TW202309062A - 抗體組成物之純化方法 - Google Patents
抗體組成物之純化方法 Download PDFInfo
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- TW202309062A TW202309062A TW111117152A TW111117152A TW202309062A TW 202309062 A TW202309062 A TW 202309062A TW 111117152 A TW111117152 A TW 111117152A TW 111117152 A TW111117152 A TW 111117152A TW 202309062 A TW202309062 A TW 202309062A
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Abstract
本發明課題為提供一種將抗體藥物的附加糖鏈的異構物去除的方法等。本發明例如提供一種抗體組成物之純化方法,其係包含:藉由將前述抗體組成物載入慣用層析裝置,使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於層析擔體,以及以溶離液讓前述擔體發生溶離,使吸附於前述擔體的前述抗體溶離而得到純化抗體組成物。
Description
本發明關於一種抗體組成物的製造方法。更詳細而言,關於一種抗體組成物之純化方法,其係在Fc區糖鏈結合共有區以外減少了附加糖鏈的異構物。
以單株抗體為有效成分的抗體藥物,被期待活用抗體分子所具有的對抗原的高結合親和性與結合特異性作為一種分子標靶藥物,一直都在進行研究開發。抗體藥物,對於以癌症或自體免疫疾病為首的各種疾病的治療來說已經是不可欠缺的,目前的狀況已達接近100項製品在世界上被承認使用(非專利文獻1、非專利文獻2)。對於能滿足未滿足需求的新穎抗體藥物開發的期待依然很高,今後也可期待研究開發出許多新抗體藥物。
抗體(IgG)中,存在於重鏈的Fc部分的附加糖鏈的共有區(Asn297-X-Ser/Thr,X為Pro以外的胺基酸)中的第297個殘基Asn殘基(Asn297)具有N-鍵結型糖鏈。已知存在於該附加糖鏈的共有區的糖鏈有助於作為抗體分子的生物活性或血中動態、安全性等(非專利文獻3、非專利文獻4)。例如,已知若將附加在Asn297的N-鍵結型糖鏈的還原末端的N-乙醯葡萄糖胺(GlcNAc)殘基的Fuc殘基(核心岩藻醣)去除,則會增強抗體依存性細胞傷害(ADCC)活性。同樣地,已知結合於Fc部分的糖鏈的非還原末端部分的半乳糖(Gal)殘基的數目愈多,與補體第一成分(C1q)的結合愈增強,結果會增強補體依存性細胞傷害(CDC)活性(非專利文獻5)。
另一方面,在抗體的Fc區的附加糖鏈的共有區以外鍵結了糖鏈的抗體(附加糖鏈的異構物)的事例已為所周知。例如,確認了SP2/0細胞所產生的抗惡性腫瘤劑西妥昔單抗(Cetuximab),在Fab部分結合了N-結合型糖鏈(非專利文獻3)。除此之外,關於附加糖鏈的異構物也已經有各項報告(非專利文獻6、非專利文獻7、非專利文獻8)。
已知附加糖鏈的異構物,會有對生物活性(非專利文獻10、非專利文獻11)、免疫原性(非專利文獻8、非專利文獻10)、血中半衰期(非專利文獻9)等各種抗體的特性造成影響的可能性。尤其在Fab區或CDR區附近結合了糖鏈的情況,會有生物活性可能降低的顧慮。同樣地,在附加糖鏈的共有區以外結合了糖鏈的抗體之附加糖鏈的異構物,也會有免疫原性等的安全性的顧慮。
在因為糖鏈附加在附加糖鏈的共有區以外,生物活性大幅降低或免疫原性大幅增加的情況,就抗體藥物的成分而言,會產生不希望有的負面影響,因此這些附加糖鏈的異構物會被視為來自目標物質的雜質。這種來自目標物質的雜質,不希望被包含而成為藥物的成分,從規制上的觀點看來,也希望儘可能去除(非專利文獻12)。
包含了在抗體的Fc區的附加糖鏈的共有區以外可能附加糖鏈的共有序列的抗體,在被發現為藥物開發的候選分子的情況,一般而言是將共有序列變更為其他胺基酸序列等來讓糖鏈不會結合在該部位。此時,也會有與原本的抗體性能相比,結合活性等較為降低的情形,可能會無法取得具有目標治療效果的抗體。
在抗體的附加糖鏈的共有區以外的Fab或CDR等的區域結合了糖鏈的附加糖鏈的異構物,可藉由進行少量且高性能的分析級別的附加糖鏈的異構物的分離來評估、區別。已知有組合高效液相層析與質量分析來對胜肽片段進行評估的方法、或對由抗體切出的糖鏈本身進行評估的方法(非專利文獻6、非專利文獻9)。另外,已知有藉由疏水性交互作用層析(HIC)來分析在抗體的Fab區結合了糖鏈的抗體的事例(非專利文獻13)。但是在這些事例中,任一者都是以分析為目的,使用了極少量的抗體,且使用了分離性能極高的分析級別的方法,無法將所分離出的組成物作為抗體藥物來利用。另外,HIC分析的總說(非專利文獻14)公開了許多抗體的分離性能的有用性,然而完全沒有說明藉由利用HIC的純化法可將附加糖鏈的異構物分離。
像這樣,將這種附加糖鏈的異構物分離、去除,調製出不含或充分減少在附加糖鏈的共有區以外附加了糖鏈的異構物之抗體組成物的方法仍為未知。亦即,在以往的技術中,附加糖鏈的異構物是停留在能夠以分析級別來評估糖鏈的附加狀況,並不存在將抗體的附加糖鏈的異構物分離、去除,製造出能夠以藥物來提供的量的抗體組成物的方法,這樣的抗體組成物也是未知。
所以,在通常存在於抗體的Fc區的附加糖鏈的共有區以外附加了糖鏈,而包含這樣的附加糖鏈的異構物的情況,這些成分不會被分離,會被包含而成為抗體藥物的一部分。關於前述西妥昔單抗,也正在以糖鏈附加在Fc部分的附加糖鏈的共有區及Fab內的非共有區兩者的混合物的組成來進行開發(非專利文獻3)。如果是附加糖鏈的異構物不具有生物活性的情況,來自目標物質的雜質會被包含而成為藥物的成分,這是不希望發生的。
此外,已有文獻報告了使用特異性地結合於既定糖鏈的凝集素的親和層析來進行附加糖鏈的異構物的分離(非專利文獻15、非專利文獻16)。已知刀豆蛋白A(Concanavalin A)作為配體來使用,分離特性很高,然而由於是利用親和性進行的分離,從使用天然物看來,並不適合於藥物的製造,可適用於製造抗體藥物的事例並不存在。
關於抗體以外實現糖鏈成分分離的技術,已知有將基因轉殖抗凝血酶利用糖鏈數的不同藉由層析來分離的事例(專利文獻1)。另外,基因轉殖紅血球生成素及其衍生物,是藉由唾液酸的加成數的不同藉由層析來進行分離(專利文獻2)。同樣地,已有使用等電點層析將卵白蛋白或運鐵蛋白分離的事例(專利文獻3)。但是,無法將這些技術直接適用於抗體,在抗體組成物方面,完全沒有將附加糖鏈的異構物分離去除的製造法的報告。
關於前述以外的技術,已知有藉由酵素處理將糖鏈去除的方法(非專利文獻17)。但是,從安全性的觀點看來,酵素的分離、去除或來源等也會成為問題,因此難以利用作為藥物的製造技術。在藉由酵素將抗體的糖鏈去除的情況,將附加糖鏈的共有區與非共有區的糖鏈區分並且去除也是不可能的。
專利文獻4所記載的抗hDLK-1抗體,是可期待其抗腫瘤活性的抗體。
[先前技術文獻]
[專利文獻]
[專利文獻1]國際公開第2008/120801號
[專利文獻2]日本特開2001-064300號公報
[專利文獻3]國際公開第2005/100379號
[專利文獻4]國際公開第2014/054820號
[非專利文獻]
[非專利文獻1]YAKUGAKU ZASSHI 137(7), 815-816 (2017)
[非專利文獻2]Bull. Natl. Inst. Health Sci., 132, 36-46 (2014)
[非專利文獻3]CHROMATOGRAPHY, 34(2), 83-88 (2013)
[非專利文獻4]Protein Cell, 9, 63-73 (2018)
[非專利文獻5]Front. Immunol., 8 (646), 1-8 (2017)
[非專利文獻6]J. Biol. Chem., 284 (47), 32493-32506 (2009)
[非專利文獻7]Embo J., 10, 2717-2723 (1991)
[非專利文獻8]J. Immunol., 196, 1435-1441 (2016)
[非專利文獻9]Anal. Biochem., 349, 197-207 (2006)
[非專利文獻10]Nature Review|Immunology, published online, 04 Feb, (2019)
[非專利文獻11]Biochem. J., 338 (2), 529-538 (1999)
[非專利文獻12]ICH Q6 B Guideline
[非專利文獻13]mAbs, 6 (4), 852-858 (2014)
[非專利文獻14]J. Pharm. Biomed. Anal., 130, 3-18 (2016)
[非專利文獻15]J. Biol. Chem., 285 (21), 16012-22 (2010)
[非專利文獻16]Oncotarget, 7 (21), 31166-76 (2016)
[非專利文獻17]J. Immunol. Methods, 467,58-62 (2019)
[發明所欲解決的課題]
結合於抗體的附加糖鏈的共有區以外的糖鏈,從生物活性或安全性的觀點看來,都是不希望存在的,正需要開發出一種技術,可將抗體藥物所含有的附加糖鏈的異構物去除至足夠的程度,能夠簡便地調製出均一的純化抗體組成物。所以,本發明課題為提供去除抗體藥物之附加糖鏈的異構物的方法。
在另一個態樣之中,本發明課題為得到更有效且更安全的抗hDLK-1抗體的純化組成物。
[用於解決課題的手段]
如前述先前技術所示般,雖然有為了分析評估的目的,藉由具有精密且高水準的分離能力的層析將附加糖鏈的異構物分離的報告,然而並沒有可將抗體藥物的附加糖鏈的異構物藉由層析來分離去除的製造技術或調製技術的報告。本發明人等為了解決上述課題而鑽研,結果令人驚訝地發現,藉由使用慣用的疏水性交互作用層析擔體,特別是讓附加糖鏈的異構物及目標物質的吸附及分離條件最適化,可調製出減少了附加糖鏈的異構物的純化抗體組成物。
另外,使用本發明人等新發現的附加糖鏈的異構物的減少方法,對於專利文獻4所記載的抗hDLK-1抗體進行附加糖鏈的異構物的分離、非附加糖鏈的異構物的純化。在抗體的CDR附近具有糖鏈的附加糖鏈的異構物,可能會對抗體的結合活性造成影響,其活性降低的程度也和糖鏈的大小或結合位置有關係,因此還考量了不影響活性的可能性。令人驚訝地發現,專利文獻4所記載的抗hDLK-1抗體之中,附加糖鏈的異構物的活性會完全消失,因此會成為藥物中的「雜質」。藉此,本發明人等成功地鑑定附加糖鏈的異構物是抗hDLK-1抗體粗純化物中的新雜質,藉由讓其可去除,達成了更有效且更安全的抗hDLK-1抗體的純化組成物的提供。
亦即,本發明關於以下的(1)~(27)。
(1) 一種抗hDLK-1抗體組成物之純化方法,該抗體之重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且該方法包含:
藉由將抗體粗純化物載入慣用層析裝置,使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於前述層析裝置的擔體,以及
以溶離液讓前述擔體發生溶離,使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物,
此處,所得到的前述純化抗體組成物中,在Fc區的附加糖鏈的共有區以外的部位附加糖鏈之附加糖鏈的異構物的含量,與前述抗體粗純化物相比,較為減少。
(2) 如(1)之純化方法,其中前述慣用層析裝置的擔體為疏水性交互作用層析擔體,或混合模式層析擔體。
(3) 如(1)或(2)之純化方法,其中前述慣用層析裝置的擔體的平均粒徑為15μm以上。
(4) 如(1)或(2)之純化方法,其中前述慣用層析裝置的擔體的平均粒徑為20~100μm。
(5) 如(1)~(4)中任一項之純化方法,其中前述慣用層析裝置的擔體具有苄基或丁基。
(6) 如(1)~(5)中任一項之純化方法,其中前述慣用層析裝置的擔體之每單位量的蛋白質載入量為20mg/mL以上。
(7) 如(1)~(6)中任一項之純化方法,其中前述慣用層析裝置的擔體為疏水性交互作用層析擔體,且包含將前述抗體粗純化物載入前述擔體之後,在溶離前以洗淨液將該擔體洗淨。
(8) 如(7)之純化方法,其中前述洗淨液的鹽濃度比前述溶離液的鹽濃度高10mM以上。
(9) 如(7)或(8)之純化方法,其中前述洗淨液的鹽濃度為0.5M以上。
(10) 如(7)或(8)之純化方法,其中前述洗淨液的鹽濃度為1.0M以上。
(11) 如(7)~(10)中任一項之純化方法,其中前述洗淨液的pH比前述溶離液的pH低0.2以上,且pH在4~8的範圍內。
(12) 如(7)~(11)中任一項之純化方法,其中前述洗淨係藉由流過2倍擔體容積以上的洗淨液來進行。
(13) 如(7)~(12)中任一項之純化方法,其中藉由使用pH及/或鹽濃度階段性或直線地變化的移動相來進行洗淨及溶離。
(14) 如(7)~(13)中任一項之純化方法,其中前述溶離液的鹽濃度在0.5M以下或不含鹽,且pH為5~7。
(15) 如(1)~(14)中任一項之純化方法,其產率為20%以上。
(16) 如(1)~(15)中任一項之純化方法,其中前述純化抗體組成物中之附加糖鏈的異構物相對於所有抗體的比例為5%以下。
(17) 如(16)之純化方法,其中前述抗體粗純化物中之附加糖鏈的異構物相對於所有抗體的比例大於5%。
(18) 一種如(1)之抗hDLK-1抗體組成物之純化方法,其係包含:
將抗體粗純化物載入使用具有苄基或丁基且粒徑為20~100μm的擔體的慣用層析裝置,
使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於前述擔體,
藉由以pH4~6的洗淨液將前述擔體洗淨1次以上而去除附加糖鏈的異構物,以及
以鹽濃度在0.5M以下或不含鹽且pH5~7的溶離液使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物,
此處,前述純化抗體組成物中,附加糖鏈的異構物相對於所有抗體的比例,與前述抗體粗純化物相比,較為減低。
(19) 一種如(1)之抗體組成物之純化方法,其係包含:
將鹽濃度調整至0.5M以上的前述抗體粗純化物載入使用具有苄基或丁基且粒徑為20~100μm的擔體的慣用層析裝置,將附加糖鏈的異構物去除至流出液,
以洗淨液將前述擔體洗淨,以及
以鹽濃度在0.5M以下或不含鹽且pH5~7的溶離液使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物,
此處,前述純化抗體組成物中,附加糖鏈的異構物相對於所有抗體的比例,與前述抗體粗純化物相比,較為減低。
(20) 一種如(1)之抗體組成物之純化方法,其係包含:
將pH調整至4~6的前述抗體粗純化物載入使用具有苄基或丁基且粒徑為20~100μm的擔體的慣用層析裝置,將附加糖鏈的異構物去除至流出液,
以洗淨液將前述擔體洗淨,以及
以鹽濃度在0.5M以下或不含鹽且pH5~7的溶離液使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物,
此處,前述純化抗體組成物中,附加糖鏈的異構物相對於所有抗體的比例,與前述抗體粗純化物相比,較為減低。
(21) 如(1)~(20)中任一項之方法,其中前述慣用層析裝置的擔體之每單位量的蛋白質載入量為20g/L以上,且前述洗淨係藉由流過5倍擔體容積以上的洗淨液來進行。
(22) 一種抗體組成物的製造方法,該抗體組成物中,在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體相對於所有抗體的比例為95%以上,該方法包含如(1)~(21)中任一項之純化方法。
(23) 一種抗hDLK-1抗體純化組成物的製造方法,該抗體之重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且該方法包含:
將抗體粗純化物載入使用具有苄基或丁基且粒徑為20~100μm的擔體的慣用層析裝置,
使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於前述擔體,
藉由以含0.5M以上的鹽的洗淨液將前述擔體洗淨1次以上而去除附加糖鏈的異構物,以及
以鹽濃度在0.5M以下或不含鹽且pH5~7的溶離液使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物,
此處,前述純化抗體組成物中,附加糖鏈的異構物相對於所有抗體的比例,與前述抗體粗純化物相比,較為減低。
(24) 一種抗體組成物,其係藉由如(22)或(23)之製造方法所製造。
(25) 一種由抗hDLK-1抗體粗純化物將在Fc區的附加糖鏈的共有區以外的部位附加糖鏈之附加糖鏈的異構物去除的方法,該抗體之重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且該方法包含:
將抗體粗純化物載入疏水性交互作用層析擔體,使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於前述擔體,
以洗淨液將前述擔體洗淨,以及
藉由以溶離液由前述擔體溶離,使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物。
(26) 一種抗hDLK-1抗體組成物,該抗體之重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且該抗體組成物含有所有抗體的95%以上之在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體。
(27) 一種抗hDLK-1抗體,其重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈。
[發明之效果]
本發明可減少、去除在抗體的附加糖鏈的共有區以外附加了糖鏈之附加糖鏈的異構物。
本發明的第一效果,可提供附加糖鏈的異構物的比例減少的醫療用抗體組成物。因此,藉由該方法純化而減少了附加糖鏈的異構物之高純度抗體組成物,可製成純度較高的藥物製劑。
本發明的第二效果,可提供去除附加糖鏈的異構物的醫療用抗體組成物。藉此,可提供活性本體的純度非常高的組成物,亦即在有效性與安全性方面優異的醫藥組成物。
尤其在本發明中純化的抗hDLK-1抗體之純化組成物,由於去除了附加糖鏈的異構物這種不具有活性的雜質,因此可提供在有效性與安全性方面優異的醫藥組成物。
以下詳細說明本發明。本發明的範圍不受這些說明拘束,除了以下的例示以外,還可在不損及本發明意旨的範圍適當地變更來實施。此外,本說明書包括成為本發明優先權主張基礎的日本特願2021-079977號說明書(令和3年(2021年)5月10日申請)的全體。在本說明書之中所引用的所有刊行物,例如先前技術文獻及公開公報、專利公報其他專利文獻,作為參考而被收錄至本說明書中。
在本說明書之中,「附加糖鏈的共有序列」或「糖鏈結合共有序列」,意指Asn-X-Ser/Thr(X為Pro以外的胺基酸)所表示的胺基酸序列,與抗體中該序列的位置及Asn存在的位置無關。在本說明書之中,「Fc區的附加糖鏈的共有區」、「附加糖鏈的共有區」或「糖鏈結合共有區」,意指存在於抗體的Fc部分、通常包含Asn297的附加糖鏈的共有序列(代表性的例子為Asn297-X-Ser/Thr(X為Pro以外的胺基酸))。編碼附加糖鏈的共有序列及附加糖鏈的共有區的鹼基序列,只要是編碼該胺基酸序列的序列,則任何序列皆包含在內。
在本說明書之中,「附加糖鏈的異構物」意指在附加糖鏈的共有區以外的胺基酸附加了糖鏈的抗體。將附加在附加糖鏈的共有區以外的胺基酸的糖鏈稱為「非共有糖鏈」。在附加糖鏈的異構物之中,結合了非共有糖鏈的區,包括Fab區、抗原結合區的Fv(可變)區、互補決定區(CDR)、附加糖鏈的共有區以外的Fc區及融合抗體的融合序列部分,代表性的例子為CDR區。非共有糖鏈在附加糖鏈的共有區以外的胺基酸的糖鏈結合樣式,可為附加糖鏈的共有序列的Asn-X-Ser/Thr(X為Pro以外的胺基酸)上的N型糖鏈,或可為Ser或Thr上的O型糖鏈。
抗體分子的構造通常為四聚物,是兩種相同多肽鏈各兩個結合而形成,因此非共有糖鏈的結合部位,理論上在抗體一分子中會存在2的倍數個。成為本發明純化對象的抗體粗純化物,只要是包含具有一個以上非共有糖鏈的抗體即可。抗體每分子的非共有糖鏈的結合數,可為1個以上、2個以上、3個以上、4個以上、5個以上、6個以上、7個以上、8個以上、9個以上或10個以上。另外,抗體是由4條多肽鏈(通常為兩條重鏈及兩條輕鏈)所形成,結合於任一條鏈的非共有糖鏈的結合數,可為1個以上、2個以上、3個以上、4個以上、5個以上、6個以上、7個以上、8個以上、9個以上或10個以上。抗體粗純化物中的非共有糖鏈的可結合區域中的糖鏈的結合比例,亦即附加糖鏈的異構物的存在比例,在將抗體一分子中單方的「重鏈與輕鏈的組合」或與其相符的構造上的非共有糖鏈為完全結合的情況定為100%時,可為每分子抗體0.1%以上200%以下,通常在有必要適用本發明的方法的情況,為1%以上50%以下。此處,200%意指在一分子抗體之中,在成對的「重鏈與輕鏈的組合」兩者(兩處)鍵結了非共有糖鏈。
在本說明書之中,「抗體」,除了全長抗體之外,還包括抗體片段、全長抗體或抗體片段與其他物質的融合物,例如包括小鼠抗體、小鼠-人類嵌合抗體、人源化抗體、人類抗體及其胺基酸修飾體、加成體、缺失體、取代體、糖鏈修飾體等。抗體的免疫球蛋白類型並未受到特別限定,可為IgG、IgM、IgA、IgE、IgD、或IgY的任一種免疫球蛋白類型(同種型),宜為IgG。另外,在本發明的抗體為IgG的情況,可為任一種亞型(IgG1、IgG2、IgG3、或IgG4)。抗體片段宜為抗原結合性片段,包括F(ab')
2、Fab'、Fab、Fab
3、單鏈Fv(以下稱為「scFv」)、(串聯)雙特異性單鏈Fv(sc(Fv)
2)、單鏈三重體、奈米抗體、二價VHH、五價VHH、微型抗體、(兩鏈)雙鏈抗體、串聯雙鏈抗體、雙特異性三抗體、雙特異性雙抗體、雙親和重定向分子(DART)、三鏈抗體(或三抗體)、四鏈抗體(或[sc(Fv)
2]
2、或(scFv-SA)4)、雙硫鍵Fv(以下稱為「dsFv」)、小型IgG、重鏈抗體、或其聚合物。抗體片段與其他物質的融合物,可列舉融合蛋白質,尤其是Fc融合蛋白質等。
尤其,本說明書中的抗體,意指重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或12所記載的胺基酸序列之抗hDLK-1抗體。宜為重鏈具有選自序列識別號4、8、16、20、24、28、32及36的胺基酸序列,且輕鏈具有序列識別號12所記載的胺基酸序列之抗hDLK-1抗體。
在本說明書之中,將純化對象之包含附加糖鏈的異構物的抗體組成物稱為「抗體粗純化物」。抗體粗純化物只要是包含附加糖鏈的異構物,則任一者皆可利用。例如抗體粗純化物,可列舉血漿等來自生物體的組成物或其處理物,以及編入抗體基因的轉形細胞的培養液(可為培養上清液,以下同樣)或其處理物。來自生物體的組成物,可列舉包含由基因轉殖非人類動物或植物等所得到的抗體的組成物等。轉形細胞只要是可結合糖鏈的細胞,則任一者皆可,具體而言,可列舉動物細胞、植物細胞或酵母細胞等的具有可和糖鏈結合的性質的細胞株,更具體而言,可列舉中國倉鼠卵巢細胞(CHO細胞)、小鼠骨髓瘤細胞的NS0細胞、SP2/0細胞、大鼠骨髓瘤細胞的YB2/0細胞、IR983F細胞、來自敘利亞倉鼠腎臟的BHK細胞、來自人類胎兒腎的293細胞、人類骨髓瘤細胞的Namalwa細胞、胚性幹細胞,或在受精卵細胞等導入抗體基因的細胞。前述細胞株也可利用由初代不死化細胞株衍生的各種亞種。例如,如果是CHO細胞,則可利用CHO K1株、CHO DG44株、CHO S株及其衍生株等(Palsson等,Nature Biotechnology 31(8), 759-765, 2013)。藉由將這些細胞以適合於生產蛋白質的培養基來培養,能夠以培養液的形式得到抗體粗純化物。所使用的培養基,可使用例如含血清的培養基、不含血清蛋白或血清分離物等的來自動物的成分的培養基、無血清培養基或無蛋白質培養基等、任一種培養基皆可使用,宜為無血清培養基、不含來自動物的原料的培養基、無蛋白質培養基、完全化學合成培養基。
此外,上述抗體粗純化物,可利用經過過濾、鹽析、一種以上的層析、pH調整、緩衝液置換、濃縮、稀釋等的處理之來自生物體的組成物或培養液,或者對於來自生物體的組成物或培養液進行純化等操作的中間體組成物等的組成物。進行純化操作的中間體組成物,還可利用用來構築抗體藥物的製造程序的任何單元操作後的溶液。例如希望是蛋白質A親和層析後、陽離子交換層析後、陰離子交換層析後、緩衝液交換後、低pH處理後、過濾後的組成物等。
在N型糖鏈的情況,關於附加糖鏈的異構物存在的可能性,可由該抗體的胺基酸序列或基因序列來推測。另外,不論糖鏈的類型,藉由抗體的胜肽圖譜及質量分析可推測出糖鏈結合位置。從較簡便的方法來說,藉由對經過胜肽-N-醣苷酶(PNGase)處理的抗體與不經過該處理的抗體實施SDS聚丙烯醯胺凝膠電泳(SDS-PAGE)並且作比較,能夠以蛋白質電泳帶的變化的方式來觀察糖鏈的結合。
抗體的種類如前述所揭示,而本方法適合作為包含治療用、診斷用或預防用抗體的抗體藥物的抗體生產過程中的純化方法。這是因為抗體藥物所含的抗體的異構物的含量必須有恆常性,來自目標物質的雜質必須儘可能減少(ICH Q6B指引)。治療用或預防用抗體的例子,可列舉結合於配體而將配體的活性中和的抗體、結合於細胞表面的受體而將配體的結合中和的抗體、結合於細胞表面對細胞本身表現出毒性的抗體。診斷用抗體的例子,可列舉結合於配體或細胞表面的受體的抗體。對細胞的毒性,可列舉抗體依存性細胞毒性(Antibody-dependent Cellular Cytotoxicity)、補體依存性細胞毒性(Complement-Dependent Cytotoxicity)、抗體依存性細胞吞噬(Antibody dependent cellular phagocytosis)活性等。另外,抗體-藥物複合體(Antibody Drug Conjugate)、放射性標識抗體(Radioisotope-labelled Antibody)等的化學修飾抗體、細胞介素等的融合抗體、多重特異性抗體(Multi-specific Antibody)(Nature, 580(16), 330-338 (2020))等,只要是具有糖鏈的抗體衍生物,即可利用本發明的技術。
更具體而言,對CD3、EGF受體、CD20、RS病毒、TNFα、CD25、IL-6受體、CD33、VEGF、IgE、補體C5、IL-12、IL-23、IL-1β、RANKL、CCR4、HER2、CD30、IL-5、IL-5受體、α4整合素、α4β7整合素、PD-1、CD52、IL-17、IL-17A、IL-17受體、CTLA-4、PCSK9、SLAMF7、BlyS、CD38、PD-L1、IL-4α受體、CD22、CD23、第IXa因子、X因子、CD19、抑硬素、DLK-1等的蛋白質抗原(宜為來自人類的蛋白質抗原)的抗體的純化可利用本方法。融合蛋白質,可列舉可溶性TNF受體Fc融合蛋白質、CTLA4改變Fc融合蛋白質、Fc-TPOR促效劑胜肽融合蛋白質、VEGF受體-Fc融合蛋白質等。這些各種抗體或融合蛋白質等的純化皆可利用本方法。
其中一個最希望的事例是抗DLK-1抗體的純化。具體而言,可列舉前揭的專利文獻4(WO2014/054820)所記載的人源化抗人類DLK-1抗體,例如包含具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列的重鏈(尤其是可變區具有這些胺基酸序列的重鏈,在本段落中,以下同樣)及具有序列識別號10或12所記載的胺基酸序列的輕鏈(尤其是可變區具有這些胺基酸序列的輕鏈,在本段落中,以下同樣)之抗體,宜為包含具有選自序列識別號4、8、16、20、24、28、32及36的胺基酸序列的重鏈及具有序列識別號12所記載的胺基酸序列的輕鏈之抗體。這樣的人源化抗人類DLK-1單株抗體,其輕鏈的可變區具有糖鏈結合共有序列。
以下的表1及2包含了先前揭示的專利文獻4(WO2014/054820)所記載的抗人類DLK-1抗體的全長序列(底線表示訊號序列,在成熟蛋白質之中,不含底線部的序列),例如在該抗體之中,以方框圍住的NSS序列的N會成為糖鏈結合共有序列。因此,在讓動物細胞等表現出該基因的情況,有可能會產生包含附加糖鏈的異構物的糖鏈組成物。同樣地,具有與該抗DLK-1抗體的序列類似的序列的DLK-1抗體,也會同樣的可能性。作為由包含這種附加糖鏈的異構物的抗體粗純化物將附加糖鏈的異構物去除或減少的方法,本方法是有用的。此外,以下的表中所記載的HuBA-1-3D-1的H鏈、HuBA-1-3D-2的H鏈、HuBA-1-3D-1 A24G的H鏈、HuBA-1-3D-2 A24G的H鏈、HuBA-1-3D-1 T73K的H鏈、HuBA-1-3D-2 T73K的H鏈、HuBA-1-3D-1 A24G/T73K的H鏈、HuBA-1-3D-2 A24G/T73K的H鏈及HuBA-1-3D的L鏈胺基酸序列,分別依序記載於序列識別號38、42、46、50、54、58、62、66、及70。另外,由這些序列將訊號序列除外的成熟蛋白質的胺基酸序列,分別依序記載於序列識別號40、44、48、52、56、60、64、68、及72。在本發明說明書等之中,「H鏈」意指重鏈,「L鏈」意指輕鏈。
HuBA-1-3D VH1及HuBA-1-3D VH2的CDR1的胺基酸序列為「DYAMH」(序列識別號73)、CDR2的胺基酸序列為「VISTYYGNTNYNQKFKG」(序列識別號74)及CDR3的胺基酸序列為「GGLREYYYAMDY」(序列識別號75)。另外,HuBA-1-3D VL的CDR1的胺基酸序列為「KSSQSLLNSSNQKNYLA」(序列識別號76)、CDR2的胺基酸序列為「FASTRES」(序列識別號77)及CDR3的胺基酸序列為「QQHYSTPPT」(序列識別號78)。本發明之抗體,可為具有這些CDR的全部或一部分的抗體。此外,這些CDR序列是依照Kabat等(Sequences of Proteins of Immunological Interests, Fifth edition, NIH Publication No.91-3242, U.S. Department of Health and Human Services, 1991)的定義。在本發明說明書等之中,「VH」意指重鏈可變區,「VL」意指輕鏈可變區。
與抗體粗純化物相比,附加糖鏈的異構物的含量較為減少的抗體組成物(在本說明書之中稱為「純化抗體組成物」),是以包含附加糖鏈的異構物的抗體粗純化物作為起始原料,供應至慣用的層析裝置而得到。所以,本發明關於一種抗體粗純化物之純化方法,其係包含:藉由將前述抗體粗純化物載入慣用層析裝置,使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於前述擔體,以及以溶離液讓前述擔體發生溶離,使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物,並且所得到的純化抗體組成物中,在Fc區的附加糖鏈的共有區以外的部位附加糖鏈之附加糖鏈的異構物的含量,與純化前的抗體粗純化物相比,較為減少。
慣用層析裝置所使用的擔體,可利用疏水性交互作用層析擔體、疏水層析擔體,或混合模式層析擔體(亦稱為多模式層析擔體,宜為具有疏水層析的性質的混合模式層析擔體)。疏水性交互作用層析擔體,可使用在基底基材上結合了甲基、乙基、丙基、丁基、辛基、己基、丙二醇基、苯基、烷基苯基、苄基、烷基苄基等的疏水性官能基而成的擔體。混合模式層析擔體,可列舉將前述疏水性官能基與離子交換性官能基以任意比例混合而成的擔體。例如可利用N-苄基-N-甲基乙醇胺等作為官能基。陽離子交換性官能基的代表性例子可列舉CM(羧甲基、-O-CH
2-COOH)、SP(磺丙基、-O-C
3H
6-SO
3H)等,陰離子交換性的代表性例子可列舉DEAE(二乙胺基乙基、
-O-C
2H
4-N-(C
2H
5)
2)、QAE(季銨化胺乙基或二乙基-(2-羥丙基)-胺乙基、-O-C
2H
4-N-(C
2H
5)
2(CH
2-CH(OH)-CH
2))等,可利用這些官能基。擔體的基材,可列舉纖維素、葡聚糖凝膠(Sephadex)、交聯瓊脂糖、聚丙烯醯胺、甲基丙烯酸酯及各種合成聚合物。有或沒有細孔的擔體,任一者皆可利用。混合模式層析擔體的其他形態,還可列舉如羥基磷灰石(Ca
10(PO
4)
6(OH)
2)或氟磷灰石(Ca
10(PO
4)
6F
2)般具有磷酸鈣等的官能基的化合物。
層析擔體可從市售品取得來利用。具體而言,可列舉Butyl-Sepharose(註冊商標) 4 Fast Flow(平均粒徑90μm)、Butyl-S Sepharose 6 Fast Flow(平均粒徑90μm)、Octyl Sepharose(註冊商標) 4 Fast Flow(平均粒徑90μm)、Phenyl Sepharose(註冊商標) 6 Fast Flow (high sub)(平均粒徑90μm)、Phenyl Sepharose(註冊商標) 6 Fast Flow(low sub)(平均粒徑90μm)、Butyl Sepharose(註冊商標) High Performance(平均粒徑90μm)、Phenyl Sepharose High Performance(平均粒徑90μm)、SOURCE 15ETH(平均粒徑15μm)、SOURCE 15ISO(平均粒徑15μm)、SOURCE 15PHE(平均粒徑15μm)、Capto Phenyl(High Sub)(平均粒徑90μm)、Capto Butyl(平均粒徑90μm)、Capto Octyl(平均粒徑90μm)、Capto Phenyl ImpRes(平均粒徑36~44μm)、Capto Butyl ImpRes(平均粒徑36~44μm)、Capto adhere(平均粒徑90μm)、Capto adhere ImpRes(平均粒徑36~44μm)、Capto MMC(平均粒徑90μm)、Capto MMC ImpRes(平均粒徑36~44μm)、Capto Core 700(平均粒徑90μm)、ReadyToProcess Adsorber Phenyl(以上為英國Cytiva公司)、TOYOPEARL(註冊商標) Butyl-600(平均粒徑40~90μm)、TOYOPEARL(註冊商標) Phenyl-600(平均粒徑40~90μm)、TOYOPEARL(註冊商標) PPG-600(平均粒徑40~90μm)、TOYOPEARL(註冊商標) Butyl-650(平均粒徑40~90μm)、TOYOPEARL(註冊商標) Phenyl-650(平均粒徑40~90μm)、TOYOPEARL(註冊商標) SuperButyl-550(平均粒徑40~90μm)、TOYOPEARL(註冊商標) Hexyl-650(平均粒徑50~150μm)、TOYOPEARL(註冊商標) Ether-650(平均粒徑40~90μm)(以上為東曹公司製)、POROS Ethyl(平均粒徑50μm)、POROS Benzyl(平均粒徑50μm)、POROS Benzyl Ultra(平均粒徑50μm)(以上,Thermo Fisher公司製)、Macro-Prep t-Butyl HIC(平均粒徑50μm)、Macro-Prep Methyl HIC(平均粒徑50μm)、陶瓷羥基磷灰石(平均粒徑20~80μm)、陶瓷氟磷灰石(平均粒徑20~80μm)、Bio-Gel HT(平均粒徑20~80μm)(以上為Bio-Rad公司製)、QMA Spherosil(平均粒徑50μm)、Methyl Ceramic Hyper D(平均粒徑50μm)(以上為Poul公司製)、Fractogel EMD Phenyl (S)(平均粒徑20~90μm)、Fractogel EMD Propyl (S)(平均粒徑20~90μm)(以上為Merck公司製)、Cellufine MAX Phenyl(平均粒徑40~130μm)、Cellufine MAX Phenyl LS(平均粒徑40~130μm)、Cellufine MAX Butyl(平均粒徑40~130μm)(以上為JNC公司製)、丁基化CHITO PEARL、苯基化CHITO PEARL(以上為富士紡Holdings公司製)等的擔體,可由這些擔體選擇較適當的擔體來使用。較佳為Capto Butyl、POROS Benzyl Ultra、POROS Butyl Ultra等的擔體。
本發明的純化目的所使用的層析擔體的平均粒徑,可定為15μm以上、20μm以上、30μm以上或40μm以上。另外,層析擔體所能夠處理的抗體粗純化物的液量,可定為100mL以上、1L以上、10L以上或100L以上。填充該擔體的層析用管柱,可使用容量100mL~1,000L左右(直徑5cm~2m左右)的管柱。提供層析純化的抗體粗純化物的量,至少為1L以上,希望為10L以上,較希望為100L以上,更希望為500L以上,最大到20,000L左右。由於必須處理這樣大量的抗體粗純化物,因此層析的線流速為1,000cm/hr以下,希望在500cm/hr以下。提供純化的抗體的量,以蛋白質質量計,為10g以上,希望為100g以上,較希望為1kg以上,更希望為10kg以上。藉由本技術所欲去除或減少的附加糖鏈的異構物,會被包含在層析所溶離出來的抗體當中前半的組分,讓所希望得到的目標抗體溶離至後半的組分,將附加糖鏈的異構物分離、去除。
將包含了附加糖鏈的異構物的抗體粗純化物載入慣用層析裝置的條件,必須是至少讓目標成分之僅在附加糖鏈的共有區附加糖鏈的抗體(非附加糖鏈的異構物抗體)可吸附於層析擔體的條件。抗體的吸附,是藉由抗體及層析擔體的疏水性、親水性的程度所形成的交互作用而發生。緩衝液可利用疏水性交互作用層析、疏水層析、混合模式層析通常使用的緩衝液。只要是抗體在層析的條件下安定,則任一種皆可,可列舉例如磷酸緩衝液、醋酸緩衝液、檸檬酸緩衝液、Tris緩衝液、甘胺酸緩衝液、硼酸緩衝液、酒石酸緩衝液、MES緩衝液、HEPES緩衝液、MOPS緩衝液、胺基酸緩衝液及這些混合緩衝液。這些緩衝液的濃度,可在0.1mM~300mM左右的一般使用範圍任意選擇。緩衝液的pH可在pH4~8的範圍任意選擇,希望pH為4~6。
在這些緩衝液中,因應必要在抗體不會產生沉澱的程度加入適量的硫酸鈉、硫酸銨、氯化鈉、檸檬酸鈉等的鹽類來增強前述層析擔體的交互作用,可進行往層析擔體的吸附。前述鹽類可任意選擇一種以上,鹽濃度可使用在300mM~2M的範圍且讓抗體安定地存在,並可讓抗體充分吸附於層析擔體的濃度。以1M左右的鹽濃度為佳,且以可吸附抗體的低鹽濃度為佳。每單位量的層析擔體可吸附的抗體量,會依照層析擔體的種類或緩衝液的條件而有各種不同,每1mg擔體可吸附10mg以上的抗體,理想的情況每1mg擔體可吸附20mg以上的抗體。層析的操作可在0℃~40℃的任意溫度來實施。希望是在室溫下操作,較希望在控制溫度的條件下操作。
在前述條件下,包含附加糖鏈的異構物的抗體粗純化物被載入層析儀之後,藉由溶離來進行分離純化。利用層析進行的附加糖鏈的異構物的分離,可藉由在讓抗體吸附於層析擔體之後,以階段性、連續性、或其組合來降低流過管柱的緩衝液的鹽濃度,或者提高pH、降低導電率、或其組合來進行。讓抗體吸附於層析擔體之後,首先,主要使附加糖鏈的異構物溶離,接下來,使僅在附加糖鏈的共有區附加糖鏈的抗體(非附加糖鏈的異構物抗體)溶離,可調製出目標純化抗體組成物。「溶離」是指減弱藉由疏水性交互作用等結合於層析擔體的抗體成分在擔體上的結合,讓該抗體成分由層析擔體排出。讓附加糖鏈的異構物溶離的條件,是附加糖鏈的異構物和層析擔體的交互作用與非附加糖鏈的異構物抗體和層析擔體的交互作用相比足夠弱的緩衝液的條件。藉由使用足量該條件的該緩衝液將層析擔體洗淨,可在僅讓目標抗體的非附加糖鏈的異構物抗體吸附於層析擔體的狀態下僅將附加糖鏈的異構物溶離去除。
用來將附加糖鏈的異構物溶離去除(洗淨)的緩衝液,只要和讓非附加糖鏈的異構物抗體溶離的緩衝液之間有10mM以上的鹽濃度差異及/或有0.2單位以上的pH差異,則任一種緩衝液皆可利用。使非附加糖鏈的異構物抗體以高鹽濃度(通常在300mM~2M的範圍)吸附於層析擔體之後,將層析擔體洗淨的洗淨液的鹽濃度,可定與溶離的鹽濃度同等以上的範圍。可定為例如與抗體溶離時的鹽濃度同等,或同等至高1M的鹽濃度的範圍,或同等至高0.5M的鹽濃度範圍,或比抗體溶離時的鹽濃度高10mM以上的鹽濃度,例如可定為0.5M以上或1.0M以上。洗淨液的pH可定為與抗體吸附時的pH同等或最大高出2單位的pH範圍,或與溶離時的pH同等或最大低2單位的範圍。例如洗淨液的pH可為比溶離液的pH低0.2單位的pH。洗淨液的pH可在pH4~8的範圍內。另外,用來將附加糖鏈的異構物溶離去除的緩衝液(洗淨液)的通液量,相對於層析擔體容積,可定為2倍擔體容積(CV)以上、3CV以上、4CV以上、5CV以上、10CV以上、15CV以上或20CV以上,或者5~20CV、5~15CV、5~10CV或10~20CV。
從載入抗體粗純化物到洗淨為止的pH及/或鹽濃度的變化,可為階段性的(一階段或其以上、二階段或其以上、三階段或其以上、數階段等),或可為連續性的。從載入抗體粗純化物到洗淨為止的pH的範圍通常在4~6的範圍內。
上述鹽濃度、pH及/或洗淨液量的條件,可因應作為原料使用的抗體粗純化物中附加糖鏈的異構物的含量、及抗體的等電點或胺基酸序列等的抗體本身的特性等適當地設定。
本說明書中,利用層析的附加糖鏈的異構物的分離方法,可包含使附加糖鏈的異構物由層析擔體通過(flow-through),接下來使僅在附加糖鏈的共有區附加糖鏈的抗體溶離,藉此可調製出非附加糖鏈的異構物的抗體純度高的純化抗體組成物。通過(flow-through)是指在將抗體粗純化物載入填充了層析擔體的管柱時,不讓附加糖鏈的異構物吸附於層析擔體,直接溶離至管柱外。此情況下,抗體粗純化物中的附加糖鏈的異構物,會有和層析擔體的交互作用弱的情形,藉由讓平衡化緩衝液(希望為1管柱容積以上)連續或間歇地通過,可將包含不需要的附加糖鏈的異構物的成分排除至管柱外。用來讓附加糖鏈的異構物特異性地通過的緩衝液,是選擇附加糖鏈的異構物和層析擔體的交互作用與僅在附加糖鏈的共有區結合了糖鏈的抗體和層析擔體的交互作用相比足夠弱的條件的緩衝液。使用足量的該緩衝液,僅讓附加糖鏈的異構物由層析擔體通過,能夠僅使目標抗體吸附於層析擔體。接下來,使用鹽濃度等的條件與前述緩衝液不同的緩衝液,使結合的抗體由層析擔體溶離,調製出所希望的純化抗體組成物。
僅讓附加糖鏈的異構物通過的緩衝液,只要是與讓僅在附加糖鏈的共有區結合糖鏈的抗體溶離的溶離液相比,有10mM以上的鹽濃度差異(高10mM以上的鹽濃度)及/或0.2單位以上的pH差異(低0.2以上的pH),則任一種緩衝液皆可利用作為通過用洗淨液。緩衝液的鹽濃度,可定為與使非附加糖鏈的異構物抗體由擔體溶離的鹽濃度同等或高1M的鹽濃度範圍(宜為高0.5M的鹽濃度範圍)。此時的鹽濃度,通常是在相對於讓抗體溶離的鹽濃度為100%以上的範圍。或者,以與抗體吸附時的pH同等或最大高出2單位的pH範圍的洗淨液,或以與溶離時的pH同等或最大低2單位的pH範圍的洗淨液來洗淨。pH及/或鹽濃度的變化,可為一階段或數階段,或可為連續的。另外,通常該緩衝液的鹽濃度或pH,會在將作為原料的抗體粗純化物供應至層析擔體前調整至適當範圍。用來讓附加糖鏈的異構物通過的緩衝液(洗淨液)的通液量,是選擇在層析擔體量的2倍以上,希望為5倍以上的條件。洗淨液的通液量更希望為10~20倍以上。上述鹽濃度、pH、及/或洗淨液量是因應原料所使用的抗體粗純化物中附加糖鏈的異構物的含量適當地設定。
載入層析擔體的抗體粗純化物中的抗體載入量,亦即蛋白質的載入量,會影響到附加糖鏈的異構物的分離能力,因此是以讓所得到的純化抗體組成物達到所希望的產率及純度(附加糖鏈的異構物的含量)來決定每單位層析擔體的蛋白質載入量。通常在層析操作中,擔體能夠吸附的蛋白質質量會有極限,以最大動態吸附容量(DBC:Dynamic Binding Capacity)等的參數為指標,可控制層析操作。一般而言,將蛋白質載入量定在最大動態吸附容量以下,蛋白質的回收率及分離能力良好,可期待所希望的層析的分離效果。另外,在本發明的方法中,宜將載入量定在一定量以上,以使不需要的附加糖鏈的異構物不會被吸附,藉由以每1g層析擔體的量或每1mL層析擔體的量至少為20mg以上、25mg以上、30mg以上或35mg以上,且在最大動態吸附容量以下來載入蛋白質,進行附加糖鏈的異構物的分離、去除。蛋白質的載入量,可依照欲純化的抗體粗純化物中的附加糖鏈的異構物的含量來決定。亦即,在層析純化前的抗體粗純化物中的附加糖鏈的異構物的含量高的情況,可定為接近最大動態吸附容量的載入量。
在一個態樣之中,本發明的方法包含:依照供分離的抗體的性質來選擇所使用的慣用層析裝置擔體之步驟、讓利用所選擇的層析擔體進行附加糖鏈的異構物的分離去除最適化之步驟。
選擇層析擔體的步驟,首先在前述鹽濃度及pH條件下,將純化對象的抗體粗純化物載入任意兩種以上的層析擔體,可選出僅在附加糖鏈的共有區附加糖鏈的抗體吸附量高的層析擔體。進一步因應必要,以鹽濃度階段性或連續降低的溶離液來溶離,測定通過擔體的溶離液中的附加糖鏈的異構物及/或僅在附加糖鏈的共有區附加糖鏈的抗體的量,可選擇能夠得到附加糖鏈的異構物的量少且僅在附加糖鏈的共有區附加糖鏈的抗體的量多的溶離液的層析擔體,作為附加糖鏈的異構物的分離較優異的層析擔體。
利用所選擇的層析擔體進行附加糖鏈的異構物的分離去除之最適化步驟,是進行載入時所使用的緩衝液的組成、濃度及pH、所添加的鹽的種類及濃度、載入層析擔體的抗體粗純化物的載入量、洗淨時的緩衝液的組成、濃度、pH、洗淨次數及洗淨液量、溶離時的緩衝液的組成、濃度及pH、所添加的鹽的種類及鹽濃度、以及其變更方法、利用附加糖鏈的異構物的吸附或通過來進行的去除等條件的檢討與選擇。該最適化的步驟,可針對各參數逐一最適化,或可使用實驗設計法等的統計解析手段,統整多項參數的交互作用來選定最合適的條件。
抗體的純化方法有各種步驟。大多數抗體的純化程序能夠以2步驟以上的不同層析模式來進行,通常利用3步驟的層析構築純化程序的情形很多。例如可將蛋白質A親和層析、陽離子交換層析、陰離子交換層析、混合模式層析、疏水性交互作用層析等之中的兩種以上組合來利用。本發明的方法可編入這些抗體純化程序中利用慣用層析裝置的抗體層析的任何步驟。具體而言,在第1步驟利用了蛋白質A親和層析、第2步驟利用了陽離子交換層析、第3步驟利用了疏水性交互作用層析的純化程序之中,能夠以第3步驟的疏水性交互作用層析作為本發明的方法。另外,在第1步驟利用了蛋白質A親和層析、第2步驟利用了混合模式層析、第3步驟利用了疏水性交互作用層析的純化程序的情況,能夠以第3步驟的疏水性交互作用層析作為本發明的方法。另外,在第1步驟利用了蛋白質A親和層析、第2步驟利用了疏水性交互作用層析、第3步驟利用了混合模式層析的純化程序的情況,能夠以第2步驟的疏水性交互作用層析作為本發明的方法。此外,如果是第1步驟利用了蛋白質A親和層析、第2步驟利用了陰離子交換層析、第3步驟利用了疏水性交互作用層析的純化程序,則能夠以第3步驟的疏水性交互作用層析作為本發明的方法。另外,在這些層析的順序當中,也能夠與疏水性交互作用層析一起使用混合模式層析的步驟,或者不採用疏水性交互作用層析,來作為本發明的方法。
藉由本發明的方法,可有效率地去除附加糖鏈的異構物,可提供包含減少至所希望的含量的量的附加糖鏈的異構物之純化抗體組成物。藉由本發明的方法純化後的純化抗體組成物中,附加糖鏈的異構物的量相對於所有抗體量的比例,可定為10%以下,較希望為5%以下、4%以下、3%以下、2%以下、1%以下、0.5%以下,或可定為0.2%以下。另外,純化前的抗體粗純化物所含有的附加糖鏈的異構物的量相對於所有抗體量的比例,可為50%以上、20%以上、10%以上或5%以上。本發明的方法,可為以20%以上、40%以上或50%以上的步驟產率得到附加糖鏈的異構物量減少的抗體的方法。
最終純化抗體組成物或純化後的抗體組成物中,附加糖鏈的異構物的含量及含有比例及純化產率,可藉由調整前述層析的參數來達成,宜為以附加糖鏈的異構物減少至任意量,可得到抗體生產所能容許的純化產率的方式來最適化。
附加糖鏈的異構物的去除狀況的確認,可藉由分析評估用的高效液相層析(HPLC:High Performance Liquid Chromatography)或超高效液相層析(UHPLC:Ultra High Performance Liquid Chromatography)來進行。進行這些分析時,使用平均粒徑為15μm以下的擔體,並藉由超高流速且超高壓的層析,可分離出附加糖鏈的異構物。例如適合為TSKgel Butyl-NPR管柱(平均粒徑2.5μm,東曹公司)、TSKgel Phenyl-5PR管柱(平均粒徑10或13μm,東曹公司)、TSKgel Ether-5PW管柱(平均粒徑10μm,東曹公司)、TSKgel BioAssist Phenyl管柱(平均粒徑10μm,東曹公司)、Protein-Pak Hi Res HIC管柱(Waters公司)、BioPro HIC管柱(平均粒徑2.3或4μm,YMC公司)、Proteomix HIC管柱(平均粒徑1.7或5μm,M&S機器公司)、AdvanceBio HIC管柱(平均粒徑3.5μm,Agilent公司)、MAbPac HIC-10LC管柱(平均粒徑5μm,ThermoFisher公司)、MAbPac HIC-20LC管柱(平均粒徑5μm,ThermoFisher公司)、MAbPac HIC-Butyl LC管柱(平均粒徑5μm,ThermoFisher公司)、Shim-pack Bio-HIC管柱(平均粒徑4μm,島津製作所公司)、Shodex HIC PH-814(平均粒徑10μm,昭光通商公司)、COSMOSIL HIC(平均粒徑5μm,nacalai tesque公司)等。
藉由本發明的方法純化後的純化抗體組成物,作為抗體藥物的活性成分,可作為人類及動物的各種疾病的治療藥、預防藥或診斷藥來使用。
以下使用實施例,進一步詳細說明本發明,然而這並不會限定本發明之範圍。
[實施例1]
在抗體的附加糖鏈的共有區以外結合了糖鏈的抗體粗純化物(培養上清液)的調製與組成比的評估
作為附加糖鏈的抗體,使用了具有國際公開第2014/054820號所記載的H鏈及L鏈(本發明中的H鏈:序列識別號64、L鏈:序列識別號72)的人源化抗人類DLK-1單株抗體(以下稱為「抗hDLK-1抗體」)。該抗體,在其輕鏈的可變區具有附加糖鏈的共有序列,因此在讓動物細胞等表現出該基因的情況,有可能會產生、混入包含附加糖鏈的異構物的糖鏈組成物。
以具有編碼抗hDLK-1抗體的胺基酸序列的基因的表現載體來將CHO細胞DG44株轉形,調製出安定表現細胞株群DGC8-R-T11-14.2d。使用該細胞株群,以1L規模的DASGIP(註冊商標) Parallel Bioreactor System (Eppendorf公司製)生物反應器來進行培養。培養是使用無血清的完全化學合成培養基,藉由13天的饋料批次式法,在pH7、34℃~37℃的條件下實施。將該培養上清液以蛋白質A層析(MabSelect SuRe擔體(Cytiva公司))來純化,得到抗體組成物。
<抗體組成物的HIC-HPLC分析>
對所得到的抗體組成物依照以下的條件進行HIC-HPLC分析。
HPLC裝置:Prominence HPLC System(島津製作所)
分析管柱:TSKgel Butyl-NPR管柱(東曹,0014947;4.6mm×3.5cm,平均粒徑2.5μm)
移動相A:含2.3M硫酸銨的0.1M磷酸鈉緩衝液(pH7.0)
移動相B:0.1M磷酸鈉緩衝液(pH7.0)
分析條件:蛋白質濃度2mg/mL×注入10μL、梯度0~3分鐘;0%B、3~15分鐘;0~100%B、15~20分鐘;100%B、流速0.5mL/min、偵測波長220nm或280nm
如圖1所表示般,分離出三個峰,在13分鐘附近的溶離時間偵測到主峰(峰3),在12.5分鐘及12.0分鐘附近的溶離時間分別偵測到峰2及峰1。峰3被認為是僅在附加糖鏈的共有區結合了糖鏈的抗體,峰2被認為在峰3的抗體上在另一處結合了糖鏈之附加糖鏈的異構物,峰1被認為是在峰3的抗體上在另兩處結合了糖鏈之附加糖鏈的異構物(參考實施例2)。由此分析結果,確認了培養上述群株細胞所得到的抗體粗純化物中,包含附加糖鏈的異構物約10%。
[實施例2]
在抗體的附加糖鏈的共有區以外結合了糖鏈之抗體粗純化物的調製
(培養上清液)與附加糖鏈的狀況的確認
分別使用具有編碼抗hDLK-1抗體的重鏈及輕鏈的胺基酸序列的基因序列的表現載體(pFUSE),以及編碼在同抗體的輕鏈的糖鏈結合共有序列(Asn-Ser-Ser)的第三個胺基酸導入點變異的變異序列(Asn-Ser-Ala)及重鏈的胺基酸序列的表現載體(pFUSE),將ExpiCHO細胞轉形,進行抗體蛋白質的暫時性表現。將其培養上清液以蛋白質A親和層析來純化,將所得到的抗體粗純化物胜肽-N-醣苷酶F(PNGase F)處理或PNGase F非處理後,以還原SDS聚丙烯醯胺凝膠電泳(SDS-PAGE)(銀染色)來分析。將變異前的抗hDLK-1抗體表示為NSS抗體,以及將輕鏈的糖鏈結合共有序列變異後的抗hDLK-1抗體表示為NSA抗體。
結果如圖2所表示般,在NSS抗體的圖中,並未以PNGase F處理的條件下,在25kDa的輕鏈電泳帶上方觀察到具有大於25kDa的分子量的電泳帶,然而藉由PNGase F的處理,該電泳帶會消失。由此確認了NSS抗體中,在具有抗體的附加糖鏈的共有區以外的共有序列的輕鏈結合了糖鏈。另一方面,在NSA的圖中,無論有無PNGase F處理,都沒有觀察到該電泳帶。由這些結果可知,暫時性表現所產生的人源化抗DLK-1單株抗體(NSS抗體)的粗純化物中,包含了在輕鏈附加糖鏈之附加糖鏈的異構物。
[實施例3]
利用疏水性交互作用層析擔體進行附加糖鏈的異構物的分離1
將包含與實施例1同樣地調製出的抗hDLK-1抗體的培養上清液以蛋白質A親和層析來純化,得到包含附加糖鏈的異構物約10%的同抗體的粗純化物。對該抗體粗純化物添加氯化鈉且使終濃度成為1.0M,然後調整成pH5.0,作為層析擔體的載入用試樣。
使用該抗體粗純化物,以吸脫附模式來評估對於填充Capto Butyl(平均粒徑90μm,Cytiva公司製)的疏水性交互作用層析管柱(擔體容積5mL)的吸附特性。管柱的試樣載入量,以每單位擔體的蛋白質質量計,定為15mg/mL,流速定為300cm/h。依照以下的移動相的條件來實施層析。
移動相A:含1M氯化鈉的20mM檸檬酸鈉-15mM Tris緩衝液(pH5.0)
移動相B:20mM檸檬酸鈉-15mM Tris緩衝液(pH5.0)
1.平衡化:100%移動相A;10管柱容積(CV)
2.載入:抗體粗純化物的試樣
3.洗淨:100%移動相A;10CV
4.線型梯度溶離:以40CV由0%移動相B到100%移動相B
5.管柱洗淨:1M氫氧化鈉;5CV
圖3表示溶離層析圖譜(HIC389)。由此結果確認了在使用Capto Butyl擔體的線型梯度的條件下,抗體成分吸附於擔體。對於載入管柱的試樣、來自Capto Butyl管柱的流出液、以及同管柱的吸附組分,以實施例1的HIC-HPLC分析法來評估,將結果表示於圖4。載入的試樣中含2.1%(Peak1)及12.1%(Peak2)的附加糖鏈的異構物,會被去除至流出液(HIC389 Flow-through),在管柱的吸附組分(HIC389 Elute)中可得到去除了附加糖鏈的異構物且純度為100%的抗體(在附加糖鏈的共有區以外沒有附加糖鏈的抗體)(Peak3)組成物。
由以上的結果可知,藉由使用疏水性交互作用層析擔體(Capto Butyl),可將附加糖鏈的異構物分離。可知若使用選定的層析擔體,則附加糖鏈的異構物會有效率地被去除至流出液,在吸附組分中可得到高純度的純化抗體組成物。
[實施例4]
利用疏水性交互作用層析擔體進行附加糖鏈的異構物的分離2
將包含與實施例1同樣地調製出的人源化抗人類DLK-1單株抗體的培養上清液以蛋白質A親和層析來純化,而得到包含附加糖鏈的異構物約10%的同抗體粗純化物。對該抗體粗純化物添加氯化鈉且使終濃度成為1.0M,然後將pH定為4.5、4.7或5.0,將導電率定為70mS/cm以下,作為層析擔體的載入用試樣。
使用該抗體粗純化物,以吸脫附模式來評估對Capto Butyl(平均粒徑90μm,Cytiva公司製)及POROS Benzyl Ultra(平均粒徑50μm,Thermo Fisher公司製)各疏水性交互作用層析管柱(擔體容積5mL)的吸附特性。各管柱的蛋白質試樣載入量,以每單位擔體的蛋白質質量計為20mg/mL來實施,流速定為300cm/h。依照以下的移動相的條件來實施層析。
移動相A:含1M氯化鈉的20mM檸檬酸鈉緩衝液(pH4.5、4.7或5.0)
移動相B:20mM檸檬酸鈉緩衝液(pH4.5、4.7或5.0)
1.平衡化:100%移動相A;5CV
2.載入:試樣溶液
3.洗淨:100%移動相A;8CV
4.洗淨1:100%移動相A;5CV
5.洗淨2:100%移動相B+0.8M氯化鈉;5CV
6.溶離:100%移動相B;10CV
7.管柱洗淨:水(適量)
8.管柱洗淨:1M氫氧化鈉(適量)
將Capto Butyl與POROS Benzyl Ultra的溶離層析圖譜分別表示於圖5A及圖5B。在任一個pH條件下,抗體組成物都會吸附於Capto Butyl擔體,然後可溶離。在POROS Benzyl Ultra方面,也確認了良好的吸附與溶離。具體而言,圖5A及圖5B的圖譜顯示,橫軸0~50mL附近的峰,是沒有被吸附、由管柱通過而溶離出來的抗體(附加糖鏈的異構物),作為50~300mL的峰而溶離出來的抗體,是在附加糖鏈的共有區以外沒有附加糖鏈的抗體,由管柱徐緩地溶離出來。在Capto Butyl的情況,顯示與pH無關,在洗淨/溶離的全部區域抗體會溶離,藉由回收溶離的中間層,能夠以高純度得到在附加糖鏈的共有區以外沒有附加糖鏈的抗體。另外,在Poros Benzyl Ultra的情況,峰會隨著pH而變動(pH4.5時,抗體良好地吸附,溶離後半會由管柱溶離,然而pH5.0時,80%左右沒有吸附,洗淨過程中抗體會溶離)看來,顯示可因應pH來控制溶離成分。以上的結果顯示,使用Capto Butyl及POROS Benzyl Ultra的任一者,皆可分離附加糖鏈的異構物,因此可使用疏水性交互作用層析擔體來進行附加糖鏈的異構物的分離。另外,此結果顯示,在疏水性交互作用層析時,藉由使洗淨/溶離液的pH、鹽濃度及液量最適化,可將抗體組成物吸附,能夠達成附加糖鏈的異構物的去除。
[實施例5]
疏水性交互作用層析擔體中,載入時的鹽濃度、洗淨時的鹽濃度及pH、溶離緩衝液的種類,對於附加糖鏈的異構物的去除效率(純度及產率)造成影響的檢討
對於Capto Butyl與POROS Benzyl Ultra層析擔體,藉由階段性的溶離,檢討是否可去除附加糖鏈的異構物。作為載入液,是使用將蛋白質A親和層析純化的實施例1的抗體粗純化物以0.5M Tris調整成pH4.5或pH5.0的液體。作為參數,檢討了試樣載入液的鹽濃度、洗淨液(洗淨液I、洗淨液II)的pH及鹽濃度,以及溶離緩衝液的種類及pH,測定抗體的產率及純化組分的非附加糖鏈的異構物抗體純度(在附加糖鏈的共有區以外沒有附加糖鏈的抗體之純度)(利用HIC-HPLC分析法所得到的附加糖鏈的異構物的含量)。將測試結果揭示於表3。
以各條件來實施的利用Capto Butyl擔體進行的純化,產率為23%~73%,利用HIC-HPLC分析所得到的純化抗體組成物的純度(不含附加糖鏈的異構物的抗體成分)為97.1%~99.3%。另一方面,POROS Benzyl Ultra擔體的純化產率雖然稍低(33%~67%),然而利用HIC-HPLC分析所得到的純化抗體組成物的純度全部為100%。尤其在使用POROS Benzyl Ultra擔體的條件下,載入含0.8M氯化鈉的試樣,以含0.8M氯化鈉的洗淨液I、含0.6M氯化鈉的洗淨液II依序洗淨,以pH6的檸檬酸鈉緩衝液溶離的條件,從純度及產率的觀點看來為最適當。
另外,由一連串的測試結果,判明了以下的事項。
(i)若提高洗淨液(洗淨液I及/或洗淨液II)的pH,則純度會提升。
(ii)若提高洗淨液(洗淨液I及/或洗淨液II)的pH,則產率會降低。
(iii)若提高溶離液的pH,則純度提升。
(iv)若提高溶離液的pH,則產率降低。
(v)若降低載入液及/或洗淨液的鹽濃度,則產率會降低。
(vi)可設定抗體組成物的純度可達到95%以上,理想的情況為99%以上的條件。
如以上的結果所示,可知對於疏水性交互作用層析擔體(例如Capto Butyl與POROS Benzyl Ultra)來說,藉由使載入液的鹽濃度及pH、洗淨液的鹽濃度及pH、洗淨液量、洗淨次數、溶離時的pH及緩衝液的種類最適化,可讓產率及附加糖鏈的異構物的混入量最適化。
[實施例6]
為了控制附加糖鏈的異構物的混入率及目標物質的純度及產率的洗淨條件的最適化
將包含與實施例1同樣地調製出的抗hDLK-1抗體的培養上清液以蛋白質A親和層析來純化,而得到包含附加糖鏈的異構物約10%的同抗體粗純化物。對該抗體粗純化物添加氯化鈉且使終濃度成為1.0M,然後適當地調整pH及導電率,作為層析擔體的載入用試樣。使用該抗體粗純化物,並使用POROS Benzyl Ultra擔體,以試樣的載入量、洗淨液I及洗淨液II的液量作為輸入參數,評估輸出參數之產率及HIC-HPLC分析所得到的純度。
結果可知,藉由將每單位層析擔體量的蛋白質載入量調整成25g/L以上,並將洗淨液I及洗淨液II調整成5~15管柱容積(CV),能夠以97~100%的純度將產率控制在40~80%(表4)。尤其,蛋白質的載入量,相較於25g/L,在30g/L或35g/L的情況,所能夠得到的純化抗體組成物會有純度高的傾向。另外,藉由增加洗淨液量,所得到的純化抗體組成物的產率雖然降低,但是純度有提升的傾向。此結果表示,在蛋白質載入量低的條件下,藉由增加洗淨液量,可提高純化抗體組成物的純度。
亦即,由此結果判明了,藉由將層析時的蛋白質載入量定在一定以上,及/或適當地控制洗淨液量,可將附加糖鏈的異構物的含量從約10%控制在減少至3~0%的範圍。將藉由實驗設計法(DoE:Design of Experiment)解析該結果的結果表示於圖6。對洗淨液I及洗淨液II的液量與產率及純度的影響以等高線圖來表示,判明了以洗淨液量可控制任意產率及純度。
純化程序內的附加糖鏈的異構物的去除步驟之位置及洗淨液量對純度的影響
將包含與實施例1同樣地調製出的抗hDLK-1抗體的培養上清液以蛋白質A親和層析來純化,而得到包含附加糖鏈的異構物約10%的同抗體粗純化物。將此抗體粗純化物在pH3~4保持一定時間,中和之後,以此作為載入層析擔體用的試樣。
對於該抗體粗純化物,以圖7所表示的兩種流程,依照在疏水性交互作用層析之後進行混合模式層析或在混合模式層析之後進行疏水性交互作用層析的順序來純化。疏水性交互作用層析擔體使用了POROS Benzyl Ultra,混合模式層析擔體使用了Capto MMC。疏水性交互作用層析的條件如以下所述。ID No. HIC446、HIC457及HIC463是在疏水層析後進行混合模式層析,ID No. HIC447是在混合模式層析後進行疏水層析。
<疏水性交互作用層析的條件>
擔體:POROS Benzyl Ultra
管柱尺寸:260mL、填充高度13.3cm
流速:300cm/h
每單位擔體的蛋白質載入量:30g/L
層析條件:
1.平衡化:含0.8M氯化鈉的20mM檸檬酸鈉(pH5.0);5CV
2.載入:抗體粗純化物的試樣
3.洗淨1:含0.8M氯化鈉的20mM檸檬酸鈉(pH5.0);6CV(HIC446/HIC447)、8CV(HIC457)或15CV(HIC463)
4.洗淨2:含0.6M氯化鈉的20mM檸檬酸鈉(pH5.0);5CV
5.溶離:20mM檸檬酸鈉(pH6.0);6.3CV
6.管柱洗淨:1M氫氧化鈉水溶液;3CV
將疏水性交互作用層析步驟所得到的純化抗體組成物的產率及純度揭示於表5及圖8。在任一純化流程之中,產率皆在60%以上,利用HIC-HPLC分析所得到的純度(代表附加糖鏈的異構物的混入量)為99%以上。另外還明白了依照洗淨1的液量(6~15CV),可控制產率及純度。亦即可知,抗體純度約為90%的組成物,藉由5CV的洗淨會提升至98%左右,藉由8CV的洗淨會提升至99%左右,藉由15CV的洗淨會提升至99.5%左右。
包含附加糖鏈的異構物之去除步驟的純化程序(POROS Benzyl Ultra)
得到包含與實施例1同樣地調製出的抗hDLK-1抗體的培養上清液約200L。確認了附加糖鏈的異構物在抗體粗純化物中約含10%。
將培養上清液過濾,經過了使用蛋白質A親和層析(MabSelect SuRe 10L;Cytiva公司)、低pH病毒去活化、POROS Benzyl Ultra擔體(Thermo Fisher公司,10L)的疏水性交互作用層析、使用Capto adhere擔體(Cytiva公司,10L)的混合模式層析、病毒過濾、利用切向流過濾(TFF)進行的處方緩衝液置換、滅菌過濾等的步驟,而得到高純度的純化抗體組成物。
POROS Benzyl Ultra層析,是將每單位擔體量的蛋白質載入量調整成35g/L、30g/L、16g/L,實施3次層析。將POROS Benzyl Ultra層析步驟的條件及POROS Benzyl Ultra層析步驟的結果分別揭示於表6及表7。
[表6]
參數 | 條件 |
管柱 | 擔體量:10L、填充高度:20cm |
平衡化 | 含0.8M氯化鈉的20mM檸檬酸鈉緩衝液(pH5.0)、4CV |
載入液 | 蛋白質濃度6.71mg/mL(合計液量約125kg、合計蛋白質質量841g) |
每單位擔體的載入量 | 載入量:35g/L-resin(載入液量約52L)、30g/L-resin(載入液量約45L)、16g/L-resin(載入液量約24L) |
洗淨1 | 含0.8M氯化鈉的20mM檸檬酸鈉緩衝液(pH5.0)、15CV |
洗淨2 | 含0.6M氯化鈉的20mM檸檬酸鈉緩衝液(pH5.0)、5CV |
溶離 | 20mM檸檬酸鈉緩衝液(pH6.0)、10CV |
洗淨3 | 1M氫氧化鈉水溶液 |
[表7]
第1次 | 第2次 | 第3次 | |
載入量 | 35g/L | 30g/L | 16g/L |
產率 | 54% | 58% | 77% |
純度 | 100% | 100% | 89.74 |
第1次及第2次層析的載入量是在規定量以上的適當範圍內,而第3次層析的載入量是16g/L的低條件。其結果,雖然產率有77%,然而指示附加糖鏈的異構物混入量的HIC-HPLC純度為89.74%的低值。因此,僅將第1次及第2次的分部收集液統合,供應至接下來的層析步驟。
經過全部的步驟,最終得到的純化抗體組成物,總產率為46%。另外,利用HIC-HPLC分析所得到的純化抗體組成物的純度為100%,沒有包含附加糖鏈的異構物。像這樣,即使在抗體的層析條件下,也可得到充分減少附加糖鏈的異構物的純化抗體組成物。另外還顯示,載入層析擔體的蛋白質載入量對於附加糖鏈的異構物的去除率是重要的。
[實施例9]
包含附加糖鏈的異構物的去除步驟的純化程序(Capto Butyl)
得到包含與實施例1同樣地調製出的抗hDLK-1抗體的培養上清液。確認了附加糖鏈的異構物在抗體粗純化物中包含約10%。
使用該培養液,依照蛋白質A親和層析步驟(擔體:MabSelect SuRe)、低pH病毒去活化步驟、疏水性交互作用層析步驟(Capto Butyl擔體)、混合模式層析步驟(Capto adhere)的順序來純化。
如表8所揭示的結果般,藉由疏水性層析步驟,可使附加糖鏈的異構物的非含有率提升至純度99.8%,能夠以總產率25%取得純化抗體組成物。
如此結果所表示般,可知即使使用POROS Benzyl Ultra擔體以外的疏水性交互作用層析擔體,也能得到高純度的純化抗體組成物。
[表8]
[實施例10]
純化步驟 | 產率 | 純度 | 條件等 |
培養上清液 | 100% | 90% | - |
蛋白質A親和層析 | >90% | 89.9% | - |
低pH | 100% | 89.9% | - |
疏水性交互作用層析 (Capto Butyl擔體) | 37% | 99.8% | 載入量20mg/mL,以含0.8M氯化鈉的20mM檸檬酸鈉緩衝液(pH4.5)、30CV洗淨後,以含50mM氯化鈉及75mM精胺酸鹽酸鹽的20mM檸檬酸鈉(pH4.5)、10CV溶離。 |
混合模式層析 (Capto adhere擔體) | >90% | 99.8% | - |
純化程序全體 | 25% | 99.8% | - |
附加糖鏈的異構物的分部收集與生物活性的測定
對於包含與實施例1同樣方式取得的附加糖鏈的異構物約10%的抗hDLK-1抗體粗純化物,使用實施例1所記載的HIC-HPLC分析系統,將各峰精密地分部收集。分部收集是分成約10次來實施,將各峰統合之後,以等張磷酸緩衝液進行緩衝液交換。此外,將分部收集所使用的HIC-HPLC移動相的條件變更為以下。
移動相A:含2.3M硫酸銨的50mM磷酸緩衝液、pH7
移動相B:50mM磷酸緩衝液、pH7
對於分部收集前的抗體粗純化物(a)、分部收集後的純化抗體組成物(b、c)使用實施例1所揭示的HIC-HPLC來分析,將結果表示於圖9。
對於這些試樣,由10μg/mL以3倍公比來稀釋,並以ADCC Reporter Bioassay套組(Promega、G7010)在E:T比率6:1(標的細胞為DLK1高表現的HEK293細胞株)、反應時間6小時的條件下測量ADCC活性。將結果表示於圖10。分部收集前的抗體粗純化物及分部收集後的主峰(圖9中的(b))可得到大致同樣的S型曲線,確認了具有同等的生物活性。另一方面,藉由分部收集所單離出的附加糖鏈的異構物(圖9中的(c)),在任一濃度都是幾乎沒有得到訊號,可知沒有生物活性。由此結果確認了,附加糖鏈的異構物為雜質,就生物藥品的成分而言必須儘可能去除的成分。
[產業上的可利用性]
藉由本發明,可調製出Fc區糖鏈結合共有區以外的附加糖鏈的異構物減少的純化抗體組成物來利用。
[圖1]為表示由產生人源化抗hDLK-1單株抗體的細胞之培養液取得的粗純化物的HIC-HPLC分析圖譜之圖形。
[圖2]為表示利用暫時性表現人源化抗hDLK-1單株抗體的CHO細胞所得到的培養上清液在蛋白質A純化後的SDS-PAGE分析結果的照片。
[圖3]為表示利用Capto Butyl疏水性交互作用層析擔體進行抗體組成物的分離曲線之圖形。
[圖4]為表示以Capto Butyl擔體所分離出的純化抗體組成物利用HIC-HPLC進行純度分析測試的結果的圖形及表。表中的Peak1及Peak2表示附加糖鏈的異構物抗體(Peak1是4條多肽鏈的1處CDR結合了糖鏈,Peak2是兩處的CDR結合了糖鏈),Peak3表示僅在附加糖鏈的共有區結合了糖鏈的抗體的比例(%)。
[圖5A]為表示利用Capto Butyl擔體進行抗體組成物的分離曲線之圖形。
[圖5B]為表示利用Poros Benzyl Ultra擔體進行抗體組成物的分離曲線的圖形。
[圖6]為表示對於使用POROS Benzyl Ultra擔體的層析條件的最適化來控制附加糖鏈的異構物含量藉由實驗設計法來解析的結果之圖形。
[圖7]為表示抗體組成物的純化程序中的層析步驟的順序之圖。
[圖8]為表示疏水性交互作用層析的洗淨液量改變時的溶離圖譜及產率與純度的圖形(上圖:洗淨液1為6CV且洗淨液2為5CV的條件,中圖:洗淨液1為8CV且洗淨液2為5CV的條件,下圖:洗淨液1為15CV且洗淨液2為5CV的條件)。
[圖9]表示由使用HIC-HPLC分析的抗體粗純化物精密分離所得到的單離成分的分析數據。(a)表示單離前的抗體粗純化物的HIC-HPLC分析的結果。(b)表示在(a)的HIC-HPLC之中,作為主峰所收集到的組分的HIC-HPLC分析結果。(c)表示在(a)的HIC-HPLC之中,作為前峰所收集到的組分的HIC-HPLC分析結果。
[圖10]為表示附加糖鏈的異構物的ADCC活性的評估結果的圖形。縱軸表示代表ADCC活性的螢光強度,橫軸表示抗體濃度。圓形為作為圖9(b)的主峰所收集到的組分(不含附加糖鏈的異構物的成分),方形為作為圖9(c)的前峰所收集到的組分(附加糖鏈的異構物),三角形表示圖9(a)的單離前的抗體粗純化物的結果。
[圖11]為表示HuBA-1-3D-1的H鏈(γ1鏈)的編碼區的鹼基序列(序列識別號37)與胺基酸序列(序列識別號38)。胺基酸是以一個字母記號來表示,終止密碼子的位置是以"・"來表示。
[圖12]為表示HuBA-1-3D-2的H鏈(γ1鏈)的編碼區的鹼基序列(序列識別號41)與胺基酸序列(序列識別號42)。胺基酸是以一個字母記號來表示,終止密碼子的位置是以"・"來表示。
[圖13]為表示L鏈(κ鏈)的編碼區的鹼基序列(序列識別號69)與胺基酸序列(序列識別號70)。圖中,胺基酸是以一個字母記號來表示,終止密碼子的位置是以"・"來表示。
[圖14]為表示HuBA-1-3D-1 T73K的H鏈(γ1鏈)的編碼區的鹼基序列(序列識別號53)與胺基酸序列(序列識別號54)。胺基酸是以一個字母記號來表示,終止密碼子的位置是以"・"來表示。上述HuBA-1-3D-1 T73K的VH的推測胺基酸序列(序列識別號18)中,由N末端至19胺基酸所形成的胜肽為訊號胜肽。HuBA-1-3D-1 T73K的VH的成熟胜肽的cDNA鹼基序列揭示於序列識別號19,其推測胺基酸序列揭示於序列識別號20。
[圖15]為表示HuBA-1-3D-1 A24G/T73K的H鏈(γ1鏈)的編碼區的鹼基序列(序列識別號61)與胺基酸序列(序列識別號62)。胺基酸是以一個字母記號來表示,終止密碼子的位置是以"・"來表示。上述HuBA-1-3D-1 A24G/T73K的VH的推測胺基酸序列(序列識別號22)中,由N末端至19胺基酸所形成的胜肽為訊號胜肽。HuBA-1-3D-1 A24G/T73K的VH的成熟胜肽的cDNA鹼基序列揭示於序列識別號23,其推測胺基酸序列揭示於序列識別號24。
[圖16]為表示HuBA-1-3D-VH1、HuBA-1-3D-VH1 A24G、HuBA-1-3D-VH1 T73K、HuBA-1-3D-VH1 A24G/ T73K、HuBA-1-3D-VH2、HuBA-1-3D-VH2 A24G、HuBA-1-3D-VH2 T73K及HuBA-1-3D-VH2 A24G/T73K的胺基酸序列比對之圖。括號內表示序列表中的序列識別號。
<![CDATA[<110> 凱依歐姆生物科學股份有限公司(CHIOME Bioscie]]>nce Inc.) <![CDATA[<120> 抗體組成物之純化方法]]> <![CDATA[<130> G2920WO]]> <![CDATA[<150> JP 2021-079977]]> <![CDATA[<151> 2021-05-10]]> <![CDATA[<160> 78 ]]> <![CDATA[<170> PatentIn 3.5 版]]> <![CDATA[<210> 1]]> <![CDATA[<211> 420]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(420)]]> <![CDATA[<400> 1]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc aag gcc aca atg act gtc gac aca tcc acc agc 288 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca 420 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 2]]> <![CDATA[<211> 140]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 2]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 3]]> <![CDATA[<211> 363]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(363)]]> <![CDATA[<400> 3]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 4]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 4]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 5]]> <![CDATA[<211> 420]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(420)]]> <![CDATA[<400> 5]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc cga gcc aca atg act gtc gac aca tcc acc agc 288 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca 420 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 6]]> <![CDATA[<211> 140]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 6]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 7]]> <![CDATA[<211> 363]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(363)]]> <![CDATA[<400> 7]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 8]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 8]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 9]]> <![CDATA[<211> 399]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(399)]]> <![CDATA[<400> 9]]> atg gaa tca cag acc cag gtc ctc atg ttt ctt ctg ctc tgg gta tct 48 Met Glu Ser Gln Thr Gln Val Leu Met Phe Leu Leu Leu Trp Val Ser 1 5 10 15 ggt gcc tgt gca gac att gtc atg aca cag tct cca gac tcc ctg gct 96 Gly Ala Cys Ala Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 gtg tca ctg gga gag agg gcc act atc aac tgc aag tcc agt cag agc 144 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35 40 45 ctt ctg aat agt agc aat caa aag aac tat ttg gcc tgg tac cag cag 192 Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 aaa cca gga cag cct cct aaa ctt ctg gtc tac ttt gca tcc act agg 240 Lys Pro Gly Gln Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg 65 70 75 80 gaa tct ggg gtc cct gat cgc ttc agt ggc agt gga tct ggg aca gat 288 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 ttc act ctt acc atc agc agt ctg cag gct gaa gat gtg gca gtt tac 336 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 100 105 110 tac tgt cag caa cat tat agc act cct ccc aca ttc ggt cag ggg acc 384 Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr 115 120 125 aag ctg gag atc aaa 399 Lys Leu Glu Ile Lys 130 <![CDATA[<210> 10]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 10]]> Met Glu Ser Gln Thr Gln Val Leu Met Phe Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Ala Cys Ala Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr 115 120 125 Lys Leu Glu Ile Lys 130 <![CDATA[<210> 11]]> <![CDATA[<211> 339]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(339)]]> <![CDATA[<400> 11]]> gac att gtc atg aca cag tct cca gac tcc ctg gct gtg tca ctg gga 48 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 gag agg gcc act atc aac tgc aag tcc agt cag agc ctt ctg aat agt 96 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 agc aat caa aag aac tat ttg gcc tgg tac cag cag aaa cca gga cag 144 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 cct cct aaa ctt ctg gtc tac ttt gca tcc act agg gaa tct ggg gtc 192 Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 cct gat cgc ttc agt ggc agt gga tct ggg aca gat ttc act ctt acc 240 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 atc agc agt ctg cag gct gaa gat gtg gca gtt tac tac tgt cag caa 288 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 cat tat agc act cct ccc aca ttc ggt cag ggg acc aag ctg gag atc 336 His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 aaa 339 Lys <![CDATA[<210> 12]]> <![CDATA[<211> 113]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 12]]> Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <![CDATA[<210> 13]]> <![CDATA[<211> 420]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(420)]]> <![CDATA[<400> 13]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc aag gcc aca atg act gtc gac aca tcc acc agc 288 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca 420 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 14]]> <![CDATA[<211> 140]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 14]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 15]]> <![CDATA[<211> 363]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(363)]]> <![CDATA[<400> 15]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 16]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 16]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 17]]> <![CDATA[<211> 420]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(420)]]> <![CDATA[<400> 17]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc 288 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca 420 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 18]]> <![CDATA[<211> 140]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 18]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 19]]> <![CDATA[<211> 363]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(363)]]> <![CDATA[<400> 19]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 20]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 20]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 21]]> <![CDATA[<211> 420]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(420)]]> <![CDATA[<400> 21]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc 288 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca 420 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 22]]> <![CDATA[<211> 140]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 22]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 23]]> <![CDATA[<211> 363]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(363)]]> <![CDATA[<400> 23]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 24]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 24]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 25]]> <![CDATA[<211> 420]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(420)]]> <![CDATA[<400> 25]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc cga gcc aca atg act gtc gac aca tcc acc agc 288 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca 420 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 26]]> <![CDATA[<211> 140]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成]]>構建體 <![CDATA[<400> 26]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 27]]> <![CDATA[<211> 363]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(363)]]> <![CDATA[<400> 27]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 28]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 28]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 29]]> <![CDATA[<211> 420]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(420)]]> <![CDATA[<400> 29]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc 288 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca 420 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 30]]> <![CDATA[<211> 140]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 30]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 31]]> <![CDATA[<211> 363]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(363)]]> <![CDATA[<400> 31]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 32]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 32]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 33]]> <![CDATA[<211> 420]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(420)]]> <![CDATA[<400> 33]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc 288 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca 420 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 34]]> <![CDATA[<211> 140]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 34]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 <![CDATA[<210> 35]]> <![CDATA[<211> 363]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 重組 DNA]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(363)]]> <![CDATA[<400> 35]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca 363 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 36]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 36]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <![CDATA[<210> 37]]> <![CDATA[<211> 1413]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1413)]]> <![CDATA[<400> 37]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc aag gcc aca atg act gtc gac aca tcc acc agc 288 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag 432 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg 480 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg 528 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg 624 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc 720 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 768 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 960 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 1104 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 1152 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1248 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 1344 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 1392 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 tcc ctg tct ccg ggt aaa tga 1413 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 38]]> <![CDATA[<211> 470]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 38]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 39]]> <![CDATA[<211> 1356]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1356)]]> <![CDATA[<400> 39]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg 384 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 576 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 768 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 816 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1008 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1056 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1104 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1152 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 1248 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1296 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 1344 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 ccg ggt aaa tga 1356 Pro Gly Lys 450 <![CDATA[<210> 40]]> <![CDATA[<211> 451]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 40]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <![CDATA[<210> 41]]> <![CDATA[<211> 1413]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1413)]]> <![CDATA[<400> 41]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc cga gcc aca atg act gtc gac aca tcc acc agc 288 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag 432 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg 480 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg 528 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg 624 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc 720 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 768 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 960 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 1104 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 1152 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1248 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 1344 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 1392 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 tcc ctg tct ccg ggt aaa tga 1413 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 42]]> <![CDATA[<211> 47]]>0 <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 42]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 43]]> <![CDATA[<211> 1356]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1356)]]> <![CDATA[<400> 43]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg 384 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 576 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 768 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 816 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1008 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1056 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1104 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1152 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 1248 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1296 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 1344 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 ccg ggt aaa tga 1356 Pro Gly Lys 450 <![CDATA[<210> 44]]> <![CDATA[<211> 451]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 44]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <![CDATA[<210> 45]]> <![CDATA[<211> 1413]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1413)]]> <![CDATA[<400> 45]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc aag gcc aca atg act gtc gac aca tcc acc agc 288 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag 432 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg 480 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg 528 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg 624 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc 720 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 768 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 960 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 1104 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 1152 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1248 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 1344 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 1392 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 tcc ctg tct ccg ggt aaa tga 1413 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 46]]> <![CDATA[<211> 470]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 46]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 47]]> <![CDATA[<211> 1356]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1356)]]> <![CDATA[<400> 47]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg 384 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 576 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 768 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 816 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1008 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1056 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1104 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1152 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 1248 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1296 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 1344 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 ccg ggt aaa tga 1356 Pro Gly Lys 450 <![CDATA[<210> 48]]> <![CDATA[<211> 451]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 48]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <![CDATA[<210> 49]]> <![CDATA[<211> 1413]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1413)]]> <![CDATA[<400> 49]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc cga gcc aca atg act gtc gac aca tcc acc agc 288 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag 432 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg 480 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg 528 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg 624 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc 720 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 768 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 960 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 1104 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 1152 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1248 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 1344 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 1392 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 tcc ctg tct ccg ggt aaa tga 1413 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 50]]> <![CDATA[<211> 470]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 50]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 51]]> <![CDATA[<211> 1356]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1356)]]> <![CDATA[<400> 51]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aca tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg 384 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 576 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 768 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 816 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1008 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1056 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1104 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1152 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 1248 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1296 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 1344 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 ccg ggt aaa tga 1356 Pro Gly Lys 450 <![CDATA[<210> 52]]> <![CDATA[<211> 451]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 52]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <![CDATA[<21]]>0> 53]]> <br/><![CDATA[<211> 1413]]> <br/><![CDATA[<212> DNA]]> <br/><![CDATA[<213> 人工序列]]> <br/> <br/><![CDATA[<220>]]> <br/><![CDATA[<223> 抗體]]> <br/> <br/> <br/><![CDATA[<220>]]> <br/><![CDATA[<221> CDS]]> <br/><![CDATA[<222> (1)..(1413)]]> <br/> <br/><![CDATA[<400> 53]]> <br/><![CDATA[atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc 288 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag 432 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg 480 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg 528 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg 624 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc 720 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 768 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 960 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 1104 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 1152 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1248 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 1344 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 1392 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 tcc ctg tct ccg ggt aaa tga 1413 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 54]]> <![CDATA[<211]]>> 470]]> <br/><![CDATA[<212> PRT]]> <br/><![CDATA[<213> 人工序列]]> <br/> <br/><![CDATA[<220>]]> <br/><![CDATA[<223> 合成構建體]]> <br/> <br/><![CDATA[<400> 54]]> <br/> <br/><![CDATA[Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 55]]> <![CDATA[<211> 1356]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1356)]]> <![CDATA[<400> 55]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg 384 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 576 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 768 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 816 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1008 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1056 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1104 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1152 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 1248 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1296 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 1344 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 ccg ggt aaa tga 1356 Pro Gly Lys 450 <![CDATA[<210> 56]]> <![CDATA[<211> 451]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 56]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <![CDATA[<210> 57]]> <![CDATA[<211> 1413]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1413)]]> <![CDATA[<400> 57]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc 288 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag 432 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg 480 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg 528 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg 624 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc 720 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 768 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 960 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 1104 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 1152 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1248 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 1344 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 1392 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 tcc ctg tct ccg ggt aaa tga 1413 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 58]]> <![CDATA[<211> 470]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 58]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 59]]> <![CDATA[<211> 1356]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1356)]]> <![CDATA[<400> 59]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag gct tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg 384 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 576 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 768 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 816 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1008 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1056 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1104 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1152 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 1248 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1296 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 1344 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 ccg ggt aaa tga 1356 Pro Gly Lys 450 <![CDATA[<210> 60]]> <![CDATA[<211> 451]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 60]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <![CDATA[<210> 61]]> <![CDATA[<211> 1413]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1413)]]> <![CDATA[<400> 61]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc 288 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag 432 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg 480 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg 528 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg 624 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc 720 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 768 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 960 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 1104 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 1152 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1248 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 1344 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 1392 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 tcc ctg tct ccg ggt aaa tga 1413 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 62]]> <![CDATA[<211> 470]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 62]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 63]]> <![CDATA[<211> 1356]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1356)]]> <![CDATA[<400> 63]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc aag gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg 384 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 576 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 768 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 816 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1008 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1056 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1104 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1152 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 1248 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1296 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 1344 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 ccg ggt aaa tga 1356 Pro Gly Lys 450 <![CDATA[<210> 64]]> <![CDATA[<211> 451]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 64]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <![CDATA[<210> 65]]> <![CDATA[<211> 1413]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1413)]]> <![CDATA[<400> 65]]> atg ggt tgg agc tgt atc atc ttc ttt ctg gta gca aca gct aca ggc 48 Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 gtg cac tcc caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag 96 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 act gat tat gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg 192 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg att gga gtt att agt act tac tat ggt aat aca aac tac aac 240 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 cag aag ttt aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc 288 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 aca gcc tat atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg 336 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 tat tac tgt gca aga gga gga ttg cga gag tat tac tat gct atg gac 384 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 tac tgg ggt caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag 432 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg 480 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg 528 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg 624 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc 720 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 768 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 960 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 1104 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 1152 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1248 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 1344 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 1392 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 tcc ctg tct ccg ggt aaa tga 1413 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 66]]> <![CDATA[<211> 470]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 66]]> Met Gly Trp Ser Cys Ile Ile Phe Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <![CDATA[<210> 67]]> <![CDATA[<211> 1356]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(1356)]]> <![CDATA[<400> 67]]> caa gtc cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aaa gtc tcc tgc aag ggt tcc ggc tac aca ttc act gat tat 96 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gct atg cac tgg gtg cga cag gcc cct gga caa ggc ctg gag tgg att 144 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga gtt att agt act tac tat ggt aat aca aac tac aac cag aag ttt 192 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 aag ggc cga gcc aca atg act gtc gac aaa tcc acc agc aca gcc tat 240 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctt agg agc ttg aga tct gac gat act gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga gga gga ttg cga gag tat tac tat gct atg gac tac tgg ggt 336 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 caa gga acc atg gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg 384 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 576 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 768 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 816 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1008 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1056 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1104 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1152 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg 1248 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1296 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 1344 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 ccg ggt aaa tga 1356 Pro Gly Lys 450 <![CDATA[<210> 68]]> <![CDATA[<211> 451]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 68]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <![CDATA[<210> 69]]> <![CDATA[<211> 723]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(723)]]> <![CDATA[<400> 69]]> atg gaa tca cag acc cag gtc ctc atg ttt ctt ctg ctc tgg gta tct 48 Met Glu Ser Gln Thr Gln Val Leu Met Phe Leu Leu Leu Trp Val Ser 1 5 10 15 ggt gcc tgt gca gac att gtc atg aca cag tct cca gac tcc ctg gct 96 Gly Ala Cys Ala Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 gtg tca ctg gga gag agg gcc act atc aac tgc aag tcc agt cag agc 144 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35 40 45 ctt ctg aat agt agc aat caa aag aac tat ttg gcc tgg tac cag cag 192 Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 aaa cca gga cag cct cct aaa ctt ctg gtc tac ttt gca tcc act agg 240 Lys Pro Gly Gln Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg 65 70 75 80 gaa tct ggg gtc cct gat cgc ttc agt ggc agt gga tct ggg aca gat 288 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 ttc act ctt acc atc agc agt ctg cag gct gaa gat gtg gca gtt tac 336 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 100 105 110 tac tgt cag caa cat tat agc act cct ccc aca ttc ggt cag ggg acc 384 Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr 115 120 125 aag ctg gag atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc 432 Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc 480 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg 528 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag 576 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 gac agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc 624 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 aaa gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat 672 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 cag ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt 720 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 tag 723 <![CDATA[<210> 70]]> <![CDATA[<211> 240]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 70]]> Met Glu Ser Gln Thr Gln Val Leu Met Phe Leu Leu Leu Trp Val Ser 1 5 10 15 Gly Ala Cys Ala Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <![CDATA[<210> 71]]> <![CDATA[<211> 663]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 抗體]]> <![CDATA[<220>]]> <![CDATA[<221> CDS]]> <![CDATA[<222> (1)..(663)]]> <![CDATA[<400> 71]]> gac att gtc atg aca cag tct cca gac tcc ctg gct gtg tca ctg gga 48 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 gag agg gcc act atc aac tgc aag tcc agt cag agc ctt ctg aat agt 96 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 agc aat caa aag aac tat ttg gcc tgg tac cag cag aaa cca gga cag 144 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 cct cct aaa ctt ctg gtc tac ttt gca tcc act agg gaa tct ggg gtc 192 Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 cct gat cgc ttc agt ggc agt gga tct ggg aca gat ttc act ctt acc 240 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 atc agc agt ctg cag gct gaa gat gtg gca gtt tac tac tgt cag caa 288 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 cat tat agc act cct ccc aca ttc ggt cag ggg acc aag ctg gag atc 336 His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat 384 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac 432 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc 480 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac 528 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac 576 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc 624 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 663 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <![CDATA[<210> 72]]> <![CDATA[<211> 220]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> 合成構建體]]> <![CDATA[<400> 72]]> Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <![CDATA[<210> 73]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> CDRH1]]> <![CDATA[<400> 73]]> Asp Tyr Ala Met His 1 5 <![CDATA[<210> 74]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> CDRH2]]> <![CDATA[<400> 74]]> Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <![CDATA[<210> 75]]> <![CDATA[<211> 12]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> CDRH3]]> <![CDATA[<400> 75]]> Gly Gly Leu Arg Glu Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 <![CDATA[<210> 76]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> CDRL1]]> <![CDATA[<400> 76]]> Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <![CDATA[<210> 77]]> <![CDATA[<211> ]]> 7 <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> CDRL2]]> <![CDATA[<400> 77]]> Phe Ala Ser Thr Arg Glu Ser 1 5 <![CDATA[<210> 78]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列]]> <![CDATA[<220>]]> <![CDATA[<223> CDRL3]]> <![CDATA[<400> 78]]> Gln Gln His Tyr Ser Thr Pro Pro Thr 1 5
Claims (27)
- 一種抗hDLK-1抗體組成物之純化方法,該抗體之重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且該方法包含: 藉由將抗體粗純化物載入慣用層析裝置,使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於前述層析裝置的擔體,以及 以溶離液讓前述擔體發生溶離,使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物, 此處,所得到的前述純化抗體組成物中,在Fc區的附加糖鏈的共有區以外的部位附加糖鏈之附加糖鏈的異構物的含量,與前述抗體粗純化物相比,較為減少。
- 如請求項1之純化方法,其中前述慣用層析裝置的擔體為疏水性交互作用層析擔體,或混合模式層析擔體。
- 如請求項1或2之純化方法,其中前述慣用層析裝置的擔體的平均粒徑為15μm以上。
- 如請求項1或2之純化方法,其中前述慣用層析裝置的擔體的平均粒徑為20~100μm。
- 如請求項1~4中任一項之純化方法,其中前述慣用層析裝置的擔體具有苄基或丁基。
- 如請求項1~5中任一項之純化方法,其中前述慣用層析裝置的擔體之每單位量的蛋白質載入量為20mg/mL以上。
- 如請求項1~6中任一項之純化方法,其中前述慣用層析裝置的擔體為疏水性交互作用層析擔體,且包含將前述抗體粗純化物載入前述擔體之後,在溶離前以洗淨液將該擔體洗淨。
- 如請求項7之純化方法,其中前述洗淨液的鹽濃度比前述溶離液的鹽濃度高10mM以上。
- 如請求項7或8之純化方法,其中前述洗淨液的鹽濃度為0.5M以上。
- 如請求項7或8之純化方法,其中前述洗淨液的鹽濃度為1.0M以上。
- 如請求項7~10中任一項之純化方法,其中前述洗淨液的pH比前述溶離液的pH低0.2以上,且pH在4~8的範圍內。
- 如請求項7~11中任一項之純化方法,其中前述洗淨係藉由流過2倍擔體容積以上的洗淨液來進行。
- 如請求項7~12中任一項之純化方法,其中藉由使用pH及/或鹽濃度階段性或直線地變化的移動相來進行洗淨及溶離。
- 如請求項7~13中任一項之純化方法,其中前述溶離液的鹽濃度在0.5M以下或不含鹽,且pH為5~7。
- 如請求項1~14中任一項之純化方法,其產率為20%以上。
- 如請求項1~15中任一項之純化方法,其中前述純化抗體組成物中之附加糖鏈的異構物相對於所有抗體的比例為5%以下。
- 如請求項16之純化方法,其中前述抗體粗純化物中之附加糖鏈的異構物相對於所有抗體的比例大於5%。
- 一種如請求項1之抗hDLK-1抗體組成物之純化方法,其係包含: 將抗體粗純化物載入使用具有苄基或丁基且粒徑為20~100μm的擔體的慣用層析裝置, 使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於前述擔體, 藉由以pH4~6的洗淨液將前述擔體洗淨1次以上而去除附加糖鏈的異構物,以及 以鹽濃度在0.5M以下或不含鹽且pH5~7的溶離液使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物, 此處,前述純化抗體組成物中,附加糖鏈的異構物相對於所有抗體的比例,與前述抗體粗純化物相比,較為降低。
- 一種如請求項1之抗體組成物之純化方法,其係包含: 將鹽濃度調整至0.5M以上的前述抗體粗純化物載入使用具有苄基或丁基且粒徑為20~100μm的擔體的慣用層析裝置,將附加糖鏈的異構物去除至流出液, 以洗淨液將前述擔體洗淨,以及 以鹽濃度在0.5M以下或不含鹽且pH5~7的溶離液使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物, 此處,前述純化抗體組成物中,附加糖鏈的異構物相對於所有抗體的比例,與前述抗體粗純化物相比,較為降低。
- 一種如請求項1之抗體組成物之純化方法,其係包含: 將pH調整至4~6的前述抗體粗純化物載入使用具有苄基或丁基且粒徑為20~100μm的擔體的慣用層析裝置,將附加糖鏈的異構物去除至流出液, 以洗淨液將前述擔體洗淨,以及 以鹽濃度在0.5M以下或不含鹽且pH5~7的溶離液使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物, 此處,前述純化抗體組成物中,附加糖鏈的異構物相對於所有抗體的比例,與前述抗體粗純化物相比,較為降低。
- 如請求項1~20中任一項之方法,其中前述慣用層析裝置的擔體之每單位量的蛋白質載入量為20g/L以上,且前述洗淨係藉由流過5倍擔體容積以上的洗淨液來進行。
- 一種抗體組成物的製造方法,該抗體組成物中,在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體相對於所有抗體的比例為95%以上,該方法包含如請求項1~21中任一項之純化方法。
- 一種抗hDLK-1抗體純化組成物的製造方法,該抗體之重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且該方法包含: 將抗體粗純化物載入使用具有苄基或丁基且粒徑為20~100μm的擔體的慣用層析裝置, 使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於前述擔體, 藉由以含0.5M以上的鹽的洗淨液將前述擔體洗淨1次以上,而去除附加糖鏈的異構物去除,以及 以鹽濃度在0.5M以下或不含鹽且pH5~7的溶離液使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物, 此處,前述純化抗體組成物中,附加糖鏈的異構物相對於所有抗體的比例,與前述抗體粗純化物相比,較為降低。
- 一種抗體組成物,其係藉由如請求項22或23之製造方法所製造。
- 一種由抗hDLK-1抗體粗純化物將在Fc區的附加糖鏈的共有區以外的部位附加糖鏈之附加糖鏈的異構物去除的方法,該抗體之重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且該方法包含: 將抗體粗純化物載入疏水性交互作用層析擔體,使在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體吸附於前述擔體, 以洗淨液將前述擔體洗淨,以及 藉由以溶離液由前述擔體溶離,使吸附於前述擔體的前述抗體溶離,而得到純化抗體組成物。
- 一種抗hDLK-1抗體組成物,該抗體之重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且該抗體組成物含有所有抗體的95%以上之在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈的抗體。
- 一種抗hDLK-1抗體,其重鏈具有選自序列識別號2、4、6、8、14、16、18、20、22、24、26、28、30、32、34及36的胺基酸序列,且輕鏈具有序列識別號10或序列識別號12所記載的胺基酸序列,並且在Fc區的附加糖鏈的共有區以外的部位沒有附加糖鏈。
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