本公開涉及一種添加有糖基或具有改善的結(jié)合親和性的經(jīng)修飾的dkk2蛋白、對其編碼的核酸、其制備方法及其用途。
背景技術(shù)：
：糖基化是糖與蛋白質(zhì)連接的工藝，主要分為n-糖基化和o-糖基化。糖基化由糖基轉(zhuǎn)移酶催化，并已經(jīng)報道有約200種糖基轉(zhuǎn)移酶。糖的種類和結(jié)構(gòu)可能會影響蛋白質(zhì)折疊、穩(wěn)定性、溶解度和對蛋白酶的敏感性、血清半衰期、抗原性、活性增加等。dkk2是wnt的阻遏蛋白，屬于dickkopf家族，并已經(jīng)報道作為wnt信號通路的抑制因子或刺激因子(wuw等,curr.biol.,10(24),第1611-1614頁,2000)。dkk2可以包含兩個特定的富含半胱氨酸的結(jié)構(gòu)域(crd)，并包括不同長度的連接區(qū)域。特別地，dkk2在具有10個半胱氨酸氨基酸的dickkopf家族成員中高度保守半胱氨酸-2區(qū)域(krupnikve等,gene,238(2),第301-313頁,1999)。dkk2是在動物細胞中因低表達效率而難以產(chǎn)生的蛋白質(zhì)，因此使用dkk2的治療劑的開發(fā)正在延遲。因此，需要能夠維持結(jié)合親和性或?qū)ζ涞孜锞哂懈纳频慕Y(jié)合親和性并且還表現(xiàn)出增加的表達效率的dkk2。技術(shù)實現(xiàn)要素：問題的解決方案一方面，提供經(jīng)修飾的dkk2多肽，其與野生型dkk2多肽的氨基酸序列相比，包含一個或多個額外糖基化位點。另一方面，提供編碼經(jīng)修飾的dkk2多肽的核酸。又另一方面，提供制備經(jīng)修飾的dkk2多肽的方法。又另一方面，提供用于促進血管生成的藥物組合物，所述組合物包含經(jīng)修飾的dkk2多肽或編碼經(jīng)修飾的dkk2多肽的核酸、以及藥學(xué)上可接受的載體。又另一方面，提供用于預(yù)防或治療血管通透性相關(guān)疾病的藥物組合物，所述組合物包括經(jīng)修飾的dkk2多肽或編碼經(jīng)修飾的dkk2多肽的核酸、以及藥學(xué)上可接受的載體。又另一方面，提供一種促進受試者的血管生成的方法，所述方法包括將經(jīng)修飾的dkk2多肽或編碼經(jīng)修飾的dkk2多肽的核酸施用于所述受試者。又另一方面，提供一種預(yù)防或治療受試者的血管通透性相關(guān)疾病的方法，所述方法包括將經(jīng)修飾的dkk2多肽或編碼經(jīng)修飾的dkk2多肽的核酸施用于受試者。發(fā)明的有益效果根據(jù)一方面的經(jīng)修飾的dkk2多肽、對其編碼的核酸、其制備方法及其用途，可以有效制備具有額外糖基或?qū)ζ涞孜飈rp6具有改善的結(jié)合親和性的經(jīng)修飾的dkk2蛋白，從而被用于促進血管生成或用于預(yù)防或治療血管通透性相關(guān)疾病。附圖說明從以下結(jié)合附圖對示例性實施方式的描述中，這些和/或其它方面將變得顯而易見和更容易理解，其中：圖1a和圖1b分別是示出了含有各種標簽的野生型dkk2蛋白和突變型dkk2蛋白的免疫印跡結(jié)果的圖像，圖1c是示出了含有fc標簽的dkk2n-gly4和dkk2n-gly5蛋白的免疫印跡結(jié)果的圖像，圖1d是示出了純化蛋白質(zhì)的電泳結(jié)果的圖像。圖2a和圖2b是示出了突變型dkk2蛋白對各200ng的mlrp6和hlrp6在490nm處的吸光度的圖，圖2c和圖2d是示出了dkk2蛋白對各100ng的mlrp6和hlrp6在490nm處的吸光度的圖；圖3a和圖3b是示出了在施用試驗材料后第14天和第21天施用突變型dkk2蛋白質(zhì)的視網(wǎng)膜的熒光強度(％)的圖；以及圖4是示出了伊文思藍血管通透性(％)根據(jù)施用dkk2蛋白質(zhì)的圖。具體實施方式一方面，提供了與野生型dickkopf(dkk)2多肽的氨基酸序列相比包含一個或多個額外糖基化位點的經(jīng)修飾的dkk2多肽。dkk2是指dickkopf-2蛋白，也稱為dickkopf相關(guān)蛋白2、富含半胱氨酸的分泌蛋白2、crsp2、crispy2或crsp2蛋白。dkk2是一種在人類中由dkk2基因編碼的蛋白質(zhì)。該基因編碼作為dickkopf家族成員的蛋白質(zhì)。已知dkk2是一種分泌蛋白質(zhì)，含有兩個富含半胱氨酸的區(qū)域，通過與wnt信號通路的相互作用而參與胚胎發(fā)育。此外，它可以作為wnt/β-連環(huán)蛋白信號傳導(dǎo)的激動劑或拮抗劑，這取決于細胞情況和輔因子kremen2的存在。dkk2可以與低密度脂蛋白受體相關(guān)蛋白6(lrp6)結(jié)合。野生型dkk2多肽可以是野生型dkk2前體多肽。野生型dkk2前體多肽可以是人類中具有基因庫登錄號為np_055236.1的氨基酸序列(序列號1)的多肽或小鼠中具有基因庫登錄號為np_064661.2的氨基酸序列的多肽。野生型dkk2前體多肽可以包括信號肽，并且信號肽可以通過共翻譯修飾或翻譯后修飾來切割。野生型dkk2多肽可以是成熟dkk2。成熟dkk2可以是包括通過從序列號1的氨基酸序列的n-末端去除位置1至33處的氨基酸序列(信號肽或前導(dǎo)肽)而得到的氨基酸序列的多肽，或包括序列號3的氨基酸序列的多肽。野生型dkk2前體多肽可以通過人類中具有基因庫登錄號為nm_014421的核苷酸序列(序列號4)的核酸以及小鼠中具有基因庫登錄號為nm_020265的核苷酸序列的核酸來編碼。糖基化是指將糖基轉(zhuǎn)移到蛋白質(zhì)的反應(yīng)。糖基化由糖基轉(zhuǎn)移酶催化。糖基化可以是n-糖基化、o-糖基化、磷酸-絲氨酸糖基化、c-甘露糖基化、糖基磷脂酰肌醇化或其組合。n-糖基化是指糖分子與天冬酰胺的酰胺基團的連接。糖基化位點是指可以連接有糖分子或糖基的位點。例如，糖基化位點可以是asn-xaa-ser/thr的共有序列中的天冬酰胺殘基(其是n-糖基化位點)。asn表示天冬酰胺，xaa表示除脯氨酸以外的氨基酸，ser表示絲氨酸，thr表示蘇氨酸，ser/thr表示絲氨酸或蘇氨酸。asn-xaa-ser/thr表示從n-末端起由天冬酰胺-氨基酸(不包括脯氨酸)-絲氨酸或氨基酸組成的多肽。糖分子與asn-xaa-ser/thr的天冬酰胺連接。野生型dkk2前體多肽可以從n-末端在位置36處包括一個糖基化位點。經(jīng)修飾的dkk2多肽的糖基化位點可以通過將天門冬酰胺(asn，n)取代序列號3的氨基酸序列中選自由5i、31g、96p、110d、44p、2l、45c、57c、62q、63g、85p、6k、98t、101i、11g、121h、135p、138k、151l、152r、166f、187k、203g、211d、213t和214y組成的組中的一種或多種氨基酸而引入。數(shù)字表示從序列號3的氨基酸序列的n-末端起氨基酸所在的位置，并且字母字符表示氨基酸的1個字母代碼。例如，‘5i’表示從序列號3的氨基酸序列的n-末端起位置5處的異亮氨酸。修飾可以是一個或多個氨基酸的取代。經(jīng)修飾的dkk2多肽可以是dkk2多肽，其中通過引入糖基化位點而額外引入糖基。盡管經(jīng)修飾的dkk2多肽可以在糖鍵、糖組成、糖基結(jié)構(gòu)或其組合中變化，但糖基化發(fā)生在dkk2多肽的氨基酸序列的相同糖基化位點。作為結(jié)果，可以增加細胞內(nèi)表達效率。例如，雖然經(jīng)修飾的dkk2多肽可以在糖鍵(例如n-糖基化和o-糖基化)、糖組成(例如唾液酸化程度的差異)和糖基結(jié)構(gòu)中根據(jù)宿主細胞(其中經(jīng)修飾的dkk2多肽被表達)的種類而變化，但糖基化可以發(fā)生在dkk2多肽的相同糖基化位點。經(jīng)修飾的dkk2多肽可以是包含一個或多個選自序列號5至30的氨基酸序列的多肽。標簽可以進一步包含在經(jīng)修飾的dkk2多肽的n-末端或c-末端。標簽可以是連接到dkk2多肽的多肽，以促進表達、純化、檢測等。標簽可以是例如fc(片段可結(jié)晶)區(qū)、多組氨酸肽或其組合。fc區(qū)可以是人fc區(qū)、小鼠fc區(qū)等。fc區(qū)可以是由序列號48的核苷酸序列編碼的多肽。fc區(qū)可以是由序列號49的核苷酸序列組成的多肽。標簽可以是本領(lǐng)域技術(shù)人員已知的。經(jīng)修飾的dkk2多肽可以進一步包括n-末端處的信號肽。信號肽是指存在于大部分新合成的蛋白質(zhì)的n-末端的肽(大約5-30個氨基酸長)，其注定分泌途徑。信號肽可以是序列號1的氨基酸序列的n-末端起位置1至33處的氨基酸序列或包含序列號2的氨基酸序列的多肽。與野生型dkk2多肽相比，經(jīng)修飾的dkk2多肽可以是具有額外糖基、lrp6結(jié)合親和性的增加、或其組合的多肽。與作為對照組的野生型dkk2多肽相比，在經(jīng)修飾的dkk2多肽中的額外糖基可以是糖基量的增加。由于額外糖基，與野生型dkk2多肽的量相比，經(jīng)修飾的dkk2多肽的量可能在細胞中增加。與編碼野生型dkk2多肽的轉(zhuǎn)錄物相比，編碼經(jīng)修飾的dkk2多肽的轉(zhuǎn)錄物的量可以在細胞中增加。多肽或轉(zhuǎn)錄物的量的增加可能意味著多肽的細胞內(nèi)表達效率增加。與野生型dkk2多肽相比，經(jīng)修飾的dkk2多肽對lrp6的結(jié)合親和性可能增加。結(jié)合親和性越高，解離常數(shù)(kd)可能越低。經(jīng)修飾的dkk2多肽對于lrp6的解離常數(shù)可以是例如約0.1nm至約100nm、約1nm至約50nm、約2nm至約40nm、約3nm至約30nm、約4nm至約20nm、約5nm至約10nm、或約5.5nm。另一方面，提供一種編碼經(jīng)修飾的dkk2多肽的核酸。該經(jīng)修飾的dkk2多肽與上述相同。核酸可以包括選自序列號43至47中的任一個核苷酸序列。核酸在編碼經(jīng)修飾的dkk2多肽的核酸的5'-末端或3'-末端處還可以包括編碼標簽的核苷酸序列。核酸在編碼經(jīng)修飾的dkk2多肽的核酸的5'-末端處還可以包括編碼信號肽的核苷酸序列。核酸可以可操作地連接到基因表達調(diào)節(jié)元件，例如啟動子、操縱子、增強子和/或轉(zhuǎn)錄終止子。另一方面，提供一種制備經(jīng)修飾的dkk2多肽的方法，所述方法包括：在培養(yǎng)基的存在下培養(yǎng)用包含編碼經(jīng)修飾的dkk2多肽的核酸的載體引入的細胞，以獲得培養(yǎng)產(chǎn)物；并從培養(yǎng)產(chǎn)物獲得經(jīng)修飾的dkk2多肽。該經(jīng)修飾的dkk2多肽和編碼經(jīng)修飾的dkk2多肽的核酸與上述相同。細胞可以是真核細胞。例如，細胞可以是動物細胞。細胞可以是例如胚胎腎細胞、卵巢細胞、骨髓瘤細胞或視網(wǎng)膜衍生細胞。胚胎腎細胞可以是人胚胎腎(hek)293細胞或幼倉鼠腎(bhk)細胞。卵巢細胞可能是中國倉鼠卵巢細胞(cho)細胞。骨髓瘤細胞可以是ns0細胞或sp2/0細胞。視網(wǎng)膜衍生細胞可以是perc6細胞。根據(jù)細胞的種類，在糖鍵(例如n-糖基化和o-糖基化)、糖組成(例如唾液酸化程度的差異)和糖基結(jié)構(gòu)中變化的dkk2多肽可以被表達。該方法還可以包括將包含編碼經(jīng)修飾的dkk2多肽的核酸的載體引入細胞。載體可以是例如質(zhì)粒、病毒載體、粘?；蛉嗽烊旧w。載體可以是包含啟動子序列的表達載體。載體可以是能夠在真核細胞中表達靶基因的載體。引入是指將核酸引入細胞。引入可以是例如通過轉(zhuǎn)化、突變、轉(zhuǎn)導(dǎo)、綴合或電穿孔引入。該方法包括在培養(yǎng)基的存在下培養(yǎng)引入的細胞以獲得培養(yǎng)產(chǎn)物。培養(yǎng)基是指含有促進細胞存活或增殖所必需或有作用的成分的培養(yǎng)基。根據(jù)細胞種類，本領(lǐng)域技術(shù)人員可以選擇培養(yǎng)基?？梢允褂帽绢I(lǐng)域技術(shù)人員已知的方法進行孵育來進行培養(yǎng)。培養(yǎng)可以在例如約37℃的溫度和5％co2條件下進行。該方法包括從培養(yǎng)產(chǎn)物得到經(jīng)修飾的dkk2多肽。培養(yǎng)產(chǎn)物可以是不包括培養(yǎng)細胞的培養(yǎng)液或培養(yǎng)細胞。得到經(jīng)修飾的dkk2多肽可以包括得到和裂解細胞，并純化或過濾多肽。得到多肽的方法可以是本領(lǐng)域技術(shù)人員已知的。另一方面，提供一種用于促進血管生成的藥物組合物，所述組合物包含經(jīng)修飾的dkk2多肽或編碼經(jīng)修飾的dkk2多肽的核酸、以及藥學(xué)上可接受的載體。該經(jīng)修飾的dkk2多肽和編碼經(jīng)修飾的dkk2多肽的核酸與上述相同。血管生成是指形成新血管的過程。血管生成包括新血管從預(yù)先存在的血管生長的過程。血管生成是傷口愈合和肉芽組織以及生長和發(fā)育中的正常和重要的過程。此外，血管生成是腫瘤從休眠狀態(tài)轉(zhuǎn)變?yōu)閻盒誀顟B(tài)的基本步驟。促進可以在體外或體內(nèi)實現(xiàn)。促進可能誘導(dǎo)具有缺血性血管疾病的受試者的新血管的形成或再生。藥物組合物可以是用于預(yù)防或治療缺血性血管疾病的藥物組合物。缺血性血管疾病可以是例如燒傷、牛皮癬、潰瘍、缺血、缺血性心臟病、缺血性腦血管疾病或勃起功能障礙。另一方面，提供一種用于預(yù)防或治療血管通透性相關(guān)疾病的藥物組合物，所述組合物包含經(jīng)修飾的dkk2多肽或編碼經(jīng)修飾的dkk2多肽的核酸、以及藥學(xué)上可接受的載體。該經(jīng)修飾的dkk2多肽和編碼經(jīng)修飾的dkk2多肽的核酸與上述相同。血管通透性相關(guān)疾病是指具有由于血管通透性增加而導(dǎo)致身體流體進入周圍組織的泄漏增加而引起的癥狀的疾病。血管通透性相關(guān)疾病可以是例如糖尿病性視網(wǎng)膜病變、糖尿病性黃斑水腫、視網(wǎng)膜靜脈閉塞后的黃斑水腫、黃斑變性(例如新生血管(濕性)年齡相關(guān)性黃斑變性)、脈絡(luò)膜新生血管形成、青光眼視網(wǎng)膜色素變性、視網(wǎng)膜病變早產(chǎn)兒(rop)、青光眼、角膜營養(yǎng)不良、視網(wǎng)膜疾病、斯塔格特氏病、常染色體顯性藥物、最佳黃斑營養(yǎng)不良、囊性黃斑水腫、缺血性視網(wǎng)膜病變、炎癥誘導(dǎo)的視網(wǎng)膜退行性疾病、x連鎖青少年視網(wǎng)膜病變、malattialeventinese(ml)或doyne蜂窩狀視網(wǎng)膜營養(yǎng)不良癥。如本文所用，術(shù)語“預(yù)防”是指通過施用藥物組合物來抑制或延緩疾病發(fā)生的所有作用。術(shù)語“治療”是指通過施用藥物組合物有利于癥狀好轉(zhuǎn)或改善的所有作用。藥物組合物可以包括藥學(xué)上可接受的載體。載體包括賦形劑、稀釋劑或輔助物質(zhì)。載體可以是例如選自乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖醇、木糖醇、赤蘚糖醇、麥芽糖醇、淀粉、金合歡膠、藻酸鹽、明膠、磷酸鈣、硅酸鈣、纖維素、甲基纖維素、聚乙烯基吡咯烷酮、水、生理鹽水溶液、緩沖液例如pbs、羥基苯甲酸甲酯、羥基苯甲酸丙酯、滑石、硬脂酸鎂和礦物油。組合物可以包括填料、抗凝集劑、潤滑劑、潤濕劑、風(fēng)味劑、乳化劑、防腐劑等。藥物組合物可以以任何制劑制備。組合物可以配制成口服劑型(例如粉劑、片劑、膠囊劑、糖漿劑、丸劑、顆粒劑)或腸胃外劑型(例如可注射制劑)。此外，組合物可以以全身劑型或局部劑型制備。藥物組合物可以包括有效量的經(jīng)修飾的dkk2多肽或編碼經(jīng)修飾的dkk2多肽的核酸。有效量可以根據(jù)本領(lǐng)域技術(shù)人員選擇的細胞或受試者適當(dāng)?shù)剡x擇。有效量可以根據(jù)疾病的嚴重程度、患者的年齡、體重、健康狀況、性別和藥物敏感性、給藥時間、給藥途徑、排泄率、治療期、和與組合物混合或共同給藥的藥物以及醫(yī)療領(lǐng)域中眾所周知的其它因素來確定?；谒幬锝M合物，有效量可以為約0.5μg至約2g、約1μg至約1g、約10μg至約500mg、約100μg至約100mg、約1mg至約50mg。藥物組合物的給藥劑量可以是例如每個成人約0.001mg/kg至約100mg/kg、約0.001mg/kg至約10mg/kg、約0.001mg/kg至約1mg/kg、約0.005mg/kg至約1mg/kg、約0.01mg/kg至約1mg/kg、或約0.1mg/kg至約1mg/kg，每天一次、每天數(shù)次、每周兩次或三次、每月一次至四次、每年一至十二次。另一方面，提供一種促進受試者的血管生成的方法，所述方法包括將經(jīng)修飾的dkk2多肽或編碼經(jīng)修飾的dkk2多肽的核酸施用于受試者。另一方面，提供一種預(yù)防或治療受試者的血管通透性相關(guān)疾病的方法，所述方法包括將經(jīng)修飾的dkk2多肽或編碼經(jīng)修飾的dkk2多肽的核酸施用于受試者。該經(jīng)修飾的dkk2多肽、編碼經(jīng)修飾的dkk2多肽的核酸、血管生成、促進、血管通透性相關(guān)疾病、預(yù)防和治療與上述相同。受試者可以是哺乳動物，例如人、牛、馬、豬、狗、綿羊、山羊或貓。受試者可能是具有缺血性疾病或高可能性具有缺血性疾病的受試者。受試者可以是具有血管通透性相關(guān)疾病或高可能性具有血管通透性相關(guān)疾病的受試者?？梢酝ㄟ^任何方式直接對受試者進行給藥，例如口服、靜脈內(nèi)、肌肉內(nèi)、透皮、粘膜、鼻內(nèi)、氣管內(nèi)或皮下給藥。給藥可以是局部或全身給藥?，F(xiàn)在將詳細參考示例性實施方式，其示例在附圖中示出，其中相同的附圖標記始終表示相同的元件。在這方面，本示例性實施方式可以具有不同的形式，并且不應(yīng)被解釋為限于本文所闡述的描述。因此，下面僅通過參考附圖描述示例性實施方式來解釋各方面。如本文所用，術(shù)語“和/或”包括一個或多個相關(guān)列出的項目的任何和所有組合。以下參照實施例對本發(fā)明進行更詳細的說明。然而，這些實施例僅用于說明的目的，本發(fā)明的范圍并不受這些實施例的限制。實施例1.n-糖基化突變型dkk2蛋白的制備和鑒定1.dkk2蛋白的n-糖基化位點的預(yù)測野生型dkk2蛋白的氨基酸序列(序列號3)從n-末端起在位置3處具有n-糖基化位點。為了人工誘導(dǎo)野生型dkk2蛋白中的n-糖基化，使用netnglyc1.0服務(wù)器(www.cbs.dtu.dk/services/netnglyc/)預(yù)測野生型dkk2蛋白的n-糖基化位點。從netnglyc1.0服務(wù)器預(yù)測了共26種dkk2蛋白的n-糖基化位點，結(jié)果如表1中所示。在表1中，氨基酸代表野生型dkk2蛋白的氨基酸(序列號3)和由其突變的天冬酰胺(asn，n)，數(shù)字表示從野生型dkk2蛋白的n-末端起突變氨基酸所在的位置。[表1]在26種dkk2蛋白的n-糖基化位點候選中，選擇了在netnglyc1.0服務(wù)器中顯示高n-糖基化潛能的5種突變型dkk2蛋白，并檢測了蛋白質(zhì)生產(chǎn)效率。所選擇的5種突變型dkk2蛋白的n-糖基化潛能在下表2中給出。[表2]突變型dkk2蛋白n-糖基化的潛能dkk2n-gly10.7342dkk2n-gly20.7206dkk2n-gly30.7010dkk2n-gly40.7541dkk2n-gly50.73722.所選擇的n-糖基化dkk2蛋白的生產(chǎn)效率的驗證為了表達實施例1.1中選擇的5種dkk2蛋白質(zhì)，將其各自插入高效表達載體中，以通過瞬時表達系統(tǒng)檢查蛋白質(zhì)生產(chǎn)效率。(1)聚合酶鏈反應(yīng)使用野生型dkk2天然形式(基因庫登錄號np_055236.1，序列號3)(由medpacto,inc.,korea提供)作為模板。設(shè)計用于dkk2n-糖基化的引物的核苷酸序列在表3中給出。[表3]對于聚合酶鏈反應(yīng)，制備以下組成的混合物。100ng模板2.5單位pfudna聚合酶(spx16-rs500,solgentco.,ltd.)10pmol正向引物10pmol反向引物1μl10mmdntp5μl10xpfu聚合酶緩沖液50μl總體積將制備的混合物在95℃下孵育2分鐘，然后重復(fù)30次循環(huán)，每次循環(huán)包括95℃下20秒、64℃下40秒、72℃下1分鐘，然后在72℃下孵育10分鐘。因此，得到如此擴增的突變型dkk2核酸。(2)擴增產(chǎn)物的克隆和宿主細胞中瞬時蛋白表達的檢測將擴增的突變型dkk2核酸或野生型dkk2核酸在10單位的限制酶sfii(目錄號r033s，enzynomics，korea)和1x反應(yīng)緩沖液的存在下在50℃下孵育6小時。將反應(yīng)產(chǎn)物在瓊脂糖凝膠上電泳，使用凝膠純化試劑盒(目錄號1014876，qiagen，usa)純化插入載體的核酸。將哺乳動物表達載體n293f-fc(atp-100，anrt)和純化的核酸以1:3的重量比混合，隨后在10單位的t4dna連接酶(目錄號m001s，enzynomics，korea)和1x反應(yīng)緩沖液的存在下在22℃下孵育4小時以上。將反應(yīng)產(chǎn)物轉(zhuǎn)化到大腸桿菌中，通過測序分析檢查編碼dkk2n-gly1、dkk2n-gly2、dkk2n-gly3、dkk2n-gly4或dkk2n-gly5(分別為序列號43至47)的核苷酸序列。選擇包含引入有突變型dkk2核酸的n293f-fc載體的克隆。從選擇的克隆得到引入有突變型dkk2核酸(mdkk2)的n293f-fc-mdkk2載體。fc核酸是編碼人免疫球蛋白g1的fc片段的核酸(序列號48)。將哺乳動物hek293f懸浮細胞以5×105個細胞/ml的密度接種于自由式培養(yǎng)基(目錄號1508027，invitrogen，usa)中，并在37℃和5％co2的條件下培養(yǎng)。培養(yǎng)細胞直到細胞的密度達到約1×106細胞/ml(約24小時)。將25μg引入有突變型dkk2核酸的n293f-fc載體、50μg聚乙烯亞胺(pei)(目錄號23966，polysciences，usa)和600μlpmi培養(yǎng)基(目錄號sh30027.01，hiclone，usa)混合，并在室溫下孵育20分鐘。向培養(yǎng)的hek293f細胞中加入反應(yīng)產(chǎn)物，并在37℃和5％co2的條件下培養(yǎng)約5天。然后，為了得到由細胞分泌的水溶性蛋白質(zhì)，從培養(yǎng)產(chǎn)物中除去細胞，僅收集不包括細胞的培養(yǎng)液。將200μl培養(yǎng)液進行10％sds-page，然后進行免疫印跡。由于得到的突變型dkk2蛋白和野生型dkk2蛋白在其n-末端包含fc-標簽、his-標簽或mfc-標簽，使用以1：4000稀釋的抗fc-hrp(目錄號31414，pierce，usa)、以1：2000稀釋的抗his-hrp(目錄號a7058，sigma，usa)或以1：2000稀釋的抗mfc(目錄號31430，pierce，usa)。作為顯色試劑，使用eclkit(目錄號0034077geusa)以得到圖像。圖1a示出了含有各種標簽的野生型dkk2蛋白的免疫印跡結(jié)果(暴露時間：1分鐘，m：標記物(kda)，1：n-fc-dkk2，2：n-fc(igg4)-dkk2，3：n-his-dkk2，4：n-mfc-dkk2)。如圖1a所示，即使暴露1分鐘，也沒有檢測到野生型dkk2蛋白。因此，證實了野生型dkk2蛋白幾乎不表達。圖1b示出了包含各種標簽的突變型dkk2蛋白的免疫印跡結(jié)果(暴露時間：1分鐘(左)或1秒(右)，m：標記(kda)，1：n-mfc-dkk2-gly1，2：n-mfc-dkk2-gly2，3：n-mfc-dkk2-gly3，4：n-fc4-dkk2-gly1，5：n-his-dkk2-gly1，6：n-fc-dkk2-gly1，7：n-his-dkk2-gly2，8：n-fc4-dkk2-gly2，9：n-fc-dkk2-gly2，10：n-fc-dkk2-gly3，11：n-his-dkk2-gly3，12：n-fc4-dkk2-gly3)。如圖1b所示，檢測到包含fc標簽的突變型dkk2蛋白，而幾乎沒有檢測到包含fc以外的標簽的突變型dkk2蛋白。因此，證實了含有fc標簽的突變型dkk2蛋白顯示出提高的表達效率。使用一次性開放柱純化表達的蛋白質(zhì)，并測量其濃度。由于含有fc標簽的蛋白質(zhì)顯示出提高的表達效率，因此獲得含有fc標簽的dkk2n-gly4和dkk2n-gly5蛋白。將獲得的蛋白質(zhì)進行免疫印跡，結(jié)果示于圖1c(曝光時間：1秒(左)或30秒(右)，m：標記(kda)，1：n-fc-dkk2-gly4，2：n-fc-dkk2-gly5)。如圖1c所示，證實了n-fc-dkk2-n-gly4比n-fc-dkk2-n-gly5顯示出更高的表達效率。(3)蛋白質(zhì)的大量生產(chǎn)和純化如實施例1(2)中所述，選擇用含有fc標簽和突變性dkk2核酸的載體轉(zhuǎn)化的細胞。將所選擇的細胞在37℃和5％co2的條件下培養(yǎng)約5天。將除細胞外的培養(yǎng)液在室溫下以4800rpm的速度離心20分鐘以得到上清液。使用0.22μm過濾器(目錄號pr02890，millipore，usa)進行過濾。制備裝有蛋白a珠(目錄號17-1279-03，gehealthcare，sweden)的5ml柱，并在4℃下將濾液以0.9ml/min的速率施加到珠子整夜。用100ml以上的pbs(磷酸緩沖鹽水)(目錄號71111，gibco，usa)洗滌珠子。此后，將0.1m甘氨酸-hcl(目錄號g7126，sigma，usa)施加到珠子以洗脫6個級分。加入1mtris(目錄號t-1503-5kg，sigma，usa)(ph9.0)以中和餾分。定量級分中的蛋白質(zhì)，并收集含有蛋白質(zhì)的級分。將級分施加到超濾離心管(目錄號ufc805024，millipore，usa)，并根據(jù)制造商的說明書離心。將1xpbs加入濃縮物中并重復(fù)離心三次，以用pbs替代作為儲存緩沖液。定量純化的蛋白質(zhì)。在40ml培養(yǎng)液中n-fc-dkk2-gly1的量為130μg，在40ml培養(yǎng)液中n-fc-dkk2-gly2的量為860μg，在40ml培養(yǎng)液中n-fc-dkk2-gly3的量為150μg，在40ml培養(yǎng)液中n-fc-dkk2-gly4的量為462μg，在40ml培養(yǎng)液中n-fc-dkk2-gly5的量為27μg。在引入有n-糖基化位點的突變型dkk2蛋白中，n-fc-dkk2-gly2顯示出最高的表達效率，n-fc-dkk2-gly4顯示出接著最高的表達效率。因此，與野生型dkk2蛋白相比，引入了n-糖基化位點的突變型dkk2蛋白質(zhì)高度表達，n-fc-dkk2-gly2和n-fc-dkk2-gly4的表達效率比野生型dkk2分別高約80倍和約50倍。為了檢查純化的蛋白質(zhì)的純度，將3μg蛋白質(zhì)通過10％sds-page電泳，結(jié)果示于圖1d(m：標記物(kda)，1：n-fc-dkk2-gly1，2：n-fc-dkk2-gly2，3：n-fc-dkk2-gly3，4：n-fc-dkk2-gly4，5：n-fc-dkk2-gly5)。如圖1d中所示，發(fā)現(xiàn)n-fc-dkk2-gly2和n-fc-dkk2-gly4的純度相似。3.突變型dkk2蛋白的結(jié)合親和性的檢測由于已知dkk2蛋白與小鼠lrp6(mlrp6)和人lrp6(hlrp6)結(jié)合，因此檢查了野生型dkk2和突變型dkk2蛋白對lrp6的結(jié)合親和性。具體地，將200nghlrp6蛋白(目錄號1505-lr-025，r&d，usa)或mlrp6蛋白(目錄號2960-lr-025，r&d，usa)應(yīng)用于elisa板(登錄號439454，nunc，denmark)的每個孔，并在4℃下孵育過夜以用該蛋白質(zhì)包覆該板。將200μl4％(w/v)脫脂乳(cat.no.232100，difco，france)/1xpbs施加到elisa板的孔中，并在室溫下孵育約1小時以進行封閉。此后，從elisa板中除去封閉溶液。將每個純化的dkk2蛋白和100μl1％(w/v)脫脂乳/1xpbs混合以制備100nm的dkk2蛋白，并將100nm的dkk2蛋白進行1/4系列稀釋。將稀釋的蛋白質(zhì)施加到制備的elisa板上，并在室溫下孵育約2小時。此后，將板用200μlpbst洗滌5次。將2μl抗人fc-hrp(目錄號31413，thermo，usa)抗體與4ml含有1％(w/v)脫脂乳的pbs混合，并向elisa板的每個孔中加入200μl該混合物，并在室溫下孵育1小時。然后，除去elisa板的二次抗體，并用200μlpbs將該板洗滌5次。將10μlh2o2溶液(目錄號h1009-100ml，sigma，usa)、10mlpc緩沖液(5.1g檸檬酸一水合物，7.3g磷酸鈉/l(ph5.0))和一片opd(目錄號p8787-100tab，sigma，usa)混合以制備混合物，并將總體積為100μl的混合物加入到每個孔中。在室溫下在黑暗中進行孵育10分鐘，隨后進行顯色。此后，向每個孔中加入50μl終止緩沖液以終止顯色。使用elisa讀數(shù)器測量490nm處的吸光度，并根據(jù)測量的吸光度計算解離常數(shù)kd值(m)。圖2a示出了突變型dkk2蛋白對200ngmlrp6在490nm處的吸光度，圖2b示出了突變型dkk2蛋白對200nghlrp6在490nm處的吸光度(■：n-fc-dkk2-gly1，▲：n-fc-dkk2-gly2，▼：n-fc-dkk2-gly3，◆：n-fc-dkk2-gly4，●：n-fc-dkk2-gly5)。如圖2a和2b中所示，n-fc-dkk2-gly4顯示出對mlrp6和hlrp6的最大改善的結(jié)合親和性。證實了n-fc-dkk2-gly2顯示出最高的表達效率，但其對mlrp6和hlrp6的結(jié)合親和性不高。因此，n-fc-dkk2-gly4被證實是表現(xiàn)出改善的表達效率和對mlrp6和hlrp6的高結(jié)合親和性的突變型dkk2蛋白。為了比較在n-fc-dkk2-gly4與野生型dkk2(目錄號6628-dk-010，r&d，usa)之間的對mlrp6和hlrp6的結(jié)合親和性，以與上述相同的方式進行elisa，不同之處在于使用了100ng的lrp6。圖2c示出了dkk2蛋白對100ngmlrp6在490nm處的吸光度，圖2d示出了dkk2蛋白對100nghlrp6在490nm處的吸光度(■：n-fc-dkk2-gly4，▲：野生型dkk2)。從測量的吸光度計算出的解離常數(shù)(kd)和r2值在表4中給出。[表4]證實了n-fc-dkk2-gly4對hlrp6和mlrp6的結(jié)合親和性比野生型dkk2高約5倍至約10倍。4.通過突變型dkk2蛋白的血管生成的檢測為了檢查突變型dkk2蛋白是否能夠誘導(dǎo)血管生成，進行角膜袋檢測(cpa)。c57bl6小鼠(雄性，9周齡，重21.81g-23.81g)購自orientbioinc.(korea)，然后在進行實驗的動物設(shè)施中馴化約7天。為了向角膜施用藥物，制備藥物顆粒。具體地，將10g蔗糖八硫酸鹽-鋁絡(luò)合物(s0652，sigma-aldrich)溶于100ml的pbs(磷酸鹽緩沖液鹽水)中以制備10％(w/v)硫糖鋁溶液。將12g聚甲基丙烯酸2-羥基乙酯(p3932，sigma-aldrich)溶于100ml無水乙醇中以制備12％(w/v)聚hema溶液。將石蠟?zāi)ぶ糜谂囵B(yǎng)皿中并在紫外線下放置約15分鐘。將5μl12％(w/v)聚hema溶液、1μl10％(w/v)硫糖鋁溶液和4μl藥物混合。將各0.2μl該混合物分配在石蠟?zāi)ど希⒎峙涞念w粒在室溫下干燥約1-2小時。將干燥的顆粒在使用前儲存在冰箱中。將適應(yīng)的小鼠(雄性，10周齡，體重2123.43g-26.11g)分為2組(每組n＝5)，并通過腹膜內(nèi)注射氯胺酮和賽拉嗪(rompuntm)，拜耳公司，德國)的4:1(v/v)混合物使其麻醉。大約10分鐘后，將0.5％的普拉卡因滴入其角膜。用vongraefe白內(nèi)障刀，在顯微鏡下在角膜上制成一個微口袋。將制備的顆粒植入角膜微口袋(第1天)。為了防止感染，將土霉素眼藥膏施用于眼睛。作為測試材料，使用n-fc-dkk2-gly4，每只小鼠施用475ng的n-fc-dkk2-gly4。作為陰性對照組，使用pbs。此后，在顯微鏡下檢查角膜，通過圖像分析儀觀察血管生成和結(jié)構(gòu)特征。在第5天，在陰性對照組中未發(fā)現(xiàn)血管生成，而在n-fc-dkk2-gly4處理組中檢測到血管生成。計算n-fc-dkk2-gly4處理組中血管生成的面積(mm2)，結(jié)果在表5中給出。[表5]序號第1天第3天第5天第7天10.000.000.000.0020.000.000.000.0030.000.000.000.0040.000.000.020.0350.000.000.060.13如表5中所示，與陰性對照組相比，在n-fc-dkk2-gly4處理組中觀察到血管生成。5.突變型dkk2蛋白的糖尿病性視網(wǎng)膜病變改善作用檢查突變型dkk2蛋白是否能夠改善糖尿病性視網(wǎng)膜病變的癥狀。栗鼠兔(雄性，10-11周齡，體重2.0kg-2.5kg)購自dreambioinc.(韓國)，然后在進行實驗的動物設(shè)施中馴化約7天。將作為糖尿病誘導(dǎo)劑的四氧嘧啶一水合物(sigma-aldrichco.)施用于適應(yīng)的兔子的耳靜脈。在給藥后第7天(第0天)測量血糖水平，選擇具有血糖水平為300mg/dl的動物。將選擇的兔子隨機分配，使得每組的平均血糖水平平均分布。通過靜脈注射zoletil50(virbac，法國)和賽拉嗪(rompuntm，bayerag，germany)使糖尿病誘導(dǎo)的兔子麻醉。作為測試材料，在第0天(施用四氧嘧啶后第7天)和第7天通過玻璃體內(nèi)注射施用n-fc-dkk2-gly4(pbs中)，以總共10μl/眼的量進行兩次給藥，每天一次。作為正常對照組，注射pbs而不是四氧嘧啶。作為糖尿病對照組，施用四氧嘧啶，作為陽性對照組，在第0天以50μl/眼的量通過玻璃體內(nèi)注射施用(bayerkorealtd.)一次。施用藥物在下表6中給出。[表6]組動物數(shù)量施用材料一次劑量或施用頻率備注17pbs0未處理27pbs0糖尿病誘導(dǎo)37eylea2000μg，一次糖尿病誘導(dǎo)47n-fc-dkk2-gly47μg，兩次糖尿病誘導(dǎo)57n-fc-dkk2-gly420μg，兩次糖尿病誘導(dǎo)67n-fc-dkk2-gly470μg，兩次糖尿病誘導(dǎo)在第0天(測試材料給藥開始的那天)、第14天和第21天，將散瞳劑(mydriacyl滴眼劑1％)滴入兔眼中，并用zoletil50(法國virbac)和賽拉嗪((rompuntm)，拜耳公司，德國)麻醉。此后，通過耳靜脈注射2％(w/v)熒光素鈉鹽溶液(sigmaaldrich)，并使用眼底照相機(trc-50ix，topcon，japan)拍攝兔子的眼睛約2分鐘或更短。通過熒光眼底圖像評估視網(wǎng)膜和功效的檢查。使用imagej軟件(nih，bethesda，md)進行圖像分析，以測量視網(wǎng)膜非血管區(qū)域的熒光強度，并基于組1(正常對照組)的平均值(100％)，計算每個受試者的測量值的相對水平(％)。計算出的熒光強度示于圖3a和3b(圖3a：第14天，圖3b：第21天；*和***：與組1相比，分別為p<0.05和p<0.001；#、##和###：與組2相比，分別為p<0.05，p<0.01和p<0.001；$：與組3相比，p<0.05)。如圖3a和3b中所示，n-fc-dkk2-gly4處理組根據(jù)給藥劑量在視網(wǎng)膜的非血管區(qū)域中顯示出顯著低的熒光強度，表明n-fc-dkk2-gly4具有抑制視網(wǎng)膜非血管區(qū)域的血管滲漏的作用。此外，在第21天，用zoletil50和賽拉嗪麻醉兔子。通過耳靜脈注射1％(v/v)伊文思藍溶液(abcam)。約10分鐘后，將兔子的眼睛切下并固定在10％(v/v)中性緩沖福爾馬林溶液中。大約24小時后，將視網(wǎng)膜與眼睛分開，并將1ml蒸餾水施加到分離的視網(wǎng)膜上，然后渦旋約10分鐘。將混合物在10000×g和室溫下離心約10分鐘。將0.3ml上清液轉(zhuǎn)移到96孔微量培養(yǎng)板中，并使用versamax酶標儀(moleculardevice，usa)測量620nm處的吸光度?；诮M1(正常對照組)的平均值(100％)，計算每個受試者的伊文思藍血管通透性。計算的通透性示于圖4(*和***：與組1相比，分別為p<0.05和p<0.001；##和###：與組2相比，分別為p<0.01和p<0.001；$$：與組3相比，p<0.01)。如圖4中所示，n-fc-dkk2-gly4處理組根據(jù)給藥劑量顯示出顯著低的血管通透性，表明n-fc-dkk2-gly4具有抑制視網(wǎng)膜血管通透性的作用。糖尿病性視網(wǎng)膜病變的特征在于視網(wǎng)膜血管生成(或新生血管形成)、玻璃體出血等。由于n-fc-dkk2-gly4抑制血管生成、玻璃體出血和視網(wǎng)膜血管通透性，證實了經(jīng)修飾的dkk2多肽例如n-fc-dkk2-gly4對糖尿病性視網(wǎng)膜病變具有預(yù)防或治療作用。應(yīng)當(dāng)理解，本文描述的示例性實施方式應(yīng)僅在描述性意義上被考慮，而不是為了限制的目的。在其它示例性實施方式中，每個示例性實施方式中的特征或方面的描述通常被認為可用于其它類似特征或方面。雖然已經(jīng)參考附圖描述了一個或多個示例性實施方式，但是本領(lǐng)域普通技術(shù)人員將會理解，在不脫離由如下權(quán)利要求所定義的精神和范圍的情況下，可以在形式和細節(jié)上進行各種改變。<110>株式會社麥迪帕克特<120>經(jīng)修飾的dkk2蛋白、對其編碼的核酸、其制備方法及其用途<130>px051158-pct<160>49<170>kopatentin2.0<210>1<211>259<212>prt<213>人工序列<220><223>人野生型dkk2前體<400>1metalaalaleumetargserlysaspsersercyscysleuleuleu151015leualaalavalleumetvalgluserserglnileglyserserarg202530alalysleuasnserilelysserserleuglyglygluthrprogly354045glnalaalaasnargseralaglymettyrglnglyleualaphegly505560glyserlyslysglylysasnleuglyglnalatyrprocysserser65707580asplysglucysgluvalglyargtyrcyshisserprohisglngly859095serseralacysmetvalcysargarglyslyslysargcyshisarg100105110aspglymetcyscysproserthrargcysasnasnglyilecysile115120125provalthrgluserileleuthrprohisileproalaleuaspgly130135140thrarghisargaspargasnhisglyhistyrserasnhisaspleu145150155160glytrpglnasnleuglyargprohisthrlysmetserhisilelys165170175glyhisgluglyaspprocysleuargserseraspcysileglugly180185190phecyscysalaarghisphetrpthrlysilecyslysprovalleu195200205hisglnglygluvalcysthrlysglnarglyslysglyserhisgly210215220leugluilepheglnargcysaspcysalalysglyleusercyslys225230235240valtrplysaspalathrtyrserserlysalaargleuhisvalcys245250255glnlysile<210>2<211>33<212>prt<213>人工序列<220><223>人野生型dkk2的信號肽<400>2metalaalaleumetargserlysaspsersercyscysleuleuleu151015leualaalavalleumetvalgluserserglnileglyserserarg202530ala<210>3<211>226<212>prt<213>人工序列<220><223>人野生型成熟dkk2<400>3lysleuasnserilelysserserleuglyglygluthrproglygln151015alaalaasnargseralaglymettyrglnglyleualapheglygly202530serlyslysglylysasnleuglyglnalatyrprocysserserasp354045lysglucysgluvalglyargtyrcyshisserprohisglnglyser505560seralacysmetvalcysargarglyslyslysargcyshisargasp65707580glymetcyscysproserthrargcysasnasnglyilecysilepro859095valthrgluserileleuthrprohisileproalaleuaspglythr100105110arghisargaspargasnhisglyhistyrserasnhisaspleugly115120125trpglnasnleuglyargprohisthrlysmetserhisilelysgly130135140hisgluglyaspprocysleuargserseraspcysilegluglyphe145150155160cyscysalaarghisphetrpthrlysilecyslysprovalleuhis165170175glnglygluvalcysthrlysglnarglyslysglyserhisglyleu180185190gluilepheglnargcysaspcysalalysglyleusercyslysval195200205trplysaspalathrtyrserserlysalaargleuhisvalcysgln210215220lysile225<210>4<211>3659<212>dna<213>人工序列<220><223>人dkk2mrna<400>4cgggagcccgcggcgagcgtagcgcaagtccgctccctaggcatcgctgcgctggcagcg60attcgctgtctcttgtgagtcaggggacaacgcttcggggcaactgtgagtgcgcgtgtg120ggggacctcgattctcttcagatctcgaggattcggtccggggacgtctcctgatcccct180actaaagcgcctgctaactttgaaaaggagcactgtgtcctgcaaagtttgacacataaa240ggataggaaaagagaggagagaaaagcaactgagttgaaggagaaggagctgatgcgggc300ctcctgatcaattaagaggagagttaaaccgccgagatcccggcgggaccaaggaggtgc360ggggcaagaaggaacggaagcggtgcgatccacagggctgggttttcttgcaccttgggt420cacgcctccttggcgagaaagcgcctcgcatttgattgcttccagttattgcagaacttc480ctgtcctggtggagaagcgggtctcgcttgggttccgctaatttctgtcctgaggcgtga540gactgagttcatagggtcctgggtccccgaaccaggaagggttgagggaacacaatctgc600aagcccccgcgacccaagtgaggggccccgtgttggggtcctccctccctttgcattccc660acccctccgggctttgcgtcttcctggggaccccctcgccgggagatggccgcgttgatg720cggagcaaggattcgtcctgctgcctgctcctactggccgcggtgctgatggtggagagc780tcacagatcggcagttcgcgggccaaactcaactccatcaagtcctctctgggcggggag840acgcctggtcaggccgccaatcgatctgcgggcatgtaccaaggactggcattcggcggc900agtaagaagggcaaaaacctggggcaggcctacccttgtagcagtgataaggagtgtgaa960gttgggaggtattgccacagtccccaccaaggatcatcggcctgcatggtgtgtcggaga1020aaaaagaagcgctgccaccgagatggcatgtgctgccccagtacccgctgcaataatggc1080atctgtatcccagttactgaaagcatcttaacccctcacatcccggctctggatggtact1140cggcacagagatcgaaaccacg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n-gly1的反向引物<400>32gcagcgggtactgttgcagcacatgccatctcggtggc38<210>33<211>42<212>dna<213>人工序列<220><223>dkk2n-gly2的正向引物<400>33aatctaggaagaaatcacactaagatgtcacatataaaaggg42<210>34<211>42<212>dna<213>人工序列<220><223>dkk2n-gly2的反向引物<400>34catcttagtgtgatttcttcctagattctgccatcccaagtc42<210>35<211>41<212>dna<213>人工序列<220><223>dkk2n-gly3的正向引物<400>35catcagggggaaaactgtaccaaacaacgcaagaagggttc41<210>36<211>40<212>dna<213>人工序列<220><223>dkk2n-gly3的反向引物<400>36ttgtttggtacagttttccccctgatggagcactggtttg40<210>37<211>39<212>dna<213>人工序列<220><223>dkk2n-gly3的正向引物<400>37atcccggctctgaatggtactcggcacagagatcgaaac39<210>38<211>39<212>dna<213>人工序列<220><223>dkk2n-gly4的的反向引物<400>38gtgccgagtaccattcagagccgggatgtgaggggttaa39<210>39<211>42<212>dna<213>人工序列<220><223>dkk2n-gly5的正向引物<400>39gggcaggcctacaattgtagcagtgataaggagtgtgaagtt42<210>40<211>39<212>dna<213>人工序列<220><223>dkk2n-gly5的反向引物<400>40atcactgctacaattgtaggcctgccccaggtttttgcc39<210>41<211>29<212>dna<213>人工序列<220><223>載體的正向引物<400>41accggtggtaccgccaccatgggatggag29<210>42<211>29<212>dna<213>人工序列<220><223>載體的反向引物<400>42ggatttatacaaggaggagaaaatgaaag29<210>43<211>678<212>dna<213>人工序列<220><223>編碼dkk2n-gly1多肽的核酸<400>43aaactcaactccaacaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcggcggcagtaagaagggcaaaaacctgggg120caggcctacccttgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcccagttactgaaagc300atcttaacccctcacatcccggctctggatggtactcggcacagagatcgaaaccacggt360cattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatgtca420catataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaagggttt480tgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaagtc540tgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgactgt600gcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccagactc660catgtgtgtcagaaaatt678<210>44<211>681<212>dna<213>人工序列<220><223>編碼dkk2n-gly2多肽的核酸<400>44aaactcaactccatcaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcaatggcagtaagaagggcaaaaacctgggg120caggcctacccttgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcccagttactgaaagc300atcttaacccctcacatcccggctctggatggaattactcggcacagagatcgaaaccac360ggtcattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatg420tcacatataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaaggg480ttttgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaa540gtctgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgac600tgtgcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccaga660ctccatgtgtgtcagaaaatt681<210>45<211>678<212>dna<213>人工序列<220><223>編碼dkk2n-gly3多肽的核酸<400>45aaactcaactccatcaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcggcggcagtaagaagggcaaaaacctgggg120caggcctacccttgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcaatgttactgaaagc300atcttaacccctcacatcccggctctggatggtactcggcacagagatcgaaaccacggt360cattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatgtca420catataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaagggttt480tgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaagtc540tgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgactgt600gcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccagactc660catgtgtgtcagaaaatt678<210>46<211>678<212>dna<213>人工序列<220><223>編碼dkk2n-gly4多肽的核酸<400>46aaactcaactccatcaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcggcggcagtaagaagggcaaaaacctgggg120caggcctacccttgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcccagttactgaaagc300atcttaacccctcacatcccggctctgaatggtactcggcacagagatcgaaaccacggt360cattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatgtca420catataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaagggttt480tgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaagtc540tgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgactgt600gcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccagactc660catgtgtgtcagaaaatt678<210>47<211>678<212>dna<213>人工序列<220><223>編碼dkk2n-gly5多肽的核酸<400>47aaactcaactccatcaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcggcggcagtaagaagggcaaaaacctgggg120caggcctacaattgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcccagttactgaaagc300atcttaacccctcacatcccggctctggatggtactcggcacagagatcgaaaccacggt360cattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatgtca420catataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaagggttt480tgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaagtc540tgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgactgt600gcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccagactc660catgtgtgtcagaaaatt678<210>48<211>696<212>dna<213>人工序列<220><223>編碼人免疫球蛋白g1的fc區(qū)的核酸<400>48gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctg60gggggaccgtcagtcttcctctttcccccaaaacccaaggacaccctcatgatctcccgg120acccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc180aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag240tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaat300ggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacc360atctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgg420gatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagc480gacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcct540cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc600aggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccac660tacacgcagaagagcctctccctgtctccgggtaaa696<210>49<211>232<212>prt<213>人工序列<220><223>免疫球蛋白g1的fc區(qū)<400>49gluprolyssercysasplysthrhisthrcysproprocysproala151015progluleuleuglyglyproservalpheleupheproprolyspro202530lysaspthrleumetileserargthrprogluvalthrcysvalval354045valaspvalserhisgluaspprogluvallyspheasntrptyrval505560aspglyvalgluvalhisasnalalysthrlysproarggluglugln65707580tyrasnserthrtyrargvalvalservalleuthrvalleuhisgln859095asptrpleuasnglylysglutyrlyscyslysvalserasnlysala100105110leuproalaproileglulysthrileserlysalalysglyglnpro115120125arggluproglnvaltyrthrleuproproserargaspgluleuthr130135140lysasnglnvalserleuthrcysleuvallysglyphetyrproser145150155160aspilealavalglutrpgluserasnglyglnprogluasnasntyr165170175lysthrthrproprovalleuaspseraspglyserphepheleutyr180185190serlysleuthrvalasplysserargtrpglnglnglyasnvalphe195200205sercysservalmethisglualaleuhisasnhistyrthrglnlys210215220serleuserleuserproglylys225230當(dāng)前第1頁12