>321091]AlaGlnVallieAsnThrAsnSerLeuSerLeulieThrGlnAsnAsn1092]1510151093]lieAsnLysAsnGlnSerAlaLeuSerSerSerlieGluArgLeuSer1094]202530
33[1095]SerGlyLeuArglieAsnSerAlaLysAspAspAlaAlaGlyGlnAla[1096]354045[1097]IleAlaAsnArgPheThrSerAsnlieLysGlyLeuThrGlnAlaAla[1098]505560[1099]ArgAsnAlaAsnAspGlylieSerValAlaGlnThrThrGluGlyAla[1100]65707580[1101]LeuSerGlulieAsnAsnAsnLeuGlnArgValArgGluLeuThrVal[1102]859095[1103]GlnAlaThrThrGlyThrAsnSerGluSerAspLeuSerSerlieGln[1104]100105110[1105]AspGlulieLysSerArgLeuAspGlulieAspArgValSerGlyGln[1106]115120125[1107]ThrGlnPheAsnGlyValAsnValLeuAlaLysAsnGlySerMetLys[1108]130135140[1109]IleGlnValGlyAlaAsnAspAsnGlnThrlieThrlieAspLeuLys[1110]145150155160[1111]GlnIleAspAlaLysThrLeuGlyLeuAspThrProLeuGlyAspThr[1112]165170175[1113]ThrHisThrSerGlyAlaSerThrProLeuGlyAspThrThrHisThr[1114]180185190[1115]SerGlyProlieValGlnAsnLeuGlnGlyGlnMetValHisGlnAla[1116]195200205[1117]IleSerProArgThrLeuAsnAlaTrpValLysValValGluGluLys[1118]210215220[1119]AlaPheSerProGluVallieProMetPheSerAlaLeuSerGluGly[1120]225230235240[1121]AlaThrProGlnAspLeuAsnThrMetLeuAsnThrValGlyGlyHis[1122]245250255[1123]GlnAlaAlaMetGlnMetLeuLysGluThrlieAsnGluGluAlaAla[1124]260265270[1125]GluTrpAspArgLeuHisProValHisAlaGlyProlieAlaProGly[1126]275280285[1127]GlnMetArgGluProArgGlySerAsplieAlaGlyThrThrSerThr[1128]290295300[1129]LeuGlnGluGlnlieGlyTrpMetThrHisAsnProProlieProVal[1130]305310315320[1131]GlyGlulieTyrLysArgTrplielieLeuGlyLeuAsnLyslieVal[1132]325330335[1133]ArgMetTyrSerProThrSerlieLeuAsplieArgGlnGlyProLys
34[1134]340345350[1135]GluProPheArgAspTyrValAspArgPheTyrLysThrLeuArgAla[1136]355360365[1137]GluGlnAlaSerGlnGluValLysAsnTrpMetThrGluThrLeuLeu[1138]370375380[1139]ValGlnAsnAlaAsnProAspCysLysThrlieLeuLysAlaLeuGly[1140]385390395400[1141]ProGlyAlaThrLeuGluGluMetMetThrAlaCysGlnGlyValGly[1142]405410415[1143]GlyProGlyHisLysAlaArgValLeuThrThrAspProLeuLysAla[1144]420425430[1145]LeuAspAspAlalieAlaSerValAspLysPheArgSerSerLeuGly[1146]435440445[1147]AlaValGlnAsnArgLeuAspSerAlaValThrAsnLeuAsnAsnThr[1148]450455460[1149]ThrThrAsnLeuSerGluAlaGlnSerArglieGlnAspAlaAspTyr[1150]465470475480[1151]AlaThrGluValSerAsnMetSerLysAlaGlnlielieGlnGlnAla[1152]485490495[1153]GlyAsnSerValLeuAlaLysAlaAsnGlnValProGlnGlnValLeu[1154]500505510[1155]SerLeuLeuGlnGlyLeuGluHisHisHisHisHisHis[1156]515520525
3權(quán)利要求
1.一種改造的非致病細(xì)菌來源的鞭毛素粘膜佐劑，其特征在于，所述鞭毛素粘膜佐劑 去除了鞭毛蛋白的主要抗原活性區(qū)。
2.根據(jù)權(quán)利要求1所述的鞭毛素粘膜佐劑，其特征在于，所述非致病細(xì)菌包括大腸桿 菌K12株。
3.根據(jù)權(quán)利要求1所述的鞭毛素粘膜佐劑，其特征在于，所述抗原活性區(qū)包括高變區(qū)。
4.根據(jù)權(quán)利要求1所述的鞭毛素粘膜佐劑，其特征在于，所述鞭毛素粘膜佐劑還含有 柔性結(jié)構(gòu)，所述柔性結(jié)構(gòu)免疫原性低。
5.根據(jù)權(quán)利要求4所述的鞭毛素粘膜佐劑，其特征在于，所述柔性結(jié)構(gòu)包括人IgG3鉸 鏈序列。
6.根據(jù)權(quán)利要求1所述的鞭毛素粘膜佐劑，其特征在于，所述抗原活性區(qū)替換為目的 抗原。
7.根據(jù)權(quán)利要求6所述的鞭毛素粘膜佐劑，其特征在于，所述目的抗原包括p24抗原。
8.根據(jù)權(quán)利要求1所述的鞭毛素粘膜佐劑，其特征在于，所述佐劑包括如下的佐劑，該 佐劑的基因序列如SEQID:1或氨基酸？序列如SEQID :22，基因序列如SEQID2或氨基酸■序列如SEQID :23，基因序列如SEQID3或氨基酸■序列如SEQID :24，基因序列如SEQID4或氨基酸■序列如SEQID :25，基因序列如SEQID5或氨基酸■序列如SEQID :26，基因序列如SEQID6或氨基酸■序列如SEQID :27，基因序列如SEQID7或氨基酸■序列如SEQID :28，基因序列如SEQID8或氨基酸■序列如SEQID :29，基因序列如SEQID9或氨基酸■序列如SEQID :30，基因序列如SEQID10或氨基丨f序列如SEQ ID:31，基因序列如SEQID11或氨基丨f序列如SEQ IDj 3 2 ο
9.一種改造的非致病細(xì)菌來源的鞭毛素粘膜佐劑的制備方法，其特征在于，去除鞭毛 蛋白片段中的主要抗原活性區(qū)。
10.根據(jù)權(quán)利要求9所述的制備方法，其特征在于，選擇的非致病細(xì)菌包括大腸桿菌 K12 株。
全文摘要
本發(fā)明提供了一種優(yōu)化改造的非致病細(xì)菌來源的鞭毛素粘膜佐劑及其制備方法，將鞭毛素粘膜佐劑去除鞭毛蛋白的主要抗原活性區(qū)，將其替換為目的抗原，加入免疫原性低的柔性結(jié)構(gòu)，用這種方法得到的改造的鞭毛素粘膜佐劑的基因序列為SEQ ID NO1-11，氨基酸序列為SEQ ID NO22-32。本發(fā)明的佐劑降低了其可能具有的潛在的危險(xiǎn)性、其自身的抗原性、免疫原性以及由其引發(fā)的炎癥反應(yīng)和毒性；提高了抗原遞呈效果；通過添加低免疫原性的柔性結(jié)構(gòu)，維持其三維結(jié)構(gòu)與天然的鞭毛素蛋白相類似，從而維持其佐劑活性。
文檔編號(hào)A61K39/39GK102000329SQ20091019428
公開日2011年4月6日 申請日期2009年11月27日 優(yōu)先權(quán)日2009年11月27日
發(fā)明者劉芳, 楊菁毅, 鄢慧民 申請人:中國科學(xué)院武漢病毒研究所