TW202221043A - PD-1/TGF-beta四價雙特異性抗體、其製備方法和用途 - Google Patents
PD-1/TGF-beta四價雙特異性抗體、其製備方法和用途 Download PDFInfo
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Abstract
本揭露描述了一種基於共同輕鏈的結構對稱的四價雙特異性抗體及其構建方法。本揭露製備的四價雙特異性抗體具有與單抗相似甚至更優的生物學活性和理化性質,可以用於治療各種炎性疾病、癌症和其它疾病。
Description
本揭露涉及抗體領域,更具體地,本揭露涉及一類四價雙特異性抗體、其製備方法和用途。
雙特異性抗體是指能同時特異性結合兩種抗原或兩種表位的抗體分子。根據對稱性,雙特異性抗體可以分為結構對稱的和不對稱的分子。根據結合位點的多少,雙特異性抗體可以分為二價、三價、四價和多價分子。
ScFv由重鏈可變區(VH)和輕鏈可變區(VL)組成,其中VH和VL通過柔性肽接頭連接在一起。在ScFv中,結構域的順序可以是VH-接頭-VL或VL-接頭-VH。已經報導了各種接頭用於連接VH和VL,例如短丙胺酸接頭,富含甘胺酸-絲胺酸的接頭,採用螺旋構象的接頭,以及衍生自各種免疫球蛋白和非免疫球蛋白分子的接頭(Ahmad Z A, Yeap S K, Ali A M, et al. scFv antibody: principles and clinical application[J]. Clinical and developmental immunology, 2012, 2012:980250.)。ScFv通常代表一個抗體的最小結合位點。可以通過接頭連接兩個ScFv來構建雙特異性抗體。如此構建成的雙抗分子是二價的,每種抗原具有一個結合位點,分子量通常在50-60kDa左右。兩個串聯的ScFv片段會獨立折疊並形成各自的抗原結合位點。雙特異的串聯scFv形式已廣泛應用於癌症免疫療法中,用於將T細胞重新靶向腫瘤細胞或腫瘤微環境中的腫瘤相關細胞。這種形式構成了雙特異性T細胞銜接子(bispecific T-cell engager,BiTE)的分子基礎,第一個被批准上市的BiTE分子是Blinatumomab(Huehls A M, Coupet T A, Sentman C L. Bispecific T-cell engagers for cancer immunotherapy[J]. Immunology and cell biology, 2015, 93(3): 290-296.)。
Diabody(Db)是由兩條鏈組成的二價分子,每條鏈包含一個VH和VL結構域,來自相同或不同的抗體。在Diabody中,兩個可變結構域通過短接頭連接,通常是5個胺基酸殘基,例如GGGGS。因為接頭顯著短於允許鏈內裝配所需的長度,這將導致兩條ScFv鏈以頭對尾方向二聚化,從而產生分子量與串聯ScFv相當的包裝緊密的Diabody分子(Wu C. Diabodies: molecular engineering and therapeutic applications[J]. Drug News Perspect, 2009, 22(8): 453.)。Diabody的兩條不同的鏈在細胞內表達時會出現可變結構域的錯誤配對。此外,由於兩條鏈之間沒有非共價鍵相連,Diabody並不穩定(Kipriyanov S M, Moldenhauer G, Braunagel M, et al. Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies[J]. Journal of molecular biology, 2003, 330(1): 99-111.)。
Diabody可以與人類Fc融合產生更類似免疫球蛋白的分子,稱為Di-diabody (Lu D, Zhang H, Koo H, et al. A fully human recombinant IgG-like bispecific antibody to both the epidermal growth factor receptor and the insulin-like growth factor receptor for enhanced antitumor activity[J]. Journal of Biological Chemistry, 2005, 280(20): 19665-19672.)。兩個串聯的ScFv也可以與人類Fc融合產生更類似免疫球蛋白的分子,稱為TaFv-Fc(Chen Z, Xie W, Acheampong D O, et al. A human IgG-like bispecific antibody co-targeting epidermal growth factor receptor and the vascular endothelial growth factor receptor 2 for enhanced antitumor activity[J]. Cancer biology & therapy, 2016, 17(2): 139-150.)。
除以上描述的Di-diabody和TaFv-Fc之外,結構對稱的四價雙特異性抗體還有(不限於)以下幾種類型。
在天然IgG的重鏈或輕鏈的N末端或C末端通過多肽接頭連接ScFv,如此可以形成IgG-ScFv形式的雙特異性抗體(Coloma M J, Morrison S L. Design and production of novel tetravalent bispecific antibodies[J]. Nature biotechnology, 1997, 15(2): 159.)。
在天然抗體的輕鏈和重鏈的N末端通過多肽接頭分別再接入另一個抗體的VL和VH,如此可以形成DVD-Ig形式的雙特異性抗體(Wu C, Ying H, Grinnell C, et al. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin[J]. Nature biotechnology, 2007, 25(11): 1290.)。
CrossMAb是一種抗體Fab臂的功能區互換的技術,是由羅氏開發的技術平臺。該技術解決了同源輕重鏈正確裝配的問題,進一步提高了裝配的成功率。基於CrossMAb的多種形式的雙特異性抗體的形式已被公開(Klein C, Schaefer W, Regula J T. The use of CrossMAb technology for the generation of bi-and multispecific antibodies[C]//MAbs. Taylor & Francis, 2016, 8(6): 1010-1020.)。
在天然抗體重鏈的N末端通過多肽接頭連接另一個抗體的輕鏈,然後VH+CH1作為獨立的短鏈與連接到N末端的輕鏈配對,如此可以形成FIT-IgG形式的雙特異性抗體(US 10266608 B2)。
IgG-TCR通過用T細胞受體(T cell receptors,TCR)的α和β鏈的恆定區分別替換重鏈的CH1和輕鏈的CL來提高相關的重鏈和輕鏈的配對效率(Wu X, Sereno A J, Huang F, et al. Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies[C]//MAbs. Taylor & Francis, 2015, 7(2): 364-376.)。WuXiBody在IgG-TCR的基礎上進行了改進,在α和β鏈的恆定區之間增加了一個工程化二硫鍵,進一步提高了相關的重鏈和輕鏈配對的效率,以及雙特異性抗體分子的穩定性(WO 2019/057122 A1)。
最近還報導了一種構建四價雙特異性抗體的方法。該方法基於晶體結構對Fab的重鏈和輕鏈之間的介面進行人工設計,並通過實驗方法篩選出一種正交的(orthogonal)Fab界面,該正交的Fab的重鏈和輕鏈能夠特異配對而不會與野生型的重鏈和輕鏈錯誤配對(Lewis S M, Wu X, Pustilnik A, et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface[J]. Nature biotechnology, 2014, 32(2): 191.;Wu X, Sereno A J, Huang F, et al. Fab-based bispecific antibody formats with robust biophysical properties and biological activity[C]//MAbs. Taylor & Francis, 2015, 7(3): 470-482.)。
另外,結構不對稱的雙特異性抗體還可能採用以下幾種結構(不限於)。
杵臼結構(knob-in-hole,KIH)的主要功能是促使雙特異性抗體的兩條不同重鏈異二聚化。其結構特點為:組成雙特異性抗體的一條重鏈的CH3區發生突變形成一個突起的「杵」的結構,另一條重鏈的CH3區發生突變形成一個凹陷的「臼」的結構,杵臼結構設計有利於兩種異源抗體重鏈的正確裝配(Merchant A M, Zhu Z, Yuan J Q, et al. An efficient route to human bispecific IgG[J]. Nature biotechnology, 1998, 16(7): 677.)。DuetMab通過使用KIH技術實現2條不同重鏈的異二聚化,並通過用工程化二硫鍵替換CH1-CL介面之間的天然二硫鍵來提高相關的重鏈和輕鏈的配對效率(Mazor Y, Oganesyan V, Yang C, et al. Improving target cell specificity using a novel monovalent bispecific IgG design[C]//MAbs. Taylor & Francis, 2015, 7(2): 377-389.)。
另外一種增強不同重鏈異二聚化的方法為靜電轉向突變(Electrostatic Steering Mutations)技術,該方法基於具有靜電相互作用的帶電殘基。該方法選擇改變了CH3介面中的電荷極性,使得靜電匹配的Fc結構域因為有利的電荷吸引作用而利於異二聚體形成,而不利的電荷排斥作用則抑制同源二聚化(US 10011858 B2;Gunasekaran K, Pentony M, Shen M, et al. Enhancing antibody Fc heterodimer formation through electrostatic steering effects applications to bispecific molecules and monovalent IgG[J]. Journal of Biological Chemistry, 2010, 285(25): 19637-19646.)。
共同輕鏈(Common Light Chain)經常被用於雙特異性抗體的構建。共同輕鏈能夠減少重鏈和輕鏈錯誤配對引起的副產物的產生,增加雙特異性抗體的產率(Brinkmann U, Kontermann R E. The making of bispecific antibodies[C]//MAbs. Taylor & Francis, 2017, 9(2): 182-212.)。通常情況下,共同輕鏈需要通過實驗方法篩選出來,一般可以通過雜交瘤或噬菌體展示技術獲得。通常情況下,需要對抗A抗體和抗B抗體的抗體庫進行高通量篩選,這要求兩個抗體庫的庫容量不能太小(Sampei Z, Igawa T, Soeda T, et al. Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity[J]. PloS one, 2013, 8(2): e57479.;US 9657102 B2)。
雙特異性抗體正在逐步成為一類新的治療性抗體,可以用於治療各種炎性疾病、癌症和其它疾病。雖然最近報導了大量新的雙特異性抗體的構造形式,然而,生產雙特異性抗體的主要技術難點在於獲得正確配對的分子。上述雙特異性抗體的形式均存在錯誤配對的問題,因此會產生一種或多種錯誤配對導致的副產物或者聚集體,從而影響目的雙特異性抗體的產率、純度和理化穩定性,進而影響雙特異性抗體在體內的安全性和有效性。
本揭露描述了一種基於共同輕鏈的結構對稱的四價雙特異性抗體及其構建方法。通常情況下,雙特異性抗體的重鏈和輕鏈之間會出現錯誤配對,重鏈和重鏈之間也會出現錯誤配對,正確裝配的效率越高錯誤配對的程度就越低。在此,本揭露用共同輕鏈解決了重鏈和輕鏈之間的錯誤配對問題,並將一個抗體的重鏈可變區VH-B與CH1連接,然後通過肽接頭與另一個抗體的重鏈可變區VH-A連接,再與重鏈恆定區CH1-CH2-CH3連接,構成一條長重鏈。長重鏈和共同輕鏈的基因在同一細胞內表達時,每條長重鏈會與兩條共同輕鏈配對,長重鏈與共同輕鏈之間不會出現錯誤配對,因為與每條長重鏈配對的兩條輕鏈是相同的;長重鏈之間會進行同源二聚化而不是異源二聚化,因此長重鏈之間也不會有錯誤配對問題。本揭露的四價雙特異性抗體不需要進行Fc修飾,製備方法簡便,具有與單抗相似甚至更優的生物學活性和理化性質。
進一步的,本申請的揭露人在對上述四價雙特異性抗體長期研究的過程中意外地發現:抗人PD-1抗體mAb1-25-Hu(609)主要通過重鏈結合PD-1分子,而對輕鏈依賴較小。因此,可以將609的重鏈可變區/重鏈與其他靶點的抗體的重鏈可變區/重鏈和輕鏈可變區/輕鏈(作為共同輕鏈)組合用於構建結合PD-1以及其他靶點的雙特異性抗體,可應用於包括但不限於本揭露的四價雙特異性抗體結構或本領域已知的其他形式的包含共同輕鏈的雙特異性抗體結構。
因此,本揭露的第一個目的在於提供一種四價雙特異性抗體。
本揭露的第二個目的在於提供一種編碼所述四價雙特異性抗體的分離的核苷酸。
本揭露的第三個目的在於提供一種包含所述核苷酸的表達載體。
本揭露的第四個目的在於提供一種包含所述表達載體的宿主細胞。
本揭露的第五個目的在於提供一種所述的四價雙特異性抗體的製備方法。
本揭露的第六個目的在於提供一種包含所述的四價雙特異性抗體的藥物組合物。
本揭露的第七個目的在於提供所述的四價雙特異性抗體或所述的藥物組合物在製備治療癌症、炎性疾病、自體免疫性疾病和其它病症的藥物中的用途。
本揭露的第八個目的在於提供一種四價雙特異性抗體的構建方法。
本揭露的第九個目的在於提供一種包含如SEQ ID NO:83所示的胺基酸序列的重鏈可變區的雙特異性抗體。
本揭露的第十個目的在於提供包含如SEQ ID NO:83所示的胺基酸序列的重鏈可變區用於構建雙特異性抗體的用途。
為了達到上述目的,本揭露提供了以下技術方案:
本揭露的第一個方面提供了一種四價雙特異性抗體,包含:
兩條多肽鏈,所述多肽鏈從N末端到C末端包含VH-B-CH1-肽接頭-VH-A-CH1-CH2-CH3,其中,所述VH-A為第一抗體的重鏈可變區,VH-B為第二抗體的重鏈可變區,所述CH1為重鏈恆定區的第一結構域,所述CH2為重鏈恆定區的第二結構域,所述CH3為重鏈恆定區的第三結構域,所述第一抗體特異性結合第一抗原,所述第二抗體特異性結合第二抗原;
四條共同輕鏈,所述共同輕鏈從N末端到C末端包含VL-CL,其中,所述VL為輕鏈可變區,所述CL為輕鏈恆定區,所述多肽鏈的VH-A-CH1和所述多肽鏈的VH-B-CH1分別與所述共同輕鏈的VL-CL配對,所述VH-A和所述VL形成第一抗原結合位點,所述VH-B與所述VL形成第二抗原結合位點。
本揭露的雙特異性抗體的結構如第1圖所示。
根據本揭露,所述共同輕鏈通過以下方法篩選獲得:
將第一抗體和第二抗體的重鏈和輕鏈分別交換獲得第一抗體的重鏈與第二抗體的輕鏈組合以及第二抗體的重鏈與第一抗體的輕鏈組合的雜合抗體,如果:
(a)第一抗體的重鏈與第二抗體的輕鏈組合成的雜合抗體能夠特異性結合第一抗原,則選擇第二抗體的輕鏈作為共同輕鏈;
(b)第二抗體的重鏈與第一抗體的輕鏈組合成的雜合抗體能夠特異性結合第二抗原,則選擇第一抗體的輕鏈作為共同輕鏈;
(c)第一抗體的重鏈與第二抗體的輕鏈組合成的雜合抗體不能特異性結合第一抗原,且第二抗體的重鏈與第一抗體的輕鏈組合成的雜合抗體不能特異性結合第二抗原,則將第一抗體的輕鏈進行回復突變使得第二抗體的重鏈與第一抗體的突變輕鏈組合的雜合抗體能夠特異性結合第二抗原,然後選擇第一抗體的突變輕鏈作為共同輕鏈;或
(d)第一抗體的重鏈與第二抗體的輕鏈組合成的雜合抗體不能特異性結合第一抗原,且第二抗體的重鏈與第一抗體的輕鏈組合成的雜合抗體不能特異性結合第二抗原,則將第二抗體的輕鏈進行回復突變使得第一抗體的重鏈與第二抗體的突變輕鏈組合的雜合抗體能夠特異性結合第一抗原,然後選擇第二抗體的突變輕鏈作為共同輕鏈。
其中,對於上述(a)和(b)的情形,當雜合抗體能夠特異性結合抗原時,作為較佳的實施方式,同樣可以選擇將輕鏈進行回復突變從而進一步提高雜合抗體與抗原結合的親和力。因此,進一步的,所述共同輕鏈通過以下方法篩選獲得:
將第一抗體和第二抗體的重鏈和輕鏈分別交換獲得第一抗體的重鏈與第二抗體的輕鏈組合以及第二抗體的重鏈與第一抗體的輕鏈組合的雜合抗體,如果:
(a’)第一抗體的重鏈與第二抗體的輕鏈組合成的雜合抗體能夠特異性結合第一抗原,則將第二抗體的輕鏈進行回復突變使得第一抗體的重鏈與第二抗體的突變輕鏈組合的雜合抗體結合第一抗原的親和力優於第一抗體的重鏈與第二抗體的輕鏈組合的雜合抗體結合第一抗原的親和力,然後選擇第二抗體的突變輕鏈作為共同輕鏈;
(b’)第二抗體的重鏈與第一抗體的輕鏈組合成的雜合抗體能夠特異性結合第二抗原,則將第一抗體的輕鏈進行回復突變使得第二抗體的重鏈與第一抗體的突變輕鏈組合的雜合抗體結合第二抗原的親和力優於第二抗體的重鏈與第一抗體的輕鏈組合的雜合抗體結合第二抗原的親和力,然後選擇第一抗體的突變輕鏈作為共同輕鏈。
根據本揭露,所述肽接頭為人工連接子。較佳的,所述人工連接子選自由以下組成的組:G、GS、SG、GGS、GSG、SGG、GGG、GGGS、SGGG、GGGGS、GGGGSGS、GGGGSGGS、GGGGSGGGGS、GGGGSGGGGSGGGGS(SEQ ID NO:150)、AKTTPKLEEGEFSEAR、AKTTPKLEEGEFSEARV、AKTTPKLGG、SAKTTPKLGG、SAKTTP、RADAAP、RADAAPTVS、RADAAAAGGPGS、SAKTTPKLEEGEFSEARV、ADAAP、ADAAPTVSIFPP、TVAAP、TVAAPSVFIFPP、QPKAAP、QPKAAPSVTLFPP、AKTTPP、AKTTPPSVTPLAP、AKTTAPSVYPLAP、ASTKGP、ASTKGPSVFPLAP、GENKVEYAPALMALS、GPAKELTPLKEAKVS、GHEAAAVMQVQYPAS等。更佳的,所述人工連接子為GGGGSGGGGSGGGGS(SEQ ID NO:150)。
根據本揭露,所述多肽鏈中靠近N末端的CH1結構域和CH1-CH2-CH3可以來自相同或不同亞型的重鏈恆定區,包括由IgG1、IgG2、IgG3、IgG4組成的組的重鏈恆定區,較佳為來自IgG1或IgG4的重鏈恆定區,較佳的,所述IgG4包含S228P(根據EU編碼)突變。
根據本揭露,所述CL為κ輕鏈恆定區或λ輕鏈恆定區。
根據本揭露的較佳實施例,所述第一抗原和所述第二抗原選自由以下組成的組:VEGF/PD-1、PD-1/VEGF、TGF-β/PD-1、PD-1/TGF-β、HER2/CD47、CD47/HER2、HER2/CD137、CD137/HER2、PD-1/CD137、CD137/PD-1、PD-1/CD40、CD40/PD-1、PD-1/EGFR、EGFR/PD-1、PD-1/HER2、HER2/PD-1、PD-1/CTLA-4、CTLA-4/PD-1、PD-1/LAG-3、LAG-3/PD-1。
根據本揭露的較佳的實施例,所述VH-A或所述VH-B具有如SEQ ID NO:83所示的序列。
根據本揭露的較佳的實施例,所述VH-A、所述VH-B和所述VL的序列選自由以下組成的組:SEQ ID NO:1、11、15;SEQ ID NO:11、1、15;SEQ ID NO:20、11、15;SEQ ID NO:11、20、15;SEQ ID NO:29、41、42;SEQ ID NO:41、29、42;SEQ ID NO:31、41、42;SEQ ID NO:41、31、42;SEQ ID NO:53、61、54;SEQ ID NO:61、53、54;SEQ ID NO:53、77、54;SEQ ID NO:77、53、54;SEQ ID NO:83、91、92;SEQ ID NO:91、83、92;SEQ ID NO:83、105、106;SEQ ID NO:105、83、106;SEQ ID NO:83、113、114;SEQ ID NO:113、83、114;SEQ ID NO:83、117、118;SEQ ID NO:117、83、118;SEQ ID NO:83、119、120;SEQ ID NO:119、83、120;SEQ ID NO:83、121、122;SEQ ID NO:121、83、122。
根據本揭露的較佳的實施例,所述多肽鏈和所述共同輕鏈的序列選自由以下組成的組:SEQ ID NO:16、13;SEQ ID NO:18、13;SEQ ID NO:21、13;SEQ ID NO:45、43;SEQ ID NO:47、43;SEQ ID NO:49、43;SEQ ID NO:51、43;SEQ ID NO:65、63;SEQ ID NO:67、63;SEQ ID NO:79、63;SEQ ID NO:81、63;SEQ ID NO:95、93;SEQ ID NO:97、93;SEQ ID NO:109、107;SEQ ID NO:111、107;SEQ ID NO:131、123;SEQ ID NO:133、125;SEQ ID NO:135、127;SEQ ID NO:137、127;SEQ ID NO:139、127;SEQ ID NO:141、127;SEQ ID NO:143、129;SEQ ID NO:145、129。
本揭露的第二個方面提供了一種分離的核苷酸,所述的核苷酸編碼所述的四價雙特異性抗體。
本揭露的第三個方面提供了一種表達載體,所述的表達載體含有所述的核苷酸。
根據本揭露的較佳的實施例,所述的核苷酸編碼所述多肽鏈和所述共同輕鏈,所述核苷酸具有選自由以下組成的組的序列:SEQ ID NO:17、14;SEQ ID NO:19、14;SEQ ID NO:22、14;SEQ ID NO:46、44;SEQ ID NO:48、44;SEQ ID NO:50、44;SEQ ID NO:52、44;SEQ ID NO:66、64;SEQ ID NO:68、64;SEQ ID NO:80、64;SEQ ID NO:82、64;SEQ ID NO:96、94;SEQ ID NO:98、94;SEQ ID NO:110、108;SEQ ID NO:112、108;SEQ ID NO:132、124;SEQ ID NO:134、126;SEQ ID NO:136、128;SEQ ID NO:138、128;SEQ ID NO:140、128;SEQ ID NO:142、128;SEQ ID NO:144、130;SEQ ID NO:146、130。
本揭露的第四個方面提供了一種宿主細胞,所述的宿主細胞含有所述的表達載體。
本揭露的第五個方面提供了所述的四價雙特異性抗體的製備方法,所述方法包含以下步驟:
(a)在表達條件下,培養所述的宿主細胞,從而表達所述的四價雙特異性抗體;
(b)分離並純化(a)所述的四價雙特異性抗體。
本揭露的第六個方面提供了一種藥物組合物,所述藥物組合物含有如上所述的四價雙特異性抗體和藥學上可接受的載體。
本揭露的第七個方面提供了所述的四價雙特異性抗體或所述的藥物組合物在製備治療癌症和炎性疾病、自體免疫性疾病和其它病症的藥物中的用途。本揭露還提供了治療癌症和炎性疾病、自體免疫性疾病和其它病症的方法,包括向有需要的受試者施用所述的四價雙特異性抗體或所述的藥物組合物。所述癌症包括但不限於:黑素瘤(例如,轉移性惡性黑素瘤)、腎癌(例如,透明細胞癌)、前列腺癌(例如,激素難治性前列腺腺癌)、胰腺癌、乳腺癌、結腸癌、肺癌(例如,非小細胞肺癌)、食道癌、頭頸鱗狀細胞癌、肝癌、卵巢癌、宮頸癌、甲狀腺癌、成膠質細胞瘤、神經膠質瘤、白血病、淋巴瘤及其它贅生性惡性疾病。所述炎性疾病、自體免疫性疾病和其它病症包括但不限於:眼科、纖維化、哮喘、類風濕性關節炎、系統性紅斑狼瘡、多發性硬化、銀屑病、特應性皮炎等。其中,所述受試者包括但不限於人。
本揭露的第八個方面提供了一種四價雙特異性抗體的構建方法,包含以下步驟:
(a)將第二抗體的重鏈可變區VH-B與重鏈恆定區的第一結構域CH1連接,所述第二抗體特異性結合第二抗原;
(b)將(a)通過肽接頭與第一抗體的重鏈可變區VH-A連接,所述第一抗體特異性結合第一抗原;
(c)將(b)與重鏈恆定區CH1-CH2-CH3連接,形成多肽鏈;
(d)將(c)與共同輕鏈VL-CL分別構建入表達載體中組合表達,從而獲得目的四價雙特異性抗體;
其中,所述CH1為重鏈恆定區的第一結構域,所述CH2為重鏈恆定區的第二結構域,所述CH3為重鏈恆定區的第三結構域,所述VL為輕鏈可變區,所述CL為輕鏈恆定區,所述VH-A-CH1和所述VH-B-CH1分別與所述VL-CL配對,所述VH-A與所述VL形成第一抗原結合位點,所述VH-B與所述VL形成第二抗原結合位點。
本揭露的第九個方面提供了一種雙特異性抗體,其中,包含至少兩個不同的重鏈可變區以及至少兩條共同輕鏈,所述共同輕鏈包含相同的輕鏈可變區,所述重鏈可變區與所述輕鏈可變區形成抗原結合位點,其中,所述重鏈可變區包含如SEQ ID NO:83所示的胺基酸序列。
本揭露的第十個方面提供了包含如SEQ ID NO:83所示的胺基酸序列的重鏈可變區用於構建雙特異性抗體的用途,其中,所述雙特異性抗體包含至少兩個不同的重鏈可變區以及至少兩條共同輕鏈,所述共同輕鏈包含相同的輕鏈可變區,所述重鏈可變區與所述輕鏈可變區形成抗原結合位點。
本揭露中,術語「抗體(Antibody,縮寫Ab)」和「免疫球蛋白G(Immunoglobulin G,縮寫IgG)」是有相同結構特徵的約150000道爾頓的異四聚糖蛋白,其由兩條相同的輕鏈(LC)和兩條相同的重鏈(HC)組成。每條輕鏈通過一個共價二硫鍵與重鏈相連,而不同免疫球蛋白同種型(isotype)的重鏈間的二硫鍵數目不同。每條重鏈和輕鏈也有規則間隔的鏈內二硫鍵。每條重鏈的一端有可變區(VH),其後是恆定區,重鏈恆定區由三個結構域CH1、CH2、以及CH3構成。每條輕鏈的一端有可變區(VL),另一端有恆定區,輕鏈恆定區包括一個結構域CL;輕鏈的恆定區與重鏈恆定區的CH1結構域配對,輕鏈的可變區與重鏈的可變區配對。恆定區不直接參與抗體與抗原的結合,但是它們表現出不同的效應功能,例如參與抗體依賴的細胞介導的細胞毒性作用(ADCC,antibody-dependent cell-mediated cytotoxicity )等。重鏈恆定區包括IgG1、IgG2、IgG3、IgG4亞型;輕鏈恆定區包括κ(Kappa)或λ(Lambda)。抗體的重鏈和輕鏈通過重鏈的CH1結構域和輕鏈的CL結構域之間的二硫鍵共價連接在一起,抗體的兩條重鏈通過鉸鏈區之間形成的多肽間二硫鍵共價連接在一起。
本揭露中,術語「雙特異性抗體(雙抗)」是指能同時特異性結合兩種抗原(靶點)或兩種表位的抗體分子。
本揭露中,術語「單株抗體(單抗)」指從一類基本均一的群體獲得的抗體,即該群體中包含的單個抗體是相同的,除少數可能存在的天然發生的突變外。單株抗體高特異性地針對單個抗原位點。而且,與常規多克隆抗體製劑(通常是具有針對不同抗原決定簇的不同抗體的混合物)不同,各單株抗體是針對抗原上的單個決定簇。除了它們的特異性外,單株抗體的好處還在於它們可以通過雜交瘤培養來合成,不會被其它免疫球蛋白污染。修飾語「單株」表示了抗體的特性,是從基本均一的抗體群中獲得的,這不應被解釋成需要用任何特殊方法來生產抗體。
本揭露中,術語「Fab」和「Fc」是指木瓜蛋白酶可將抗體裂解為兩個完全相同的Fab段和一個Fc段。Fab段由抗體的重鏈的VH和CH1以及輕鏈的VL和CL結構域組成。Fc段即可結晶片段(fragment crystallizable,Fc),由抗體的CH2和CH3結構域組成。Fc段無抗原結合活性,是抗體與效應分子或細胞相互作用的部位。
本揭露中,術語「可變」表示抗體中可變區的某些部分在序列上有所不同,它形成各種特定抗體對其特定抗原的結合和特異性。然而,可變性並不均勻地分佈在整個抗體可變區中。它集中於重鏈可變區和輕鏈可變區中稱為互補決定區(complementarity-determining region,CDR)或超變區中的三個片段中。可變區中較保守的部分稱為框架區(frame region,FR)。天然重鏈和輕鏈的可變區中各自包含四個FR區,它們大致上呈β-折疊構型,由形成連接環的三個CDR相連,在某些情況下可形成部分β折疊結構。每條鏈中的CDR通過FR區緊密地靠在一起並與另一鏈的CDR一起形成了抗體的抗原結合部位(參見 Kabat等,NIH Publ.No.91-3242,卷I,647-669頁(1991))。
本揭露中,術語「鼠源抗體」是指來源於大鼠或小鼠的抗體,較佳為小鼠。
本揭露中,術語「人源化抗體」是指其CDR來源於非人物種(較佳為小鼠)抗體,抗體分子中殘餘的部分(包括框架區和恆定區)來源於人抗體。此外,框架區殘基可被改變以維持結合親和性。
本揭露中,術語「特異性結合」和「結合」是指兩分子間的非隨機的結合反應,如抗體和其所針對的抗原之間的反應。通常,抗體以小於大約10-7M,例如小於大約10-8M、10-9M、10-10M、10-11M或更小的平衡解離常數(KD)結合該抗原。本揭露中,術語「KD」是指特定抗體-抗原相互作用的平衡解離常數,其用於描述抗體與抗原之間的結合親和力。平衡解離常數越小,抗體-抗原結合越緊密,抗體與抗原之間的親和力越高。例如,使用表面等離子體共振術(Surface Plasmon Resonance,縮寫SPR)在BIACORE儀中測定抗體與抗原的結合親和力或使用ELISA測定抗體與抗原結合的相對親和力。
本揭露中,術語「價」是指抗體分子中存在指定數量的抗原結合位點。較佳的,本揭露的雙特異性抗體具有四個抗原結合位點,是四價的。本揭露中,抗原結合位點包含重鏈可變區(VH)和輕鏈可變區(VL)。
本揭露中,術語「表位」是指與抗體特異性結合的多肽決定簇。本揭露的表位是抗原中被抗體結合的區域。
本揭露中,術語「肽接頭」是指具有胺基酸序列的肽。本揭露的肽接頭是天然連接子或人工連接子。較佳的,本揭露的肽接頭是人工連接子。本揭露的多肽接頭可以選自G、GS、SG、GGS、GSG、SGG、GGG、GGGS、SGGG、GGGGS、GGGGSGS、GGGGSGGS、GGGGSGGGGS、GGGGSGGGGSGGGGS(SEQ ID NO:150)、AKTTPKLEEGEFSEAR、AKTTPKLEEGEFSEARV、AKTTPKLGG、SAKTTPKLGG、SAKTTP、RADAAP、RADAAPTVS、RADAAAAGGPGS、SAKTTPKLEEGEFSEARV、ADAAP、ADAAPTVSIFPP、TVAAP、TVAAPSVFIFPP、QPKAAP、QPKAAPSVTLFPP、AKTTPP、AKTTPPSVTPLAP、AKTTAPSVYPLAP、ASTKGP、ASTKGPSVFPLAP、GENKVEYAPALMALS、GPAKELTPLKEAKVS和GHEAAAVMQVQYPAS等。接頭還可以是在體內可斷裂的肽接頭、蛋白酶(如MMP)敏感性接頭、可以通過還原而斷裂的基於二硫鍵的接頭等,參見先前所描述的(Fusion Protein Technologies for Biopharmaceuticals:Applications andChallenges,由Stefan R .Schmidt編輯),或本領域已知的任何可斷裂的接頭。這些可斷裂的接頭可以用於在體內釋放分子頂部的Fab,以便提高組織滲透和分佈,增強與靶標的結合,減少潛在副作用,以及調節2個不同的Fab區的體內功能和半衰期。最佳的,本揭露的人工連接子為GGGGSGGGGSGGGGS(SEQ ID NO:150)。
本揭露中,術語「共同輕鏈」是指包含相同的輕鏈可變區和輕鏈恆定區的輕鏈,其能夠與結合第一抗原的第一抗體的重鏈配對,形成特異性結合第一抗原的結合位點,也能夠與結合第二抗原的第二抗體的重鏈配對,形成特異性結合第二抗原的結合位點。進一步的,共同輕鏈的輕鏈可變區與第一抗體的重鏈可變區形成第一抗原結合位點,共同輕鏈的輕鏈可變區與第二抗體的重鏈可變區形成第二抗原結合位點。
本揭露中,術語「表達載體」可以為pTT5,pSECtag系列,pCGS3系列,pcDNA系列載體等,以及其它用於哺乳動物表達系統的載體等,表達載體中包括連接有合適的轉錄和翻譯調節序列的融合DNA序列。
本揭露中,術語「宿主細胞」是指適用於表達上述表達載體的細胞,可以是真核細胞,如哺乳動物或昆蟲宿主細胞培養系統均可用於本揭露的融合蛋白的表達,CHO(中國倉鼠卵巢,Chinese Hamster Ovary),HEK293,COS,BHK以及上述細胞的衍生細胞均可適用於本揭露。
本揭露中,術語「藥物組合物」是指本揭露的雙特異性四價抗體可以和藥學上可以接受的載體一起組成藥物製劑組合物從而更穩定地發揮療效,這些製劑可以保證本揭露公開的雙特異性四價抗體的胺基酸核心序列的構象完整性,同時還保護蛋白質的多官能團防止其降解(包括但不限於凝聚、脫胺或氧化)。
本揭露描述了一種基於共同輕鏈的結構對稱的四價雙特異性抗體及其構建方法。本揭露製備的雙特異性抗體具有與單抗相似甚至更優的生物學活性和理化性質,可以用於治療各種炎性疾病、癌症和其它疾病。
以下實施例中使用的表達純化抗體方法說明如下:將外源基因構建到pcDNA3.4(購自Thermo Fisher Scientific)表達載體中,利用PEI(Polyethylenimine)轉染法將表達載體的組合轉入HEK293F細胞(購自Thermo Fisher Scientific)中以表達抗體,然後用ProteinA親和層析純化抗體。
以下實施例中使用的ELISA檢測方法說明如下:將重組蛋白分別包被酶標板,用含有1%牛血清白蛋白的PBST(PBST為含0.05% Tween-20的磷酸鹽緩衝液)封閉酶標板。將待測抗體進行梯度稀釋,然後轉移到上述包被重組蛋白的酶標板中,室溫孵育半小時後洗板;加入適當稀釋的HRP(Horseradish Peroxidase)標記的羊抗人抗體(Fc或Fab-specific,購自Sigma),室溫孵育半小時後洗板;每孔加入100μL以TMB(3,3’,5,5’-Tetramethylbenzidine)為底物的顯色液,室溫孵育1-5分鐘;加50μL終止液(2M H
2SO
4)終止反應;酶標儀(SpectraMax 190)讀取OD450,用GraphPad Prism6進行作圖和資料分析,並計算EC50。
以下實施例中使用的評估增強混合淋巴細胞反應(Mixed Lymphocyte Reaction,MLR)的能力方法說明如下:用Histopaque(購自Sigma)從人血液中分離出外周血單個核細胞(Peripheral Blood Mononuclear Cell,縮寫PBMC),然後將PBMC中的單核細胞通過貼壁法分離出來,然後用IL-4(25 ng/mL)聯合GM-CSF(25 ng/mL)誘導單核細胞分化成誘導的樹突細胞。七天之後,消化收集上述誘導的樹突細胞。用上述方法從另外供體的血液中分離出PBMC,然後用MACS磁鐵和CD4 MicroBeads(購自Miltenyi biotec)從中分離CD4+T細胞。將誘導的樹突狀細胞(10
4/孔)和分離出的CD4+T細胞(10
5/孔)按比例混勻後接種到96孔板中,每孔150μL;數小時後,在上述96孔板中加入50μL梯度稀釋的抗體;將96孔板置於37°C細胞培養箱中孵育3天。上述實驗過程中使用AIM-V培養基(Thermo Fisher Scientific)培養細胞。然後按照標準操作流程檢測IL-2和IFN-γ的分泌。IL-2和IFN-γ的檢測使用標準的雙抗夾心法(相關檢測用的配對抗體購自BD Biosciences)。用酶標儀(SpectraMax 190)讀取OD450,用GraphPad Prism6進行作圖並計算EC50。
以下實施例中使用的理化性質檢測方法說明如下:
HPLC-SEC
抗體是高分子量蛋白質,具有高度複雜的二級和三級結構。由於翻譯後修飾、聚集和降解等變化,抗體在生物化學和生物物理特性方面是異質的。當通過分離技術分析雙特異性抗體時,通常會觀察到變體、聚集體和降解片段,它們的存在可能會損害安全性和有效性。在生產和存儲抗體的過程中容易出現聚集體、降解片段和不完整組裝的分子。本揭露使用高效液相色譜-尺寸排阻色譜(High-performance liquid chromatography–size exclusion chromatography,HPLC-SEC)檢測樣品中上述雜質的含量。聚集體的分子量要大於單體,因此相應峰的保留時間較短;降解片段或不完整組裝分子的分子量要小於單體,因此相應峰的保留時間較長。HPLC-SEC所用色譜儀為Dionex Ultimate 3000;流動相配製方法如下:取適量20mM磷酸二氫鈉母液,用20mM磷酸氫二鈉調節PH至6.8±0.1;進樣量:20µg;色譜柱為TSK G3000SWXL,規格為7.8×300mm x 5μm;流速0.5 mL/min,洗脫時間30分鐘;柱溫25°C,樣品室溫度10°C;檢測波長214nm。
HPLC-IEC
許多翻譯後修飾(例如N醣基化、C末端賴胺酸殘基修飾、N末端穀胺醯胺或谷胺酸環化、天冬醯胺脫醯胺化、天冬胺酸異構化和胺基酸殘基氧化等)會直接或間接地引起抗體表面電荷的改變,導致電荷異質性的產生。基於所帶電荷可對電荷變體進行分離和分析,常用的分析方法有陽離子交換色譜法(cation exchange chromatography,CEX)和陰離子交換色譜法(anionexchange chromatography,AEX)。當通過基於色譜法的方法分析時,酸性種類(acidic species)和鹼性種類(basic species)基於它們相對於主峰(main peak)的保留時間來定義。酸性種類是早於CEX的主峰或晚於AEX的主峰洗脫出來的變體,而鹼性種類是晚於CEX的主峰或早於AEX的主峰洗脫出來的變體。酸性種類和鹼性種類所對應的峰分別稱作酸性峰和鹼性峰。在生產和存儲抗體的過程中容易產生電荷變體。在此使用高效液相色譜-離子交換色譜(High-performance liquid chromatography-ionexchange chromatography,HPLC-IEC)分析樣品的電荷異質性。HPLC-IEC所用色譜儀為Dionex Ultimate 3000;流動相A:20mM PB pH6.3,流動相B:20mM PB+200mM NaCl pH6.3,兩種流動相混合的比例按照預先設置的程式隨時間而改變,流速1.0 mL/min;色譜柱:Thermo PropacTM WCX-10;柱溫30°C,樣品室溫度10°C;進樣量:20µg;檢測波長:214nm。
CE-SDS
本揭露使用CE-SDS(Capillary Electrophoresis-Sodium Dodecyl Sulfate)分析樣品中降解片段或不完整組裝的分子的含量。CE分為非還原和還原兩種類型,用於前者的樣品在變性時不需要用還原劑DTT將分子內的二硫鍵破壞,而用於後者的樣品在變性時需要用還原劑DTT將分子內的二硫鍵破壞。非還原和還原CE-SDS分別記作NR-CE-SDS和R-CE-SDS。所用毛細管電泳儀為ProteomeLab
TMPA800 plus(Beckman Coulter),配備UV 214nm檢測器,毛細管型號為Bare Fused-Silica Capillary,規格30.7cm×50μm,有效長度20.5cm;其它相關試劑購自Beckman Coulter。儀器關鍵參數設置如下:毛細管和樣品室溫度為20±2°C,分離電壓為15 kV。
DSC
差示掃描量熱法(Differential Scanning Calorimeter,DSC)主要通過在可控的升溫或降溫過程檢測生物分子中的熱量變化來反映樣品的熱穩定性。通過加熱,蛋白樣品的去折疊會吸收熱量,而消除樣品池溫差所需的補充能量則通過設備記錄下來,這些熱量變化會在圖譜上形成一個峰形,其中蛋白質樣品發生去折疊時所對應的峰頂溫度作為熔融溫度Tm。Tm是蛋白熱穩定性的一個重要指示,Tm越高,蛋白的穩定性越好。
本揭露中涉及的序列資訊總結在表1中。
表1、本揭露的抗體的序列資訊
SEQ ID NO: | 序列名稱 |
1 | Bevacizumab(601)的重鏈可變區的胺基酸序列 |
2 | Bevacizumab(601)的輕鏈可變區的胺基酸序列 |
3 | 鼠源20號抗體的重鏈可變區的胺基酸序列 |
4 | 鼠源20號抗體的輕鏈可變區的胺基酸序列 |
5 | 鼠源20號抗體的重鏈互補決定區H-CDR1的胺基酸序列 |
6 | 鼠源20號抗體的重鏈互補決定區H-CDR2的胺基酸序列 |
7 | 鼠源20號抗體的重鏈互補決定區H-CDR3的胺基酸序列 |
8 | 鼠源20號抗體的輕鏈互補決定區L-CDR1的胺基酸序列 |
9 | 鼠源20號抗體的輕鏈互補決定區L-CDR2的胺基酸序列 |
10 | 鼠源20號抗體的輕鏈互補決定區L-CDR3的胺基酸序列 |
11 | 20-Hu的重鏈可變區的胺基酸序列 |
12 | 20-Hu的輕鏈可變區的胺基酸序列 |
13 | 601-LC-V94L的胺基酸序列 |
14 | 601-LC-V94L的核苷酸序列 |
15 | 601-LC-V94L的輕鏈可變區的胺基酸序列 |
16 | 20-Fab-601-IgG4的胺基酸序列 |
17 | 20-Fab-601-IgG4的核苷酸序列 |
18 | 601-Fab-20-IgG4的胺基酸序列 |
19 | 601-Fab-20-IgG4的核苷酸序列 |
20 | Y0313-1的重鏈可變區的胺基酸序列 |
21 | 20-Fab-0313-IgG4的胺基酸序列 |
22 | 20-Fab-0313-IgG4的核苷酸序列 |
23 | 鼠源1D11號抗體的重鏈互補決定區H-CDR1的胺基酸序列 |
24 | 鼠源1D11號抗體的重鏈互補決定區H-CDR2的胺基酸序列 |
25 | 鼠源1D11號抗體的重鏈互補決定區H-CDR3的胺基酸序列 |
26 | 鼠源1D11號抗體的輕鏈互補決定區L-CDR1的胺基酸序列 |
27 | 鼠源1D11號抗體的輕鏈互補決定區L-CDR2的胺基酸序列 |
28 | 鼠源1D11號抗體的輕鏈互補決定區L-CDR3的胺基酸序列 |
29 | 1D11-Hu的重鏈可變區的胺基酸序列 |
30 | 1D11-Hu的輕鏈可變區的胺基酸序列 |
31 | mAb127的重鏈可變區的胺基酸序列 |
32 | mAb127的輕鏈可變區的胺基酸序列 |
33 | 鼠源14號抗體的重鏈可變區的胺基酸序列 |
34 | 鼠源14號抗體的輕鏈可變區的胺基酸序列 |
35 | 鼠源14號抗體的重鏈互補決定區H-CDR1的胺基酸序列 |
36 | 鼠源14號抗體的重鏈互補決定區H-CDR2的胺基酸序列 |
37 | 鼠源14號抗體的重鏈互補決定區H-CDR3的胺基酸序列 |
38 | 鼠源14號抗體的輕鏈互補決定區L-CDR1的胺基酸序列 |
39 | 鼠源14號抗體的輕鏈互補決定區L-CDR2的胺基酸序列 |
40 | 鼠源14號抗體的輕鏈互補決定區L-CDR3的胺基酸序列 |
41 | 14-Hu的重鏈可變區的胺基酸序列 |
42 | 14-Hu的輕鏈可變區的胺基酸序列 |
43 | 14-Hu-LC的胺基酸序列 |
44 | 14-Hu-LC的核苷酸序列 |
45 | 14-Fab-1D11-IgG4的胺基酸序列 |
46 | 14-Fab-1D11-IgG4的核苷酸序列 |
47 | 1D11-Fab-14-IgG4的胺基酸序列 |
48 | 1D11-Fab-14-IgG4的核苷酸序列 |
49 | 14-Fab-127-IgG4的胺基酸序列 |
50 | 14-Fab-127-IgG4的核苷酸序列 |
51 | 127-Fab-14-IgG4的胺基酸序列 |
52 | 127-Fab-14-IgG4的核苷酸序列 |
53 | 19H6-Hu的重鏈可變區的胺基酸序列 |
54 | 19H6-Hu的輕鏈可變區的胺基酸序列 |
55 | Anti-CD47B的重鏈互補決定區H-CDR1的胺基酸序列 |
56 | Anti-CD47B的重鏈互補決定區H-CDR2的胺基酸序列 |
57 | Anti-CD47B的重鏈互補決定區H-CDR3的胺基酸序列 |
58 | Anti-CD47B的輕鏈互補決定區L-CDR1的胺基酸序列 |
59 | Anti-CD47B的輕鏈互補決定區L-CDR2的胺基酸序列 |
60 | Anti-CD47B的輕鏈互補決定區L-CDR3的胺基酸序列 |
61 | Anti-CD47B-Hu的重鏈可變區的胺基酸序列 |
62 | Anti-CD47B-Hu的輕鏈可變區的胺基酸序列 |
63 | 19H6-Hu-LC的胺基酸序列 |
64 | 19H6-Hu-LC的核苷酸序列 |
65 | CD47B-Fab-19H6-IgG1的胺基酸序列 |
66 | CD47B-Fab-19H6-IgG1的核苷酸序列 |
67 | 19H6-Fab-CD47B-IgG1的胺基酸序列 |
68 | 19H6-Fab-CD47B-IgG1的核苷酸序列 |
69 | 鼠源94號抗體的重鏈可變區的胺基酸序列 |
70 | 鼠源94號抗體的輕鏈可變區的胺基酸序列 |
71 | 鼠源94號抗體的重鏈互補決定區H-CDR1的胺基酸序列 |
72 | 鼠源94號抗體的重鏈互補決定區H-CDR2的胺基酸序列 |
73 | 鼠源94號抗體的重鏈互補決定區H-CDR3的胺基酸序列 |
74 | 鼠源94號抗體的輕鏈互補決定區L-CDR1的胺基酸序列 |
75 | 鼠源94號抗體的輕鏈互補決定區L-CDR2的胺基酸序列 |
76 | 鼠源94號抗體的輕鏈互補決定區L-CDR3的胺基酸序列 |
77 | 94-Hu的重鏈可變區的胺基酸序列 |
78 | 94-Hu的輕鏈可變區的胺基酸序列 |
79 | 94-Fab-19H6-IgG1-LALA 的胺基酸序列 |
80 | 94-Fab-19H6-IgG1-LALA 的核苷酸序列 |
81 | 19H6-Fab-94-IgG1-LALA的胺基酸序列 |
82 | 19H6-Fab-94-IgG1-LALA的核苷酸序列 |
83 | mAb1-25-Hu(609)的重鏈可變區的胺基酸序列 |
84 | mAb1-25-Hu(609)的輕鏈可變區的胺基酸序列 |
85 | Anti-CD137的重鏈互補決定區H-CDR1的胺基酸序列 |
86 | Anti-CD137的重鏈互補決定區H-CDR2的胺基酸序列 |
87 | Anti-CD137的重鏈互補決定區H-CDR3的胺基酸序列 |
88 | Anti-CD137的輕鏈互補決定區L-CDR1的胺基酸序列 |
89 | Anti-CD137的輕鏈互補決定區L-CDR2的胺基酸序列 |
90 | Anti-CD137的輕鏈互補決定區L-CDR3的胺基酸序列 |
91 | Anti-CD137-Hu的重鏈可變區的胺基酸序列 |
92 | Anti-CD137-Hu的輕鏈可變區的胺基酸序列 |
93 | Anti-CD137-Hu-LC的胺基酸序列 |
94 | Anti-CD137-Hu-LC的核苷酸序列 |
95 | 609-Fab-137-IgG4的胺基酸序列 |
96 | 609-Fab-137-IgG4的核苷酸序列 |
97 | 137-Fab-609-IgG4的胺基酸序列 |
98 | 137-Fab-609-IgG4的核苷酸序列 |
99 | Anti-CD40的重鏈互補決定區H-CDR1的胺基酸序列 |
100 | Anti-CD40的重鏈互補決定區H-CDR2的胺基酸序列 |
101 | Anti-CD40的重鏈互補決定區H-CDR3的胺基酸序列 |
102 | Anti-CD40的輕鏈互補決定區L-CDR1的胺基酸序列 |
103 | Anti-CD40的輕鏈互補決定區L-CDR2的胺基酸序列 |
104 | Anti-CD40的輕鏈互補決定區L-CDR3的胺基酸序列 |
105 | Anti-CD40-Hu的重鏈可變區的胺基酸序列 |
106 | Anti-CD40-Hu的輕鏈可變區的胺基酸序列 |
107 | Anti-CD40-Hu-LC的胺基酸序列 |
108 | Anti-CD40-Hu-LC的核苷酸序列 |
109 | 609-Fab-40-IgG4的胺基酸序列 |
110 | 609-Fab-40-IgG4的核苷酸序列 |
111 | 40-Fab-609-IgG4的胺基酸序列 |
112 | 40-Fab-609-IgG4的核苷酸序列 |
113 | Cetuximab的重鏈可變區的胺基酸序列 |
114 | Cetuximab的輕鏈可變區的胺基酸序列 |
115 | Trastuzumab的重鏈可變區的胺基酸序列 |
116 | Trastuzumab的輕鏈可變區的胺基酸序列 |
117 | Pertuzumab的重鏈可變區的胺基酸序列 |
118 | Pertuzumab的輕鏈可變區的胺基酸序列 |
119 | 10D1(Ipilimumab)的重鏈可變區的胺基酸序列 |
120 | 10D1(Ipilimumab)的輕鏈可變區的胺基酸序列 |
121 | 5E7-Hu的重鏈可變區的胺基酸序列 |
122 | 5E7-Hu的輕鏈可變區的胺基酸序列 |
123 | Cetuximab-LC的胺基酸序列 |
124 | Cetuximab-LC的核苷酸序列 |
125 | Pertuzumab-LC的胺基酸序列 |
126 | Pertuzumab-LC的核苷酸序列 |
127 | Ipilimumab-LC的胺基酸序列 |
128 | Ipilimumab-LC的核苷酸序列 |
129 | 5E7-Hu-LC的胺基酸序列 |
130 | 5E7-Hu-LC的核苷酸序列 |
131 | 609-Fab-Cetuximab-IgG4的胺基酸序列 |
132 | 609-Fab-Cetuximab-IgG4的核苷酸序列 |
133 | 609-Fab-Pertuzumab-IgG4的胺基酸序列 |
134 | 609-Fab-Pertuzumab-IgG4的核苷酸序列 |
135 | 609-Fab-Ipilimumab-IgG1的胺基酸序列 |
136 | 609-Fab-Ipilimumab-IgG1的核苷酸序列 |
137 | Ipilimumab-Fab-609-IgG1的胺基酸序列 |
138 | Ipilimumab-Fab-609-IgG1的核苷酸序列 |
139 | 609-Fab-Ipilimumab-IgG4的胺基酸序列 |
140 | 609-Fab-Ipilimumab-IgG4的核苷酸序列 |
141 | Ipilimumab-Fab-609-IgG4的胺基酸序列 |
142 | Ipilimumab-Fab-609-IgG4的核苷酸序列 |
143 | 609-Fab-5E7-IgG4的胺基酸序列 |
144 | 609-Fab-5E7-IgG4的核苷酸序列 |
145 | 5E7-Fab-609-IgG4的胺基酸序列 |
146 | 5E7-Fab-609-IgG4的核苷酸序列 |
147 | IgG1重鏈恒定區的胺基酸序列 |
148 | IgG4(S228P)重鏈恒定區的胺基酸序列 |
149 | κ輕鏈恒定區的胺基酸序列 |
150 | 連接子(GGGGSGGGGSGGGGS) |
151 | 重鏈第四個框架區(WGQGTLVTVSS) |
152 | 輕鏈第四個框架區(FGQGTKVEIK) |
153 | 輕鏈第四個框架區(FGGGTKVELK) |
以下實施例、實驗例是對本揭露進行進一步的說明,不應理解為對本揭露的限制。實施例不包括對傳統方法的詳細描述,如那些用於構建載體和質粒的方法,將編碼蛋白的基因插入到這樣的載體和質粒的方法或將質粒引入宿主細胞的方法。這樣的方法對於本領域中具有普通技術的人員是眾所周知的,並且在許多出版物中都有所描述,包括Sambrook, J., Fritsch, E.F. and Maniais, T.(1989) Molecular Cloning: A Laboratory Manual,2nd edition, Cold spring Harbor Laboratory Press。
實施例1構建抗PD-1和VEGF的雙特異性抗體
實施例1.1 序列
從公開的文獻資料(Magdelaine-Beuzelin C, Kaas Q, Wehbi V, et al. Structure–function relationships of the variable domains of monoclonal antibodies approved for cancer treatment[J]. Critical reviews in oncology/hematology, 2007, 64(3): 210-225.)中獲得Bevacizumab(後文用601替代)抗體的重鏈可變區和輕鏈可變區序列(SEQ ID NO:1和2)。由上海生工生物工程有限公司合成編碼上述可變區的DNA。601的重鏈可變區(601-VH)和輕鏈可變區(601-VL)分別與人IgG1重鏈恆定區(SEQ ID NO:147)和人Kappa輕鏈恆定區(SEQ ID NO:149)相連,構建成全長的601抗體的重鏈和輕鏈基因,分別命名為601-HC和601-LC。
根據WO2018/137576A1中實施例1-5所述,依據篩選結果,最終挑取20號鼠源抗人PD-1單抗作為先導抗體,並獲得其重鏈可變區核苷酸序列和輕鏈可變區核苷酸序列,並翻譯成胺基酸序列(SEQ ID NO:3和4)。
對20號抗體的重鏈可變區和輕鏈可變區胺基酸序列進行分析,依據Kabat規則分別確定20號抗體重鏈和輕鏈的抗原互補決定區和框架區。20號抗體重鏈CDR的胺基酸序列為H-CDR1:NYDMS(SEQ ID NO:5)、H-CDR2:TISGGGGYTYYSDSVKG(SEQ ID NO:6)和H-CDR3:PYGHYGFEY(SEQ ID NO:7),輕鏈CDR的胺基酸序列為L-CDR1:SASQGISNFLS(SEQ ID NO:8)、L-CDR2:YTSSLHS(SEQ ID NO:9)和L-CDR3:QQYSNLPWT(SEQ ID NO:10)。
在https://www.ncbi.nlm.nih.gov/igblast/,將鼠源20號抗體的重鏈可變區與人IgG胚系序列進行同源性比較,選擇IGHV3-21*01為重鏈CDR移植範本,將鼠源的20號抗體的重鏈CDR移植入IGHV3-21*01骨架區,並在H-CDR3之後加入WGQGTLVTVSS(SEQ ID NO:151)作為第四個框架區,獲得CDR移植重鏈可變區序列。同樣地,將鼠源20號抗體的輕鏈可變區與人IgG胚系序列同源性比較,選擇IGKV1-39*01為輕鏈CDR移植範本,將鼠源20號抗體的輕鏈CDR移植入IGKV1-39*01的骨架區,並在L-CDR3之後加入FGQGTKVEIK(SEQ ID NO:152)作為第四個框架區,獲得CDR移植輕鏈可變區序列。在CDR移植可變區的基礎上,對一些框架區的胺基酸位點進行回復突變(回復突變就是將人源框架區的某些胺基酸突變成鼠源框架區同一位置的胺基酸,回復突變的位點一般對維持抗體的結構和/或親和力是至關重要的)。在進行回復突變時,將胺基酸序列進行Kabat編碼,位元點的位置由Kabat碼指示。
較佳的,對於CDR移植重鏈可變區,根據Kabat編碼,將第28位元的T回復突變為鼠源的V,將第44位的G回復為R,第94位的R回復為S。對於CDR移植輕鏈可變區,將第43位的A回復為T,第44位的P回復為V,第71位的F回復為Y。
上述帶有回復突變位點的重鏈可變區和輕鏈可變區分別定義為人源化的重鏈可變區和輕鏈可變區(SEQ ID NO:11和12)。由上海生工生物工程有限公司合成編碼上述人源化的重鏈和輕鏈可變區的DNA。將合成的人源化重鏈可變區與人IgG4(鉸鏈區含有S228P點突變)恆定區(SEQ ID NO:148)相連,獲得全長的人源化重鏈基因,命名為20-Hu-HC;將人源化輕鏈可變區與人Kappa鏈恆定區(SEQ ID NO:149)相連,獲得全長的人源化輕鏈基因,命名為20-Hu-LC。
上述抗體的重鏈和輕鏈基因分別構建到pcDNA3.4表達載體中,利用PEI轉染法將所得重鏈和輕鏈表達載體一起轉入HEK293F細胞中以表達抗體。HEK293F細胞在Free Style 293 Expression Medium中培養5天後收取細胞上清,利用Protein A親和層析法純化抗體。20-Hu-HC與20-Hu-LC組合後獲得的抗體命名為20-Hu。
實施例1.2 共同輕鏈的選擇
用BLAST對20-Hu輕鏈可變區與601輕鏈可變區的胺基酸序列進行對比分析,結果顯示,兩者之間完全相同的胺基酸占比89%(Identities),性質相似的胺基酸占比94%(Positives)。
將601-HC和601-LC的基因序列分別構建到pcDNA3.4表達載體中。將20-Hu-HC、20-Hu-LC、601-HC和601-LC的表達載體按照下述方式進行組合:20-Hu-HC+20-Hu-LC、601-HC+601-LC、20-Hu-HC+601-LC和601-HC+20-Hu-LC,表達純化抗體,所得的抗體分別命名為20-Hu、601、20-Hu-HC+601-LC和601-HC+20-Hu-LC。
ELISA檢測方法如下:自製帶有6*His標籤的人PD-1的胞外區蛋白(PD-1的胞外區來源如WO2018/137576A1中所述),將這種重組蛋白記作PD1-His。自製帶有6*His標籤的人VEGF165(序列來自NCBI,Accession:AAM03108),將這種重組蛋白記作VEGF165-His。用PD1-His和VEGF165-His分別包被酶標板,包被濃度分別為20ng/孔和10ng/孔。
如第2A圖所示,20-Hu和20-Hu-HC+601-LC能夠有效結合PD1-His,EC50分別是0.2062nM和0.9747nM;而601和601-HC+20-Hu-LC不能有效結合PD1-His,無法準確計算出EC50。如第2B圖所示,601和601-HC+20-Hu-LC能夠有效結合VEGF165-His,EC50為0.4681nM和8.217nM,而20-Hu和20-Hu-HC+601-LC不能有效結合VEGF165-His。
與20-Hu相比,20-Hu-HC+601-LC對PD1-His的相對親和力明顯下降;與601相比,601-HC+20-Hu-LC對VEGF165-His的相對親和力也明顯下降。在此嘗試用回復突變的方式,使20-Hu-HC+601-LC對PD1-His的相對親和力得到增強。分析發現,601輕鏈可變區和20-Hu輕鏈可變區之間有12個胺基酸殘基存在差異,其中28、32、34、46、50、71、93和94位上的胺基酸殘基(按照Kabat規則編碼)對維持抗體的親和力可能是至關重要的。在此,通過定點誘變,將601-LC上述位置的胺基酸殘基分別突變成20-Hu-LC對應位置的胺基酸殘基,這些帶有點突變的601-LC分別記作601-LC-D28G、601-LC-Y32F、601-LC-N34S、601-LC-V46L、601-LC-F50Y、601-LC-F71Y、601-LC-T93N和601-LC-V94L。
將上述輕鏈的基因序列分別構建到pcDNA3.4表達載體中。將20-Hu-HC與上述帶有點突變的601-LC的表達載體分別進行組合,表達純化抗體,所得的抗體分別命名為20-Hu-HC+601-LC-D28G、20-Hu-HC+601-LC-Y32F、20-Hu-HC+601-LC-N34S、20-Hu-HC+601-LC-V46L、20-Hu-HC+601-LC-F50Y、20-Hu-HC+601-LC-F71Y、20-Hu-HC+601-LC-T93N、20-Hu-HC+601-LC-V94L。用上述實施例中描述的ELISA評估上述抗體結合PD-1的相對親和力,20-Hu-HC+601-LC作為參照。ELISA結果顯示,上述突變體和20-Hu-HC+601-LC的EC50分別為0.4849nM、0.4561nM、0.1751nM、0.5333nM、0.5255nM、1.0345nM、0.4859nM、0.3079nM和0.6251nM;與20-Hu-HC+601-LC相比,20-Hu-HC+601-LC-N34S和20-Hu-HC+601-LC-V94L對PD-1的相對親和力明顯更高;其它突變抗體的相對親和力與20-Hu-HC+601-LC基本相同甚至更低。
將601-HC與上述帶有點突變的601-LC的表達載體分別進行組合,表達純化抗體,所得的抗體分別命名為601-HC+601-LC-D28G、601-HC+601-LC-Y32F、601-HC+601-LC-N34S、601-HC+601-LC-V46L、601-HC+601-LC-F50Y、601-HC+601-LC-F71Y、601-HC+601-LC-T93N、601-HC+601-LC-V94L。用上述實施例中描述的ELISA評估上述抗體結合VEGF165的相對親和力,601作為參照。ELISA結果顯示,上述突變體和601的EC50分別為0.1328nM、0.1254nM、0.2081nM、0.3256nM、0.1400nM、0.1481nM、0.1259nM、0.1243nM和0.1291nM;與601相比,601-HC+601-LC-N34S和601-HC+601-LC-V46L對VEGF165的相對親和力明顯降低;其它突變抗體的相對親和力與601基本相同。
綜上所述,V94L突變能夠增強20-Hu-HC+601-LC對PD-1的相對親和力,同時又不降低601對VEGF165的相對親和力。在此選擇601-LC-V94L(SEQ ID NO:13和14)作為共同輕鏈構建雙特異性抗體。
實施例1.3雙特異性抗體的構建
將20-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接601的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈基因命名為20-Fab-601-IgG4(SEQ ID NO:16和17)。相似地,將601的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接20-Hu的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈基因命名為601-Fab-20-IgG4(SEQ ID NO:18和19)。
將上述序列分別構建到pcDNA3.4表達載體中,將20-Fab-601-IgG4和601-Fab-20-IgG4表達載體分別與601-LC-V94L表達載體組合,表達純化抗體,所得的抗體分別命名為20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L。
實施例1.4ELISA測定相對親和力
如圖第3A圖所示,20-Hu-HC+601-LC-V94L、20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L能夠有效結合PD1-His,EC50分別是0.3314nM、0.4768nM和1.772nM。如第3B圖所示,601-HC+601-LC-V94L、20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L能夠有效結合VEGF165-His,EC50為0.01872nM、0.05859nM和0.03886nM。20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L既能夠結合PD-1又能結合VEGF,這說明它們是雙特異性抗體。
實施例1.5 Biacore測定親和力
在此通過Biacore 8K(GE healthcare)檢測上述抗體與PD-1或VEGF之間的親和力。在Biacore 8K上,使用偶聯有ProteinA/G的晶片分別捕獲各種抗體,再將重組蛋白PD1-His或VEGF165-His進樣,得到結合-解離曲線,用6M鹽酸胍再生緩衝液洗脫後重複下一個迴圈;利用Biacore 8K Evaluation Software對資料進行分析。結果如表2所示。
表2-1.對PD-1的結合和解離動力學參數以及平衡解離常數
表2-2.對VEGF的結合和解離動力學參數以及平衡解離常數
樣品名稱 | Kon (1/Ms) | Koff (1/s) | KD(M) |
20-Hu-HC+601-LC-V94L | 2.86E+04 | 4.54E-05 | 15.9E-10 |
20-Fab-601-IgG4-V94L | 7.52E+04 | 6.66E-05 | 8.85E-10 |
樣品名稱 | Kon (1/Ms) | Koff (1/s) | KD(M) |
601-HC+601-LC-V94L | 3.90E+06 | 2.91E-05 | 7.46E-12 |
20-Fab-601-IgG4-V94L | 1.81E+06 | 1.67E-05 | 9.26E-12 |
表2-1顯示,20-Hu-HC+601-LC-V94L和20-Fab-601-IgG4-V94L對PD-1的平衡解離常數(KD)十分相近,KD分別是1.59E-09和8.85E-10。表2-2顯示,601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L對VEGF165-His的結合常數(Kon)和解離常數(Koff)十分相近,平衡解離常數(KD)也基本相當,KD分別是7.46E-12和9.26E-12。平衡解離常數(KD)與親和力高低成反比。
實施例1.6物理化學性質的表徵
實施例1.6.1 HPLC-SEC
第4A圖表示單抗601-HC+601-LC-V94L的HPLC-SEC圖譜,其中存在2個明顯的峰,分別是Peak1和Peak2,占比分別為0.7%和99.3%。其中Peak1的保留時間短於主峰Peak2,說明Peak1可能是聚集體產生的;圖中沒有出現可能代表降解片段或不完整組裝分子的峰。第4B圖表示20-Fab-601-IgG4-V94L的HPLC-SEC圖譜,其中存在兩個明顯的峰,分別是Peak1和Peak2,占比分別為0.7%和99.3%。其中Peak1的保留時間短於主峰Peak2,說明Peak1可能是聚集體產生的;圖中沒有出現可能代表降解片段或不完整組裝分子的峰。
實施例1.6.2HPLC-IEC
第5A圖和第5B圖表示601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L的HPLC-IEC圖譜,它們的主峰分別占比79.31%和80.64%,該結果表明20-Fab-601-IgG4-V94L的電荷異質性與601-HC+601-LC-V94L相當。
實施例1.6.3 CE-SDS
第6A圖和第6B圖分別表示601-HC+601-LC-V94L的NR-CE-SDS和R-CE-SDS的圖譜,NR-CE-SDS圖譜中主峰Peak9占比97.90%;R-CE-SDS圖譜中兩個主峰Peak6(對應輕鏈)和Peak12(對應重鏈)分別占比30.92%和65.27%,兩者峰面積之比為1:2.1。第6C圖和第6D圖分別表示20-Fab-601-IgG4-V94L的NR-CE-SDS和R-CE-SDS的圖譜,NR-CE-SDS圖譜中主峰Peak13占比96.74%;R-CE-SDS圖譜中兩個主峰Peak3(對應輕鏈)和Peak12(對應重鏈)分別占比38.42%和59.74%,兩者峰面積之比為2:3.1。NR-CE-SDS中,601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L的主峰占比十分相近;R-CE-SDS中,601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L的輕鏈和重鏈的峰面積之比均符合預期。
實施例1.6.4 DSC
第7A圖和第7B圖分別表示601-HC+601-LC-V94L和20-Fab-601-IgG4-V94L的DSC圖譜。其中601-HC+601-LC-V94L的TmOnset和Tm分別為66.46°C和75.37°C,20-Fab-601-IgG4-V94L的TmOnset和Tm分別為65.92°C和74.28°C,該結果表明20-Fab-601-IgG4-V94L的熱穩定性與601-HC+601-LC-V94L非常相近。
實施例1.7改進型雙特異性抗體的構建
實施例1.7.1雙特異性抗體的構建
在美國專利申請US20020032315A1中,相關發明人利用噬菌體展示法對Bevacizumab的重鏈可變區和輕鏈可變區進行改造,獲得了親和力和中和活性更高的重鏈可變區胺基酸序列Y0313-1(US20020032315A1中的SEQ ID NO:114,即本揭露中的SEQ ID NO:20)。
在此用Y0313-1置換了20-Fab-601-IgG4-V94L中的601(Bevacizumab)重鏈可變區。方法如下:將20-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接Y0313-1的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈基因命名為20-Fab-0313-IgG4(SEQ ID NO:21和22)。
將上述序列構建到pcDNA3.4表達載體中,將20-Fab-0313-IgG4與601-LC-V94L表達載體組合,表達純化抗體,所得的抗體分別命名為20-Fab-0313-IgG4-V94L。
實施例1.7.2測定對VEGF生物活性的中和能力
人臍靜脈內皮細胞(Human Umbilical Vein Endothelial Cells,HUVEC)購自澳賽爾斯(AllCells)生物技術有限公司。用臍靜脈內皮細胞完全培養基(購自AllCells,貨號和規格:H-004/500mL)對HUVEC進行培養和傳代。待HUVEC生長至對數生長期時,用胰酶消化使細胞脫落,離心後用完全培養基重懸細胞,然後按照8000細胞/孔的密度接種於96孔細胞培養板中;24小時後,將96孔板中的完全培養基置換為基礎培養基(購自AllCells,貨號和規格:H-004B/500mL),150μL/孔;在含有400ng/mL重組VEGF165(購自Acrobiosystems,貨號:VE5-H4210)的基礎培養中對抗體進行梯度稀釋,然後將VEGF165和抗體的混合溶液加入96孔板中,50μL/孔;在37°C、5%CO
2細胞培養箱中繼續孵育3天;3天後每孔加入20μL CCK-8(Dojindo)溶液,在培養箱中繼續孵育4小時;用酶標儀讀取OD450;GraphPad Prism6進行資料分析,作圖並計算IC50。
如第8圖所示,601、20-Fab-601-IgG4-V94L、601-Fab-20-IgG4-V94L和20-Fab-0313-IgG4-V94L均能有效抑制VEGF165誘導的HUVEC細胞增殖,它們的IC50分別為4.422nM、9.039nM、3.84nM和1.632nM。上述結果顯示,20-Fab-0313-IgG4-V94L中和VEGF165生物活性的能力最強。
實施例1.7.3評估增強MLR的功能活性
如第9A圖和第9B圖所示,20-Humanized(即20-Hu)、20-Hu-HC+601-LC-V94L和20-Fab-0313-IgG4-V94L均能有效刺激MLR分泌IL-2和IFN-γ,刺激MLR分泌IL-2的EC50分別為0.2571nM、0.3703nM和0.7554nM,刺激MLR分泌IFN-γ的EC50分別為0.1426nM、0.247nM和1.036nM。
實施例1.7.4 測定抗PD-1和VEGF雙特異性抗體同時結合兩種抗原的能力
用VEGF165(購自Acrobiosystems,貨號:VE5-H4210)包被微孔板。用含1%牛血清白蛋白的PBST將待測抗體進行梯度稀釋,然後轉移到上述微孔板中,室溫孵育半小時左右;PBST洗板三遍;將生物素化的人PD-1胞外段重組蛋白(購自Sino Biological,貨號:10377-H08H-B)稀釋到200 ng/mL,轉移到微孔板中,室溫孵育半小時左右;PBST洗板三遍;將Streptavidin-HRP(購自BD Biosciences,貨號:554066)稀釋1000倍,轉移到微孔板中,室溫孵育半小時左右;PBST洗板三遍;加入TMB顯色液(100 μL/孔),室溫孵育1-5分鐘;加終止液(50 μL/孔),終止顯色反應;用酶標儀讀取OD450;用GraphPad Prism6進行資料分析和作圖,並計算EC50。
如第10圖所示,20-Fab-0313-IgG4-V94L在結合VEGF165之後仍然能夠有效地繼續結合人PD-1,EC50為0.3293 nM。601和20-Hu-HC+601-LC-V94L都不能同時結合PD-1和VEGF165。
實施例1.7.5 測定抗PD-1和VEGF雙特異性抗體在大鼠體內的藥代動力學
採用SD(Sprague-Dawley)大鼠(購自浙江維通利華實驗動物技術有限公司)進行雙特異性抗體的藥代動力學研究。每組四隻大鼠,體重200公克左右,每隻通過靜脈注射(Intravenous Injection,I.V.)劑量為1mg的抗體;分別在給藥後的特定時間眼眶取血,血液自然凝固後離心取血清。
血清中目的抗體濃度的測量方法如下:用雙特異性抗體對應的兩種相關抗原(VEGF165購自Acrobiosystems,貨號:VE5-H4210;帶有6*His標籤的人PD-1的胞外區重組蛋白的來源如實施例1.2中所述)分別包被酶標板,然後用含有1%牛血清白蛋白的PBST封閉酶標板。將適當稀釋的大鼠血清分別轉移到上述包被兩種相關抗原的酶標板中;經過1小時的室溫孵育之後洗板,然後加入HRP標記的羊抗人(Fc-Specific)抗體(購自Sigma;該抗體經過種屬交叉吸附處理,不識別大鼠抗體);經過半小時的室溫孵育之後洗板,每孔加入100μL以TMB為底物的顯色液,室溫孵育1-5分鐘;加50μL終止液(2M H2SO4)終止反應;酶標儀讀取OD450,並用標準曲線將OD450換算成抗體血清濃度;用GraphPad Prism6進行資料分析和作圖;用Phoenix軟體計算抗體藥物在大鼠體內的半衰期。
如第11圖所示,計算出20-Fab-0313-IgG4-V94L的半衰期為16.9天(以PD-1為抗原的檢測結果)和17.3天(以VEGF165為抗原的檢測結果)。上述結果表明,20-Fab-0313-IgG4-V94L具有良好的藥代動力學性質。
實施例1.7.6抗PD-1和VEGF雙特異性抗體在小鼠體內的抗腫瘤作用
利用人外周血單個核細胞(PBMC)在NSG小鼠體內重建人源免疫系統,並在此小鼠上建立人肺癌NCI-H460皮下移植瘤模型。該小鼠模型同時存在表達人PD-1蛋白的T細胞,以及表達人VEGF的人類腫瘤細胞,因此可以用來評價抗PD-1和VEGF雙特異性抗體的體內抗腫瘤活性。具體實施步驟如下:收集體外培養的人非小細胞肺癌NCI-H460細胞(ATCC
®HTB-177
™),將細胞懸液濃度調整為1×10
8/mL,與Matrigel(購自BD Biosciences,貨號:356234)以等體積混合。體外復甦購買的PBMC(購自Allcells,貨號:PB005-C),用PBS重懸PBMC細胞,將PBMC懸液濃度調整為1×10
7/mL。將混合好的腫瘤細胞懸液和PBMC懸液以等體積混合。在無菌條件下,接種200μL細胞混合懸液於M-NSG小鼠(購自上海南方模式生物研究中心)右側上背部皮下。當天將接種混合細胞的小鼠按體重隨機分組,每組10隻小鼠。各組小鼠藥物處理情況如下:對照組,僅注射生理鹽水;Avastin組,注射10 mg/kg的抗VEGF陽性對照抗體Avastin(由羅氏製藥生產);Opdivo組,注射10 mg/kg的抗PD-1陽性對照抗體Opdivo(由百時美施貴寶生產);20-Fab-0313-IgG4-V94L組,注射16 mg/kg的20-Fab-0313-IgG4-V94L。考慮到雙特異性抗體和單株抗體的分子量存在差異,本實驗中藥物劑量按照等物質的量進行設定。隨後,按照上述設計好的方案給藥,每週給藥2次,共給藥10次,每週測定腫瘤體積2次。最終,測定的各組腫瘤隨時間的生長曲線如第12圖所示。
結果顯示,在第31天實驗結束時,Avastin、Opdivo和20-Fab-0313-IgG4-V94L各組的抑瘤率分別為84.5%、35.8%和96.6%(抑瘤率=(對照組平均體積-實驗組平均體積)/對照組平均體積×100%)。與Avastin和Opdivo相比,20-Fab-0313-IgG4-V94L能夠更加有效地抑制腫瘤生長。
實施例2構建抗PD-1和TGF-Beta的雙特異性抗體
實施例2.1 序列
US5571714A公開了一系列抗TGF-β(Transforming growth factor beta)單株抗體,其中鼠源單抗1D11.16(後續簡稱1D11)能夠有效結合TGF-β1和-β2。相關揭露人已將1D11對應的雜交瘤存儲到美國典型培養物保藏中心(ATCC
®HB-9849
™)。從US20180244763A1獲得鼠源1D11序列。
對1D11號抗體的重鏈可變區和輕鏈可變區胺基酸序列進行分析,依據Kabat規則分別確定1D11號抗體重鏈和輕鏈的抗原互補決定區和框架區。1D11號抗體重鏈CDR的胺基酸序列為H-CDR1:TYWMN(SEQ ID NO:23)、H-CDR2:QIFPASGSTNYNEMFEG(SEQ ID NO:24)和H-CDR3:GDGNYALDAMDY(SEQ ID NO:25),輕鏈CDR的胺基酸序列為L-CDR1:RASESVDSYGNSFMH(SEQ ID NO:26)、L-CDR2:LASNLES(SEQ ID NO:27)和L-CDR3:QQNNEDPLT(SEQ ID NO:28)。
在https://www.ncbi.nlm.nih.gov/igblast/,將鼠源1D11號抗體的重鏈可變區與人IgG胚系序列進行同源性比較,選擇IGHV1-46*01為重鏈CDR移植範本,將鼠源的1D11號抗體的重鏈CDR移植入IGHV1-46*01骨架區,並在H-CDR3之後加入WGQGTLVTVSS(SEQ ID NO:151)作為第四個框架區,獲得CDR移植重鏈可變區序列。同樣地,將鼠源1D11號抗體的輕鏈可變區與人IgG胚系序列同源性比較,選擇IGKV7-3*01為輕鏈CDR移植範本,將鼠源1D11號抗體的輕鏈CDR移植入IGKV7-3*01的骨架區,並在L-CDR3之後加入FGGGTKVELK(SEQ ID NO:153)作為第四個框架區,獲得CDR移植輕鏈可變區序列。在CDR移植可變區的基礎上,對一些框架區的胺基酸位點進行回復突變。在進行回復突變時,將胺基酸序列進行Kabat編碼,位元點的位置由Kabat碼指示。
較佳的,對於CDR移植重鏈可變區,將第28位的T回復為鼠源的I,第30位的T回復為I,第48位的M回復為I,第67位的V回復為A,第69位的M回復為L,第71位的R回復為V,第78位的V回復為A。對於CDR移植輕鏈可變區,將第81位的N回復為D。
上述人源化的重鏈可變區和輕鏈可變區(SEQ ID NO:29和30)由上海生工生物工程有限公司合成編碼上述可變區的DNA。將合成的人源化重鏈可變區與人IgG4(鉸鏈區含有S228P點突變)重鏈恆定區(SEQ ID NO:148)相連,獲得全長的人源化重鏈基因,命名為1D11-Hu-HC;將人源化輕鏈可變區與人Kappa輕鏈恆定區(SEQ ID NO:149)相連,獲得全長的人源化輕鏈基因,命名為1D11-Hu-LC。
上述抗體的重鏈和輕鏈基因分別構建到pcDNA3.4表達載體中,表達純化抗體。1D11-Hu-HC與1D11-Hu-LC組合後獲得的抗體定義為1D11-Hu。
US20100136021A1也公開了一系列抗TGF-β抗體,其中單抗mAb12.7(後續簡稱mAb127)能夠有效結合並中和TGF-β1、TGF-β2和TGF-β3。從US20100136021A1中獲得mAb127的重鏈可變區和輕鏈可變區序列(SEQ ID NO:31和32)。
由上海生工生物工程有限公司合成編碼上述可變區的DNA。將合成的重鏈可變區與人IgG4(鉸鏈區含有S228P點突變)重鏈恆定區(SEQ ID NO:148)相連,獲得全長的重鏈基因,命名為mAb127-HC;將輕鏈可變區與人Kappa輕鏈恆定區(SEQ ID NO:149)相連,獲得全長的輕鏈基因,命名為mAb127-LC。
根據WO2018/137576A1中實施例1-5所述,挑取14號鼠源單抗作為先導抗體並獲得其重鏈可變區核苷酸序列和輕鏈可變區核苷酸序列,並翻譯成胺基酸序列(SEQ ID NO:33和34)。
對14號抗體的重鏈可變區和輕鏈可變區胺基酸序列進行分析,依據Kabat規則分別確定14號抗體重鏈和輕鏈的抗原互補決定區和框架區。14號抗體重鏈CDR的胺基酸序列為H-CDR1:GYTMN(SEQ ID NO:35)、H-CDR2:LINPYNGDTSYNQKFKG(SEQ ID NO:36)和H-CDR3:WRYTMDY(SEQ ID NO:37),輕鏈CDR的胺基酸序列為L-CDR1:RASESVDNYGNSFMN(SEQ ID NO:38)、L-CDR2:FASNLES(SEQ ID NO:39)和L-CDR3:QQNNEAPPT(SEQ ID NO:40)。
在https://www.ncbi.nlm.nih.gov/igblast/,將鼠源14號抗體的重鏈可變區與人IgG胚系序列進行同源性比較,選擇IGHV1-46*01為重鏈CDR移植範本,將鼠源的14號抗體的重鏈CDR移植入IGHV1-46*01骨架區,並在H-CDR3之後加入WGQGTLVTVSS(SEQ ID NO:151)作為第四個框架區,獲得CDR移植重鏈可變區序列。同樣地,將鼠源14號抗體的輕鏈可變區與人IgG胚系序列進行同源性比較,選擇IGKV7-3*01為輕鏈CDR移植範本,將鼠源14號抗體的輕鏈CDR移植入IGKV7-3*01的骨架區,並在L-CDR3之後加入FGQGTKVEIK(SEQ ID NO:152)作為第四個框架區,獲得CDR移植輕鏈可變區序列。在CDR移植可變區的基礎上,對一些框架區的胺基酸位點進行回復突變。在進行回復突變時,將胺基酸序列進行Kabat編碼,位元點的位置由Kabat碼指示。
較佳的,對於CDR移植重鏈可變區,將第28位元(Kabat編碼)的T回復為鼠源的S,第48位的M回復為I,第67位的V回復為A,第69位的M回復為V,第71位的R回復為V,第73位的T回復為K,第78位的V回復為A。對於CDR移植輕鏈可變區,將第46位的L回復為P,第68位的G回復為R,第81位的N回復為D。
上述帶有回復突變位點的重鏈和輕鏈可變區分別定義為人源化的重鏈可變區和輕鏈可變區(SEQ ID NO:41和42)。由上海生工生物工程有限公司合成編碼上述人源化的重鏈和輕鏈可變區的DNA。將合成的人源化重鏈可變區與人IgG4(鉸鏈區含有S228P點突變)重鏈恆定區(SEQ ID NO:148)相連,獲得全長的人源化重鏈基因,命名為14-Hu-HC;將人源化輕鏈可變區與人Kappa輕鏈恆定區(SEQ ID NO:149)相連,獲得全長的人源化輕鏈基因,命名為14-Hu-LC。
上述抗體的重鏈和輕鏈基因分別構建到pcDNA3.4表達載體中,表達純化抗體。14-Hu-HC與14-Hu-LC組合後獲得的抗體定義為14-Hu。
實施例2.2 共同輕鏈的選擇
用BLAST對14-Hu輕鏈可變區與1D11-Hu輕鏈可變區的胺基酸序列進行對比分析,結果顯示,兩者之間完全相同的胺基酸占比92%(Identities),性質相似的胺基酸占比94%(Positives)。
將14-Hu-HC、14-Hu-LC、1D11-Hu-HC和1D11-Hu-LC的表達載體按照下述方式進行組合:14-Hu-HC+14-Hu-LC、1D11-Hu-HC+1D11-Hu-LC、14-Hu-HC+1D11-Hu-LC和1D11-Hu-HC+14-Hu-LC,表達純化抗體,所得的抗體分別命名為14-Hu、1D11-Hu、14-Hu-HC+1D11-Hu-LC和1D11-Hu-HC+14-Hu-LC。
ELISA檢測方法如下:自製帶有6*His標籤的人PD-1的胞外區蛋白(PD-1胞外區來源如WO2018/137576A1中所述),將這種重組蛋白記作PD1-His並用其包被酶標板(20 ng/孔);用TGF-β1(購自Sino Biological)包被酶標板(5 ng/孔)。
如第13A圖所示,14-Hu能夠有效結合PD1-His,EC50是0.3924nM;而1D11-Hu、14-Hu-HC+1D11-Hu-LC和1D11-Hu-HC+14-Hu-LC不能有效結合PD1-His。如第13B圖所示,1D11-Hu能夠有效結合TGF-β1,EC50為0.06624nM,1D11-Hu-HC+14-Hu-LC也能結合TGF-β1,EC50為0.5255 nM,而14-Hu和14-Hu-HC+1D11-Hu-LC不能結合TGF-β1。在此選擇14-Hu-LC(SEQ ID NO:43和44)作為共同輕鏈構建雙特異性抗體。
用BLAST對14-Hu輕鏈可變區與mAb127輕鏈可變區的胺基酸序列進行對比分析,結果顯示,兩者之間完全相同的胺基酸占比75%(Identities),性質相似的胺基酸占比83%(Positives)。
將14-Hu-LC、mAb127-HC和mAb127-LC的表達載體按照下述方式進行組合:mAb127-HC+mAb127-LC和mAb127-HC+14-Hu-LC,表達純化抗體,所得的抗體分別命名為mAb127和mAb127-HC+14-Hu-LC。
用上述實施例中描述的ELISA檢測1D11-Hu、1D11-Hu-HC+14-Hu-LC、mAb127和mAb127-HC+14-Hu-LC對TGF-β1的相對親和力,用GraphPad Prism6進行作圖和資料分析,並計算EC50。
如第14圖所示,1D11-Hu、mAb127和mAb127-HC+14-Hu-LC均能夠有效結合TGF-β1,EC50分別是0.1338nM、0.04136 nM和0.07105 nM。與1D11-Hu相比,mAb127和mAb127-HC+14-Hu-LC有更低的EC50和更高的平臺,因此兩者對TGF-β1具有更高的親和力。在此選擇14-Hu-LC(SEQ ID NO:43和44)作為共同輕鏈構建雙特異性抗體。
實施例2.3雙特異性抗體的構建
將14-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接1D11的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈基因命名為14-Fab-1D11-IgG4(SEQ ID NO:45和46)。相似地,將1D11的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接14-Hu的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈基因命名為1D11-Fab-14-IgG4(SEQ ID NO:47和48)。
按照上述實施例中描述的方法,將14-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接mAb127的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈基因命名為14-Fab-127-IgG4(SEQ ID NO:49和50)。相似地,將mAb127的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接14-Hu的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈基因命名為127-Fab-14-IgG4(SEQ ID NO:51和52)。
將上述序列分別構建到pcDNA3.4表達載體中,將14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4表達載體分別與14-Hu-LC表達載體組合,表達純化抗體,所得的抗體分別命名為14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4。
實施例2.4 ELISA測定相對親和力
如第15A圖所示,14-Hu、14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4均能夠結合PD1-His,EC50分別是0.4321 nM、0.4367 nM、1.996 nM、0.3873 nM和3.955 nM;與14-Fab-1D11-IgG4和14-Fab-127-IgG4相比,1D11-Fab-14-IgG4和127-Fab-14-IgG4對PD1-His的相對親和力更弱,這可能是由空間位阻造成的。如第15B圖所示,1D11-Hu-HC+14-Hu-LC、mAb127-HC+14-Hu-LC、14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4均能結合TGF-β1,EC50分別是1.267 nM、0.0803 nM、0.6985 nM、0.3628 nM、0.1525 nM和0.1083 nM。與1D11-Hu-HC+14-Hu-LC、14-Fab-1D11-IgG4和1D11-Fab-14-IgG4相比,mAb127-HC+14-Hu-LC、14-Fab-127-IgG4和127-Fab-14-IgG4對TGF-β1的相對親和力更強。
實施例2.5評估增強MLR的功能活性
如第16A圖和第16B圖所示,14-Hu和14-Fab-127-IgG4均能有效刺激MLR分泌IL-2和IFN-γ,刺激MLR分泌IL-2的EC50分別為0.1008nM和0.3185 nM,刺激MLR分泌IFN-γ的EC50分別為0.04716 nM和0.5871 nM。另外,實驗結果顯示,在較高濃度下,與14-Hu相比,14-Fab-127-IgG4能夠刺激MLR分泌更多IL-2和IFN-γ。
實施例2.6 測定抗PD-1和TGF-Beta雙特異性抗體同時結合兩種抗原的能力
用TGF-β1(購自Sino Biological,貨號:10804-HNAC)包被微孔板。用含1%牛血清白蛋白的PBST將待測抗體進行梯度稀釋,然後轉移到上述微孔板中,室溫孵育半小時左右。後續實驗步驟與實施例1.7.4相同。
如第17圖所示,14-Fab-127-IgG4在結合TGF-β1之後仍然能夠有效地繼續結合人PD-1,EC50為0.2784 nM。14-Hu和mAb127-HC+14-Hu-LC都不能同時結合PD-1和TGF-β1。
實施例2.7 抗PD-1和TGF-beta雙特異性抗體在小鼠體內的抗腫瘤作用
人PD-1轉基因小鼠(種系背景為C57BL/6)和MC38小鼠結直腸癌細胞購自上海南方模式生物研究中心,該轉基因小鼠中用人源PD-1基因的胞外段替換了小鼠的同源部分。本揭露的雙特異性抗體能夠識別該轉基因小鼠中的PD-1分子,也可以結合小鼠內源的TGF-beta。具體實施步驟如下:將MC38在體外培養,培養基為含有10%血清的DMEM(血清和培養基購自Gibco);將培養的MC38細胞接種於人PD-1轉基因小鼠中,每隻小鼠皮下接種2×10
6個細胞;待接種的腫瘤細胞生長至體積接近100mm
3時,將小鼠隨機分組,每組8隻小鼠。各組小鼠藥物處理情況如下:對照組,僅注射生理鹽水;Keytruda組(兩個劑量組),注射2 mg/kg或10 mg/kg的抗PD-1陽性對照抗體Keytruda(由默沙東公司生產);14-Fab-127-IgG4組(兩個劑量組),注射3.2mg/kg或16 mg/kg的14-Fab-127-IgG4。考慮到雙特異性抗體和單株抗體的分子量存在差異,本實驗中藥物劑量按照等物質的量進行設定。隨後,按照上述設計好的方案給藥,每週給藥2次,共給藥6次,每週測定腫瘤體積2次。最終,測定的各組腫瘤隨時間的生長曲線如第18圖所示。
結果顯示,在第25天實驗結束時,Keytruda(兩個劑量組)和14-Fab-127-IgG4(兩個劑量組)各組的抑瘤率分別為79.2%(2 mg/kg)、77.7%(10 mg/kg)、85.1%(3.2mg/kg)和100%(16mg/kg)(抑瘤率=(對照組平均體積-實驗組平均體積)/對照組平均體積×100%)。與Keytruda相比,14-Fab-127-IgG4能夠更加有效地抑制腫瘤生長,並且14-Fab-127-IgG4在高劑量時能夠導致所有小鼠的腫瘤完全消退。
實施例3 構建抗HER-2和CD47的雙特異性抗體
實施例3.1 序列
19H6-Hu是人源化的抗人HER2單抗,其重鏈可變區和輕鏈可變區序列來自於WO2020/025013A1,人源化的重鏈可變區和輕鏈可變區分別命名為19H6-Hu-VH和19H6-Hu-VL(SEQ ID NO:53和54)。
由上海生工生物工程有限公司合成編碼上述人源化的重鏈可變區和輕鏈可變區的DNA。將合成的人源化重鏈可變區與人IgG1重鏈恆定區(SEQ ID NO:147)相連,獲得全長的人源化重鏈基因,命名為19H6-Hu-HC;將人源化輕鏈可變區與人Kappa鏈恆定區(SEQ ID NO:149)相連,獲得全長的人源化輕鏈基因,命名為19H6-Hu-LC。將19H6-Hu-HC和19H6-Hu-LC基因分別構建到pcDNA3.4表達載體中,表達純化抗體,所得抗體命名為19H6-Hu。
MABL-2(後文中稱為Anti-CD47B)是抗人CD47的鼠源單抗,其重鏈可變區和輕鏈可變區胺基酸序列來自於US20030108546A中的SEQ ID NO:12和SEQ ID NO:10。
對Anti-CD47B抗體的重鏈可變區和輕鏈可變區胺基酸序列進行分析,依據Kabat規則分別確定Anti-CD47B抗體重鏈和輕鏈的抗原互補決定區和框架區。Anti-CD47B抗體重鏈CDR的胺基酸序列為H-CDR1:NHVIH(SEQ ID NO:55)、H-CDR2:YIYPYNDGTKYNEKFKD(SEQ ID NO:56)和H-CDR3:GGYYTYDD(SEQ ID NO:57),輕鏈CDR的胺基酸序列為L-CDR1:RSSQSLVHSNGKTYLH(SEQ ID NO:58)、L-CDR2:KVSNRFS(SEQ ID NO:59)和L-CDR3:SQSTHVPYT(SEQ ID NO:60)。
在https://www.ncbi.nlm.nih.gov/igblast/,將鼠源Anti-CD47B抗體的重鏈可變區與人IgG胚系序列進行同源性比較,選擇IGHV1-46*01為重鏈CDR移植範本,將鼠源的Anti-CD47B抗體的重鏈CDR移植入IGHV1-46*01骨架區,並在H-CDR3之後加入WGQGTLVTVSS(SEQ ID NO:151)作為第四個框架區,獲得CDR移植重鏈可變區序列。同樣地,將鼠源Anti-CD47B抗體的輕鏈可變區與人IgG胚系序列同源性比較,選擇IGKV2-30*01為輕鏈CDR移植範本,將鼠源Anti-CD47B抗體的輕鏈CDR移植入IGKV2-30*01的骨架區,並在L-CDR3之後加入FGQGTKVEIK(SEQ ID NO:152)作為第四個框架區,獲得CDR移植輕鏈可變區序列。在CDR移植可變區的基礎上,對一些框架區的胺基酸位點進行回復突變。在進行突變時,將胺基酸序列進行Kabat編碼,位元點的位置由Kabat碼指示。
較佳的,對於CDR移植重鏈可變區,根據Kabat編碼,將第30位元的T突變為A,將第69位的M突變為L,將第71位的R突變為S,將第73位的T突變為K。對於CDR移植輕鏈可變區,將第36位的F突變為Y,第46位的R突變為L。
上述帶有突變位點的重鏈可變區和輕鏈可變區分別定義為人源化的重鏈可變區和輕鏈可變區,分別命名為Anti-CD47B-Hu-VH和Anti-CD47B-Hu-VL(SEQ ID NO:61和62)。
由上海生工生物工程有限公司合成編碼上述人源化的重鏈可變區和輕鏈可變區的DNA。將合成的人源化重鏈可變區與人IgG1重鏈恆定區(SEQ ID NO:147)相連,獲得全長的人源化重鏈基因,命名為Anti-CD47B-Hu-HC;將人源化輕鏈可變區與人Kappa鏈恆定區(SEQ ID NO:149)相連,獲得全長的人源化輕鏈基因,命名為Anti-CD47B-Hu-LC。將Anti-CD47B-Hu-HC和Anti-CD47B-Hu-LC基因分別構建到pcDNA3.4表達載體中,表達純化抗體,所得抗體命名為Anti-CD47B-Hu。
實施例3.2 共同輕鏈的選擇
用BLAST對19H6-Hu輕鏈可變區與Anti-CD47B-Hu輕鏈可變區的胺基酸序列進行對比分析,結果顯示,兩者之間完全相同的胺基酸占比96%(Identities),性質相似的胺基酸占比99%(Positives)。
將19H6-Hu和Anti-CD47B-Hu的重鏈和輕鏈基因按照下述方式進行組合:19H6-Hu-HC+Anti-CD47B-Hu-LC和Anti-CD47B-Hu-HC+19H6-Hu-LC,表達純化抗體,所得的抗體分別命名為19H6-Hu-HC+Anti-CD47B-Hu-LC和Anti-CD47B-Hu-HC+19H6-Hu-LC。
ELISA檢測方法如下:帶有多聚組胺酸標籤的人Her-2胞外段的重組蛋白購自ACROBiosystems(貨號:HE2-H5225),帶多聚組胺酸標籤的人CD47胞外段的重組蛋白購自Sino Biological(貨號:12283-H08H),將這兩種重組蛋白名稱分別記作HER2-ECD-His和CD47-ECD-His。用HER2-ECD-His和CD47-ECD-His分別包被酶標板,包被濃度均為10 ng/孔。
如第19A圖所示,19H6-Hu和19H6-Hu-HC+Anti-CD47B-Hu-LC均能夠有效結合HER2-ECD-His,EC50分別是0.07701 nM和0.1388 nM;而Anti-CD47B-Hu和Anti-CD47B-Hu-HC+19H6-Hu-LC不能有效結合HER2-ECD-His。如第19B圖所示,Anti-CD47B-Hu和Anti-CD47B-Hu-HC+19H6-Hu-LC均能有效結合CD47-ECD-His,EC50分別是0.04276nM和0.0541nM,而19H6-Hu和19H6-Hu-HC+Anti-CD47B-Hu-LC不能有效結合CD47-ECD-His。在此選擇19H6-Hu-LC(SEQ ID NO:63和64)作為共同輕鏈構建雙特異性抗體。
實施例3.3雙特異性抗體的構建
將Anti-CD47B-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接19H6-Hu的重鏈可變區,最後再連接人IgG1的重鏈恆定區(CH1+CH2+CH3),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為CD47B-Fab-19H6-IgG1(SEQ ID NO:65和66)。相似地,將19H6-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接Anti-CD47B-Hu的重鏈可變區,最後再連接人IgG1的重鏈恆定區(CH1+CH2+CH3),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為19H6-Fab-CD47B-IgG1(SEQ ID NO:67和68)。
將上述序列分別構建到pcDNA3.4表達載體中,將CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1表達載體分別與19H6-Hu-LC表達載體組合,表達純化抗體,所得的抗體分別命名為CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1(為簡明起見,此處只取重鏈的名字作為抗體的名稱)。
實施例3.4 ELISA測定相對親和力
如第20A圖所示,19H6-Hu、CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1均能有效結合HER2-ECD-His,EC50分別是0.1262 nM、0.1057 nM和0.1543 nM。如第20B圖所示,Anti-CD47B-Hu-HC+19H6-Hu-LC、CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1均能有效結合CD47-ECD-His,EC50分別是0.06166 nM、0.07817 nM和0.1945 nM。上述結果顯示,CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1既能夠結合HER-2又能結合CD47,這說明它們是雙特異性抗體。
實施例4構建抗HER-2和CD137的雙特異性抗體
實施例4.1 序列
19H6-Hu是人源化的抗人HER2單抗,其來源如實施例3.1所述。
94號抗體是本公司實驗室內部製備的鼠源抗人CD137抗體,製備方法參照WO2018/137576A1中實施例1-5所述,不同之處在於使用人CD137重組蛋白免疫小鼠和使用人CD137重組蛋白對雜交瘤細胞進行ELISA篩選,其重鏈可變區胺基酸序列和輕鏈可變區胺基酸序列分別如SEQ ID NO:69和70所示。
對94號抗體的重鏈可變區和輕鏈可變區胺基酸序列進行分析,依據Kabat規則分別確定94號抗體重鏈和輕鏈的抗原互補決定區和框架區。94號抗體重鏈CDR的胺基酸序列為H-CDR1:SYDIS(SEQ ID NO:71)、H-CDR2:VIWTGGGTNYNSAFMS(SEQ ID NO:72)和H-CDR3:MDY(SEQ ID NO:73),輕鏈CDR的胺基酸序列為L-CDR1:RSSQSLLHSNGNTYLH(SEQ ID NO:74)、L-CDR2:KVSNRFS(SEQ ID NO:75)和L-CDR3:SQSTHVPWT(SEQ ID NO:76)。
在https://www.ncbi.nlm.nih.gov/igblast/,將鼠源94號抗體的重鏈可變區與人IgG胚系序列進行同源性比較,選擇IGHV4-59*01為重鏈CDR移植範本,將鼠源的94號抗體的重鏈CDR移植入IGHV4-59*01骨架區,並在H-CDR3之後加入WGQGTLVTVSS(SEQ ID NO:151)作為第四個框架區,獲得CDR移植重鏈可變區序列。同樣地,將鼠源94號抗體的輕鏈可變區與人IgG胚系序列同源性比較,選擇IGKV2-30*01為輕鏈CDR移植範本,將鼠源94號抗體的輕鏈CDR移植入IGKV2-30*01的骨架區,並在L-CDR3之後加入FGQGTKVEIK(SEQ ID NO:152)作為第四個框架區,獲得CDR移植輕鏈可變區序列。在CDR移植可變區的基礎上,對一些框架區的胺基酸位點進行回復突變。在進行突變時,將胺基酸序列進行Kabat編碼,位元點的位置由Kabat碼指示。
較佳的,對於CDR移植重鏈可變區,根據Kabat編碼,將第27位元的G突變為F,將第29位的I突變為L,將第48位的I突變為L,將第71位的R突變為S,將第71位的V突變為K,將第73位的T突變為N,將第76位的N突變為S,將第78位的F突變為V,將第93位的A突變為V。對於CDR移植輕鏈可變區,將第36位的F突變為Y,第46位的R突變為L,將第48位的I突變為F,將第87位的Y突變為F。
上述帶有突變位點的重鏈可變區和輕鏈可變區分別定義為人源化的重鏈可變區和輕鏈可變區,分別命名為94-Hu-VH和94-Hu-VL(SEQ ID NO:77和78)。
由上海生工生物工程有限公司合成編碼上述人源化的重鏈可變區和輕鏈可變區的DNA。將合成的人源化重鏈可變區與人IgG1重鏈恆定區(SEQ ID NO:147)相連,獲得全長的人源化重鏈基因,命名為94-Hu-HC;將人源化輕鏈可變區與人Kappa鏈恆定區(SEQ ID NO:149)相連,獲得全長的人源化輕鏈基因,命名為94-Hu-LC。將94-Hu-HC和94-Hu-LC基因分別構建到pcDNA3.4表達載體中,表達純化抗體,所得抗體命名為94-Hu。
實施例4.2 共同輕鏈的選擇
用BLAST對19H6-Hu輕鏈可變區與94-Hu輕鏈可變區的胺基酸序列進行對比分析,結果顯示,兩者之間完全相同的胺基酸占比97%(Identities),性質相似的胺基酸占比99%(Positives)。
將19H6-Hu和94-Hu的重鏈和輕鏈基因按照下述方式進行組合:19H6-Hu-HC+94-Hu-LC和94-Hu-HC+19H6-Hu-LC,表達純化抗體,所得的抗體分別命名為19H6-Hu-HC+94-Hu-LC和94-Hu-HC+19H6-Hu-LC。
ELISA檢測方法如下:帶有多聚組胺酸標籤的人Her-2胞外段的重組蛋白購自ACROBiosystems(貨號:HE2-H5225),帶Fc標籤的人CD137胞外段的重組蛋白購自Sino Biological(貨號:10041-H02H),將這兩種重組蛋白名稱分別記作HER2-ECD-His和CD137-ECD-Fc。用HER2-ECD-His和CD137-ECD-Fc分別包被酶標板,包被濃度均為10ng/孔。
如第21A圖所示,19H6-Hu和19H6-Hu-HC+94-Hu-LC均能夠有效結合HER2-ECD-His,EC50分別是0.1222 nM和0.1391 nM;而94-Hu和94-Hu-HC+19H6-Hu-LC不能有效結合HER2-ECD-His。如第21B圖所示,94-Hu和94-Hu-HC+19H6-Hu-LC均能有效結合CD137-ECD-Fc,EC50分別是0.3913 nM和0.634 nM,而19H6-Hu和19H6-Hu-HC+94-Hu-LC不能有效結合CD137-ECD-Fc。在此選擇19H6-Hu-LC(SEQ ID NO:63和64)作為共同輕鏈構建雙特異性抗體(實際上也可以選擇94-Hu-LC作為共同輕鏈構建雙特異性抗體)。
實施例4.3雙特異性抗體的構建
將94-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接19H6-Hu的重鏈可變區,最後再連接人IgG1的重鏈恆定區(CH1+CH2+CH3),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為94-Fab-19H6-IgG1。相似地,將19H6-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接94-Hu的重鏈可變區,最後再連接人IgG1的重鏈恆定區(CH1+CH2+CH3),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為19H6-Fab-94-IgG1。
為降低上述雙抗分子的Fc段與FcγRs(Fc gamma receptors)之間的相互作用,將它們Fc段的第234的亮胺酸和235位的亮胺酸都突變為丙胺酸,將該突變標記為LALA(參考文獻:Wang X, Mathieu M, Brezski R J. IgG Fc engineering to modulate antibody effector functions[J]. Protein & cell, 2018, 9(1): 63-73.)。突變後的上述雙抗分子的名稱分別為94-Fab-19H6-IgG1-LALA(SEQ ID NO:79和80)和19H6-Fab-94-IgG1-LALA(SEQ ID NO:81和82)。在此,LALA突變的目的主要是降低潛在的體內毒性(參考文獻:Ho S K, Xu Z, Thakur A, et al. Epitope and Fc-mediated Crosslinking, but not High Affinity, Are Critical for Antitumor Activity of CD137 Agonist Antibody with Reduced Liver Toxicity[J]. Molecular Cancer Therapeutics, 2020.pp. 1040–1051.)。
將上述序列分別構建到pcDNA3.4表達載體中,將94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA表達載體分別與19H6-Hu-LC表達載體組合,表達純化抗體,所得的抗體分別命名為94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA(為簡明起見,此處只取重鏈的名字作為抗體的名稱)。
實施例4.4 ELISA測定相對親和力
如第22A圖所示,19H6-Hu、94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA均能有效結合HER2-ECD-His,EC50分別是0.1933 nM、0.1579 nM和0.1201 nM。如第22B圖所示,94-Hu-HC+19H6-Hu-LC和94-Fab-19H6-IgG1-LALA均能有效結合CD137-ECD-Fc,EC50分別是0.634nM和0.2411nM;19H6-Fab-94-IgG1-LALA對CD137-ECD-Fc的結合較弱,EC50為和27.56 nM。上述結果顯示,94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA既能夠結合HER-2又能結合CD137,這說明它們是雙特異性抗體。
實施例5構建抗PD-1和CD137的雙特異性抗體
實施例5.1 序列
mAb1-25-Hu(後文中簡稱為609)是人源化的抗人PD-1單抗,其重鏈可變區和輕鏈可變區序列來自於WO2018/137576A1,人源化的重鏈可變區和輕鏈可變區(SEQ ID NO:83和84)分別與人IgG4(S228P)重鏈恆定區(SEQ ID NO:148)和Kappa輕鏈恆定區(SEQ ID NO:149)相連,最終獲得完整的人源化mAb1-25-Hu單抗(609)的重鏈和輕鏈基因。
4B4-1-1(後文中稱為Anti-CD137)是抗人CD137的鼠源單抗,其重鏈可變區和輕鏈可變區胺基酸序列來自於US5928893中的SEQ ID NO:10和SEQ ID NO:11。
對Anti-CD137抗體的重鏈可變區和輕鏈可變區胺基酸序列進行分析,依據Kabat規則分別確定Anti-CD137抗體重鏈和輕鏈的抗原互補決定區和框架區。Anti-CD137抗體重鏈CDR的胺基酸序列為H-CDR1:SYWMH(SEQ ID NO:85)、H-CDR2:EINPGNGHTNYNEKFKS(SEQ ID NO:86)和H-CDR3:SFTTARGFAY(SEQ ID NO:87),輕鏈CDR的胺基酸序列為L-CDR1:RASQTISDYLH(SEQ ID NO:88)、L-CDR2:YASQSIS(SEQ ID NO:89)和L-CDR3:QDGHSFPPT(SEQ ID NO:90)。
在https://www.ncbi.nlm.nih.gov/igblast/,將鼠源Anti-CD137抗體的重鏈可變區與人IgG胚系序列進行同源性比較,選擇IGHV1-46*01為重鏈CDR移植範本,將鼠源的Anti-CD137抗體的重鏈CDR移植入IGHV1-46*01骨架區,並在H-CDR3之後加入WGQGTLVTVSS(SEQ ID NO:151)作為第四個框架區,獲得CDR移植重鏈可變區序列。同樣地,將鼠源Anti-CD137抗體的輕鏈可變區與人IgG胚系序列同源性比較,選擇IGKV6-21*02為輕鏈CDR移植範本,將鼠源Anti-CD137抗體的輕鏈CDR移植入IGKV6-21*02的骨架區,並在L-CDR3之後加入FGQGTKVEIK(SEQ ID NO:152)作為第四個框架區,獲得CDR移植輕鏈可變區序列。在CDR移植可變區的基礎上,對一些框架區的胺基酸位點進行回復突變。在進行突變時,將胺基酸序列進行Kabat編碼,位元點的位置由Kabat碼指示。
較佳的,對於CDR移植重鏈可變區,根據Kabat編碼,將第30位元的T突變為S,將第69位的M突變為L,將第71位的R突變為V,將第73位的T突變為K。對於CDR移植輕鏈可變區,將第4位的L突變為M,將第58位的V突變為I,第69位的T突變為S。
上述帶有突變位點的重鏈可變區和輕鏈可變區分別定義為人源化的重鏈可變區和輕鏈可變區,分別命名為Anti-CD137-Hu-VH和Anti-CD137-Hu-VL(SEQ ID NO:91和92)。
由上海生工生物工程有限公司合成編碼上述人源化的重鏈可變區和輕鏈可變區的DNA。將合成的人源化重鏈可變區與人IgG4(S228P)重鏈恆定區(SEQ ID NO:148)相連,獲得全長的人源化重鏈基因,命名為Anti-CD137-Hu-HC;將人源化輕鏈可變區與人Kappa鏈恆定區(SEQ ID NO:149)相連,獲得全長的人源化輕鏈基因,命名為Anti-CD137-Hu-LC。將Anti-CD137-Hu-HC和Anti-CD137-Hu-LC基因分別構建到pcDNA3.4表達載體中,表達純化抗體,所得抗體命名為Anti-CD137-Hu。
實施例5.2 共同輕鏈的選擇
用BLAST對609輕鏈可變區與Anti-CD137-Hu輕鏈可變區的胺基酸序列進行對比分析,結果顯示,兩者之間完全相同的胺基酸占比73% (Identities),性質相似的胺基酸占比88% (Positives)。
將609和Anti-CD137-Hu的重鏈和輕鏈基因按照下述方式進行組合:609-HC+Anti-CD137-Hu-LC和Anti-CD137-Hu-HC+609-LC,表達純化抗體,所得的抗體分別命名為609-HC+Anti-CD137-Hu-LC和Anti-CD137-Hu-HC+609-LC。
ELISA檢測方法如下:帶有多聚組胺酸標籤的人PD-1胞外段的重組蛋白購自Sino Biological(貨號:10377-H08H),帶Fc標籤的人CD137胞外段的重組蛋白購自Sino Biological(貨號:10041-H02H),將這兩種重組蛋白名稱分別記作PD1-ECD-His和CD137-ECD-Fc。用PD1-ECD-His和CD137-ECD-Fc分別包被酶標板,包被濃度均為10 ng/孔。
如第23A圖所示,609和609-HC+Anti-CD137-Hu-LC均能夠有效結合PD1-ECD-His,EC50分別是0.1358 nM和0.2067 nM;而Anti-CD137-Hu和Anti-CD137-Hu-HC+609-LC不能有效結合PD1-ECD-His。如第23B圖所示,Anti-CD137-Hu能有效結合CD137-ECD-Fc,EC50分別為0.461 nM,而609、609-HC+Anti-CD137-Hu-LC和Anti-CD137-Hu-HC+609-LC不能有效結合CD137-ECD-Fc。在此選擇Anti-CD137-Hu-LC(SEQ ID NO:93和94)作為共同輕鏈構建雙特異性抗體。
實施例5.3雙特異性抗體的構建
將609的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接Anti-CD137-Hu的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為609-Fab-137-IgG4(SEQ ID NO:95和96)。相似地,將Anti-CD137-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接609的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為137-Fab-609-IgG4(SEQ ID NO:97和98)。
將上述序列分別構建到pcDNA3.4表達載體中,將609-Fab-137-IgG4和137-Fab-609-IgG4表達載體分別與Anti-CD137-Hu-LC表達載體組合,表達純化抗體,所得的抗體分別命名為609-Fab-137-IgG4和137-Fab-609-IgG4(為簡明起見,此處只取重鏈的名字作為抗體的名稱)。
實施例5.4 ELISA測定相對親和力
如第24A圖所示,609-HC+Anti-CD137-Hu-LC、609-Fab-137-IgG4和137-Fab-609-IgG4均能有效結合PD1-ECD-His,EC50分別是0.2067 nM、0.2293 nM和1.415 nM。如第24B圖所示,Anti-CD137-Hu、609-Fab-137-IgG4和137-Fab-609-IgG4均能有效結合CD137-ECD-Fc,EC50分別是0.461 nM、0.3572 nM和0.2424 nM。上述結果顯示,609-Fab-137-IgG4和137-Fab-609-IgG4既能夠結合PD-1又能結合CD137,這表明它們是雙特異性抗體。
實施例6構建抗PD-1和CD40的雙特異性抗體
實施例6.1 序列
609是人源化的抗人PD-1單抗,其來源如實施例5.1所述。
MAb2.220(後文中稱為Anti-CD40)是抗人CD40的鼠源單抗,其重鏈可變區和輕鏈可變區胺基酸序列來自於US6312693中的SEQ ID NO:2和SEQ ID NO:1。
對Anti-CD40抗體的重鏈可變區和輕鏈可變區胺基酸序列進行分析,依據Kabat規則分別確定Anti-CD40抗體重鏈和輕鏈的抗原互補決定區和框架區。Anti-CD40抗體重鏈CDR的胺基酸序列為H-CDR1:TTGMQ(SEQ ID NO:99)、H-CDR2:WINTHSGVPKYVEDFKG(SEQ ID NO:100)和H-CDR3:SGNGNYDLAYFAY(SEQ ID NO:101),輕鏈CDR的胺基酸序列為L-CDR1:RASQSISDYLH(SEQ ID NO:102)、L-CDR2:YASHSIS(SEQ ID NO:103)和L-CDR3:QHGHSFPWT(SEQ ID NO:104)。
在https://www.ncbi.nlm.nih.gov/igblast/,將鼠源Anti-CD40抗體的重鏈可變區與人IgG胚系序列進行同源性比較,選擇IGHV7-4-1*02為重鏈CDR移植範本,將鼠源的Anti-CD40抗體的重鏈CDR移植入IGHV7-4-1*02骨架區,並在H-CDR3之後加入WGQGTLVTVSS(SEQ ID NO:151)作為第四個框架區,獲得CDR移植重鏈可變區序列。同樣地,將鼠源Anti-CD40抗體的輕鏈可變區與人IgG胚系序列同源性比較,選擇IGKV3-11*01為輕鏈CDR移植範本,將鼠源Anti-CD40抗體的輕鏈CDR移植入IGKV3-11*01的骨架區,並在L-CDR3之後加入FGQGTKVEIK(SEQ ID NO:152)作為第四個框架區,獲得CDR移植輕鏈可變區序列。在CDR移植可變區的基礎上,對一些框架區的胺基酸位點進行回復突變。在進行突變時,將胺基酸序列進行Kabat編碼,位元點的位置由Kabat碼指示。
較佳的,對於CDR移植重鏈可變區,根據Kabat編碼,將第2位元的V突變為I,將第28位的T突變為A,將第39位的Q突變為E,將第48位的M突變為I,將第76位的S突變為N,將第93位的A突變為V。對於CDR移植輕鏈可變區,將第43位的A突變為S,將第49位的Y突變為K,將第69位的T突變為S。
上述帶有突變位點的重鏈可變區和輕鏈可變區分別定義為人源化的重鏈可變區和輕鏈可變區,分別命名為Anti-CD40-Hu-VH和Anti-CD40-Hu-VL(SEQ ID NO:105和106)。
由上海生工生物工程有限公司合成編碼上述人源化的重鏈可變區和輕鏈可變區的DNA。將合成的人源化重鏈可變區與人IgG4(S228P)重鏈恆定區(SEQ ID NO:148)相連,獲得全長的人源化重鏈基因,命名為Anti-CD40-Hu-HC;將人源化輕鏈可變區與人Kappa鏈恆定區(SEQ ID NO:149)相連,獲得全長的人源化輕鏈基因,命名為Anti-CD40-Hu-LC。將Anti-CD40-Hu-HC和Anti-CD40-Hu-LC基因分別構建到pcDNA3.4表達載體中,表達純化抗體,所得抗體命名為Anti-CD40-Hu。
實施例6.2 共同輕鏈的選擇
用BLAST對609輕鏈可變區與Anti-CD40-Hu輕鏈可變區的胺基酸序列進行對比分析,結果顯示,兩者之間完全相同的胺基酸占比90% (Identities),性質相似的胺基酸占比96% (Positives)。
將609和Anti-CD40-Hu的重鏈和輕鏈基因按照下述方式進行組合:609-HC+Anti-CD40-Hu-LC和Anti-CD40-Hu-HC+609-LC,表達純化抗體,所得的抗體分別命名為609-HC+Anti-CD40-Hu-LC和Anti-CD40-Hu-HC+609-LC。
ELISA檢測方法如下:帶有多聚組胺酸標籤的人PD-1胞外段的重組蛋白購自Sino Biological(貨號:10377-H08H),帶多聚組胺酸標籤的人CD40胞外段的重組蛋白購自Sino Biological(貨號:10774-H08H),將這兩種重組蛋白名稱分別記作PD1-ECD-His和CD40-ECD-His。用PD1-ECD-His和CD40-ECD-His分別包被酶標板,包被濃度均為10 ng/孔。
如第25A圖所示,609和609-HC+Anti-CD40-Hu-LC均能夠有效結合PD1-ECD-His,EC50分別是0.1263nM和0.1387nM;而Anti-CD40-Hu和Anti-CD40-Hu-HC+609-LC不能有效結合PD1-ECD-His。如第25B圖所示,Anti-CD40-Hu能有效結合CD40-ECD-His,EC50是0.1104nM,而609、609-HC+Anti-CD40-Hu-LC和Anti-CD40-Hu-HC+609-LC均不能有效結合CD40-ECD-His。在此選擇Anti-CD40-Hu-LC(SEQ ID NO:107和108)作為共同輕鏈構建雙特異性抗體。
實施例6.3雙特異性抗體的構建
將609的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接Anti-CD40-Hu的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為609-Fab-40-IgG4(SEQ ID NO:109和110)。相似地,將Anti-CD40-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接609的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為40-Fab-609-IgG4(SEQ ID NO:111和112)。
將上述序列分別構建到pcDNA3.4表達載體中,將609-Fab-40-IgG4和40-Fab-609-IgG4表達載體分別與Anti-CD40-Hu-LC表達載體組合,表達純化抗體,所得的抗體分別命名為609-Fab-40-IgG4和40-Fab-609-IgG4(為簡明起見,此處只取重鏈的名字作為抗體的名稱)。
實施例6.4 ELISA測定相對親和力
如第26A圖所示,609-HC+Anti-CD40-Hu-LC、609-Fab-40-IgG4和40-Fab-609-IgG4均能有效結合PD1-ECD-His,EC50分別是0.1387nM、0.1723nM和1.017nM。如第26B圖所示,Anti-CD40-Hu、609-Fab-40-IgG4和40-Fab-609-IgG4均能有效結合CD40-ECD-His,EC50分別是0.1104 nM、0.1047 nM和0.09556 nM。上述結果顯示,609-Fab-40-IgG4和40-Fab-609-IgG4既能夠結合PD-1又能結合CD40,這表明它們是雙特異性抗體。
實施例7構建抗PD-1和其它靶點的雙特異性抗體
實施例7.1 序列
609是人源化的抗人PD-1單抗,其來源如實施例5.1所述。
從公開的文獻資料(Magdelaine-Beuzelin C, Kaas Q, Wehbi V, et al. Structure–function relationships of the variable domains of monoclonal antibodies approved for cancer treatment[J]. Critical reviews in oncology/hematology, 2007, 64(3): 210-225.)中獲得Cetuximab、Bevacizumab、Trastuzumab、Pertuzumab等抗體的重鏈可變區和輕鏈可變區胺基酸序列(SEQ ID NO:1-2、113-118)。10D1(後文中稱為Ipilimumab)是抗人CTLA-4單抗,其重鏈可變區和輕鏈可變區胺基酸序列來自於US20020086014A1中的SEQ ID NO:17和SEQ ID NO:7(即本揭露中的SEQ ID NO:119和120)。
由上海生工生物工程有限公司合成編碼上述重鏈可變區和輕鏈可變區的DNA。重鏈可變區和輕鏈可變區編碼序列分別與人IgG1重鏈恆定區(SEQ ID NO:147)和人Kappa輕鏈恆定區(SEQ ID NO:149)相連,構建成全長的抗體重鏈和輕鏈基因。上述抗體的重鏈基因分別命名為Cetuximab-HC、Bevacizumab-HC、Trastuzumab-HC、Pertuzumab-HC和Ipilimumab-HC,上述抗體的輕鏈基因分別命名為Cetuximab-LC、Bevacizumab-LC、Trastuzumab-LC、Pertuzumab-LC和Ipilimumab-LC。將上述重鏈和輕鏈基因分別構建到pcDNA3.4表達載體中,將名稱對應的重鏈和輕鏈基因進行組合,表達純化抗體,所得抗體分別命名為Cetuximab-IgG1、Bevacizumab-IgG1、Trastuzumab-IgG1、Pertuzumab-IgG1和Ipilimumab-IgG1。
5E7-Hu是人源化的抗人LAG-3抗體,其重鏈可變區和輕鏈可變區序列來自於PCT/CN2020/076023中的SEQ ID NO:26和SEQ ID NO:28(即本揭露中的SEQ ID NO:121和122),由上海生工生物工程有限公司合成編碼上述人源化的重鏈可變區和輕鏈可變區的DNA。將合成的人源化重鏈可變區與人IgG4(S228P)重鏈恆定區(SEQ ID NO:148)相連,獲得全長的人源化重鏈基因,命名為5E7-Hu-HC;將人源化輕鏈可變區與人Kappa鏈恆定區(SEQ ID NO:149)相連,獲得全長的人源化輕鏈基因,命名為5E7-Hu-LC。將5E7-Hu-HC和5E7-Hu-LC基因分別構建到pcDNA3.4表達載體中,表達純化抗體,所得抗體重新命名為5E7-Hu。
實施例7.2 共同輕鏈的選擇
實施例7.2.1 檢測雜合抗體與抗原結合的親和力
將609的重鏈和上述抗體的輕鏈基因分別組合,表達純化抗體,所得的抗體分別命名為609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC。
ELISA檢測方法如下:帶有多聚組胺酸標籤的人PD-1胞外段的重組蛋白購自Sino Biological(貨號:10377-H08H),將這種重組蛋白名稱記作PD1-ECD-His。用PD1-ECD-His包被酶標板,包被濃度均為10 ng/孔。如第27圖所示,Opdivo(購自Bristol-Myers Squibb)、609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC均能夠有效結合PD1-ECD-His,EC50分別是0.2887 nM、0.1100 nM、0.2424 nM、0.1530 nM、0.2244 nM、0.1547 nM和0.1709 nM;而609-HC+Trastuzumab-LC結合PD1-ECD-His的相對親和力較弱,EC50為0.7219 nM。因此,在此可以選擇Cetuximab-LC(SEQ ID NO:123和124)、Bevacizumab-LC、Pertuzumab-LC(SEQ ID NO:125和126)、Ipilimumab-LC(SEQ ID NO:127和128)和5E7-Hu-LC(SEQ ID NO:129和130)作為共同輕鏈構建相應的雙特異性抗體。其中,同型對照抗體為不結合PD-1的人IgG4抗體。
實施例7.2.2 檢測雜合抗體阻斷PD-1/PD-L1相互作用的能力
抗體阻斷PD-1/PD-L1相互作用的能力的檢測方法如下:自製帶有人Fc標籤的人PD-1和PD-L1的胞外區融合蛋白(製備方法如WO2018/137576A1中所述),將這兩種重組蛋白分別記作PD1-ECD-hFc和PD-L1-ECD-hFc。用無水DMSO將Biotin化標記試劑Biotin N-hydroxysuccinimide ester(購自Sigma,貨號/規格:H1759-100MG)配製成100mM的母液;根據PD-L1-ECD-hFc的分子量和濃度計算相應的物質的量濃度;取適當體積的PD-L1-ECD-hFc融合蛋白,計算物質的量之後,分別與Biotin N-hydroxysuccinimide ester按照1:20的比例混勻,室溫標記1小時;透析之後用紫外分光光度法測定蛋白濃度。用包被緩衝液將人PD-1-ECD-hFc稀釋到2 μg/mL,用排槍加到96孔ELISA酶標板中,100 μL/孔,室溫孵育4小時;用PBST清洗1次,用含1%BSA的PBST封閉,200 μL/孔,室溫孵育2小時;棄封閉液,拍乾,於4°C備用。在96孔板中將Biotin化的PD-L1-ECD-hFc用PBST+1%BSA(含有1%牛血清白蛋白的PBST溶液)稀釋至500 ng/mL;用上述稀釋好的Biotin化融合蛋白分別梯度稀釋抗PD-1抗體;將上述稀釋好的抗體和Biotin化融合蛋白的混合溶液轉移到上述用人PD-1-ECD-hFc包被好的ELISA板中,室溫孵育1小時;PBST洗板3次;加入用PBST+1%BSA以1:1000稀釋的Streptavidin-HRP(購自BD Biosciences),室溫孵育45分鐘;PBST洗板3次;加顯色液(TMB底物溶液),100 μL/孔,室溫孵育1-5分鐘;加終止液終止顯色反應,50 μL/孔;用酶標儀讀取OD450值;用GraphPad Prism6進行資料整理分析和作圖,計算IC50。
如第28圖所示,Opdivo、609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC均能有效阻斷PD-1和PD-L1之間的相互作用,IC50分別為0.05729 nM、0.1309 nM、0.1199 nM、0.1191 nM、0.1162 nM、0.09876 nM、0.1052 nM和0.1312 nM。其中,同型對照抗體為不結合PD-1的人IgG4抗體。
實施例7.2.3 測定雜合抗體增強混合淋巴細胞反應的能力
然後測定上述抗體增強混合淋巴細胞反應的能力。如第29圖所示,609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC均能有效刺激混合淋巴細胞反應分泌IL-2,EC50分別為0.08623 nM、0.2510 nM、0.1211 nM、0.5171 nM、0.2040 nM和0.09101 nM。其中,609-HC+Trastuzumab-LC不能有效刺激混合淋巴細胞反應分泌IL-2。同型對照抗體為不結合PD-1的人IgG4抗體。
實施例7.2.4 流式細胞法測定雜合抗體對細胞表面PD-1的結合能力
使用流式細胞法測定雜合抗體對細胞表面PD-1的結合能力。表達PD-1的TF-1細胞的建立過程如下:將全長的人PD-1基因(序列來自UniProt,Entry:Q15116)構建到慢病毒表達載體pLVX-Puro(購自Clontech)中。用Lipofectamine 3000(購自Thermo Fisher Scientific,貨號:L3000001)將慢病毒包裝載體和裝載目的基因的pLVX-Puro轉染入HEK293FT細胞中(購自Thermo Fisher Scientific,貨號:R70007),細胞培養箱中孵育48小時之後收集細胞培養上清,用0.45微米濾膜過濾除去細胞殘骸。用上述含有病毒顆粒的上清液感染TF-1細胞(購自ATCC
®,貨號CRL-2003
™),48小時之後用Puromycin處理細胞,篩選出穩定表達目的基因的細胞群體。穩定表達PD-1的TF-1細胞株命名為TF1-PD1。
流式細胞法檢測抗體對細胞結合的方法如下:將TF1-PD1細胞接種到圓底96孔板中(每孔20萬個細胞);離心後吸掉上清,加入梯度稀釋的抗體,室溫孵育半小時左右;細胞用PBS洗滌2遍;離心後吸掉上清,每孔加入以適當稀釋的Anti-Human IgG (Fc specific)-FITC抗體(購自Sigma,貨號:F9512),室溫孵育半小時左右;細胞用PBS洗滌2遍;吸掉上清後加入Fix Buffer I(購自BD Biosciences)以固定細胞,室溫孵育5分鐘;細胞用PBS洗滌2遍,最終用200μL PBS重懸細胞;在流式細胞儀上檢測FITC通道的螢光強度;用流式細胞儀自帶軟體處理實驗資料並匯出到Excel;用GraphPad Prism6進行資料分析和作圖,計算EC50。
如第30圖所示,609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC均能有效結合細胞表面的PD-1,EC50分別為0.3761nM、0.577 nM、0.5193 nM、0.4302 nM、0.4773 nM和0.3864 nM。其中,609-HC+Trastuzumab-LC對TF1-PD1的結合作用明顯弱於其它雜合抗體。同型對照抗體為不結合PD-1的人IgG4抗體。
實施例7.2.5 丙胺酸掃描研究609輕鏈可變區CDR在609結合PD-1中的作用
上述實驗結果顯示,609的重鏈與許多其它靶點抗體的輕鏈組合之後產生的雜合抗體仍然能夠有效結合PD-1分子,並且具有阻斷PD-1/PD-L1相互作用、刺激混合淋巴細胞反應和結合細胞表面PD-1的能力。在此,用丙胺酸掃描(Alanine Scanning)研究609輕鏈可變區CDR在609結合PD-1中的作用。方法如下:將609輕鏈CDR中的胺基酸分別突變為丙胺酸(CDR中原有的丙胺酸不做改變),然後609重鏈分別與這些輕鏈突變體組合後,按照上述實施例中的方法表達並純化抗體,然後按照上述實施例中的ELISA方法,測定上述抗體對PD-1的相對親和力。如表3中所示,609-HC+609-LC-R24A中的R24A表示第24位的精胺酸突變為丙胺酸,突變胺基酸的位置由Kabat編碼指示,其餘以此類推。
表3、609輕鏈可變區的丙胺酸掃描結果
如第31圖和表3所示,丙胺酸掃描結果顯示,輕鏈CDR胺基酸分別突變為丙胺酸後均沒有顯著影響抗體對PD-1的結合,這說明609主要通過重鏈結合PD-1分子,而對輕鏈依賴較小。
實施例7.3 抗PD-1和EGFR的雙特異性抗體的構建
將609的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接Cetuximab-IgG1的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為609-Fab-Cetuximab-IgG4(SEQ ID NO:131和132)。
將上述序列分別構建到pcDNA3.4表達載體中,將609-Fab-Cetuximab-IgG4與Cetuximab-LC表達載體組合,表達純化抗體,所得的抗體命名為609-Fab-Cetuximab-IgG4(為簡明起見,此處只取重鏈的名字作為抗體的名稱)。
ELISA檢測方法如下:帶有多聚組胺酸標籤的人PD-1胞外段的重組蛋白購自Sino Biological(貨號:10377-H08H),帶多聚組胺酸標籤的人EGFR胞外段的重組蛋白購自Sino Biological(貨號:10001-H08H),將這兩種重組蛋白名稱分別記作PD1-ECD-His和EGFR-ECD-His。用PD1-ECD-His和EGFR-ECD-His分別包被酶標板,包被濃度均分別為10 ng/孔和20 ng/孔。
如第32A圖所示,609-HC+Cetuximab-LC和609-Fab-Cetuximab-IgG4均能有效結合PD1-ECD-His,EC50分別是0.7172 nM和0.2616 nM。如第32B圖所示,Cetuximab-IgG1和609-Fab-Cetuximab-IgG4均能有效結合EGFR-ECD-His,EC50分別是0.07609 nM和0.09327 nM。上述結果顯示,609-Fab-Cetuximab-IgG4既能夠結合PD-1又能結合EGFR,這說明它是雙特異性抗體。
實施例7.4 抗PD-1和HER-2的雙特異性抗體的構建
將609的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接Pertuzumab-IgG1的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為609-Fab-Pertuzumab-IgG4(SEQ ID NO:133和134)。
將上述序列分別構建到pcDNA3.4表達載體中,將609-Fab-Pertuzumab-IgG4與Pertuzumab-LC表達載體組合,表達純化抗體,所得的抗體命名為609-Fab-Pertuzumab-IgG4(為簡明起見,此處只取重鏈的名字作為抗體的名稱)。
ELISA檢測方法如下:帶有多聚組胺酸標籤的人PD-1胞外段的重組蛋白購自Sino Biological(貨號:10377-H08H),帶有多聚組胺酸標籤的人Her-2胞外段的重組蛋白購自ACROBiosystems(貨號:HE2-H5225),將這兩種重組蛋白名稱分別記作PD1-ECD-His和HER2-ECD-His。用PD1-ECD-His和HER2-ECD-His分別包被酶標板,包被濃度均為10 ng/孔。
如第33A圖所示,609-HC+Pertuzumab-LC和609-Fab-Pertuzumab-IgG4均能有效結合PD1-ECD-His,EC50分別是0.1422 nM和0.1196 nM。如第33B圖所示,Pertuzumab-IgG1和609-Fab-Pertuzumab-IgG4均能有效結合HER2-ECD-His,EC50分別是0.5352 nM和2.616 nM。上述結果顯示,609-Fab-Pertuzumab-IgG4既能夠結合PD-1又能結合HER-2,這說明它是雙特異性抗體。
實施例7.5 抗PD-1和CTLA-4的雙特異性抗體的構建
實施例7.5.1雙特異性抗體的構建
將609的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接Ipilimumab-IgG1的重鏈可變區,最後再連接人IgG1的重鏈恆定區(CH1+CH2+CH3),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈基因命名為609-Fab-Ipilimumab-IgG1(SEQ ID NO:135和136)。相似地,將Ipilimumab-IgG1的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接609的重鏈可變區,最後再連接人IgG1的重鏈恆定區(CH1+CH2+CH3),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈基因命名為Ipilimumab-Fab-609-IgG1(SEQ ID NO:137和138)。
將609的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接Ipilimumab-IgG1的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為609-Fab-Ipilimumab-IgG4(SEQ ID NO:139和140)。相似地,將Ipilimumab-IgG1的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接609的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為Ipilimumab-Fab-609-IgG4(SEQ ID NO:141和142)。
將上述序列分別構建到pcDNA3.4表達載體中,將609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4分別與Ipilimumab-LC表達載體組合,表達純化抗體,所得的抗體分別命名為609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4(為簡明起見,此處只取重鏈的名字作為抗體的名稱)。
ELISA檢測方法如下:帶有多聚組胺酸標籤的人PD-1胞外段的重組蛋白購自Sino Biological(貨號:10377-H08H),帶有人Fc標籤的人CTLA-4胞外段的重組蛋白購自Sino Biological(貨號:11159-H31H5),將這兩種重組蛋白名稱分別記作PD1-ECD-His和CTLA4-ECD-Fc。用PD1-ECD-His和CTLA4-ECD-Fc分別包被酶標板,包被濃度均為10 ng/孔。
如第34A圖所示,609-HC+Ipilimumab-LC、609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4均能有效結合PD1-ECD-His,EC50分別是0.2337 nM、0.1734 nM、0.7954 nM、0.2078 nM和0.9643 nM。如第34B圖所示,Ipilimumab-IgG1、609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4均能有效結合CTLA4-ECD-Fc,EC50分別是0.8354 nM、2.123 nM、0.3376 nM、2.626 nM和0.392 nM。上述結果顯示,609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4既能夠結合PD-1又能結合CTLA-4,這說明它們是雙特異性抗體。
實施例7.5.2 測定抗PD-1和CTLA-4雙特異性抗體的功能活性
在RPMI1640中加入以下添加劑製成RPMI 1640完全培養基:10%胎牛血清;1% MEM Non-Essential Amino Acids Solution;1% Sodium Pyruvate;1%HEPES;1‰ 2-Mercaptoethanol;1% Penicillin-Streptomycin;1% GlutaMAX(上述添加劑購自Thermo Fisher Scientific)。用上述RPMI 1640完全培養基將新鮮分離的PBMC(購自Allcells,貨號:PB005-C)洗滌並重懸,加入一定量的超抗原金黃色葡萄球菌腸毒素B(staphylococcal enterotoxin B,SEB)。該超抗原為內部製備(SEB胺基酸序列來自https://www.uniprot.org/uniprot/P01552),使用大腸桿菌生產。將PBMC細胞懸液接種到圓底96孔細胞培養板中,每孔150μL懸液和20萬個細胞;在上述96孔板中加入50μL梯度稀釋的抗體;將96孔板置於37°C細胞培養箱中孵育4天。從96孔板中取適量細胞培養上清。然後按照標準操作流程檢測IL-2分泌。用雙抗體夾心法(sandwich ELISA)檢測上清中的IL-2(相關檢測用的配對抗體購自BD Biosciences)。用酶標儀(SpectraMax 190)讀取OD450,用GraphPad Prism6進行作圖並計算EC50。
表4、抗PD-1和CTLA-4雙特異性抗體的功能活性參數
抗體 | EC50 (nM) | Top |
609 | 0.0682 | 9269 |
609-HC+Ipilimumab-LC | 0.06578 | 8546 |
Ipilimumab-IgG1 | 0.07534 | 9700 |
Ipilimumab-Fab-609-IgG4 | 0.5292 | 9115 |
609-Fab-Ipilimumab-IgG4 | 0.1267 | 10441 |
Ipilimumab-Fab-609-IgG1 | 0.1173 | 11416 |
609-Fab-Ipilimumab-IgG1 | 0.05802 | 15237 |
609-HC+Ipilimumab-LC/Ipilimumab-IgG1 | 0.06863 | 14447 |
如第35圖和表4所示,609、609-HC+Ipilimumab-LC和Ipilimumab-IgG1具有相當的EC50和Top(高平臺),表明這三個抗體具有相近的功能活性。由高到低,抗PD-1和CTLA-4雙特異性抗體的功能活性排序如下:609-Fab-Ipilimumab-IgG1>Ipilimumab-Fab-609-IgG1>609-Fab-Ipilimumab-IgG4>Ipilimumab-Fab-609-IgG4。609-Fab-Ipilimumab-IgG1和Ipilimumab-Fab-609-IgG1的功能活性明顯優於單株抗體609和Ipilimumab-IgG1,以及雜合抗體609-HC+Ipilimumab-LC,而與609-HC+Ipilimumab-LC和Ipilimumab-IgG1聯合應用(609-HC+Ipilimumab-LC/Ipilimumab-IgG1表示兩種抗體以物質的量之比1:1聯合應用)的效果近似。
實施例7.5.3 測定抗PD-1和CTLA-4雙特異性抗體的ADCC活性
抗體依賴細胞介導的細胞毒作用(antibody-dependent cell-mediated cytotoxicity,ADCC)是人IgG抗體的普遍功能,ADCC強弱與抗體亞型相關。IgG1亞型的抗PD-1和CTLA-4雙特異性抗體可能會對表達PD-1的細胞產生潛在細胞毒性。在此,用新鮮分離的PBMC(購自Allcells,貨號:PB005-C)作為效應細胞,表達PD-1的TF-1細胞作為靶細胞,測定ADCC。
如第36圖所示,IgG4同型對照抗體(為不結合PD-1和CTLA-4的單抗)沒有體現出明顯的ADCC。Ipilimumab-IgG1也沒有體現出明顯的ADCC作用,因為TF1-PD1細胞不表達CTLA-4。609具有較弱的ADCC(重鏈恆定區為IgG4亞型,靶細胞裂解最高10%左右),609-IgG1具有較強的ADCC(將609的重鏈恆定區替換為IgG1亞型,靶細胞裂解最高50%左右),因為兩者的重鏈恆定區分別為IgG4和IgG1,而IgG1通常具有比IgG4更強的ADCC活性。609-Fab-Ipilimumab-IgG1的ADCC與609相似,Ipilimumab-Fab-609-IgG1的ADCC與609-IgG1相似。609-Fab-Ipilimumab-IgG1的ADCC明顯弱於Ipilimumab-Fab-609-IgG1,可能與雙特異性抗體的空間排序方式有關聯。
實施例7.5.4 抗PD-1和CTLA-4雙特異性抗體在大鼠體內的藥代動力學
採用實施例1.7.5中描述的方法測定抗PD-1和CTLA-4雙特異性抗體在大鼠體內的藥代動力學。不同之處在於,用抗PD-1和CTLA-4雙特異性抗體對應的兩種相關抗原(PD1-ECD-His和CTLA4-ECD-Fc的來源如實施例7.5.1中所述)分別包被酶標板。
如第37圖所示,計算出609-Fab-Ipilimumab-IgG1的半衰期為15.2天(以PD-1為抗原的檢測結果)和14.6天(以CTLA-4為抗原的檢測結果)。上述結果表明,609-Fab-Ipilimumab-IgG1具有良好的藥代動力學性質。
實施例7.5.5抗PD-1和CTLA-4雙特異性抗體在小鼠體內的抗腫瘤作用
人PD-1/CTLA-4雙轉基因小鼠(種系背景為C57BL/6)購自北京百奧賽圖科技股份有限公司,MC38小鼠結直腸癌細胞購自廣州吉妮歐生物科技有限公司。PD-1/CTLA-4雙轉基因小鼠中用人源PD-1及CTLA-4基因的胞外段替換了小鼠的同源部分,因此本揭露的雙特異性抗體能夠識別該轉基因小鼠中的PD-1和CTLA-4。具體實施步驟如下:將MC38在體外培養,培養基為含有10%血清的DMEM(血清和培養基購自Gibco);將培養的MC38細胞接種於人PD-1轉基因小鼠中,每隻小鼠皮下接種2×10
6個細胞;待接種的腫瘤細胞生長至體積接近100mm
3時,將小鼠隨機分組,每組8隻小鼠。各組小鼠藥物處理情況如下:對照組,僅注射生理鹽水;609組,注射10 mg/kg的抗PD-1抗體609;Yervoy組,注射10 mg/kg的抗CTLA-4陽性對照抗體Yervoy(由百時美施貴寶生產);609+Yervoy組,注射10 mg/kg的609和10 mg/kg的Yervoy;609-Fab-Ipilimumab-IgG1組,注射16 mg/kg的609-Fab-Ipilimumab-IgG1。考慮到雙特異性抗體和單株抗體的分子量存在差異,本實驗中藥物劑量按照等物質的量進行設定。隨後,按照上述設計好的方案給藥,每週給藥2次,共給藥4次,每週測定腫瘤體積2次。最終,測定的各組腫瘤隨時間的生長曲線如第38圖所示。
結果顯示,在第14天實驗結束時,609、Yervoy、609 + Yervoy和609-Fab-Ipilimumab-IgG1各組的抑瘤率分別為48.6%、79.1%、85.9%和92.2%(抑瘤率=(對照組平均體積-實驗組平均體積)/對照組平均體積×100%)。與609和Yervoy單藥相比,609-Fab-Ipilimumab-IgG1能夠更加有效地抑制腫瘤生長。609-Fab-Ipilimumab-IgG1與609和Yervoy聯合應用的治療效果相當。
實施例7.6 抗PD-1和LAG-3的雙特異性抗體的構建
實施例7.6.1 雙特異性抗體的構建
將609的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接5E7-Hu的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為609-Fab-5E7-IgG4(SEQ ID NO:143和144)。相似地,將5E7-Hu的重鏈可變區與人IgG4的CH1結構域相連,然後再通過人工連接子(在此使用的連接子是三個串聯的GGGGS)連接609的重鏈可變區,最後再連接人IgG4的重鏈恆定區(CH1+CH2+CH3,鉸鏈區含有S228P突變),通過此程式構建成的含有兩個重鏈可變區和兩個CH1結構域的長重鏈命名為5E7-Fab-609-IgG4(SEQ ID NO:145和146)。
將上述序列分別構建到pcDNA3.4表達載體中,將609-Fab-5E7-IgG4和5E7-Fab-609-IgG4分別與5E7-Hu-LC表達載體組合,利用上述實施例中描述的方法表達並純化抗體,所得的抗體分別命名為609-Fab-5E7-IgG4和5E7-Fab-609-IgG4(為簡明起見,此處只取重鏈的名字作為抗體的名稱)。
ELISA檢測方法如下:帶有多聚組胺酸標籤的人PD-1胞外段的重組蛋白購自Sino Biological(貨號:10377-H08H),帶有多聚組胺酸標籤的人LAG-3胞外段的重組蛋白購自Sino Biological(貨號:16498-H08H),將這兩種重組蛋白名稱分別記作PD1-ECD-His和LAG3-ECD-His。用PD1-ECD-His和LAG3-ECD-His分別包被酶標板,包被濃度均為10ng/孔。
如第39A圖所示,609-HC+5E7-Hu-LC、609-Fab-5E7-IgG4和5E7-Fab-609-IgG4均能有效結合PD1-ECD-His,EC50分別是0.1523 nM、0.161 nM和0.8138 nM。如第39B圖所示,5E7-Hu、609-Fab-5E7-IgG4和5E7-Fab-609-IgG4均能有效結合LAG3-ECD-His,EC50分別是0.1472 nM、0.2082 nM和0.1529 nM。上述結果顯示,609-Fab-5E7-IgG4和5E7-Fab-609-IgG4既能夠結合PD-1又能結合LAG-3,這說明它們是雙特異性抗體。
實施例7.6.2 測定抗PD-1和LAG-3雙特異性抗體同時結合兩種抗原的能力
用LAG3-ECD-His包被微孔板。用含1%牛血清白蛋白的PBST將待測抗體進行梯度稀釋,然後轉移到上述微孔板中,室溫孵育半小時左右。後續實驗步驟與實施例1.7.4相同。
如第40圖所示,609-Fab-5E7-IgG4在結合LAG-3之後仍然能夠有效地繼續結合人PD-1,EC50為0.5294 nM。5E7-Hu和609-HC+5E7-Hu-LC都不能同時結合PD-1和LAG-3。
實施例7.6.3 測定抗PD-1和LAG-3雙特異性抗體的功能活性
在此,按照實施例7.5.2中所述的方法測定抗PD-1和LAG-3雙特異性抗體的功能活性。
表5、抗PD-1和LAG-3雙特異性抗體的功能活性參數
抗體 | EC50(nM) | Top |
5E7-Hu | 0.2835 | 3851 |
609 | 0.0271 | 7473 |
609-HC+5E7-Hu-LC | 0.0247 | 6836 |
609-Fab-5E7-IgG4 | 0.1333 | 12765 |
5E7-Hu/609-HC+5E7-Hu-LC | 0.0719 | 11661 |
如第41圖和表5所示,5E7-Hu僅能微弱刺激IL-2的分泌。609和609-HC+5E7-Hu-LC具有相當的EC50和Top(高平臺),表明這二者具有相近的功能活性。在濃度大於1nM時,609-Fab-5E7-IgG4刺激IL-2分泌的能力明顯高於單株抗體5E7-Hu和609,以及雜合抗體609-HC+5E7-Hu-LC,而與5E7-Hu和609-HC+5E7-Hu-LC聯合應用(5E7-Hu/609-HC+5E7-Hu-LC表示兩種抗體以物質的量之比1:1聯合應用)的效果近似。
實施例7.6.4抗PD-1和LAG-3雙特異性抗體在小鼠體內的抗腫瘤作用
人PD-1/LAG-3雙轉基因小鼠(種系背景為C57BL/6)購自北京百奧賽圖科技股份有限公司,MC38小鼠結直腸癌細胞購自廣州吉妮歐生物科技有限公司。PD-1/LAG-3雙轉基因小鼠中用人源PD-1及LAG-3基因的胞外段替換了小鼠的同源部分,因此本揭露的雙特異性抗體能夠識別該轉基因小鼠中的PD-1和LAG-3。具體實施步驟如下:將MC38在體外培養,培養基為含有10%血清的DMEM(血清和培養基購自Gibco);將培養的MC38細胞接種於人PD-1轉基因小鼠中,每隻小鼠皮下接種2×10
6個細胞;待接種的腫瘤細胞生長至體積接近100mm3時,將小鼠隨機分組,每組8隻小鼠。各組小鼠藥物處理情況如下:對照組,僅注射生理鹽水;609組,注射20 mg/kg的抗PD-1抗體609;5E7-Hu組,注射20 mg/kg的抗LAG-3抗體5E7-Hu;609 + 5E7-Hu組,注射20 mg/kg的609和20 mg/kg的5E7-Hu;609-Fab-5E7-IgG4組,注射32 mg/kg的609-Fab-5E7-IgG4。考慮到雙特異性抗體和單株抗體的分子量存在差異,本實驗中藥物劑量按照等物質的量進行設定。隨後,按照上述設計好的方案給藥,每週給藥2次,共給藥4次,每週測定腫瘤體積2次。最終,測定的各組腫瘤隨時間的生長曲線如第42圖所示。
結果顯示,在第14天實驗結束時,609、5E7-Hu、609 + 5E7-Hu和609-Fab-5E7-IgG4各組的抑瘤率分別為70.8%、13.1%、71.5%和82.8%(抑瘤率=(對照組平均體積-實驗組平均體積)/對照組平均體積×100%)。與609和5E7-Hu單藥相比,609-Fab-5E7-IgG4能夠更加有效地抑制腫瘤生長。609和5E7-Hu聯合沒有比609單藥的藥效更優,因此可以推測,609-Fab-5E7-IgG4作為抗PD-1和LAG-3的雙特異性抗體展現出協同效應。
實施例7.7 檢驗雜合抗體的特異性
ELISA檢測方法如下:用上述實施例中提到的相關抗原(EGFR-ECD-His、VEGF165-His、HER2-ECD-His、LAG3-ECD-His和CTLA4-ECD-Fc)分別包被酶標板,實驗條件如前所述,然後檢測609重鏈與其它靶點抗體輕鏈的雜合抗體能否識別這些靶點。Cetuximab-IgG1、601、Trastuzumab-IgG1、5E7-Hu和Ipilimumab-IgG1等抗體的來源如上述實施例中所述(其中Trastuzumab-IgG1的可變區來源如實施例7.1中所述,恆定區與Ipilimumab-IgG1相同,製備方法與其它抗體相同),在此分別作為結合各種抗原的陽性對照抗體。
第43A圖- 第43E圖顯示,609重鏈與其它靶點抗體輕鏈的雜合抗體不能識別其它靶點,這表明這些雜合抗體具有較好的特異性。
實施例7.8 HPLC-SEC
第44A圖表示609-Fab-Cetuximab-IgG4的HPLC-SEC圖譜,主峰占比99.13%。第44B圖表示609-Fab-Pertuzumab-IgG4的HPLC-SEC圖譜,主峰占比99.2%。第44C圖表示609-Fab-Ipilimumab-IgG1的HPLC-SEC圖譜,主峰占比99.3%。第44D圖表示Ipilimumab-Fab-609-IgG1的HPLC-SEC圖譜,主峰占比99.2%。第44E圖表示609-Fab-Ipilimumab-IgG4的HPLC-SEC圖譜,主峰占比99.3%。第44F圖表示Ipilimumab-Fab-609-IgG4的HPLC-SEC圖譜,主峰占比99.1%。圖44G表示609-Fab-5E7-IgG4的HPLC-SEC圖譜,主峰占比99.2%。第44H圖表示5E7-Fab-609-IgG4的HPLC-SEC圖譜,主峰占比99.0%。
實施例7.9 HPLC-IEC
第45A圖表示609-Fab-Ipilimumab-IgG1的HPLC-IEC圖譜,主峰占比83.52%。該結果表明,609-Fab-Ipilimumab-IgG1電荷異質性良好。
第45B圖表示609-Fab-5E7-IgG4的HPLC-IEC圖譜,主峰占比85.43%。該結果表明,609-Fab-5E7-IgG4電荷異質性良好。
實施例7.10 CE-SDS
第46A圖和第46B圖分別表示609-Fab-Ipilimumab-IgG1的NR-CE-SDS和R-CE-SDS的圖譜,NR-CE-SDS圖譜中主峰Peak13占比97.02%;R-CE-SDS圖譜中兩個主峰Peak2(對應輕鏈)和Peak12(對應長重鏈)分別占比39.14%和59.13%,兩者峰面積之比為2:3.0。R-CE-SDS中,609-Fab-Ipilimumab-IgG1的輕鏈和長重鏈的峰面積之比符合預期。
第46C圖和第46D圖分別表示609-Fab-5E7-IgG4的NR-CE-SDS和R-CE-SDS的圖譜,NR-CE-SDS圖譜中主峰Peak11占比94.73%;R-CE-SDS圖譜中兩個主峰Peak7(對應輕鏈)和Peak16(對應長重鏈)分別占比38.32%和59.58%,兩者峰面積之比為2:3.1。R-CE-SDS中,609-Fab-5E7-IgG4的輕鏈和長重鏈的峰面積之比符合預期。
無
第1圖為本揭露的雙特異性抗體的結構示意圖,其中,VH-A表示第一抗體的重鏈可變區,VH-B表示第二抗體的重鏈可變區,VL表示共同輕鏈的輕鏈可變區,CH1、CH2和CH3是重鏈恆定區的三個結構域,CL是共同輕鏈的輕鏈恆定區,兩條重鏈之間的線段表示二硫鍵,重鏈和輕鏈之間的線段也表示二硫鍵,靠近多肽鏈N末端的CH1和VH-A之間的線段表示人工設計的連接子,靠近多肽鏈C末端的CH1和CH2之間的線段表示抗體天然的連接子和鉸鏈區(如果重鏈是人IgG4亞型,鉸鏈區會含有S228P點突變)。
第2A圖和第2B圖為601和20-Hu及其雜合抗體的ELISA結果。
第3A圖和第3B圖為20-Fab-601-IgG4-V94L和601-Fab-20-IgG4-V94L的ELISA結果。
第4A圖和第4B圖為20-Fab-601-IgG4-V94L的HPLC-SEC圖譜。
第5A圖和第5B圖為20-Fab-601-IgG4-V94L的HPLC-IEC圖譜。
第6A圖-第6D圖為20-Fab-601-IgG4-V94L的CE-SDS圖譜。
第7A圖和第7B圖為20-Fab-601-IgG4-V94L的DSC圖譜。
第8圖為測定20-Fab-0313-IgG4-V94L對VEGF生物活性的中和能力的結果。
第9A圖和第9B圖為評估20-Fab-0313-IgG4-V94L增強MLR的功能活性的結果。
第10圖為評估20-Fab-0313-IgG4-V94L同時結合PD-1和VEGF165的能力。
第11圖為20-Fab-0313-IgG4-V94L的藥代動力學性質。
第12圖為抗PD-1和VEGF雙特異性抗體在小鼠體內的抗腫瘤生長曲線。
第13A圖和第13B圖為1D11-Hu 和14-Hu及其雜合抗體的ELISA結果。
第14圖為mAb127和14-Hu及其雜合抗體的ELISA結果。
第15A圖和第15B圖為14-Fab-1D11-IgG4、1D11-Fab-14-IgG4、14-Fab-127-IgG4和127-Fab-14-IgG4的ELISA結果。
第16A圖和第16B圖為評估14-Fab-127-IgG4增強MLR的功能活性的結果。
第17圖為評估14-Fab-127-IgG4同時結合TGF-β1和PD-1的能力。
第18圖為抗PD-1和TGF-beta雙特異性抗體在小鼠體內的抗腫瘤生長曲線。
第19A圖和第19B圖為19H6-Hu和Anti-CD47B-Hu及其雜合抗體的ELISA結果。
第20A圖和第20B圖為CD47B-Fab-19H6-IgG1和19H6-Fab-CD47B-IgG1的ELISA結果。
第21A圖和第21B圖為19H6-Hu和94-Hu及其雜合抗體的ELISA結果。
第22A圖和第22B圖為94-Fab-19H6-IgG1-LALA和19H6-Fab-94-IgG1-LALA的ELISA結果。
第23A圖和第23B圖為609和Anti-CD137-Hu及其雜合抗體的ELISA結果。
第24A圖和第24B圖為609-Fab-137-IgG4和137-Fab-609-IgG4的ELISA結果。
第25A圖和第25B圖為609和Anti-CD40-Hu及其雜合抗體的ELISA結果。
第26A圖和第26B圖為609-Fab-40-IgG4和40-Fab-609-IgG4的ELISA結果。
第27圖為609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC的ELISA結果。
第28圖為609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC阻斷PD-1/PD-L1相互作用的能力結果。
第29圖為609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Trastuzumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC增強混合淋巴細胞反應的能力結果。
第30圖為流式細胞法測定609、609-HC+Cetuximab-LC、609-HC+Bevacizumab-LC、609-HC+Pertuzumab-LC、609-HC+Ipilimumab-LC和609-HC+5E7-Hu-LC對細胞表面PD-1的結合能力。
第31圖為609輕鏈可變區的丙胺酸掃描結果。
第32A圖和第32B圖為609-Fab-Cetuximab-IgG4的ELISA結果。
第33A圖和第33 B圖為609-Fab-Pertuzumab-IgG4的ELISA結果。
第34A圖和第34B圖為609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4和Ipilimumab-Fab-609-IgG4的ELISA結果。
第35圖為抗PD-1和CTLA-4雙特異性抗體的功能活性測定結果。
第36圖為抗PD-1和CTLA-4雙特異性抗體的ADCC活性測定結果。
第37圖為抗PD-1和CTLA-4雙特異性抗體在大鼠體內的藥代動力學。
第38圖為抗PD-1和CTLA-4雙特異性抗體在小鼠體內的抗腫瘤生長曲線。
第39A圖和第39B圖為609-Fab-5E7-IgG4和5E7-Fab-609-IgG4的ELISA結果。
第4 0圖為測定PD-1和LAG-3雙特異性抗體同時結合兩種抗原的能力。
第41圖為抗PD-1和LAG-3雙特異性抗體的功能活性測定。
第42圖為抗PD-1和LAG-3雙特異性抗體在小鼠體內的抗腫瘤生長曲線。
第43A圖-第43E圖為雜合抗體的特異性檢驗。
第44A圖-第44H圖為609-Fab-Cetuximab-IgG4、609-Fab-Pertuzumab-IgG4、609-Fab-Ipilimumab-IgG1、Ipilimumab-Fab-609-IgG1、609-Fab-Ipilimumab-IgG4、Ipilimumab-Fab-609-IgG4、609-Fab-5E7-IgG4和5E7-Fab-609-IgG4的HPLC-SEC圖譜。
第45A圖和第45B圖為609-Fab-Ipilimumab-IgG1和609-Fab-5E7-IgG4的HPLC-IEC圖譜。
第46A圖-第46D圖為609-Fab-Ipilimumab-IgG1和609-Fab-5E7-IgG4的NR-CE-SDS和R-CE-SDS的圖譜。
國內寄存資訊(請依寄存機構、日期、號碼順序註記)
無
國外寄存資訊(請依寄存國家、機構、日期、號碼順序註記)
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[序列表] <![CDATA[<110> 三生國健藥業(上海)股份有限公司]]> <![CDATA[<120> PD-1/TGF-beta四價雙特異性抗體、其製備方法和用途]]> <![CDATA[<150> CN 202010357134.4]]> <![CDATA[<151> 2020-04-29]]> <![CDATA[<160> 153 ]]> <![CDATA[<170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211> 123]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 1]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 2]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 2]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 3]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 3]]> Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Glu Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Leu Phe Tyr Cys 85 90 95 Ala Ser Pro Tyr Gly His Tyr Gly Phe Glu Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <![CDATA[<210> 4]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 4]]> Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Phe 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg 100 105 <![CDATA[<210> 5]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 5]]> Asn Tyr Asp Met Ser 1 5 <![CDATA[<210> 6]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 6]]> Thr Ile Ser Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly <![CDATA[<210> 7]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 7]]> Pro Tyr Gly His Tyr Gly Phe Glu Tyr 1 5 <![CDATA[<210> 8]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 8]]> Ser Ala Ser Gln Gly Ile Ser Asn Phe Leu Ser 1 5 10 <![CDATA[<210> 9]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 9]]> Tyr Thr Ser Ser Leu His Ser 1 5 <![CDATA[<210> 10]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 10]]> Gln Gln Tyr Ser Asn Leu Pro Trp Thr 1 5 <![CDATA[<210> 11]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 11]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Tyr Gly His Tyr Gly Phe Glu Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 12]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 12]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Phe 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 13]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 13]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 14]]> <![CDATA[<211> 642]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 14]]> gacatccaga tgacccagtc ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgct ccgcctccca ggacatctcc aactacctga actggtacca gcagaagccc 120 ggcaaggccc ccaaggtgct gatctacttc acctcctccc tgcactccgg cgtgcccagc 180 aggttctccg gcagcggctc cggcaccgac ttcaccctga ccatctccag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcag tactccaccc tgccctggac cttcggccag 300 ggcaccaagg tggagatcaa gaggaccgtg gccgccccct ccgtgttcat cttccccccc 360 tccgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caactcccag 480 gagagcgtga ccgagcagga ctccaaggac tccacctaca gcctgagctc caccctgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600 ctgtcctccc ccgtgaccaa gtccttcaac aggggcgagt gc 642 <![CDATA[<210> 15]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 15]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 16]]> <![CDATA[<211> 681]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 16]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Tyr Gly His Tyr Gly Phe Glu Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly 225 230 235 240 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 245 250 255 Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro 260 265 270 Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu 275 280 285 Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp 290 295 300 Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 305 310 315 320 Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro His Tyr Tyr Gly Ser 325 330 335 Ser His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 340 345 350 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 355 360 365 Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 370 375 380 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 385 390 395 400 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 405 410 415 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 420 425 430 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 435 440 445 Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 450 455 460 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 465 470 475 480 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 485 490 495 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 500 505 510 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 515 520 525 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 530 535 540 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 545 550 555 560 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 565 570 575 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 580 585 590 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 595 600 605 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 610 615 620 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 625 630 635 640 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 645 650 655 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 660 665 670 Lys Ser Leu Ser Leu Ser Leu Gly Lys 675 680 <![CDATA[<210> 17]]> <![CDATA[<211> 2043]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 17]]> gaggtccagc tggtggagag cggaggagga ctggtgcagc ccggcggctc tctgaggctg 60 agctgtgccg ctagcggctt cgtcttctcc aactacgaca tgagctgggt gaggcaagcc 120 cccggcaaga ggctggagtg ggtggctaca atcagcggcg gcggaggcta cacatactac 180 tccgactccg tgaagggaag gttcactatc tctagggaca acgccaagaa ctctctgtat 240 ctgcagatga actctctgag agccgaggat acagctgtgt actactgtgc ctccccatac 300 ggccactacg gcttcgagta ctggggccaa ggcactctgg tcactgtgag cagcgcaagt 360 accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 420 gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 480 tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 540 tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 600 tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtggg tggaggcggt 660 tcaggcggag gtggcagcgg aggtggcggg agtgaggtgc agctggtgga gtctggcggc 720 ggactggtgc agcccggcgg cagcctgcgc ctgtcctgcg ctgcctccgg ctacaccttc 780 accaactacg gcatgaactg ggtgaggcag gcccccggaa agggcctgga gtgggtgggc 840 tggatcaaca cctacaccgg cgagcccacc tacgccgctg actttaagcg caggtttacc 900 ttctccctgg acacctccaa gtccaccgcc tacctgcaga tgaacagcct gagggccgag 960 gacaccgccg tgtactactg cgccaagtac ccccactact atggctccag ccactggtac 1020 tttgacgtgt ggggccaggg caccctggtc acagtgtcta gtgcctccac caagggccca 1080 tcggtcttcc ccctggcacc ctgctccagg agcacctctg agtccacagc ggccctgggc 1140 tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 1200 accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 1260 agcgtggtga ccgtgccctc cagcagcttg ggcaccaaga catatacctg taatgtggat 1320 cacaagcctt ccaatacaaa agtggacaag agagttgagt ccaagtacgg cccaccatgt 1380 cctccatgtc cagcccctga atttttgggc gggccttctg tctttctgtt tcctcctaaa 1440 cctaaagata ccctgatgat cagccgcaca cccgaagtca cttgtgtggt cgtggatgtg 1500 tctcaggaag atcccgaagt gcagtttaac tggtatgtcg atggcgtgga agtgcataat 1560 gccaaaacta agccccgcga agaacagttc aacagcactt atcgggtcgt gtctgtgctc 1620 acagtcctcc atcaggattg gctgaatggg aaagaatata agtgcaaggt gagcaataag 1680 ggcctcccca gcagcatcga gaagactatt agcaaagcca aagggcagcc acgggaaccc 1740 caggtgtaca ctctgccccc ctctcaggag gagatgacta aaaatcaggt ctctctgact 1800 tgtctggtga aagggtttta tcccagcgac attgccgtgg agtgggagtc taatggccag 1860 cccgagaata attataagac aactcccccc gtcctggact ctgacggcag ctttttcctg 1920 tattctcggc tgacagtgga caaaagtcgc tggcaggagg gcaatgtctt tagttgcagt 1980 gtcatgcatg aggccctgca caatcactat acacagaaaa gcctgtctct gagtctgggc 2040 aaa 2043 <![CDATA[<210> 18]]> <![CDATA[<211> 681]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 18]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 225 230 235 240 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 245 250 255 Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Asn Tyr Asp Met Ser Trp 260 265 270 Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser 275 280 285 Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val Lys Gly Arg Phe 290 295 300 Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn 305 310 315 320 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Pro Tyr 325 330 335 Gly His Tyr Gly Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 340 345 350 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 355 360 365 Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 370 375 380 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 385 390 395 400 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 405 410 415 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 420 425 430 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 435 440 445 Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 450 455 460 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 465 470 475 480 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 485 490 495 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 500 505 510 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 515 520 525 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 530 535 540 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 545 550 555 560 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 565 570 575 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 580 585 590 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 595 600 605 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 610 615 620 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 625 630 635 640 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 645 650 655 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 660 665 670 Lys Ser Leu Ser Leu Ser Leu Gly Lys 675 680 <![CDATA[<210> 19]]> <![CDATA[<211> 2043]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 19]]> gaggtgcagc tggtggagtc tggcggcgga ctggtgcagc ccggcggcag cctgcgcctg 60 tcctgcgctg cctccggcta caccttcacc aactacggca tgaactgggt gaggcaggcc 120 cccggaaagg gcctggagtg ggtgggctgg atcaacacct acaccggcga gcccacctac 180 gccgctgact ttaagcgcag gtttaccttc tccctggaca cctccaagtc caccgcctac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caagtacccc 300 cactactatg gctccagcca ctggtacttt gacgtgtggg gccagggcac cctggtgact 360 gtgagcagcg caagtaccaa gggacctagt gttttccctc ttgcaccttg ctccaggtca 420 acatcagagt ccacagctgc tcttggatgt ctcgttaagg actacttccc agagccagtt 480 accgtatcct ggaactccgg agctttgaca agcggcgttc atacattccc agctgtgttg 540 cagagttctg ggttgtacag tttgagctca gtggtgaccg tgccttcatc ttctttgggc 600 actaagacct acacctgcaa cgtggatcac aagccaagca acaccaaggt ggataagagg 660 gtgggtggag gcggttcagg cggaggtggc agcggaggtg gcgggagtga ggtccagctg 720 gtggagagcg gaggaggact ggtgcagccc ggcggctctc tgaggctgag ctgtgccgct 780 agcggcttcg tcttctccaa ctacgacatg agctgggtga ggcaagcccc cggcaagagg 840 ctggagtggg tggctacaat cagcggcggc ggaggctaca catactactc cgactccgtg 900 aagggaaggt tcactatctc tagggacaac gccaagaact ctctgtatct gcagatgaac 960 tctctgagag ccgaggatac agctgtgtac tactgtgcct ccccatacgg ccactacggc 1020 ttcgagtact ggggccaagg cactctggtc acagtgtcta gtgcctccac caagggccca 1080 tcggtcttcc ccctggcacc ctgctccagg agcacctctg agtccacagc ggccctgggc 1140 tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 1200 accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 1260 agcgtggtga ccgtgccctc cagcagcttg ggcaccaaga catatacctg taatgtggat 1320 cacaagcctt ccaatacaaa agtggacaag agagttgagt ccaagtacgg cccaccatgt 1380 cctccatgtc cagcccctga atttttgggc gggccttctg tctttctgtt tcctcctaaa 1440 cctaaagata ccctgatgat cagccgcaca cccgaagtca cttgtgtggt cgtggatgtg 1500 tctcaggaag atcccgaagt gcagtttaac tggtatgtcg atggcgtgga agtgcataat 1560 gccaaaacta agccccgcga agaacagttc aacagcactt atcgggtcgt gtctgtgctc 1620 acagtcctcc atcaggattg gctgaatggg aaagaatata agtgcaaggt gagcaataag 1680 ggcctcccca gcagcatcga gaagactatt agcaaagcca aagggcagcc acgggaaccc 1740 caggtgtaca ctctgccccc ctctcaggag gagatgacta aaaatcaggt ctctctgact 1800 tgtctggtga aagggtttta tcccagcgac attgccgtgg agtgggagtc taatggccag 1860 cccgagaata attataagac aactcccccc gtcctggact ctgacggcag ctttttcctg 1920 tattctcggc tgacagtgga caaaagtcgc tggcaggagg gcaatgtctt tagttgcagt 1980 gtcatgcatg aggccctgca caatcactat acacagaaaa gcctgtctct gagtctgggc 2040 aaa 2043 <![CDATA[<210> 20]]> <![CDATA[<211> 123]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 20]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 21]]> <![CDATA[<211> 681]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 21]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Gly Tyr Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Tyr Gly His Tyr Gly Phe Glu Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly 225 230 235 240 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 245 250 255 Gly Tyr Asp Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro 260 265 270 Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu 275 280 285 Pro Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp 290 295 300 Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 305 310 315 320 Asp Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr 325 330 335 Ser His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 340 345 350 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 355 360 365 Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 370 375 380 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 385 390 395 400 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 405 410 415 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 420 425 430 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 435 440 445 Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 450 455 460 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 465 470 475 480 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 485 490 495 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 500 505 510 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 515 520 525 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 530 535 540 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 545 550 555 560 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 565 570 575 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 580 585 590 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 595 600 605 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 610 615 620 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 625 630 635 640 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 645 650 655 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 660 665 670 Lys Ser Leu Ser Leu Ser Leu Gly Lys 675 680 <![CDATA[<210> 22]]> <![CDATA[<211> 2043]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 22]]> gaggtccagc tggtggagag cggaggagga ctggtgcagc ccggcggctc tctgaggctg 60 agctgtgccg ctagcggctt cgtcttctcc aactacgaca tgagctgggt gaggcaagcc 120 cccggcaaga ggctggagtg ggtggctaca atcagcggcg gcggaggcta cacatactac 180 tccgactccg tgaagggaag gttcactatc tctagggaca acgccaagaa ctctctgtat 240 ctgcagatga actctctgag agccgaggat acagctgtgt actactgtgc ctccccatac 300 ggccactacg gcttcgagta ctggggccaa ggcactctgg tcactgtgag cagcgcaagt 360 accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 420 gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 480 tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 540 tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 600 tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtggg tggaggcggt 660 tcaggcggag gtggcagcgg aggtggcggg agtgaggtgc agctggtgga gtctggagga 720 ggcctggtgc agcctggcgg ctctctgaga ctgtcttgcg ctgctagtgg atatgatttt 780 acacattatg gcatgaactg ggtgagacag gctccaggca agggcctgga atgggtgggc 840 tggattaata cctatacagg cgaacctacc tacgccgctg attttaagag aagattcacc 900 ttttctctgg atacatctaa gagcacagct tacctgcaga tgaacagcct gcgggccgag 960 gacaccgccg tgtactactg cgccaagtac ccctactact acggcacctc ccactggtac 1020 ttcgacgtgt ggggccaggg caccctggtg accgtgtcct ccgcctccac caagggccca 1080 tcggtcttcc ccctggcacc ctgctccagg agcacctctg agtccacagc ggccctgggc 1140 tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 1200 accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 1260 agcgtggtga ccgtgccctc cagcagcttg ggcaccaaga catatacctg taatgtggat 1320 cacaagcctt ccaatacaaa agtggacaag agagttgagt ccaagtacgg cccaccatgt 1380 cctccatgtc cagcccctga atttttgggc gggccttctg tctttctgtt tcctcctaaa 1440 cctaaagata ccctgatgat cagccgcaca cccgaagtca cttgtgtggt cgtggatgtg 1500 tctcaggaag atcccgaagt gcagtttaac tggtatgtcg atggcgtgga agtgcataat 1560 gccaaaacta agccccgcga agaacagttc aacagcactt atcgggtcgt gtctgtgctc 1620 acagtcctcc atcaggattg gctgaatggg aaagaatata agtgcaaggt gagcaataag 1680 ggcctcccca gcagcatcga gaagactatt agcaaagcca aagggcagcc acgggaaccc 1740 caggtgtaca ctctgccccc ctctcaggag gagatgacta aaaatcaggt ctctctgact 1800 tgtctggtga aagggtttta tcccagcgac attgccgtgg agtgggagtc taatggccag 1860 cccgagaata attataagac aactcccccc gtcctggact ctgacggcag ctttttcctg 1920 tattctcggc tgacagtgga caaaagtcgc tggcaggagg gcaatgtctt tagttgcagt 1980 gtcatgcatg aggccctgca caatcactat acacagaaaa gcctgtctct gagtctgggc 2040 aaa 2043 <![CDATA[<210> 23]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 23]]> Thr Tyr Trp Met Asn 1 5 <![CDATA[<210> 24]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 24]]> Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn Tyr Asn Glu Met Phe Glu 1 5 10 15 Gly <![CDATA[<210> 25]]> <![CDATA[<211> 12]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 25]]> Gly Asp Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr 1 5 10 <![CDATA[<210> 26]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 26]]> Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His 1 5 10 15 <![CDATA[<210> 27]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 27]]> Leu Ala Ser Asn Leu Glu Ser 1 5 <![CDATA[<210> 28]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 28]]> Gln Gln Asn Asn Glu Asp Pro Leu Thr 1 5 <![CDATA[<210> 29]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 29]]> 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 Ile Phe Ile Thr Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn Tyr Asn Glu Met Phe 50 55 60 Glu Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 30]]> <![CDATA[<211> 111]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 30]]> Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Leu Lys 100 105 110 <![CDATA[<210> 31]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 31]]> 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 Ser Glu 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gln Ile Phe Pro Ala Leu Gly Ser Thr Asn Tyr Asn Glu Met Tyr 50 55 60 Glu Gly Arg Val Thr Met Thr Thr 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 Ile Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 32]]> <![CDATA[<211> 111]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 32]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Phe Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Ile 85 90 95 Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <![CDATA[<210> 33]]> <![CDATA[<211> 116]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 33]]> Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Thr Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Val Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[<210> 34]]> <![CDATA[<211> 111]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 34]]> Asn Ile Ala Leu Thr Gln Ser Pro Ala Ser Val Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Asn Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Pro Leu Ile Tyr Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Ala Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <![CDATA[<210> 35]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 35]]> Gly Tyr Thr Met Asn 1 5 <![CDATA[<210> 36]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 36]]> Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <![CDATA[<210> 37]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 37]]> Trp Arg Tyr Thr Met Asp Tyr 1 5 <![CDATA[<210> 38]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 38]]> Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Asn Ser Phe Met Asn 1 5 10 15 <![CDATA[<210> 39]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 39]]> Phe Ala Ser Asn Leu Glu Ser 1 5 <![CDATA[<210> 40]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 40]]> Gln Gln Asn Asn Glu Ala Pro Pro Thr 1 5 <![CDATA[<210> 41]]> <![CDATA[<211> 116]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 41]]> 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 Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Val Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[<210> 42]]> <![CDATA[<211> 111]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 42]]> Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Asn Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Pro Leu Ile Tyr Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Ala Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <![CDATA[<210> 43]]> <![CDATA[<211> 218]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 43]]> Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Asn Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Pro Leu Ile Tyr Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Ala Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <![CDATA[<210> 44]]> <![CDATA[<211> 654]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 44]]> gatatcgtgc tgactcagag cccagcctct ctggctgtga gccccggcca gagagccaca 60 atcacttgta gggccagcga gagcgtggac aactacggca actccttcat gaattggtac 120 cagcagaagc ccggccagcc tccaaagcct ctgatctact tcgcctccaa tctggaaagc 180 ggcgtgccag ctaggtttag cggctccggc tctaggacag acttcactct gactatcaac 240 ccagtggagg ccgatgacac agccaactac tactgccagc agaacaacga ggcccctcca 300 actttcggcc aaggcactaa ggtcgagatc aagagaaccg tcgccgctcc cagcgtcttc 360 atcttccccc ccagcgatga gcagctgaag agcggaaccg ccagcgtggt gtgcctgctg 420 aacaacttct accccaggga ggccaaggtg caatggaagg tggacaacgc cctacagagc 480 ggcaactccc aggagagcgt gaccgagcag gacagcaagg atagcaccta cagcctgagc 540 agcaccctca ccctgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg 600 acccatcagg gcctgagcag ccctgtgacc aagagcttca acaggggcga gtgc 654 <![CDATA[<210> 45]]> <![CDATA[<211> 677]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 45]]> 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 Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Val Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 225 230 235 240 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 245 250 255 Ile Phe Ile Thr Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln 260 265 270 Gly Leu Glu Trp Ile Gly Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn 275 280 285 Tyr Asn Glu Met Phe Glu Gly Arg Ala Thr Leu Thr Val Asp Thr Ser 290 295 300 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 305 310 315 320 Ala Val Tyr Tyr Cys Ala Arg Gly Asp Gly Asn Tyr Ala Leu Asp Ala 325 330 335 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 340 345 350 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 355 360 365 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 370 375 380 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 385 390 395 400 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 405 410 415 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 420 425 430 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 435 440 445 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 450 455 460 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 465 470 475 480 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 485 490 495 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 500 505 510 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 515 520 525 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 530 535 540 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 545 550 555 560 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 565 570 575 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 580 585 590 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 595 600 605 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 610 615 620 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 625 630 635 640 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 645 650 655 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 660 665 670 Leu Ser Leu Gly Lys 675 <![CDATA[<210> 46]]> <![CDATA[<211> 2031]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 46]]> caagtgcagc tggtccagag cggcgctgag gtgaagaagc ccggcgccag cgtgaaagtc 60 agctgcaaag ctagcggcta ctccttcact ggctacacta tgaactgggt gaggcaagcc 120 cccggccaag gcctcgagtg gatcggactg atcaacccat acaacggcga cacaagctac 180 aaccagaagt tcaagggaag ggccacagtg actgtggaca agtccacaag cactgcctac 240 atggaactga gctctctgag gagcgaggat acagccgtgt actactgcgc taggtggaga 300 tacacaatgg actattgggg ccaaggcact ctggtcactg tgagcagcgc aagtaccaag 360 ggacctagtg ttttccctct tgcaccttgc tccaggtcaa catcagagtc cacagctgct 420 cttggatgtc tcgttaagga ctacttccca gagccagtta ccgtatcctg gaactccgga 480 gctttgacaa gcggcgttca tacattccca gctgtgttgc agagttctgg gttgtacagt 540 ttgagctcag tggtgaccgt gccttcatct tctttgggca ctaagaccta cacctgcaac 600 gtggatcaca agccaagcaa caccaaggtg gataagaggg tgggtggagg cggttcaggc 660 ggaggtggca gcggaggtgg cgggagtcag gtgcagctgg tgcagtctgg agctgaggtg 720 aagaagcctg gcgcttctgt gaaggtgtct tgtaaggctt ctggatatat ctttattacc 780 tattggatga attgggtgag acaggctcct ggccagggcc tggagtggat cggacagatt 840 tttccagctt ctggctccac aaattataat gagatgtttg agggcagagc tacactgaca 900 gtggatacat ctacatctac cgcctacatg gaactgtctt ctctgagatc tgaggataca 960 gctgtgtact attgtgctag aggcgatggc aattatgctc tggatgctat ggattattgg 1020 ggccagggaa cactggtgac cgtgtcttct gcctccacca agggcccatc ggtcttcccc 1080 ctggcaccct gctccaggag cacctctgag tccacagcgg ccctgggctg cctggtcaag 1140 gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 1200 cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 1260 gtgccctcca gcagcttggg caccaagaca tatacctgta atgtggatca caagccttcc 1320 aatacaaaag tggacaagag agttgagtcc aagtacggcc caccatgtcc tccatgtcca 1380 gcccctgaat ttttgggcgg gccttctgtc tttctgtttc ctcctaaacc taaagatacc 1440 ctgatgatca gccgcacacc cgaagtcact tgtgtggtcg tggatgtgtc tcaggaagat 1500 cccgaagtgc agtttaactg gtatgtcgat ggcgtggaag tgcataatgc caaaactaag 1560 ccccgcgaag aacagttcaa cagcacttat cgggtcgtgt ctgtgctcac agtcctccat 1620 caggattggc tgaatgggaa agaatataag tgcaaggtga gcaataaggg cctccccagc 1680 agcatcgaga agactattag caaagccaaa gggcagccac gggaacccca ggtgtacact 1740 ctgcccccct ctcaggagga gatgactaaa aatcaggtct ctctgacttg tctggtgaaa 1800 gggttttatc ccagcgacat tgccgtggag tgggagtcta atggccagcc cgagaataat 1860 tataagacaa ctccccccgt cctggactct gacggcagct ttttcctgta ttctcggctg 1920 acagtggaca aaagtcgctg gcaggagggc aatgtcttta gttgcagtgt catgcatgag 1980 gccctgcaca atcactatac acagaaaagc ctgtctctga gtctgggcaa a 2031 <![CDATA[<210> 47]]> <![CDATA[<211> 677]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 47]]> 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 Ile Phe Ile Thr Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Phe Pro Ala Ser Gly Ser Thr Asn Tyr Asn Glu Met Phe 50 55 60 Glu Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 225 230 235 240 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 245 250 255 Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg 260 265 270 Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Leu Ile Asn Pro Tyr 275 280 285 Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Ala Thr Val 290 295 300 Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu 305 310 315 320 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg Tyr Thr 325 330 335 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 340 345 350 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 355 360 365 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 370 375 380 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 385 390 395 400 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 405 410 415 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 420 425 430 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 435 440 445 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 450 455 460 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 465 470 475 480 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 485 490 495 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 500 505 510 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 515 520 525 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 530 535 540 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 545 550 555 560 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 565 570 575 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 580 585 590 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 595 600 605 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 610 615 620 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 625 630 635 640 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 645 650 655 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 660 665 670 Leu Ser Leu Gly Lys 675 <![CDATA[<210> 48]]> <![CDATA[<211> 2031]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 48]]> caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggcgcttc tgtgaaggtg 60 tcttgtaagg cttctggata tatctttatt acctattgga tgaattgggt gagacaggct 120 cctggccagg gcctggagtg gatcggacag atttttccag cttctggctc cacaaattat 180 aatgagatgt ttgagggcag agctacactg acagtggata catctacatc taccgcctat 240 atggagctgt cttctctgag atctgaagat accgctgtgt attattgtgc tagaggagat 300 ggcaactatg ctctggatgc catggattac tggggccagg gcaccctggt gactgtgagc 360 agcgcaagta ccaagggacc tagtgttttc cctcttgcac cttgctccag gtcaacatca 420 gagtccacag ctgctcttgg atgtctcgtt aaggactact tcccagagcc agttaccgta 480 tcctggaact ccggagcttt gacaagcggc gttcatacat tcccagctgt gttgcagagt 540 tctgggttgt acagtttgag ctcagtggtg accgtgcctt catcttcttt gggcactaag 600 acctacacct gcaacgtgga tcacaagcca agcaacacca aggtggataa gagggtgggt 660 ggaggcggtt caggcggagg tggcagcgga ggtggcggga gtcaagtgca gctggtccag 720 agcggcgctg aggtgaagaa gcccggcgcc agcgtgaaag tcagctgcaa agctagcggc 780 tactccttca ctggctacac tatgaactgg gtgaggcaag cccccggcca aggcctcgag 840 tggatcggac tgatcaaccc atacaacggc gacacaagct acaaccagaa gttcaaggga 900 agggccacag tgactgtgga caagtccaca agcactgcct acatggaact gagctctctg 960 aggagcgagg atacagccgt gtactactgc gctaggtgga gatacacaat ggactattgg 1020 ggccaaggca ctctggtcac agtgtctagt gcctccacca agggcccatc ggtcttcccc 1080 ctggcaccct gctccaggag cacctctgag tccacagcgg ccctgggctg cctggtcaag 1140 gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 1200 cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 1260 gtgccctcca gcagcttggg caccaagaca tatacctgta atgtggatca caagccttcc 1320 aatacaaaag tggacaagag agttgagtcc aagtacggcc caccatgtcc tccatgtcca 1380 gcccctgaat ttttgggcgg gccttctgtc tttctgtttc ctcctaaacc taaagatacc 1440 ctgatgatca gccgcacacc cgaagtcact tgtgtggtcg tggatgtgtc tcaggaagat 1500 cccgaagtgc agtttaactg gtatgtcgat ggcgtggaag tgcataatgc caaaactaag 1560 ccccgcgaag aacagttcaa cagcacttat cgggtcgtgt ctgtgctcac agtcctccat 1620 caggattggc tgaatgggaa agaatataag tgcaaggtga gcaataaggg cctccccagc 1680 agcatcgaga agactattag caaagccaaa gggcagccac gggaacccca ggtgtacact 1740 ctgcccccct ctcaggagga gatgactaaa aatcaggtct ctctgacttg tctggtgaaa 1800 gggttttatc ccagcgacat tgccgtggag tgggagtcta atggccagcc cgagaataat 1860 tataagacaa ctccccccgt cctggactct gacggcagct ttttcctgta ttctcggctg 1920 acagtggaca aaagtcgctg gcaggagggc aatgtcttta gttgcagtgt catgcatgag 1980 gccctgcaca atcactatac acagaaaagc ctgtctctga gtctgggcaa a 2031 <![CDATA[<210> 49]]> <![CDATA[<211> 677]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 49]]> 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 Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Val Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 225 230 235 240 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 245 250 255 Thr Phe Thr Ser Glu Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln 260 265 270 Gly Leu Glu Trp Met Gly Gln Ile Phe Pro Ala Leu Gly Ser Thr Asn 275 280 285 Tyr Asn Glu Met Tyr Glu Gly Arg Val Thr Met Thr Thr Asp Thr Ser 290 295 300 Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr 305 310 315 320 Ala Val Tyr Tyr Cys Ala Arg Gly Ile Gly Asn Tyr Ala Leu Asp Ala 325 330 335 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 340 345 350 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 355 360 365 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 370 375 380 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 385 390 395 400 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 405 410 415 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 420 425 430 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 435 440 445 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 450 455 460 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 465 470 475 480 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 485 490 495 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 500 505 510 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 515 520 525 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 530 535 540 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 545 550 555 560 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 565 570 575 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 580 585 590 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 595 600 605 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 610 615 620 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 625 630 635 640 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 645 650 655 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 660 665 670 Leu Ser Leu Gly Lys 675 <![CDATA[<210> 50]]> <![CDATA[<211> 2031]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 50]]> caagtgcagc tggtccagag cggcgctgag gtgaagaagc ccggcgccag cgtgaaagtc 60 agctgcaaag ctagcggcta ctccttcact ggctacacta tgaactgggt gaggcaagcc 120 cccggccaag gcctcgagtg gatcggactg atcaacccat acaacggcga cacaagctac 180 aaccagaagt tcaagggaag ggccacagtg actgtggaca agtccacaag cactgcctac 240 atggaactga gctctctgag gagcgaggat acagccgtgt actactgcgc taggtggaga 300 tacacaatgg actattgggg ccaaggcact ctggtcactg tgagcagcgc aagtaccaag 360 ggacctagtg ttttccctct tgcaccttgc tccaggtcaa catcagagtc cacagctgct 420 cttggatgtc tcgttaagga ctacttccca gagccagtta ccgtatcctg gaactccgga 480 gctttgacaa gcggcgttca tacattccca gctgtgttgc agagttctgg gttgtacagt 540 ttgagctcag tggtgaccgt gccttcatct tctttgggca ctaagaccta cacctgcaac 600 gtggatcaca agccaagcaa caccaaggtg gataagaggg tgggtggagg cggttcaggc 660 ggaggtggca gcggaggtgg cgggagtcaa gtgcagctgg tgcagagcgg cgctgaggtc 720 aaaaagcccg gcgcctccgt gaaggtgagc tgtaaggcca gcggctacac attcactagc 780 gagtggatga actgggtgag acaagccccc ggccaaggac tggaatggat gggccagatc 840 ttcccagctc tgggctccac taactacaac gagatgtacg agggaagggt cactatgact 900 acagacacta gcactagcac tgcctacatg gaactgaggt ctctgagaag cgacgataca 960 gccgtgtact actgcgccag aggcatcggc aactatgctc tggatgccat ggactactgg 1020 ggccaaggca ctctcgtgac tgtgagctcc gcctccacca agggcccatc ggtcttcccc 1080 ctggcaccct gctccaggag cacctctgag tccacagcgg ccctgggctg cctggtcaag 1140 gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 1200 cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 1260 gtgccctcca gcagcttggg caccaagaca tatacctgta atgtggatca caagccttcc 1320 aatacaaaag tggacaagag agttgagtcc aagtacggcc caccatgtcc tccatgtcca 1380 gcccctgaat ttttgggcgg gccttctgtc tttctgtttc ctcctaaacc taaagatacc 1440 ctgatgatca gccgcacacc cgaagtcact tgtgtggtcg tggatgtgtc tcaggaagat 1500 cccgaagtgc agtttaactg gtatgtcgat ggcgtggaag tgcataatgc caaaactaag 1560 ccccgcgaag aacagttcaa cagcacttat cgggtcgtgt ctgtgctcac agtcctccat 1620 caggattggc tgaatgggaa agaatataag tgcaaggtga gcaataaggg cctccccagc 1680 agcatcgaga agactattag caaagccaaa gggcagccac gggaacccca ggtgtacact 1740 ctgcccccct ctcaggagga gatgactaaa aatcaggtct ctctgacttg tctggtgaaa 1800 gggttttatc ccagcgacat tgccgtggag tgggagtcta atggccagcc cgagaataat 1860 tataagacaa ctccccccgt cctggactct gacggcagct ttttcctgta ttctcggctg 1920 acagtggaca aaagtcgctg gcaggagggc aatgtcttta gttgcagtgt catgcatgag 1980 gccctgcaca atcactatac acagaaaagc ctgtctctga gtctgggcaa a 2031 <![CDATA[<210> 51]]> <![CDATA[<211> 677]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 51]]> 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 Ser Glu 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gln Ile Phe Pro Ala Leu Gly Ser Thr Asn Tyr Asn Glu Met Tyr 50 55 60 Glu Gly Arg Val Thr Met Thr Thr 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 Ile Gly Asn Tyr Ala Leu Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 225 230 235 240 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 245 250 255 Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg 260 265 270 Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Leu Ile Asn Pro Tyr 275 280 285 Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Ala Thr Val 290 295 300 Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu 305 310 315 320 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Arg Tyr Thr 325 330 335 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 340 345 350 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 355 360 365 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 370 375 380 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 385 390 395 400 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 405 410 415 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 420 425 430 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 435 440 445 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 450 455 460 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 465 470 475 480 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 485 490 495 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 500 505 510 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 515 520 525 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 530 535 540 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 545 550 555 560 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 565 570 575 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 580 585 590 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 595 600 605 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 610 615 620 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 625 630 635 640 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 645 650 655 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 660 665 670 Leu Ser Leu Gly Lys 675 <![CDATA[<210> 52]]> <![CDATA[<211> 2031]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 52]]> caagtgcagc tggtgcagag cggcgctgag gtcaaaaagc ccggcgcctc cgtgaaggtg 60 agctgtaagg ccagcggcta cacattcact agcgagtgga tgaactgggt gagacaagcc 120 cccggccaag gactggaatg gatgggccag atcttcccag ctctgggctc cactaactac 180 aacgagatgt acgagggaag ggtcactatg actacagaca ctagcactag cactgcctac 240 atggaactga ggtctctgag aagcgacgat acagccgtgt actactgcgc cagaggcatc 300 ggcaactatg ctctggatgc catggactac tggggccaag gcactctcgt gactgtgagc 360 tccgcaagta ccaagggacc tagtgttttc cctcttgcac cttgctccag gtcaacatca 420 gagtccacag ctgctcttgg atgtctcgtt aaggactact tcccagagcc agttaccgta 480 tcctggaact ccggagcttt gacaagcggc gttcatacat tcccagctgt gttgcagagt 540 tctgggttgt acagtttgag ctcagtggtg accgtgcctt catcttcttt gggcactaag 600 acctacacct gcaacgtgga tcacaagcca agcaacacca aggtggataa gagggtgggt 660 ggaggcggtt caggcggagg tggcagcgga ggtggcggga gtcaagtgca gctggtccag 720 agcggcgctg aggtgaagaa gcccggcgcc agcgtgaaag tcagctgcaa agctagcggc 780 tactccttca ctggctacac tatgaactgg gtgaggcaag cccccggcca aggcctcgag 840 tggatcggac tgatcaaccc atacaacggc gacacaagct acaaccagaa gttcaaggga 900 agggccacag tgactgtgga caagtccaca agcactgcct acatggaact gagctctctg 960 aggagcgagg atacagccgt gtactactgc gctaggtgga gatacacaat ggactattgg 1020 ggccaaggca ctctggtcac agtgtctagt gcctccacca agggcccatc ggtcttcccc 1080 ctggcaccct gctccaggag cacctctgag tccacagcgg ccctgggctg cctggtcaag 1140 gactacttcc ccgaaccggt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 1200 cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 1260 gtgccctcca gcagcttggg caccaagaca tatacctgta atgtggatca caagccttcc 1320 aatacaaaag tggacaagag agttgagtcc aagtacggcc caccatgtcc tccatgtcca 1380 gcccctgaat ttttgggcgg gccttctgtc tttctgtttc ctcctaaacc taaagatacc 1440 ctgatgatca gccgcacacc cgaagtcact tgtgtggtcg tggatgtgtc tcaggaagat 1500 cccgaagtgc agtttaactg gtatgtcgat ggcgtggaag tgcataatgc caaaactaag 1560 ccccgcgaag aacagttcaa cagcacttat cgggtcgtgt ctgtgctcac agtcctccat 1620 caggattggc tgaatgggaa agaatataag tgcaaggtga gcaataaggg cctccccagc 1680 agcatcgaga agactattag caaagccaaa gggcagccac gggaacccca ggtgtacact 1740 ctgcccccct ctcaggagga gatgactaaa aatcaggtct ctctgacttg tctggtgaaa 1800 gggttttatc ccagcgacat tgccgtggag tgggagtcta atggccagcc cgagaataat 1860 tataagacaa ctccccccgt cctggactct gacggcagct ttttcctgta ttctcggctg 1920 acagtggaca aaagtcgctg gcaggagggc aatgtcttta gttgcagtgt catgcatgag 1980 gccctgcaca atcactatac acagaaaagc ctgtctctga gtctgggcaa a 2031 <![CDATA[<210> 53]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 53]]> 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 Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile 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 Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[<210> 54]]> <![CDATA[<211> 112]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 54]]> Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <![CDATA[<210> 55]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 55]]> Asn His Val Ile His 1 5 <![CDATA[<210> 56]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 56]]> Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Asp <![CDATA[<210> 57]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 57]]> Gly Gly Tyr Tyr Thr Tyr Asp Asp 1 5 <![CDATA[<210> 58]]> <![CDATA[<211> 16]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 58]]> Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His 1 5 10 15 <![CDATA[<210> 59]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 59]]> Lys Val Ser Asn Arg Phe Ser 1 5 <![CDATA[<210> 60]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 60]]> Ser Gln Ser Thr His Val Pro Tyr Thr 1 5 <![CDATA[<210> 61]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 61]]> 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 Ala Asn His 20 25 30 Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[<210> 62]]> <![CDATA[<211> 112]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 62]]> Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Lys Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <![CDATA[<210> 63]]> <![CDATA[<211> 219]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 63]]> Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <![CDATA[<210> 64]]> <![CDATA[<211> 657]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 64]]> gacgtggtga tgacccagag ccctttatct ttacccgtta cactgggaca gcccgccagc 60 atcagctgtc gtagcagcca gtctttagtg cacagcaacg gcaacaccta tttacactgg 120 taccagcaga gacccggcca gagccccaga ctgctgatct acaaggtgag caatcgtttc 180 tccggcgtgc ccgacagatt cagcggcagc ggaagcggca ccgacttcac tttaaagatc 240 agcagagtgg aggccgagga cgtgggcgtg tacttctgca gccagagcac ccacatccct 300 tggaccttcg gccaaggtac caaggtggag atcaagagaa ccgtcgccgc tcccagcgtc 360 ttcatcttcc cccccagcga tgagcagctg aagagcggaa ccgccagcgt ggtgtgcctg 420 ctgaacaact tctaccccag ggaggccaag gtgcaatgga aggtggacaa cgccctacag 480 agcggcaact cccaggagag cgtgaccgag caggacagca aggatagcac ctacagcctg 540 agcagcaccc tcaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600 gtgacccatc agggcctgag cagccctgtg accaagagct tcaacagggg cgagtgc 657 <![CDATA[<210> 65]]> <![CDATA[<211> 677]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 65]]> 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 Ala Asn His 20 25 30 Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 225 230 235 240 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Gly Ser Gly 245 250 255 Tyr Thr Phe Thr Asp Tyr Ala Ile His Trp Val Arg Gln Ala Pro Gly 260 265 270 Gln Ser Leu Glu Trp Ile Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile 275 280 285 Asn Tyr Asn Gln Lys Phe Lys Gly Arg Ala Thr Met Thr Val Asp Lys 290 295 300 Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp 305 310 315 320 Thr Ala Val Tyr Tyr Cys Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr 325 330 335 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 340 345 350 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 355 360 365 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 370 375 380 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 385 390 395 400 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 405 410 415 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 420 425 430 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 435 440 445 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 450 455 460 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 465 470 475 480 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 485 490 495 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 500 505 510 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 515 520 525 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 530 535 540 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 545 550 555 560 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 565 570 575 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 580 585 590 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 595 600 605 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 610 615 620 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 625 630 635 640 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 645 650 655 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 660 665 670 Leu Ser Pro Gly Lys 675 <![CDATA[<210> 66]]> <![CDATA[<211> 2031]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 66]]> caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggcgcctc cgtgaaagtg 60 agctgcaagg catccggcta caccttcgcc aaccacgtga tccactgggt gcggcaggcc 120 cccggccagg gcctggagtg gatgggctac atctatcctt ataatgatgg cacaaagtat 180 aatgagaagt ttaaggatag ggtaacactg acatctgata agtctacatc tacagtgtac 240 atggagctgt cctctctgag gtccgaggac accgccgtgt actactgcgc cagaggcggc 300 tactacacct acgacgactg gggccagggc accctggtga ccgtgtcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt caagttcagc tggtgcagag cggagctgag 720 gtgaagaagc ccggcgccag cgtgaaggtg agctgtaagg gcagcggcta caccttcacc 780 gactacgcca tccactgggt gagacaagct cccggtcagt ctttagaatg gatcggcgtg 840 ttcagcatct actacgagaa catcaactat aaccagaagt tcaagggtcg tgccaccatg 900 accgtggaca agagcaccag caccgcctac atggagctga ggtctttaag gagcgacgac 960 accgccgtgt actactgcgc tcgtagggac ggcggcacca tcaactactg gggccaaggt 1020 actttagtga cagtgagcag cgcgagcacc aagggacctt ccgtgtttcc cctcgccccc 1080 agctccaaaa gcaccagcgg cggaacagct gctctcggct gtctcgtcaa ggattacttc 1140 cccgagcccg tgaccgtgag ctggaacagc ggagccctga caagcggcgt ccacaccttc 1200 cctgctgtcc tacagtcctc cggactgtac agcctgagca gcgtggtgac agtccctagc 1260 agctccctgg gcacccagac atatatttgc aacgtgaatc acaagcccag caacaccaag 1320 gtcgataaga aggtggagcc taagtcctgc gacaagaccc acacatgtcc cccctgtccc 1380 gctcctgaac tgctgggagg cccttccgtg ttcctgttcc cccctaagcc caaggacacc 1440 ctgatgattt ccaggacacc cgaggtgacc tgtgtggtgg tggacgtcag ccacgaggac 1500 cccgaggtga aattcaactg gtacgtcgat ggcgtggagg tgcacaacgc taagaccaag 1560 cccagggagg agcagtacaa ttccacctac agggtggtgt ccgtgctgac cgtcctccat 1620 caggactggc tgaacggcaa agagtataag tgcaaggtga gcaacaaggc cctccctgct 1680 cccatcgaga agaccatcag caaagccaag ggccagccca gggaacctca agtctatacc 1740 ctgcctccca gcagggagga gatgaccaag aaccaagtga gcctcacatg cctcgtcaag 1800 ggcttctatc cttccgatat tgccgtcgag tgggagtcca acggacagcc cgagaacaac 1860 tacaagacaa caccccccgt gctcgattcc gatggcagct tcttcctgta ctccaagctg 1920 accgtggaca agtccagatg gcaacaaggc aacgtcttca gttgcagcgt catgcatgag 1980 gccctccaca accactacac ccagaaaagc ctgtctctga gtcctggcaa a 2031 <![CDATA[<210> 67]]> <![CDATA[<211> 677]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 67]]> 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 Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile 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 Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 225 230 235 240 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 245 250 255 Tyr Thr Phe Ala Asn His Val Ile His Trp Val Arg Gln Ala Pro Gly 260 265 270 Gln Gly Leu Glu Trp Met Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr 275 280 285 Lys Tyr Asn Glu Lys Phe Lys Asp Arg Val Thr Leu Thr Ser Asp Lys 290 295 300 Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 305 310 315 320 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp 325 330 335 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 340 345 350 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 355 360 365 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 370 375 380 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 385 390 395 400 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 405 410 415 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 420 425 430 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 435 440 445 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 450 455 460 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 465 470 475 480 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 485 490 495 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 500 505 510 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 515 520 525 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 530 535 540 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 545 550 555 560 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 565 570 575 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 580 585 590 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 595 600 605 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 610 615 620 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 625 630 635 640 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 645 650 655 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 660 665 670 Leu Ser Pro Gly Lys 675 <![CDATA[<210> 68]]> <![CDATA[<211> 2031]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 68]]> caagttcagc tggtgcagag cggagctgag gtgaagaagc ccggcgccag cgtgaaggtg 60 agctgtaagg gcagcggcta caccttcacc gactacgcca tccactgggt gagacaagct 120 cccggtcagt ctttagaatg gatcggcgtg ttcagcatct actacgagaa catcaactat 180 aaccagaagt tcaagggtcg tgccaccatg accgtggaca agagcaccag caccgcctac 240 atggagctga ggtctttaag gagcgacgac accgccgtgt actactgcgc tcgtagggac 300 ggcggcacca tcaactactg gggccaaggt actttagtga cagtgagcag cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt caggtgcagc tggtgcagtc cggcgccgag 720 gtgaagaagc ccggcgcctc cgtgaaagtg agctgcaagg catccggcta caccttcgcc 780 aaccacgtga tccactgggt gcggcaggcc cccggccagg gcctggagtg gatgggctac 840 atctatcctt ataatgatgg cacaaagtat aatgagaagt ttaaggatag ggtaacactg 900 acatctgata agtctacatc tacagtgtac atggagctgt cctctctgag gtccgaggac 960 accgccgtgt actactgcgc cagaggcggc tactacacct acgacgactg gggccagggc 1020 accctggtga ccgtgtcctc cgcgagcacc aagggacctt ccgtgtttcc cctcgccccc 1080 agctccaaaa gcaccagcgg cggaacagct gctctcggct gtctcgtcaa ggattacttc 1140 cccgagcccg tgaccgtgag ctggaacagc ggagccctga caagcggcgt ccacaccttc 1200 cctgctgtcc tacagtcctc cggactgtac agcctgagca gcgtggtgac agtccctagc 1260 agctccctgg gcacccagac atatatttgc aacgtgaatc acaagcccag caacaccaag 1320 gtcgataaga aggtggagcc taagtcctgc gacaagaccc acacatgtcc cccctgtccc 1380 gctcctgaac tgctgggagg cccttccgtg ttcctgttcc cccctaagcc caaggacacc 1440 ctgatgattt ccaggacacc cgaggtgacc tgtgtggtgg tggacgtcag ccacgaggac 1500 cccgaggtga aattcaactg gtacgtcgat ggcgtggagg tgcacaacgc taagaccaag 1560 cccagggagg agcagtacaa ttccacctac agggtggtgt ccgtgctgac cgtcctccat 1620 caggactggc tgaacggcaa agagtataag tgcaaggtga gcaacaaggc cctccctgct 1680 cccatcgaga agaccatcag caaagccaag ggccagccca gggaacctca agtctatacc 1740 ctgcctccca gcagggagga gatgaccaag aaccaagtga gcctcacatg cctcgtcaag 1800 ggcttctatc cttccgatat tgccgtcgag tgggagtcca acggacagcc cgagaacaac 1860 tacaagacaa caccccccgt gctcgattcc gatggcagct tcttcctgta ctccaagctg 1920 accgtggaca agtccagatg gcaacaaggc aacgtcttca gttgcagcgt catgcatgag 1980 gccctccaca accactacac ccagaaaagc ctgtctctga gtcctggcaa a 2031 <![CDATA[<210> 69]]> <![CDATA[<211> 111]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 69]]> Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Lys Tyr Tyr Cys Val 85 90 95 Arg Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 100 105 110 <![CDATA[<210> 70]]> <![CDATA[<211> 112]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 70]]> Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Phe Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <![CDATA[<210> 71]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 71]]> Ser Tyr Asp Ile Ser 1 5 <![CDATA[<210> 72]]> <![CDATA[<211> 16]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 72]]> Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met Ser 1 5 10 15 <![CDATA[<210> 73]]> <![CDATA[<211> 3]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 73]]> Met Asp Tyr 1 <![CDATA[<210> 74]]> <![CDATA[<211> 16]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 74]]> Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <![CDATA[<210> 75]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 75]]> Lys Val Ser Asn Arg Phe Ser 1 5 <![CDATA[<210> 76]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 76]]> Ser Gln Ser Thr His Val Pro Trp Thr 1 5 <![CDATA[<210> 77]]> <![CDATA[<211> 111]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 77]]> Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <![CDATA[<210> 78]]> <![CDATA[<211> 112]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 78]]> Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Phe Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <![CDATA[<210> 79]]> <![CDATA[<211> 671]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 79]]> Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 100 105 110 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser 115 120 125 Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 130 135 140 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 145 150 155 160 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 165 170 175 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr 180 185 190 Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 195 200 205 Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 225 230 235 240 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 245 250 255 Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 260 265 270 Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe 275 280 285 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 290 295 300 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 305 310 315 320 Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu 325 330 335 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 340 345 350 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 355 360 365 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 370 375 380 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 385 390 395 400 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 405 410 415 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 420 425 430 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 435 440 445 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 450 455 460 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 465 470 475 480 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 485 490 495 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 500 505 510 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 515 520 525 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 530 535 540 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 545 550 555 560 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 565 570 575 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 580 585 590 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 595 600 605 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 610 615 620 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 625 630 635 640 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 645 650 655 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 660 665 670 <![CDATA[<210> 80]]> <![CDATA[<211> 2013]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 80]]> caggtgcagc tgcaggaatc tggcccaggc ctggtgaagc cttctgagac actgtctttg 60 acctgtacag tgtctggctt ttctctgtct tcttatgata tttcctggat tagacagcct 120 cctggcaagg gcctggagtg gctgggagtg atttggacag gaggcggaac aaattataac 180 tctgctttta tgtctaggct gaccatctct aaggataata gtaaatctca ggtgtctctg 240 aagctgtctt ctgtgaccgc tgctgataca gccgtgtact attgtgttag aatggattat 300 tggggccagg gcacactggt gaccgtgtct tctgcaagta ccaagggacc tagtgttttc 360 cctcttgcac cttgctccag gtcaacatca gagtccacag ctgctcttgg atgtctcgtt 420 aaggactact tcccagagcc agttaccgta tcctggaact ccggagcttt gacaagcggc 480 gttcatacat tcccagctgt gttgcagagt tctgggttgt acagtttgag ctcagtggtg 540 accgtgcctt catcttcttt gggcactaag acctacacct gcaacgtgga tcacaagcca 600 agcaacacca aggtggataa gagggtgggt ggaggcggtt caggcggagg tggcagcgga 660 ggtggcggga gtcaagttca gctggtgcag agcggagctg aggtgaagaa gcccggcgcc 720 agcgtgaagg tgagctgtaa gggcagcggc tacaccttca ccgactacgc catccactgg 780 gtgagacaag ctcccggtca gtctttagaa tggatcggcg tgttcagcat ctactacgag 840 aacatcaact ataaccagaa gttcaagggt cgtgccacca tgaccgtgga caagagcacc 900 agcaccgcct acatggagct gaggtcttta aggagcgacg acaccgccgt gtactactgc 960 gctcgtaggg acggcggcac catcaactac tggggccaag gtactttagt gacagtgagc 1020 agcgcgagca ccaagggacc ttccgtgttt cccctcgccc ccagctccaa aagcaccagc 1080 ggcggaacag ctgctctcgg ctgtctcgtc aaggattact tccccgagcc cgtgaccgtg 1140 agctggaaca gcggagccct gacaagcggc gtccacacct tccctgctgt cctacagtcc 1200 tccggactgt acagcctgag cagcgtggtg acagtcccta gcagctccct gggcacccag 1260 acatatattt gcaacgtgaa tcacaagccc agcaacacca aggtcgataa gaaggtggag 1320 cctaagtcct gcgacaagac ccacacatgt cccccctgtc ccgctcctga agctgctgga 1380 ggcccttccg tgttcctgtt cccccctaag cccaaggaca ccctgatgat ttccaggaca 1440 cccgaggtga cctgtgtggt ggtggacgtc agccacgagg accccgaggt gaaattcaac 1500 tggtacgtcg atggcgtgga ggtgcacaac gctaagacca agcccaggga ggagcagtac 1560 aattccacct acagggtggt gtccgtgctg accgtcctcc atcaggactg gctgaacggc 1620 aaagagtata agtgcaaggt gagcaacaag gccctccctg ctcccatcga gaagaccatc 1680 agcaaagcca agggccagcc cagggaacct caagtctata ccctgcctcc cagcagggag 1740 gagatgacca agaaccaagt gagcctcaca tgcctcgtca agggcttcta tccttccgat 1800 attgccgtcg agtgggagtc caacggacag cccgagaaca actacaagac aacacccccc 1860 gtgctcgatt ccgatggcag cttcttcctg tactccaagc tgaccgtgga caagtccaga 1920 tggcaacaag gcaacgtctt cagttgcagc gtcatgcatg aggccctcca caaccactac 1980 acccagaaga gcctctccct gagccctgga aag 2013 <![CDATA[<210> 81]]> <![CDATA[<211> 671]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 81]]> 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 Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile 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 Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly 225 230 235 240 Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 245 250 255 Phe Ser Leu Ser Ser Tyr Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly 260 265 270 Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Thr Gly Gly Gly Thr Asn 275 280 285 Tyr Asn Ser Ala Phe Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser 290 295 300 Lys Ser Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr 305 310 315 320 Ala Val Tyr Tyr Cys Val Arg Met Asp Tyr Trp Gly Gln Gly Thr Leu 325 330 335 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 340 345 350 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 355 360 365 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 370 375 380 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 385 390 395 400 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 405 410 415 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 420 425 430 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 435 440 445 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 450 455 460 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 465 470 475 480 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 485 490 495 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 500 505 510 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 515 520 525 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 530 535 540 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 545 550 555 560 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 565 570 575 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 580 585 590 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 595 600 605 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 610 615 620 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 625 630 635 640 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 645 650 655 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 660 665 670 <![CDATA[<210> 82]]> <![CDATA[<211> 2013]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 82]]> caagttcagc tggtgcagag cggagctgag gtgaagaagc ccggcgccag cgtgaaggtg 60 agctgtaagg gcagcggcta caccttcacc gactacgcca tccactgggt gagacaagct 120 cccggtcagt ctttagaatg gatcggcgtg ttcagcatct actacgagaa catcaactat 180 aaccagaagt tcaagggtcg tgccaccatg accgtggaca agagcaccag caccgcctac 240 atggagctga ggtctttaag gagcgacgac accgccgtgt actactgcgc tcgtagggac 300 ggcggcacca tcaactactg gggccaaggt actttagtga cagtgagcag cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt caggtgcagc tgcaggaatc tggcccaggc 720 ctggtgaagc cttctgagac actgtctttg acctgtacag tgtctggctt ttctctgtct 780 tcttatgata tttcctggat tagacagcct cctggcaagg gcctggagtg gctgggagtg 840 atttggacag gaggcggaac aaattataac tctgctttta tgtctaggct gaccatctct 900 aaggataata gtaaatctca ggtgtctctg aagctgtctt ctgtgaccgc tgctgataca 960 gccgtgtact attgtgttag aatggattat tggggccagg gcacactggt gaccgtgtct 1020 tctgcgagca ccaagggacc ttccgtgttt cccctcgccc ccagctccaa aagcaccagc 1080 ggcggaacag ctgctctcgg ctgtctcgtc aaggattact tccccgagcc cgtgaccgtg 1140 agctggaaca gcggagccct gacaagcggc gtccacacct tccctgctgt cctacagtcc 1200 tccggactgt acagcctgag cagcgtggtg acagtcccta gcagctccct gggcacccag 1260 acatatattt gcaacgtgaa tcacaagccc agcaacacca aggtcgataa gaaggtggag 1320 cctaagtcct gcgacaagac ccacacatgt cccccctgtc ccgctcctga agctgctgga 1380 ggcccttccg tgttcctgtt cccccctaag cccaaggaca ccctgatgat ttccaggaca 1440 cccgaggtga cctgtgtggt ggtggacgtc agccacgagg accccgaggt gaaattcaac 1500 tggtacgtcg atggcgtgga ggtgcacaac gctaagacca agcccaggga ggagcagtac 1560 aattccacct acagggtggt gtccgtgctg accgtcctcc atcaggactg gctgaacggc 1620 aaagagtata agtgcaaggt gagcaacaag gccctccctg ctcccatcga gaagaccatc 1680 agcaaagcca agggccagcc cagggaacct caagtctata ccctgcctcc cagcagggag 1740 gagatgacca agaaccaagt gagcctcaca tgcctcgtca agggcttcta tccttccgat 1800 attgccgtcg agtgggagtc caacggacag cccgagaaca actacaagac aacacccccc 1860 gtgctcgatt ccgatggcag cttcttcctg tactccaagc tgaccgtgga caagtccaga 1920 tggcaacaag gcaacgtctt cagttgcagc gtcatgcatg aggccctcca caaccactac 1980 acccagaaga gcctctccct gagccctgga aag 2013 <![CDATA[<210> 83]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 83]]> Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[<210> 84]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 84]]> Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 85]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 85]]> Ser Tyr Trp Met His 1 5 <![CDATA[<210> 86]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 86]]> Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <![CDATA[<210> 87]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 87]]> Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr 1 5 10 <![CDATA[<210> 88]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 88]]> Arg Ala Ser Gln Thr Ile Ser Asp Tyr Leu His 1 5 10 <![CDATA[<210> 89]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 89]]> Tyr Ala Ser Gln Ser Ile Ser 1 5 <![CDATA[<210> 90]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 90]]> Gln Asp Gly His Ser Phe Pro Pro Thr 1 5 <![CDATA[<210> 91]]> <![CDATA[<211> 119]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 91]]> 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 Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 92]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 92]]> Glu Ile Val Met Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asp Gly His Ser Phe Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 93]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 93]]> Glu Ile Val Met Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asp Gly His Ser Phe Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 94]]> <![CDATA[<211> 642]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 94]]> gagatcgtga tgacccagtc tcctgatttt cagtctgtga cccctaagga gaaggtgaca 60 atcacctgta gagcttctca gaccatctct gattatctgc attggtatca gcagaagcct 120 gatcagtctc ctaagctgct gatcaagtat gcttctcagt ctatctctgg cattccttct 180 aggttttccg gctctggctc tggctctgat tttacactga ccatcaattc tctggaggct 240 gaggatgctg ccacctatta ttgtcaggac ggccactctt ttcctcctac ctttggccag 300 ggaaccaagg tggagatcaa gagaaccgtc gccgctccca gcgtcttcat cttccccccc 360 agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480 gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600 ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642 <![CDATA[<210> 95]]> <![CDATA[<211> 676]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 95]]> Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 225 230 235 240 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 245 250 255 Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly 260 265 270 Gln Gly Leu Glu Trp Met Gly Glu Ile Asn Pro Gly Asn Gly His Thr 275 280 285 Asn Tyr Asn Glu Lys Phe Lys Ser Arg Val Thr Leu Thr Val Asp Lys 290 295 300 Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 305 310 315 320 Thr Ala Val Tyr Tyr Cys Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe 325 330 335 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 340 345 350 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 355 360 365 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 370 375 380 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 385 390 395 400 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 405 410 415 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 420 425 430 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 435 440 445 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 450 455 460 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 465 470 475 480 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 485 490 495 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 500 505 510 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 515 520 525 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535 540 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 545 550 555 560 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 565 570 575 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 580 585 590 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 595 600 605 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 610 615 620 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 625 630 635 640 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 645 650 655 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670 Ser Leu Gly Lys 675 <![CDATA[<210> 96]]> <![CDATA[<211> 2028]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 96]]> gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60 agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120 cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180 cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240 ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300 ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt caggtgcagc tggtgcagag cggcgctgaa 720 gtgaagaagc caggcgcttc tgtaaaggtg tcttgtaagg cttctggcta tacattttct 780 tcttattgga tgcactgggt gagacaggct cctggccagg gactggagtg gatgggtgag 840 atcaatcctg gcaatggcca tactaactac aacgagaagt tcaagtccag ggtgaccctg 900 accgtggaca agtccacctc caccgtgtac atggagctga gctccctgag gtccgaggac 960 accgccgtgt actactgcgc ccggagcttc actaccgccc gcggcttcgc ctattggggc 1020 cagggcaccc tggtgaccgt gagctccgcc tccaccaagg gcccatcggt cttccccctg 1080 gcaccctgct ccaggagcac ctctgagtcc acagcggccc tgggctgcct ggtcaaggac 1140 tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 1200 accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 1260 ccctccagca gcttgggcac caagacatat acctgtaatg tggatcacaa gccttccaat 1320 acaaaagtgg acaagagagt tgagtccaag tacggcccac catgtcctcc atgtccagcc 1380 cctgaatttt tgggcgggcc ttctgtcttt ctgtttcctc ctaaacctaa agataccctg 1440 atgatcagcc gcacacccga agtcacttgt gtggtcgtgg atgtgtctca ggaagatccc 1500 gaagtgcagt ttaactggta tgtcgatggc gtggaagtgc ataatgccaa aactaagccc 1560 cgcgaagaac agttcaacag cacttatcgg gtcgtgtctg tgctcacagt cctccatcag 1620 gattggctga atgggaaaga atataagtgc aaggtgagca ataagggcct ccccagcagc 1680 atcgagaaga ctattagcaa agccaaaggg cagccacggg aaccccaggt gtacactctg 1740 cccccctctc aggaggagat gactaaaaat caggtctctc tgacttgtct ggtgaaaggg 1800 ttttatccca gcgacattgc cgtggagtgg gagtctaatg gccagcccga gaataattat 1860 aagacaactc cccccgtcct ggactctgac ggcagctttt tcctgtattc tcggctgaca 1920 gtggacaaaa gtcgctggca ggagggcaat gtctttagtt gcagtgtcat gcatgaggcc 1980 ctgcacaatc actatacaca gaaaagcctg tctctgagtc tgggcaaa 2028 <![CDATA[<210> 97]]> <![CDATA[<211> 676]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 97]]> 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 Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly 210 215 220 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly 225 230 235 240 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 245 250 255 Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala 260 265 270 Pro Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg 275 280 285 Tyr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg 290 295 300 Asp Asn Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala 305 310 315 320 Glu Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe 325 330 335 Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 340 345 350 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 355 360 365 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 370 375 380 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 385 390 395 400 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 405 410 415 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 420 425 430 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 435 440 445 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 450 455 460 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 465 470 475 480 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 485 490 495 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 500 505 510 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 515 520 525 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535 540 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 545 550 555 560 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 565 570 575 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 580 585 590 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 595 600 605 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 610 615 620 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 625 630 635 640 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 645 650 655 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670 Ser Leu Gly Lys 675 <![CDATA[<210> 98]]> <![CDATA[<211> 2028]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 98]]> caggtgcagc tggtgcagag cggcgctgaa gtgaagaagc caggcgcttc tgtaaaggtg 60 tcttgtaagg cttctggcta tacattttct tcttattgga tgcactgggt gagacaggct 120 cctggccagg gactggagtg gatgggtgag atcaatcctg gcaatggcca tactaactac 180 aacgagaagt tcaagtccag ggtgaccctg accgtggaca agtccacctc caccgtgtac 240 atggagctga gctccctgag gtccgaggac accgccgtgt actactgcgc ccggagcttc 300 actaccgccc gcggcttcgc ctattggggc cagggcaccc tggtgaccgt gagctccgca 360 agtaccaagg gacctagtgt tttccctctt gcaccttgct ccaggtcaac atcagagtcc 420 acagctgctc ttggatgtct cgttaaggac tacttcccag agccagttac cgtatcctgg 480 aactccggag ctttgacaag cggcgttcat acattcccag ctgtgttgca gagttctggg 540 ttgtacagtt tgagctcagt ggtgaccgtg ccttcatctt ctttgggcac taagacctac 600 acctgcaacg tggatcacaa gccaagcaac accaaggtgg ataagagggt gggtggaggc 660 ggttcaggcg gaggtggcag cggaggtggc gggagtgagg tcaagctggt ggaaagcggc 720 ggcggcctgg tgcagcctgg aggatccctg cggctgagct gcgctgcctc cggcttcgct 780 ttcagctcct atgacatgtc ctgggtgagg caggcccctg gaaagaggct ggagtgggtg 840 gctaccatct ccggaggcgg aaggtacacc tactaccccg acacagtgaa gggaaggttc 900 accatcagcc gggataacgc caaaaacagc cactatctcc agatgaactc cctgagggcc 960 gaagatacag ccgtgtattt ctgtgcctcc ccctacggag gctattttga cgtgtgggga 1020 cagggcaccc tggtgaccgt ctcctccgcc tccaccaagg gcccatcggt cttccccctg 1080 gcaccctgct ccaggagcac ctctgagtcc acagcggccc tgggctgcct ggtcaaggac 1140 tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 1200 accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 1260 ccctccagca gcttgggcac caagacatat acctgtaatg tggatcacaa gccttccaat 1320 acaaaagtgg acaagagagt tgagtccaag tacggcccac catgtcctcc atgtccagcc 1380 cctgaatttt tgggcgggcc ttctgtcttt ctgtttcctc ctaaacctaa agataccctg 1440 atgatcagcc gcacacccga agtcacttgt gtggtcgtgg atgtgtctca ggaagatccc 1500 gaagtgcagt ttaactggta tgtcgatggc gtggaagtgc ataatgccaa aactaagccc 1560 cgcgaagaac agttcaacag cacttatcgg gtcgtgtctg tgctcacagt cctccatcag 1620 gattggctga atgggaaaga atataagtgc aaggtgagca ataagggcct ccccagcagc 1680 atcgagaaga ctattagcaa agccaaaggg cagccacggg aaccccaggt gtacactctg 1740 cccccctctc aggaggagat gactaaaaat caggtctctc tgacttgtct ggtgaaaggg 1800 ttttatccca gcgacattgc cgtggagtgg gagtctaatg gccagcccga gaataattat 1860 aagacaactc cccccgtcct ggactctgac ggcagctttt tcctgtattc tcggctgaca 1920 gtggacaaaa gtcgctggca ggagggcaat gtctttagtt gcagtgtcat gcatgaggcc 1980 ctgcacaatc actatacaca gaaaagcctg tctctgagtc tgggcaaa 2028 <![CDATA[<210> 99]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 99]]> Thr Thr Gly Met Gln 1 5 <![CDATA[<210> 100]]> <![CDATA[<211> 17]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 100]]> Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Val Glu Asp Phe Lys 1 5 10 15 Gly <![CDATA[<210> 101]]> <![CDATA[<211> 13]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 101]]> Ser Gly Asn Gly Asn Tyr Asp Leu Ala Tyr Phe Ala Tyr 1 5 10 <![CDATA[<210> 102]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 102]]> Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His 1 5 10 <![CDATA[<210> 103]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 103]]> Tyr Ala Ser His Ser Ile Ser 1 5 <![CDATA[<210> 104]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 小鼠(Mus musculus)]]> <![CDATA[<400> 104]]> Gln His Gly His Ser Phe Pro Trp Thr 1 5 <![CDATA[<210> 105]]> <![CDATA[<211> 122]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 105]]> Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Thr 20 25 30 Gly Met Gln Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Val Glu Asp Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Asn Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Asn Gly Asn Tyr Asp Leu Ala Tyr Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 106]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 106]]> Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser His Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Gly His Ser Phe Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 107]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 107]]> Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser His Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Gly His Ser Phe Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 108]]> <![CDATA[<211> 642]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 108]]> gaaatcgtgc tgacacagtc tcctgctaca ctgtctctga gtcctggaga aagagctaca 60 ctgtcttgta gagcttccca gtctatctcc gattatctgc actggtacca gcagaagcct 120 ggccagtctc ctagactgct gatcaagtat gcttctcatt ctatttctgg catcccagct 180 aggtttagtg gatctggctc tggaagcgac tttacactga ccatttcttc tctggagcct 240 gaggattttg ctgtgtatta ctgtcagcac ggccattctt ttccttggac ctttggccag 300 ggcacaaagg tggagatcaa gagaaccgtc gccgctccca gcgtcttcat cttccccccc 360 agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480 gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600 ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642 <![CDATA[<210> 109]]> <![CDATA[<211> 679]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 109]]> Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Ile Gln Leu Val Gln Ser Gly Ser Glu 225 230 235 240 Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 245 250 255 Tyr Ala Phe Thr Thr Thr Gly Met Gln Trp Val Arg Glu Ala Pro Gly 260 265 270 Gln Gly Leu Glu Trp Ile Gly Trp Ile Asn Thr His Ser Gly Val Pro 275 280 285 Lys Tyr Val Glu Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr 290 295 300 Ser Val Asn Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp 305 310 315 320 Thr Ala Val Tyr Tyr Cys Val Arg Ser Gly Asn Gly Asn Tyr Asp Leu 325 330 335 Ala Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 340 345 350 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 355 360 365 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 370 375 380 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 385 390 395 400 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 405 410 415 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 420 425 430 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 435 440 445 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 450 455 460 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 465 470 475 480 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 485 490 495 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 500 505 510 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 515 520 525 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 530 535 540 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 545 550 555 560 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 565 570 575 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 580 585 590 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 595 600 605 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 610 615 620 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 625 630 635 640 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 645 650 655 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 660 665 670 Leu Ser Leu Ser Leu Gly Lys 675 <![CDATA[<210> 110]]> <![CDATA[<211> 2037]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 110]]> gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60 agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120 cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180 cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240 ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300 ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt cagatccagc tggtgcagtc cggctccgag 720 ctgaagaagc ccggcgcctc cgtcaaggtg tcctgcaagg ccagtggcta cgcctttacc 780 accaccggca tgcagtgggt gagggaggcc ccaggccagg gcctggagtg gatcggctgg 840 atcaacacac attctggagt gcctaagtat gtggaagact ttaagggcag attcgtgttt 900 tctctggata cctctgtgaa taccgcttac ctgcagattt cttctctgaa ggctgaggac 960 acagctgtgt attactgtgt gagatctgga aatggaaact atgatctggc ttattttgcc 1020 tattggggcc agggcacact ggtgaccgtg tcttctgcct ccaccaaggg cccatcggtc 1080 ttccccctgg caccctgctc caggagcacc tctgagtcca cagcggccct gggctgcctg 1140 gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc cctgaccagc 1200 ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct cagcagcgtg 1260 gtgaccgtgc cctccagcag cttgggcacc aagacatata cctgtaatgt ggatcacaag 1320 ccttccaata caaaagtgga caagagagtt gagtccaagt acggcccacc atgtcctcca 1380 tgtccagccc ctgaattttt gggcgggcct tctgtctttc tgtttcctcc taaacctaaa 1440 gataccctga tgatcagccg cacacccgaa gtcacttgtg tggtcgtgga tgtgtctcag 1500 gaagatcccg aagtgcagtt taactggtat gtcgatggcg tggaagtgca taatgccaaa 1560 actaagcccc gcgaagaaca gttcaacagc acttatcggg tcgtgtctgt gctcacagtc 1620 ctccatcagg attggctgaa tgggaaagaa tataagtgca aggtgagcaa taagggcctc 1680 cccagcagca tcgagaagac tattagcaaa gccaaagggc agccacggga accccaggtg 1740 tacactctgc ccccctctca ggaggagatg actaaaaatc aggtctctct gacttgtctg 1800 gtgaaagggt tttatcccag cgacattgcc gtggagtggg agtctaatgg ccagcccgag 1860 aataattata agacaactcc ccccgtcctg gactctgacg gcagcttttt cctgtattct 1920 cggctgacag tggacaaaag tcgctggcag gagggcaatg tctttagttg cagtgtcatg 1980 catgaggccc tgcacaatca ctatacacag aaaagcctgt ctctgagtct gggcaaa 2037 <![CDATA[<210> 111]]> <![CDATA[<211> 679]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 111]]> Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Thr 20 25 30 Gly Met Gln Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Val Glu Asp Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Asn Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Gly Asn Gly Asn Tyr Asp Leu Ala Tyr Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val 225 230 235 240 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 245 250 255 Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val 260 265 270 Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly 275 280 285 Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr 290 295 300 Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser 305 310 315 320 Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly 325 330 335 Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 340 345 350 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 355 360 365 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 370 375 380 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 385 390 395 400 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 405 410 415 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 420 425 430 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 435 440 445 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 450 455 460 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 465 470 475 480 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 485 490 495 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 500 505 510 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 515 520 525 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 530 535 540 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 545 550 555 560 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 565 570 575 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 580 585 590 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 595 600 605 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 610 615 620 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 625 630 635 640 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 645 650 655 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 660 665 670 Leu Ser Leu Ser Leu Gly Lys 675 <![CDATA[<210> 112]]> <![CDATA[<211> 2037]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 112]]> cagatccagc tggtgcagtc cggctccgag ctgaagaagc ccggcgcctc cgtcaaggtg 60 tcctgcaagg ccagtggcta cgcctttacc accaccggca tgcagtgggt gagggaggcc 120 ccaggccagg gcctggagtg gatcggctgg atcaacacac attctggagt gcctaagtat 180 gtggaagact ttaagggcag attcgtgttt tctctggata cctctgtgaa taccgcttac 240 ctgcagattt cttctctgaa ggctgaggac acagctgtgt attactgtgt gagatctgga 300 aatggaaact atgatctggc ttattttgcc tattggggcc agggcacact ggtgaccgtg 360 tcttctgcaa gtaccaaggg acctagtgtt ttccctcttg caccttgctc caggtcaaca 420 tcagagtcca cagctgctct tggatgtctc gttaaggact acttcccaga gccagttacc 480 gtatcctgga actccggagc tttgacaagc ggcgttcata cattcccagc tgtgttgcag 540 agttctgggt tgtacagttt gagctcagtg gtgaccgtgc cttcatcttc tttgggcact 600 aagacctaca cctgcaacgt ggatcacaag ccaagcaaca ccaaggtgga taagagggtg 660 ggtggaggcg gttcaggcgg aggtggcagc ggaggtggcg ggagtgaggt caagctggtg 720 gaaagcggcg gcggcctggt gcagcctgga ggatccctgc ggctgagctg cgctgcctcc 780 ggcttcgctt tcagctccta tgacatgtcc tgggtgaggc aggcccctgg aaagaggctg 840 gagtgggtgg ctaccatctc cggaggcgga aggtacacct actaccccga cacagtgaag 900 ggaaggttca ccatcagccg ggataacgcc aaaaacagcc actatctcca gatgaactcc 960 ctgagggccg aagatacagc cgtgtatttc tgtgcctccc cctacggagg ctattttgac 1020 gtgtggggac agggcaccct ggtgaccgtc tcctccgcct ccaccaaggg cccatcggtc 1080 ttccccctgg caccctgctc caggagcacc tctgagtcca cagcggccct gggctgcctg 1140 gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc cctgaccagc 1200 ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct cagcagcgtg 1260 gtgaccgtgc cctccagcag cttgggcacc aagacatata cctgtaatgt ggatcacaag 1320 ccttccaata caaaagtgga caagagagtt gagtccaagt acggcccacc atgtcctcca 1380 tgtccagccc ctgaattttt gggcgggcct tctgtctttc tgtttcctcc taaacctaaa 1440 gataccctga tgatcagccg cacacccgaa gtcacttgtg tggtcgtgga tgtgtctcag 1500 gaagatcccg aagtgcagtt taactggtat gtcgatggcg tggaagtgca taatgccaaa 1560 actaagcccc gcgaagaaca gttcaacagc acttatcggg tcgtgtctgt gctcacagtc 1620 ctccatcagg attggctgaa tgggaaagaa tataagtgca aggtgagcaa taagggcctc 1680 cccagcagca tcgagaagac tattagcaaa gccaaagggc agccacggga accccaggtg 1740 tacactctgc ccccctctca ggaggagatg actaaaaatc aggtctctct gacttgtctg 1800 gtgaaagggt tttatcccag cgacattgcc gtggagtggg agtctaatgg ccagcccgag 1860 aataattata agacaactcc ccccgtcctg gactctgacg gcagcttttt cctgtattct 1920 cggctgacag tggacaaaag tcgctggcag gagggcaatg tctttagttg cagtgtcatg 1980 catgaggccc tgcacaatca ctatacacag aaaagcctgt ctctgagtct gggcaaa 2037 <![CDATA[<210> 113]]> <![CDATA[<211> 119]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 113]]> Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <![CDATA[<210> 114]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 114]]> Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <![CDATA[<210> 115]]> <![CDATA[<211> 120]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 115]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 116]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 116]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 117]]> <![CDATA[<211> 119]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 117]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 118]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 118]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 119]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 119]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 120]]> <![CDATA[<211> 108]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 120]]> Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 121]]> <![CDATA[<211> 116]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 121]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asp 20 25 30 Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser His Gly Gly Asp Tyr Ile Tyr Tyr Ala Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ser Arg Asp Arg Arg Ser Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[<210> 122]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 122]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 123]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 123]]> Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 124]]> <![CDATA[<211> 642]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 124]]> gacatcctgc tgacccagtc ccccgtgatc ctgtccgtgt cccctggcga gagggtgtcc 60 ttctcctgca gggcctccca gtccatcggc accaacatcc actggtacca gcagaggacc 120 aacggctccc ccaggctgct gatcaagtac gcctccgaga gcatcagcgg catcccctcc 180 aggttttccg gctccggctc cggaaccgac ttcaccctgt ccatcaactc cgtggagtcc 240 gaggacatcg ccgactacta ctgccagcag aacaacaact ggcccaccac ctttggcgcc 300 ggcaccaagc tggagctgaa gaggaccgtg gccgccccct ccgtgttcat ttttccccct 360 agcgacgagc agctgaagag cggcaccgct agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca gtggaaagtg gacaacgccc tgcagagcgg caactcccag 480 gagtccgtga ccgagcagga cagcaaggac agcacctact ccctgtcctc caccctgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gtgaggtgac acaccagggc 600 ctgtcctccc ccgtgacaaa gtccttcaac aggggcgagt gc 642 <![CDATA[<210> 125]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 125]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 126]]> <![CDATA[<211> 642]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 126]]> gacatccaga tgacccagtc tccttcttct ctgtctgctt ctgtgggcga tagagtgacc 60 atcacctgta aggcttctca ggatgtgtct atcggcgtgg cctggtatca gcagaagcct 120 ggaaaggctc ctaagctgct gatctactct gcttcttaca gatatactgg cgtgccttct 180 agattttctg gctctggctc tggcacagat tttacactga ccatctcttc tctgcagcct 240 gaggacttcg ctacctatta ctgtcagcag tactacatct atccttatac ctttggccag 300 ggcaccaagg tggagatcaa gagaaccgtc gccgctccca gcgtcttcat cttccccccc 360 agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480 gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600 ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642 <![CDATA[<210> 127]]> <![CDATA[<211> 215]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 127]]> Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <![CDATA[<210> 128]]> <![CDATA[<211> 645]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 128]]> gagatcgtgc tgactcagag ccccggcact ctgtctctga gccccggcga aagggccaca 60 ctgagctgta gggctagcca gagcgtgggc agcagctatc tggcttggta ccagcagaag 120 cccggccaag cccctagact gctgatctac ggcgccttct ctagggctac tggcatccca 180 gataggttta gcggcagcgg atccggcaca gacttcactc tgacaatctc taggctggag 240 ccagaagact tcgccgtgta ctattgccag cagtacggca gcagcccatg gactttcggc 300 caaggcacaa aggtcgagat caagagaacc gtcgccgctc ccagcgtctt catcttcccc 360 cccagcgatg agcagctgaa gagcggaacc gccagcgtgg tgtgcctgct gaacaacttc 420 taccccaggg aggccaaggt gcaatggaag gtggacaacg ccctacagag cggcaactcc 480 caggagagcg tgaccgagca ggacagcaag gatagcacct acagcctgag cagcaccctc 540 accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccatcag 600 ggcctgagca gccctgtgac caagagcttc aacaggggcg agtgc 645 <![CDATA[<210> 129]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 129]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 130]]> <![CDATA[<211> 642]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 130]]> gacatccaga tgacccagtc cccttcctcc ctgtccgctt ccgtgggcga cagggtgacc 60 atcacctgca gggccagcca ggacatctcc aactacctgt cctggtacca gcagaagccc 120 ggcaagacca tcaagctgct gatctactac acctccaggc tgcacagcgg cgtgcctagc 180 aggttctccg gttctggctc cggcaccgac tacaccttca ccatcagctc cctgcagccc 240 gaggacatcg ccacctactt ctgccagcag ggcaagaccc tgccctacac cttcggccag 300 ggcaccaagg tggagatcaa gcgcactgtg gctgccccca gtgttttcat atttcccccc 360 agtgatgagc aactgaagtc cggcacagcc tctgttgtat gtctgctgaa taatttttat 420 ccacgggagg ccaaggtgca gtggaaggtg gacaatgccc tgcagtctgg gaactctcaa 480 gagagtgtga cagagcagga cagtaaagac agcacctata gcctcagcag caccctgacc 540 ctgtctaaag ccgactatga aaaacacaaa gtgtatgcct gcgaagtgac ccatcagggg 600 ctcagctctc ccgttaccaa gagctttaac cgaggcgaat gt 642 <![CDATA[<210> 131]]> <![CDATA[<211> 676]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 131]]> Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly 225 230 235 240 Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly 245 250 255 Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly 260 265 270 Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp 275 280 285 Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser 290 295 300 Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr 305 310 315 320 Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe 325 330 335 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr 340 345 350 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 355 360 365 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 370 375 380 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 385 390 395 400 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 405 410 415 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 420 425 430 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 435 440 445 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 450 455 460 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 465 470 475 480 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 485 490 495 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 500 505 510 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 515 520 525 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535 540 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 545 550 555 560 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 565 570 575 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 580 585 590 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 595 600 605 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 610 615 620 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 625 630 635 640 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 645 650 655 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670 Ser Leu Gly Lys 675 <![CDATA[<210> 132]]> <![CDATA[<211> 2028]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 132]]> gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60 agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120 cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180 cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240 ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300 ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt caggtgcagc tgaagcagtc cggacctggc 720 ctggtgcagc cttcccagtc cctgtccatc acctgcaccg tgtccggctt ttccctgacc 780 aactacggcg tgcactgggt gaggcagtcc cctggcaagg gcctggaatg gctgggcgtg 840 atctggtccg gcggcaacac cgactacaac acccccttca cctcccggct gtccatcaac 900 aaggacaaca gcaagtccca ggtgttcttc aagatgaact ccctgcagag caacgacacc 960 gccatctact actgcgccag agccctgacc tattacgact acgagttcgc ctactggggc 1020 cagggcacac tggtgaccgt gtccgccgcc tccaccaagg gcccatcggt cttccccctg 1080 gcaccctgct ccaggagcac ctctgagtcc acagcggccc tgggctgcct ggtcaaggac 1140 tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 1200 accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 1260 ccctccagca gcttgggcac caagacatat acctgtaatg tggatcacaa gccttccaat 1320 acaaaagtgg acaagagagt tgagtccaag tacggcccac catgtcctcc atgtccagcc 1380 cctgaatttt tgggcgggcc ttctgtcttt ctgtttcctc ctaaacctaa agataccctg 1440 atgatcagcc gcacacccga agtcacttgt gtggtcgtgg atgtgtctca ggaagatccc 1500 gaagtgcagt ttaactggta tgtcgatggc gtggaagtgc ataatgccaa aactaagccc 1560 cgcgaagaac agttcaacag cacttatcgg gtcgtgtctg tgctcacagt cctccatcag 1620 gattggctga atgggaaaga atataagtgc aaggtgagca ataagggcct ccccagcagc 1680 atcgagaaga ctattagcaa agccaaaggg cagccacggg aaccccaggt gtacactctg 1740 cccccctctc aggaggagat gactaaaaat caggtctctc tgacttgtct ggtgaaaggg 1800 ttttatccca gcgacattgc cgtggagtgg gagtctaatg gccagcccga gaataattat 1860 aagacaactc cccccgtcct ggactctgac ggcagctttt tcctgtattc tcggctgaca 1920 gtggacaaaa gtcgctggca ggagggcaat gtctttagtt gcagtgtcat gcatgaggcc 1980 ctgcacaatc actatacaca gaaaagcctg tctctgagtc tgggcaaa 2028 <![CDATA[<210> 133]]> <![CDATA[<211> 676]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 133]]> Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 225 230 235 240 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 245 250 255 Phe Thr Phe Thr Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly 260 265 270 Lys Gly Leu Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser 275 280 285 Ile Tyr Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg 290 295 300 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 305 310 315 320 Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe 325 330 335 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 340 345 350 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 355 360 365 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 370 375 380 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 385 390 395 400 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 405 410 415 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 420 425 430 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 435 440 445 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 450 455 460 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 465 470 475 480 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 485 490 495 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 500 505 510 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 515 520 525 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535 540 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 545 550 555 560 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 565 570 575 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 580 585 590 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 595 600 605 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 610 615 620 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 625 630 635 640 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 645 650 655 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670 Ser Leu Gly Lys 675 <![CDATA[<210> 134]]> <![CDATA[<211> 2028]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 134]]> gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60 agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120 cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180 cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240 ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300 ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt gaagtgcagc tggtggagtc tggaggcgga 720 ctggtgcagc ctggaggctc tctgagactg tcttgtgctg cttctggctt tacctttacc 780 gattatacca tggattgggt gagacaggcc cctggaaagg gactggagtg ggtggctgat 840 gtgaacccta actctggagg atctatctat aatcagaggt ttaagggcag attcaccctg 900 tctgtggata gatctaagaa tacactgtat ctgcagatga actctctgag agctgaagat 960 acagctgtgt attactgcgc tagaaatctg ggcccttcct tttactttga ttactggggc 1020 cagggaacac tggtgaccgt gtcttctgcc tccaccaagg gcccatcggt cttccccctg 1080 gcaccctgct ccaggagcac ctctgagtcc acagcggccc tgggctgcct ggtcaaggac 1140 tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac 1200 accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg 1260 ccctccagca gcttgggcac caagacatat acctgtaatg tggatcacaa gccttccaat 1320 acaaaagtgg acaagagagt tgagtccaag tacggcccac catgtcctcc atgtccagcc 1380 cctgaatttt tgggcgggcc ttctgtcttt ctgtttcctc ctaaacctaa agataccctg 1440 atgatcagcc gcacacccga agtcacttgt gtggtcgtgg atgtgtctca ggaagatccc 1500 gaagtgcagt ttaactggta tgtcgatggc gtggaagtgc ataatgccaa aactaagccc 1560 cgcgaagaac agttcaacag cacttatcgg gtcgtgtctg tgctcacagt cctccatcag 1620 gattggctga atgggaaaga atataagtgc aaggtgagca ataagggcct ccccagcagc 1680 atcgagaaga ctattagcaa agccaaaggg cagccacggg aaccccaggt gtacactctg 1740 cccccctctc aggaggagat gactaaaaat caggtctctc tgacttgtct ggtgaaaggg 1800 ttttatccca gcgacattgc cgtggagtgg gagtctaatg gccagcccga gaataattat 1860 aagacaactc cccccgtcct ggactctgac ggcagctttt tcctgtattc tcggctgaca 1920 gtggacaaaa gtcgctggca ggagggcaat gtctttagtt gcagtgtcat gcatgaggcc 1980 ctgcacaatc actatacaca gaaaagcctg tctctgagtc tgggcaaa 2028 <![CDATA[<210> 135]]> <![CDATA[<211> 678]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 135]]> Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly 225 230 235 240 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 245 250 255 Phe Thr Phe Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly 260 265 270 Lys Gly Leu Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys 275 280 285 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 290 295 300 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 305 310 315 320 Thr Ala Ile Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp 325 330 335 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 340 345 350 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 355 360 365 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 370 375 380 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 385 390 395 400 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 405 410 415 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 420 425 430 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 435 440 445 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 450 455 460 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 465 470 475 480 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 485 490 495 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 500 505 510 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 515 520 525 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 530 535 540 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 545 550 555 560 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 565 570 575 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 580 585 590 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 595 600 605 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 610 615 620 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 625 630 635 640 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 645 650 655 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 660 665 670 Ser Leu Ser Pro Gly Lys 675 <![CDATA[<210> 136]]> <![CDATA[<211> 2034]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 136]]> gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60 agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120 cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180 cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240 ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300 ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt caagtgcagc tggtcgaaag cggcggagga 720 gtcgtccagc ccggcagatc tctgagactg agctgtgctg cctccggctt cacattcagc 780 tcctacacta tgcactgggt gaggcaagcc cccggcaagg gactggagtg ggtgactttc 840 atcagctacg acggcaacaa caagtactac gccgacagcg tgaagggaag gttcactatc 900 tctagggaca acagcaagaa cactctgtat ctgcagatga actctctgag ggccgaggat 960 actgccatct actactgcgc taggactggc tggctgggcc ctttcgatta ctggggccaa 1020 ggcactctgg tcactgtgag cagcgcgagc accaagggac cttccgtgtt tcccctcgcc 1080 cccagctcca aaagcaccag cggcggaaca gctgctctcg gctgtctcgt caaggattac 1140 ttccccgagc ccgtgaccgt gagctggaac agcggagccc tgacaagcgg cgtccacacc 1200 ttccctgctg tcctacagtc ctccggactg tacagcctga gcagcgtggt gacagtccct 1260 agcagctccc tgggcaccca gacatatatt tgcaacgtga atcacaagcc cagcaacacc 1320 aaggtcgata agaaggtgga gcctaagtcc tgcgacaaga cccacacatg tcccccctgt 1380 cccgctcctg aactgctggg aggcccttcc gtgttcctgt tcccccctaa gcccaaggac 1440 accctgatga tttccaggac acccgaggtg acctgtgtgg tggtggacgt cagccacgag 1500 gaccccgagg tgaaattcaa ctggtacgtc gatggcgtgg aggtgcacaa cgctaagacc 1560 aagcccaggg aggagcagta caattccacc tacagggtgg tgtccgtgct gaccgtcctc 1620 catcaggact ggctgaacgg caaagagtat aagtgcaagg tgagcaacaa ggccctccct 1680 gctcccatcg agaagaccat cagcaaagcc aagggccagc ccagggaacc tcaagtctat 1740 accctgcctc ccagcaggga ggagatgacc aagaaccaag tgagcctcac atgcctcgtc 1800 aagggcttct atccttccga tattgccgtc gagtgggagt ccaacggaca gcccgagaac 1860 aactacaaga caacaccccc cgtgctcgat tccgatggca gcttcttcct gtactccaag 1920 ctgaccgtgg acaagtccag atggcaacaa ggcaacgtct tcagttgcag cgtcatgcat 1980 gaggccctcc acaaccacta cacccagaaa agcctgtctc tgagtcctgg caaa 2034 <![CDATA[<210> 137]]> <![CDATA[<211> 678]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 137]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly 225 230 235 240 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 245 250 255 Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro 260 265 270 Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr 275 280 285 Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 290 295 300 Asn Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 305 310 315 320 Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp 325 330 335 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 340 345 350 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 355 360 365 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 370 375 380 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 385 390 395 400 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 405 410 415 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 420 425 430 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 435 440 445 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 450 455 460 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 465 470 475 480 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 485 490 495 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 500 505 510 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 515 520 525 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 530 535 540 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 545 550 555 560 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 565 570 575 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 580 585 590 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 595 600 605 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 610 615 620 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 625 630 635 640 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 645 650 655 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 660 665 670 Ser Leu Ser Pro Gly Lys 675 <![CDATA[<210> 138]]> <![CDATA[<211> 2034]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 138]]> caagtgcagc tggtcgaaag cggcggagga gtcgtccagc ccggcagatc tctgagactg 60 agctgtgctg cctccggctt cacattcagc tcctacacta tgcactgggt gaggcaagcc 120 cccggcaagg gactggagtg ggtgactttc atcagctacg acggcaacaa caagtactac 180 gccgacagcg tgaagggaag gttcactatc tctagggaca acagcaagaa cactctgtat 240 ctgcagatga actctctgag ggccgaggat actgccatct actactgcgc taggactggc 300 tggctgggcc ctttcgatta ctggggccaa ggcactctgg tcactgtgag cagcgcaagt 360 accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 420 gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 480 tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 540 tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 600 tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtggg tggaggcggt 660 tcaggcggag gtggcagcgg aggtggcggg agtgaggtca agctggtgga aagcggcggc 720 ggcctggtgc agcctggagg atccctgcgg ctgagctgcg ctgcctccgg cttcgctttc 780 agctcctatg acatgtcctg ggtgaggcag gcccctggaa agaggctgga gtgggtggct 840 accatctccg gaggcggaag gtacacctac taccccgaca cagtgaaggg aaggttcacc 900 atcagccggg ataacgccaa aaacagccac tatctccaga tgaactccct gagggccgaa 960 gatacagccg tgtatttctg tgcctccccc tacggaggct attttgacgt gtggggacag 1020 ggcaccctgg tgaccgtctc ctccgcgagc accaagggac cttccgtgtt tcccctcgcc 1080 cccagctcca aaagcaccag cggcggaaca gctgctctcg gctgtctcgt caaggattac 1140 ttccccgagc ccgtgaccgt gagctggaac agcggagccc tgacaagcgg cgtccacacc 1200 ttccctgctg tcctacagtc ctccggactg tacagcctga gcagcgtggt gacagtccct 1260 agcagctccc tgggcaccca gacatatatt tgcaacgtga atcacaagcc cagcaacacc 1320 aaggtcgata agaaggtgga gcctaagtcc tgcgacaaga cccacacatg tcccccctgt 1380 cccgctcctg aactgctggg aggcccttcc gtgttcctgt tcccccctaa gcccaaggac 1440 accctgatga tttccaggac acccgaggtg acctgtgtgg tggtggacgt cagccacgag 1500 gaccccgagg tgaaattcaa ctggtacgtc gatggcgtgg aggtgcacaa cgctaagacc 1560 aagcccaggg aggagcagta caattccacc tacagggtgg tgtccgtgct gaccgtcctc 1620 catcaggact ggctgaacgg caaagagtat aagtgcaagg tgagcaacaa ggccctccct 1680 gctcccatcg agaagaccat cagcaaagcc aagggccagc ccagggaacc tcaagtctat 1740 accctgcctc ccagcaggga ggagatgacc aagaaccaag tgagcctcac atgcctcgtc 1800 aagggcttct atccttccga tattgccgtc gagtgggagt ccaacggaca gcccgagaac 1860 aactacaaga caacaccccc cgtgctcgat tccgatggca gcttcttcct gtactccaag 1920 ctgaccgtgg acaagtccag atggcaacaa ggcaacgtct tcagttgcag cgtcatgcat 1980 gaggccctcc acaaccacta cacccagaaa agcctgtctc tgagtcctgg caaa 2034 <![CDATA[<210> 139]]> <![CDATA[<211> 675]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 139]]> Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly 225 230 235 240 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 245 250 255 Phe Thr Phe Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly 260 265 270 Lys Gly Leu Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys 275 280 285 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 290 295 300 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 305 310 315 320 Thr Ala Ile Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp 325 330 335 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 340 345 350 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 355 360 365 Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 370 375 380 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 385 390 395 400 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 405 410 415 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 420 425 430 Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser 435 440 445 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 450 455 460 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 465 470 475 480 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 485 490 495 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 500 505 510 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 515 520 525 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 530 535 540 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 545 550 555 560 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 565 570 575 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 580 585 590 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 595 600 605 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 610 615 620 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 625 630 635 640 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 645 650 655 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 660 665 670 Leu Gly Lys 675 <![CDATA[<210> 140]]> <![CDATA[<211> 2025]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 140]]> gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60 agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120 cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180 cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240 ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300 ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt caagtgcagc tggtcgaaag cggcggagga 720 gtcgtccagc ccggcagatc tctgagactg agctgtgctg cctccggctt cacattcagc 780 tcctacacta tgcactgggt gaggcaagcc cccggcaagg gactggagtg ggtgactttc 840 atcagctacg acggcaacaa caagtactac gccgacagcg tgaagggaag gttcactatc 900 tctagggaca acagcaagaa cactctgtat ctgcagatga actctctgag ggccgaggat 960 actgccatct actactgcgc taggactggc tggctgggcc ctttcgatta ctggggccaa 1020 ggcactctgg tcactgtgag cagcgcctcc accaagggcc catcggtctt ccccctggca 1080 ccctgctcca ggagcacctc tgagtccaca gcggccctgg gctgcctggt caaggactac 1140 ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 1200 ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 1260 tccagcagct tgggcaccaa gacatatacc tgtaatgtgg atcacaagcc ttccaataca 1320 aaagtggaca agagagttga gtccaagtac ggcccaccat gtcctccatg tccagcccct 1380 gaatttttgg gcgggccttc tgtctttctg tttcctccta aacctaaaga taccctgatg 1440 atcagccgca cacccgaagt cacttgtgtg gtcgtggatg tgtctcagga agatcccgaa 1500 gtgcagttta actggtatgt cgatggcgtg gaagtgcata atgccaaaac taagccccgc 1560 gaagaacagt tcaacagcac ttatcgggtc gtgtctgtgc tcacagtcct ccatcaggat 1620 tggctgaatg ggaaagaata taagtgcaag gtgagcaata agggcctccc cagcagcatc 1680 gagaagacta ttagcaaagc caaagggcag ccacgggaac cccaggtgta cactctgccc 1740 ccctctcagg aggagatgac taaaaatcag gtctctctga cttgtctggt gaaagggttt 1800 tatcccagcg acattgccgt ggagtgggag tctaatggcc agcccgagaa taattataag 1860 acaactcccc ccgtcctgga ctctgacggc agctttttcc tgtattctcg gctgacagtg 1920 gacaaaagtc gctggcagga gggcaatgtc tttagttgca gtgtcatgca tgaggccctg 1980 cacaatcact atacacagaa aagcctgtct ctgagtctgg gcaaa 2025 <![CDATA[<210> 141]]> <![CDATA[<211> 675]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 141]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly 225 230 235 240 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 245 250 255 Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro 260 265 270 Gly Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr 275 280 285 Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 290 295 300 Asn Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 305 310 315 320 Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp 325 330 335 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 340 345 350 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 355 360 365 Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 370 375 380 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 385 390 395 400 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 405 410 415 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 420 425 430 Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser 435 440 445 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 450 455 460 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 465 470 475 480 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 485 490 495 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 500 505 510 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 515 520 525 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 530 535 540 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 545 550 555 560 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 565 570 575 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 580 585 590 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 595 600 605 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 610 615 620 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 625 630 635 640 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 645 650 655 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 660 665 670 Leu Gly Lys 675 <![CDATA[<210> 142]]> <![CDATA[<211> 2025]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 142]]> caagtgcagc tggtcgaaag cggcggagga gtcgtccagc ccggcagatc tctgagactg 60 agctgtgctg cctccggctt cacattcagc tcctacacta tgcactgggt gaggcaagcc 120 cccggcaagg gactggagtg ggtgactttc atcagctacg acggcaacaa caagtactac 180 gccgacagcg tgaagggaag gttcactatc tctagggaca acagcaagaa cactctgtat 240 ctgcagatga actctctgag ggccgaggat actgccatct actactgcgc taggactggc 300 tggctgggcc ctttcgatta ctggggccaa ggcactctgg tcactgtgag cagcgcaagt 360 accaagggac ctagtgtttt ccctcttgca ccttgctcca ggtcaacatc agagtccaca 420 gctgctcttg gatgtctcgt taaggactac ttcccagagc cagttaccgt atcctggaac 480 tccggagctt tgacaagcgg cgttcataca ttcccagctg tgttgcagag ttctgggttg 540 tacagtttga gctcagtggt gaccgtgcct tcatcttctt tgggcactaa gacctacacc 600 tgcaacgtgg atcacaagcc aagcaacacc aaggtggata agagggtggg tggaggcggt 660 tcaggcggag gtggcagcgg aggtggcggg agtgaggtca agctggtgga aagcggcggc 720 ggcctggtgc agcctggagg atccctgcgg ctgagctgcg ctgcctccgg cttcgctttc 780 agctcctatg acatgtcctg ggtgaggcag gcccctggaa agaggctgga gtgggtggct 840 accatctccg gaggcggaag gtacacctac taccccgaca cagtgaaggg aaggttcacc 900 atcagccggg ataacgccaa aaacagccac tatctccaga tgaactccct gagggccgaa 960 gatacagccg tgtatttctg tgcctccccc tacggaggct attttgacgt gtggggacag 1020 ggcaccctgg tgaccgtctc ctccgcctcc accaagggcc catcggtctt ccccctggca 1080 ccctgctcca ggagcacctc tgagtccaca gcggccctgg gctgcctggt caaggactac 1140 ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 1200 ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 1260 tccagcagct tgggcaccaa gacatatacc tgtaatgtgg atcacaagcc ttccaataca 1320 aaagtggaca agagagttga gtccaagtac ggcccaccat gtcctccatg tccagcccct 1380 gaatttttgg gcgggccttc tgtctttctg tttcctccta aacctaaaga taccctgatg 1440 atcagccgca cacccgaagt cacttgtgtg gtcgtggatg tgtctcagga agatcccgaa 1500 gtgcagttta actggtatgt cgatggcgtg gaagtgcata atgccaaaac taagccccgc 1560 gaagaacagt tcaacagcac ttatcgggtc gtgtctgtgc tcacagtcct ccatcaggat 1620 tggctgaatg ggaaagaata taagtgcaag gtgagcaata agggcctccc cagcagcatc 1680 gagaagacta ttagcaaagc caaagggcag ccacgggaac cccaggtgta cactctgccc 1740 ccctctcagg aggagatgac taaaaatcag gtctctctga cttgtctggt gaaagggttt 1800 tatcccagcg acattgccgt ggagtgggag tctaatggcc agcccgagaa taattataag 1860 acaactcccc ccgtcctgga ctctgacggc agctttttcc tgtattctcg gctgacagtg 1920 gacaaaagtc gctggcagga gggcaatgtc tttagttgca gtgtcatgca tgaggccctg 1980 cacaatcact atacacagaa aagcctgtct ctgagtctgg gcaaa 2025 <![CDATA[<210> 143]]> <![CDATA[<211> 673]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 143]]> Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 225 230 235 240 Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 245 250 255 Phe Thr Phe Ser Asp Asp Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly 260 265 270 Lys Arg Leu Glu Trp Val Ala Ser Ile Ser His Gly Gly Asp Tyr Ile 275 280 285 Tyr Tyr Ala Asp Asn Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 290 295 300 Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 305 310 315 320 Thr Ala Val Tyr Phe Cys Ser Arg Asp Arg Arg Ser Ile Asp Tyr Trp 325 330 335 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 340 345 350 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 355 360 365 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 370 375 380 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 385 390 395 400 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 405 410 415 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 420 425 430 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 435 440 445 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 450 455 460 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 465 470 475 480 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 485 490 495 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 500 505 510 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 515 520 525 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 530 535 540 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 545 550 555 560 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 565 570 575 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 580 585 590 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 595 600 605 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 610 615 620 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 625 630 635 640 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 645 650 655 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 660 665 670 Lys <![CDATA[<210> 144]]> <![CDATA[<211> 2019]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 144]]> gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60 agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120 cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180 cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240 ctccagatga actccctgag ggccgaagat acagccgtgt atttctgtgc ctccccctac 300 ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt gaagtgcagc tggtggagtc cggaggcgga 720 ctggtgaagc ctggaggctc cctgaggctg tcctgtgccg cttccggctt caccttctcc 780 gacgactaca tggcctggtt caggcaggcc cctggaaaga ggctggagtg ggtggcttcc 840 atctcccacg gcggcgacta catctactac gccgacaacc tgaagggcag gttcaccatc 900 tccagggaca acgccaagaa ctccctgtac ctgcagatga actccctgag ggccgaggac 960 accgccgtgt acttctgctc cagggacagg aggtccatcg actattgggg ccagggcacc 1020 ctggtgacag tgtcctccgc ctccaccaag ggcccatcgg tcttccccct ggcaccctgc 1080 tccaggagca cctctgagtc cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 1140 gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 1200 gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 1260 agcttgggca ccaagacata tacctgtaat gtggatcaca agccttccaa tacaaaagtg 1320 gacaagagag ttgagtccaa gtacggccca ccatgtcctc catgtccagc ccctgaattt 1380 ttgggcgggc cttctgtctt tctgtttcct cctaaaccta aagataccct gatgatcagc 1440 cgcacacccg aagtcacttg tgtggtcgtg gatgtgtctc aggaagatcc cgaagtgcag 1500 tttaactggt atgtcgatgg cgtggaagtg cataatgcca aaactaagcc ccgcgaagaa 1560 cagttcaaca gcacttatcg ggtcgtgtct gtgctcacag tcctccatca ggattggctg 1620 aatgggaaag aatataagtg caaggtgagc aataagggcc tccccagcag catcgagaag 1680 actattagca aagccaaagg gcagccacgg gaaccccagg tgtacactct gcccccctct 1740 caggaggaga tgactaaaaa tcaggtctct ctgacttgtc tggtgaaagg gttttatccc 1800 agcgacattg ccgtggagtg ggagtctaat ggccagcccg agaataatta taagacaact 1860 ccccccgtcc tggactctga cggcagcttt ttcctgtatt ctcggctgac agtggacaaa 1920 agtcgctggc aggagggcaa tgtctttagt tgcagtgtca tgcatgaggc cctgcacaat 1980 cactatacac agaaaagcct gtctctgagt ctgggcaaa 2019 <![CDATA[<210> 145]]> <![CDATA[<211> 673]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 145]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asp 20 25 30 Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser His Gly Gly Asp Tyr Ile Tyr Tyr Ala Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ser Arg Asp Arg Arg Ser Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu 225 230 235 240 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 245 250 255 Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys 260 265 270 Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr 275 280 285 Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 290 295 300 Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 305 310 315 320 Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp 325 330 335 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 340 345 350 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 355 360 365 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 370 375 380 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 385 390 395 400 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 405 410 415 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 420 425 430 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 435 440 445 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 450 455 460 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 465 470 475 480 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 485 490 495 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 500 505 510 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 515 520 525 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 530 535 540 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 545 550 555 560 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 565 570 575 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 580 585 590 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 595 600 605 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 610 615 620 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 625 630 635 640 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 645 650 655 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 660 665 670 Lys <![CDATA[<210> 146]]> <![CDATA[<211> 2019]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 146]]> gaagtgcagc tggtggagtc cggaggcgga ctggtgaagc ctggaggctc cctgaggctg 60 tcctgtgccg cttccggctt caccttctcc gacgactaca tggcctggtt caggcaggcc 120 cctggaaaga ggctggagtg ggtggcttcc atctcccacg gcggcgacta catctactac 180 gccgacaacc tgaagggcag gttcaccatc tccagggaca acgccaagaa ctccctgtac 240 ctgcagatga actccctgag ggccgaggac accgccgtgt acttctgctc cagggacagg 300 aggtccatcg actattgggg ccagggcacc ctggtgacag tgtcctccgc aagtaccaag 360 ggacctagtg ttttccctct tgcaccttgc tccaggtcaa catcagagtc cacagctgct 420 cttggatgtc tcgttaagga ctacttccca gagccagtta ccgtatcctg gaactccgga 480 gctttgacaa gcggcgttca tacattccca gctgtgttgc agagttctgg gttgtacagt 540 ttgagctcag tggtgaccgt gccttcatct tctttgggca ctaagaccta cacctgcaac 600 gtggatcaca agccaagcaa caccaaggtg gataagaggg tgggtggagg cggttcaggc 660 ggaggtggca gcggaggtgg cgggagtgag gtcaagctgg tggaaagcgg cggcggcctg 720 gtgcagcctg gaggatccct gcggctgagc tgcgctgcct ccggcttcgc tttcagctcc 780 tatgacatgt cctgggtgag gcaggcccct ggaaagaggc tggagtgggt ggctaccatc 840 tccggaggcg gaaggtacac ctactacccc gacacagtga agggaaggtt caccatcagc 900 cgggataacg ccaaaaacag ccactatctc cagatgaact ccctgagggc cgaagataca 960 gccgtgtatt tctgtgcctc cccctacgga ggctattttg acgtgtgggg acagggcacc 1020 ctggtgaccg tctcctccgc ctccaccaag ggcccatcgg tcttccccct ggcaccctgc 1080 tccaggagca cctctgagtc cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 1140 gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 1200 gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 1260 agcttgggca ccaagacata tacctgtaat gtggatcaca agccttccaa tacaaaagtg 1320 gacaagagag ttgagtccaa gtacggccca ccatgtcctc catgtccagc ccctgaattt 1380 ttgggcgggc cttctgtctt tctgtttcct cctaaaccta aagataccct gatgatcagc 1440 cgcacacccg aagtcacttg tgtggtcgtg gatgtgtctc aggaagatcc cgaagtgcag 1500 tttaactggt atgtcgatgg cgtggaagtg cataatgcca aaactaagcc ccgcgaagaa 1560 cagttcaaca gcacttatcg ggtcgtgtct gtgctcacag tcctccatca ggattggctg 1620 aatgggaaag aatataagtg caaggtgagc aataagggcc tccccagcag catcgagaag 1680 actattagca aagccaaagg gcagccacgg gaaccccagg tgtacactct gcccccctct 1740 caggaggaga tgactaaaaa tcaggtctct ctgacttgtc tggtgaaagg gttttatccc 1800 agcgacattg ccgtggagtg ggagtctaat ggccagcccg agaataatta taagacaact 1860 ccccccgtcc tggactctga cggcagcttt ttcctgtatt ctcggctgac agtggacaaa 1920 agtcgctggc aggagggcaa tgtctttagt tgcagtgtca tgcatgaggc cctgcacaat 1980 cactatacac agaaaagcct gtctctgagt ctgggcaaa 2019 <![CDATA[<210> 147]]> <![CDATA[<211> 330]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 147]]> Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <![CDATA[<210> 148]]> <![CDATA[<211> 327]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 148]]> Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <![CDATA[<210> 149]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 149]]> Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <![CDATA[<210> 150]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 150]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <![CDATA[<210> 151]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 151]]> Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <![CDATA[<210> 152]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 152]]> Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <![CDATA[<210> 153]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 智人]]> <![CDATA[<400> 153]]> Phe Gly Gly Gly Thr Lys Val Glu Leu Lys 1 5 10
Claims (9)
- 一種PD-1/TGF-beta四價雙特異性抗體,其中該PD-1/TGF-beta四價雙特異性抗體包含兩條多肽鏈和四條共同輕鏈,該兩條多肽鏈的胺基酸序列如SEQ ID NO.49所示,該四條共同輕鏈的胺基酸序列如SEQ ID NO.43所示。
- 一種分離的核苷酸,其中該分離的核苷酸編碼如請求項1所述的PD-1/TGF-beta四價雙特異性抗體。
- 如請求項2所述的分離的核苷酸,其中該分離的核苷酸編碼該兩條多肽鏈的核苷酸的序列如SEQ ID NO.50所示,編碼該四條共同輕鏈的核苷酸的序列如SEQ ID NO.44所示。
- 一種表達載體,其中該表達載體含有如請求項2或3所述的分離的核苷酸。
- 一種宿主細胞,其中該宿主細胞含有如請求項2或3所述的分離的核苷酸或請求項4所述的表達載體。
- 一種藥物組合物,其中該藥物組合物含有如請求項1所述的PD-1/TGF-beta四價雙特異性抗體。
- 一種如請求項1所述的PD-1/TGF-beta四價雙特異性抗體或請求項6所述的藥物組合物在製備治療癌症、炎性疾病、自體免疫性疾病的藥物中的用途。
- 如請求項7所述的用途,其中該PD-1/TGF-beta四價雙特異性抗體或含有該PD-1/TGF-beta四價雙特異性抗體的該藥物組合物用於治療癌症。
- 一種如請求項1所述的PD-1/TGF-beta四價雙特異性抗體的製備方法,其中該製備方法包含以下步驟: (a) 構建如請求項4所述的表達載體,並轉化宿主細胞; (b) 在表達條件下,培養步驟 (a) 所述的該宿主細胞,表達該PD-1/TGF-beta四價雙特異性抗體; (c) 分離並純化 (b) 所述的該PD-1/TGF-beta四價雙特異性抗體。
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CN113563473A (zh) | 2021-10-29 |
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CN114349866A (zh) | 2022-04-15 |
TW202221044A (zh) | 2022-06-01 |
CN114262379B (zh) | 2023-06-02 |
CN114349866B (zh) | 2023-06-02 |
JP2023159379A (ja) | 2023-10-31 |
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CN113993901A (zh) | 2022-01-28 |
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JP2022540319A (ja) | 2022-09-15 |
EP3967711A4 (en) | 2023-08-16 |
EP4050031A1 (en) | 2022-08-31 |
EP3967711A1 (en) | 2022-03-16 |
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