TW202214677A - Il-2突變體及其應用 - Google Patents
Il-2突變體及其應用 Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本發明公開了IL-2突變體及其應用,具體公開IL-2突變體及相應的融合蛋白、綴合物、核酸片段、載體、宿主細胞、所述突變體或融合蛋白的製備方法、根據所述方法製備而成的IL-2突變體或融合蛋白、藥物組合物、製藥用途、疾病治療方法和優先刺激調節性T細胞的方法。本發明所述IL-2突變體與野生型IL-2相比,Tm值升高,改善了IL-2蛋白的穩定性;或與野生型相比,本發明所述IL-2突變體產量升高,或者其與IL-2Rβγ複合物的結合活性改變。
Description
本發明涉及生物醫藥領域,具體而言,涉及IL-2突變體及其應用。
白細胞介素2(interleukin-2,IL-2),最初被鑒定為T細胞生長因數(TCGF),後續研究發現L-2可結合其受體,並啟動T細胞、NK細胞等免疫細胞的增殖和活化。
IL-2受體包括IL-2Rα亞基(CD25)、IL-2Rβ亞基(CD122)和IL-2Rγ亞基(CD132),不同的亞基之間可形成親和力不同的受體複合物,包括高親和力受體IL-2Rαβγ,中間親和力受體IL-2Rβγ,低親和力受體IL-2Rα或IL-2Rαβ。不同細胞表達的IL-2R亞基類型有所不同,例如,靜息條件下的傳統T細胞CD4
+T、CD8
+T表面一般表達IL-2受體β(IL-2Rβ,CD122)、IL-2受體γ(IL-2Rγ,CD132),幾乎不表達IL-2受體α(IL-2Rα,CD25),而調節性T細胞(Treg)除了表達IL-2Rβ、IL-2Rγ之外,組成性高表達IL-2Rα。
目前,研究人員嘗試借助IL-2或其突變體啟動免疫細胞或某個亞群的免疫細胞,從而治療腫瘤或者自身免疫性疾病。例如,高劑量的IL-2被批准用於治療惡性黑色素瘤或轉移性腎細胞癌,IL-2聚乙二醇偶聯藥物NKTR-358被批准開展自身免疫性疾病的臨床試驗。
而提高IL-2的穩定性、產量和/或改變其與某類受體複合物的結合能力,對IL-2類型藥物的開發具有重要意義。有鑑於此,特提出本發明。
本發明公開提供IL-2突變體、融合蛋白、綴合物、核酸片段、載體、宿主細胞、製備突變體或融合蛋白的方法、根據所述方法製備而成的突變體或融合蛋白、藥物組合物、製藥用途、治療方法和優先刺激調節性T細胞的方法。
在第一方面,本發明公開提供一種IL-2突變體,與野生型IL-2相比,所述IL-2突變體包括發生在Q13、L18、G27、Y31、A73、H79、P82、I89、N90、V91、V93、F117或R120的一個或多個突變。
在一些具體的實施方式中,所述突變為插入、缺失或替換,優選為替換。
在一些具體的實施方案中,所述IL-2突變體包括Q13L、L18I、G27W、Y31V、A73L、H79Q、P82L、I89L、N90Y、V91A、V93I、F117W或R120F的一個或多個突變。
在一些具體的實施方案中,所述IL-2突變體包括(a)~(h)組中至少一組突變:
(a)Y31/A73/H79突變;優選地,Y31V/A73L/H79Q突變;
(b)Q13突變;優選地,Q13L突變;
(c)R120突變,優選地,R120F突變;
(d)L18/V91/F117突變;優選地,L18I/V91A/F117W突變;
(e)L18/I89/V93突變;優選地,L18I/I89L/V93I突變;
(f)G27/R120突變;優選地,G27W/R120F突變;
(g)P82/R120突變;優選地,P82L/R120F突變;
(h)N90/R120突變;優選地,N90Y/R120F突變。
在一些具體的實施方案中,所述IL-2突變具有如SEQ ID NO:2~9任一項所示氨基酸序列。
在一些具體的實施方案中,所述IL-2突變體的Tm值高於野生型IL-2。
在一些具體的實施方案中,所述野生型IL-2具有如SEQ ID NO:60或SEQ ID NO:1所示的氨基酸序列。
在一些具體的實施方案中,所述IL-2突變體還包括選自下組的一個或多個突變:H16、D20、N88、V91或Q126突變,例如H16E突變,D20A、D20H或D20Y突變,N88A、N88I、N88G、N88R或N88D突變,V91R或V91K突變,Q126L或Q126F突變;
優選地,所述IL-2突變體還包括選自(ⅰ)~(ⅳ)中至少一組的突變:
(ⅰ)、H16突變,優選H16E突變;
(ⅱ)、D20突變,優選D20A突變;
(ⅲ)、V 91突變,優選V91R突變;
(ⅳ)、H16/V91突變,優選H16E/V91R突變。
在一些具體的實施方案中,所述IL-2突變體包括(a)~(n)組中至少一組突變:
(a)、H16/Y31/A73/H79突變;優選為H16E/Y31V/A73L/H79Q突變;
(b)、H16/R120突變;優選H16E/R120F突變;
(c)、H16/L18/V91/F117突變;優選H16E/L18I/V91A/F117W突變;
(d)、H16/L18/I89/V93突變;優選H16E/L18I/I89L/V93I突變;
(e)、H16/G27/R120突變;優選H16E/G27W/R120F突變;
(f)、H16/P82/R120突變;優選H16E/P82L/R120F突變;
(g)、D20/Y31/A73/H79突變;優選D20A/Y31V/A73L/H79Q突變;
(h)、D20/R120突變;優選D20A/R120F突變;
(i)、V91/Y31/A73/H79突變;優選V91R/Y31V/A73L/H79Q突變;
(j)、V91/Q13突變;優選V91R/Q13L突變;
(k)、V91/R120突變;優選V91R/R120F突變;
(l)、V91/L18/I89/V93突變;優選V91R/L18I/I89L/V93I突變;
(m)、H16/V91/Y31/A73/H79突變;優選H16E/V91R/Y31V/A73L/H79Q突變;
(n)、H16/V91/L18/I89/V93突變;優選H16E/V91R/L18I/I89L/V93I突變。
在一些具體的實施方式中,所述IL-2突變體具有如SEQ ID NO:22~27、29~30、32~35或37~38任一項所述的氨基酸序列。
在一些具體的實施方式中,所述IL-2突變體還包括選自下組的一個或多個突變:N26、N29、N30、N71、Q11、L132、L70、P82、G27或F28突變;
優選地,所述IL-2突變體還包括選自下組的一個或多個突變:N26Q、N29S、N30S、N71Q、Q11C、L132C、L70C、P82C、G27C、F78C突變;
更優選地,所述IL-2突變體還包括(a)~(g)組中至少一組突變:
(a)、N26Q突變;
(b)、N29S突變;
(c)、N30S突變;
(d)、N71Q突變;
(e)、Q11C/L132C突變;
(f)L70C/P82C突變;
(g)G27C/F78C突變。
在一些具體的實施方式中,所述IL-2突變體還包括選自下組中的一個或多個突變:F42、Y45或L72突變,優選F42A、Y45A或L72G突變。
在一些具體的實施方式中,與野生型IL-2相比,所述突變體與IL-2Rβγ亞基複合物的結合能力下降;優選地,結合力
IL-2Rβγ 亞基複合物/結合力
IL-2αβγ 亞基複合物下降。
在一些具體的實施方式中,與野生型IL-2相比,所述突變體刺激非調節性T細胞或NK(自然殺傷)細胞的能力下降,所述刺激可選自:胞內STAT5磷酸化或細胞增殖。
在一些具體的實施方式中,與非調節性T細胞或NK(自然殺傷)細胞相比,所述突變體優先刺激外周血或T細胞群體中的調節性T細胞(Treg);所述優先刺激可選自:優先刺激調節性T細胞內STAT5磷酸化、優先刺激調節性T細胞增殖、提高調節性T細胞與非調節性T細胞的比率,或提高調節性T細胞與NK細胞的比率。
在第二方面,本發明公開一種IL-2突變體,與野生型IL-2相比,所述IL-2突變體包括發生在H16、D20或V91中的一個或多個突變;優選地,所述IL-2突變體包括選自下組的突變:
(ⅰ)、H16突變,優選H16E突變;
(ⅱ)、D20突變,優選D20A突變;
(ⅲ)、V 91突變,優選V91R突變;
(ⅳ)、H16/V91突變,優選H16E/V91R突變。
在一些具體的實施方式中,所述IL-2突變體具有如SEQ ID NO:21、28、31或36所示的氨基酸序列。
在一些具體的實施方式中,所述IL-2突變體還包括選自下組的一個或多個突變:N26、N29、N30、N71、Q11、L132、L70、P82、G27或F28突變;
優選地,所述IL-2突變體還包括選自下組的一個或多個突變:N26Q、N29S、N30S、N71Q、Q11C、L132C、L70C、P82C、G27C或F78C突變;
更優選地,所述IL-2突變體還包括(a)~(g)組中的至少一組突變:
(a)、N26Q突變;
(b)、N29S突變;
(c)、N30S突變;
(d)、N71Q突變;
(e)、Q11C/L132C突變;
(f)L70C/P82C突變;
(g)G27C/F78C突變。
在一些具體的實施方式中,與野生型IL-2相比,所述突變體與IL-2Rβγ亞基複合物的結合能力下降;優選地,結合力
IL-2Rβγ 亞基複合物/結合力
IL-2αβγ 亞基複合物下降。
在一些具體的實施方式中,與野生型IL-2相比,所述突變體刺激非調節性T細胞或NK(自然殺傷)細胞的能力下降,所述刺激可選自:胞內STAT5磷酸化或細胞增殖。
在一些具體的實施方式中,與非調節性T細胞或NK細胞相比,所述突變體優先刺激外周血或T細胞群體中的調節性T細胞(Treg);所述優先刺激可選自:優先刺激調節性T細胞內STAT5磷酸化、優先刺激調節性T細胞增殖、提高調節性T細胞與非調節性T細胞的比率,或提高調節性T細胞與NK細胞的比率。
在一些具體的實施方式中,所述突變包括缺失、插入或替換,優選為替換。
在一些具體的實施方式中,所述野生型IL-2具有如SEQ ID NO:60或SEQ ID NO:1所示的氨基酸序列。
在協力廠商面,本發明公開一種融合蛋白,所述融合蛋白包括第一多肽和第二多肽,其中,所述第一多肽為前述IL-2突變體,所述第二多肽為非IL-2多肽。
在一些具體的實施方式中,所述第二多肽為Fc、腫瘤抗原結合分子或IL-2受體亞基;
可選地,所述Fc為人IgG Fc,例如人IgG1 Fc;
優選地,所述人IgG1 Fc包括選自(a)~(i)組中的至少一組突變:
(a)、C220S;
(b)、N297G;
(c)C220S和N297G;
(d)、A327Q;
(e)、L234A和L235A;
(f)、A287C和L306C;
(g)、A259C和L306C;
(h)、R292C和V302C;
(i)、V323C和I332C;
更優選地,所述人Ig G1 Fc具有如SEQ ID NO:11所示的氨基酸序列;
可選地,所述腫瘤抗原包括:EDB-FN(extra domain of fibronectin)、Muc1、p53、FAP、GD2、EpCAM、 tenascin-C、CD20、CEA、MAdCAM-1或WT1(Wilms tumor protein1);可選地,所述腫瘤抗原結合分子為抗體,例如scFv,sdFv、Fab、Fab’、F(ab’)2或Fv;
可選地,所述IL-2受體亞基為IL-2受體α亞基。
在一些具體的實施方式中,所述第一多肽的C端通過或不通過連接肽與所述第二多肽的N端連接;或所述第一多肽的N端通過或不通過連接肽與所述第二多肽的C端連接;
所述連接肽優選選自:(G
4S)
n、(GGNGT)
n或(YGNGT)
n,所述n選自1、2、3、4或5;
更優選地,所述第一多肽的C端與所述第二多肽的N端通過連接肽(G
4S)
3連接。
在一些具體的實施方式中,所述融合蛋白包括SEQ ID NO:13~20或SEQ ID NO:39~56任一項所述的氨基酸序列。
在第四方面,本發明公開一種綴合物,所述綴合物包括前述突變體或融合蛋白,還包括綴合至所述突變體或融合蛋白的穩定劑、藥物或者示蹤分子,其中所述穩定劑可選自聚乙二醇,例如單甲氧基聚乙二醇。
在第五方面,本發明公開一種分離的核酸片段,所述核酸片段編碼前述突變體或融合蛋白。
在第六方面,本發明公開一種載體,所述載體包括前述核酸片段。
在第七方面,本發明公開一種宿主細胞,所述宿主細胞包含前述載體。
在一些具體的實施方式中,所述宿主細胞是原核細胞或真核細胞;所述原核細胞或真核細胞可選自大腸桿菌、酵母、昆蟲細胞或哺乳動物細胞,所述哺乳動物細胞可選自CHO細胞系或HEK293細胞系。
在第八方面,本發明公開一種製備前述IL-2突變體或融合蛋白的方法,所述方法包括培養前述宿主細胞,以及分離所述宿主細胞表達的IL-2突變體或融合蛋白。
在第九方面,本發明公開根據前述方法製備而成的IL-2突變體或融合蛋白。
在第十方面,本發明公開一種藥物組合物,所述藥物組合物包括前述突變體、融合蛋白、綴合物、核酸片段、載體或宿主細胞;以及藥學上可接受的運載體、稀釋劑或助劑;
優選地,所述藥物組合物為可注射藥物組合物,例如靜脈注射藥物組合物或皮下注射藥物組合物;更優選地,每劑所述藥物組合物包含能夠給與受試者有效量的融合蛋白;最優選地,所述有效量為0.001~10mpk,例如0.001mpk、0.002mpk、0.003mpk、0.004mpk、0.005mpk、0.006mpk、0.007mpk、0.008mpk、0.009mpk、0.01mpk、0.02mpk、0.03mpk、0.04mpk、0.05mpk、0.06mpk、0.07mpk、0.08mpk、0.09mpk、0.1 mpk、0.2mpk、0.3mpk、0.4mpk、0.5mpk、0.6mpk、0.7mpk、0.8mpk、0.9mpk、1mpk、2mpk、3mpk、4mpk、5mpk、6mpk、7mpk、8mpk、9mpk或10mpk。
在第十一方面,本發明公開前述突變體、融合蛋白、綴合物、核酸片段、載體或宿主細胞在製備藥物中的用途;
優選地,所述藥物為注射藥物,例如靜脈注射藥物或皮下注射藥物;
優選地,每劑所述藥物包含能夠給與受試者有效量的融合蛋白;最優選地,所述有效量為0.001~10mpk,例如0.001mpk、0.002mpk、0.003mpk、0.004mpk、0.005mpk、0.006mpk、0.007mpk、0.008mpk、0.009mpk、0.01mpk、0.02mpk、0.03mpk、0.04mpk、0.05mpk、0.06mpk、0.07mpk、0.08mpk、0.09mpk、0.1 mpk、0.2mpk、0.3mpk、0.4mpk、0.5mpk、0.6mpk、0.7mpk、0.8mpk、0.9mpk、1mpk、2mpk、3mpk、4mpk、5mpk、6mpk、7mpk、8mpk、9mpk或10mpk;
優選地,所述藥物用於治療自身免疫性疾病,或增生性疾病,或病毒感染。
更優選地,所述自身免疫性疾病包括類風濕關節炎、強直性脊柱炎、系統性紅斑狼瘡、皮膚紅斑狼瘡、狼瘡性腎炎、IgA腎病、乾燥綜合征、多肌炎、皮肌炎、硬皮病、銀屑病、斑塊狀銀屑病、斑禿、多發性硬化症、肌萎縮側索硬化症、炎性腸病、潰瘍性結腸炎、克羅恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、濕疹或哮喘;
更優選地,所述增生性疾病包括贅生物、實體瘤、血液瘤、惡性腹水或惡性胸水;其中,所述實體瘤可為良性或惡性,原發性或轉移性,所述惡性實體瘤可為癌或肉瘤,例如,上皮細胞癌、內皮細胞癌、鱗狀細胞癌、畸胎瘤、肺部腫瘤、乳頭瘤病毒引起的癌、腺癌、癌腫、黑色素瘤、血管肉瘤、神經母細胞瘤、轉移性肺癌、非小細胞肺癌、小細胞肺癌、乳腺癌、Merkel細胞癌、卵巢癌、腎細胞癌、轉移性腎癌、頭頸癌、膀胱癌、非肌層浸潤性膀胱癌;所述血液瘤可選自白血病、淋巴瘤、多發性骨髓瘤,例如B細胞淋巴瘤、T細胞淋巴瘤、皮膚T細胞淋巴瘤、T細胞大顆粒淋巴細胞白血病;
更優選地,所述病毒感染選自HIV感染、新型冠狀病毒感染或HPV病毒感染。
在第十二方面,本發明公開一種治療自身免疫性疾病,或增生性疾病,或病毒感染的方法,所述方法包括向受試者施用有效量的前述IL-2突變體、融合蛋白、綴合物、核酸片段、載體、宿主細胞或藥物組合物;
優選地,施用方式為注射,例如靜脈注射或者皮下注射;
優選地,所述有效量為0.001~10mpk,例如0.001mpk、0.002mpk、0.003mpk、0.004mpk、0.005mpk、0.006mpk、0.007mpk、0.008mpk、0.009mpk、0.01mpk、0.02mpk、0.03mpk、0.04mpk、0.05mpk、0.06mpk、0.07mpk、0.08mpk、0.09mpk、0.1 mpk、0.2mpk、0.3mpk、0.4mpk、0.5mpk、0.6mpk、0.7mpk、0.8mpk、0.9mpk、1mpk、2mpk、3mpk、4mpk、5mpk、6mpk、7mpk、8mpk、9mpk或10mpk;
優選地,所述自身免疫性疾病包括類風濕關節炎、強直性脊柱炎、系統性紅斑狼瘡、皮膚紅斑狼瘡、狼瘡性腎炎、IgA腎病、乾燥綜合征、多肌炎、皮肌炎、硬皮病、銀屑病、斑塊狀銀屑病、斑禿、多發性硬化症、肌萎縮側索硬化症、炎性腸病、潰瘍性結腸炎、克羅恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、濕疹或哮喘;
優選地,所述增生性疾病包括贅生物、實體瘤、血液瘤、惡性腹水或惡性胸水;其中,所述實體瘤可為良性或惡性,原發性或轉移性,所述惡性實體瘤可為癌或肉瘤,例如,上皮細胞癌、內皮細胞癌、鱗狀細胞癌、畸胎瘤、肺部腫瘤、乳頭瘤病毒引起的癌、腺癌、癌腫、黑色素瘤、血管肉瘤、神經母細胞瘤、轉移性肺癌、非小細胞肺癌、小細胞肺癌、乳腺癌、Merkel細胞癌、卵巢癌、腎細胞癌、轉移性腎癌、頭頸癌、膀胱癌、非肌層浸潤性膀胱癌;所述血液瘤可選自白血病、淋巴瘤、多發性骨髓瘤,例如B細胞淋巴瘤、T細胞淋巴瘤、皮膚T細胞淋巴瘤、T細胞大顆粒淋巴細胞白血病;
更優選地,所述病毒感染選自HIV感染、新型冠狀病毒感染或HPV病毒感染。
在第十三方面,本發明公開一種優先刺激T細胞群體或外周血中調節性T細胞的方法,其中,所述方法包括將所述T細胞群體或外周血與前述的IL-2突變體或融合蛋白,或綴合物,或核酸片段,或載體,或宿主細胞,或的藥物組合物接觸;
優選地,所述所述優先刺激包括:
(a)與非調節性T細胞或NK細胞相比,優先刺激調節性T細胞的STAT5磷酸化;
(b)與非調節性T細胞或NK細胞相比,優先刺激調節性T細胞增殖;
和/或(c)提高調節性T細胞與非調節性T細胞的比率,或提高調節性T細胞與NK細胞的比率。
術語定義和說明
除非本發明另外定義,與本發明相關的科學和技術術語應具有本領域普通技術人員所理解的含義。
除非另有說明,本發明所述術語“IL2”或“IL-2”,是指來自任何脊椎動物源,包括哺乳動物例如靈長類(例如人)和齧齒動物(例如小鼠和大鼠)和馴養或農業哺乳動物的任何天然或重組IL-2。本發明所述“IL2”或“IL-2”包括未加工成熟的IL-2(例如包含N端信號肽的IL-2)以及產生在細胞中加工成熟的IL-2的任何形式。本發明所述“IL2”或“IL-2”還包括IL-2的天然變體和片段,例如剪切變體或等位基因變體。本發明所述“IL2”或“IL-2”還包括非天然存在的突變體,例如通過基因工程人工改造的IL-2突變體。
“野生型IL-2”是在其他方面與IL-2突變體相同,只是在IL-2突變體的每個突變氨基酸位置保持野生型氨基酸的IL-2形式。示例性地,如果IL-2突變體是未加工成熟的IL-2,那麼該突變體的野生型形式是未加工成熟的IL-2;如果IL-2突變體是加工成熟的IL-2,那麼該突變體的野生型形式是加工成熟的IL-2;如果IL-2突變體是IL-2的截短形式,那麼該突變體的野生型形式是具有野生型序列的相應的IL-2截短形式。示例性地,本發明所述“野生型IL-2”可具有如下所示氨基酸序列:
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF
X QSIISTLT(SEQ ID NO:60);其中第125位氨基酸殘基“X”代表C、S、A或V。
本發明術語“突變”包含氨基酸取代、缺失、插入或其任意組合。本發明所述“突變”可以使用本領域公知的遺傳學或化學方法生成氨基酸突變,包括但不限於定點誘變、PCR、基因合成等方法。
本發明所述IL-2突變體的“突變位點”編號從SEQ ID NO:60所示“野生型IL-2”的第1位氨基酸殘基A開始計數。示例性地,本發明“Q13突變”是指IL-2突變體在SEQ ID NO:60所示野生型IL-2第13位氨基酸殘基(Gln,Q)處發生突變。示例性地,本發明“Q13L突變”是指IL-2突變體在SEQ ID NO:60所示野生型IL-2第13位處氨基酸殘基由Q(Gln)突變位L(Leu)。
本發明用於突變位點間的術語“/”表示“和”的意思,是指“/”前後的突變在同一個IL-2突變體中同時存在。示例性地,“Y31/A73/H79”是指在同一個IL-2突變體同時在Y31位點、A73位點和H79位點發生突變,“Y31V/A73L/H79Q”是指在同一個IL-2突變體同時存在Y31V、A73L和H79Q突變。
本發明術語“Tm”(melting temperature,熔點),是指50%的蛋白質發生變性時的溫度。本發明所述“Tm”可採用本領域公知的方法測定蛋白的Tm值,示例性地,可採用本發明實施例3或實施例6所示方法測定蛋白的Tm值。
本發明術語“融合蛋白”(fusion protein)是指,通過基因重組方法、化學方法或其它適當方法將兩個或多個基因的編碼區連接,在同一調控序列控制下表達基因重組所得的蛋白質產物。本發明的融合蛋白中,兩個或多個基因的編碼區之間可由編碼連接肽的序列於一個或數個位置融合。連接肽可用於構建本發明的融合蛋白。
本發明術語“連接肽(Linker)”是指,在本發明中用於連接IL-2與另一蛋白分子或蛋白片段,以保證蛋白的正確折疊和穩定性的肽。所述另一分子包括但不限於Fc。本發明的“連接肽”優選為(GGGGS)n,其中n可以為0、1、2、3、4、5。如果連接肽序列太短,可能影響兩蛋白高級結構的折疊,從而相互干擾;如果連接肽序列太長,又涉及免疫原性的問題,因為連接肽序列本身就是新的抗原。
本發明術語“第二多肽”可為單鏈多肽,例如抗體中的scFv。所述“第二多肽”還包括多鏈多肽,其中的至少一條多肽與IL-2或其突變體融合,其他多肽鏈通過共價鍵或非共價鍵連接至發生融合的所述至少一條多肽上,例如抗體中的Fab,其中Fab的重鏈可與IL-2或其突變體融合,而輕鏈通過二硫鍵連接至所述重鏈上。
本發明術語“Fc”是指,免疫球蛋白鏈恒定區,特別是免疫球蛋白重鏈恒定區的羧基端或其中的一部分,無抗原結合活性,是抗體分子與效應分子或細胞相互作用的部位。本發明所述“Fc”可以是任何Fc或其變體,來源於人或非人哺乳動物。例如,免疫球蛋白Fc可包括重鏈CH1、CH2、CH3、CH4的兩個或更多結構域與免疫球蛋白鉸鏈區的組合。Fc可以來源於不同的種屬,優選人的免疫球蛋白。根據重鏈恒定區的氨基酸序列,免疫球蛋白可以分為不同的種類,主要有5類免疫球蛋白:IgA、IgD、IgE、IgG和IgM。其中一些還可進一步分成亞類(同種型),如IgG-1、IgG-2、IgG-3、IgG-4;IgA-1和IgA-2。“Fc”優選包括至少一個免疫球蛋白絞鏈區,以及IgG的CH2和CH3結構域。更優選包括IgG1的一個CH2結構域,一個CH3結構域和一個免疫球蛋白絞鏈區,鉸鏈區起始氨基酸位置可以變動。除非另外說明,否則本發明所述Fc、恒定區或抗體中的氨基酸殘基根據如Kabat等人,Sequences of Proteins of Immunological Interes,第5版,Public Health Service,National Institutes of Health,Bethesda,MD,1991中所述的EU編號體系,也稱作EU索引,進行編號。
本發明“抗體”(antibody)在本發明中以最廣義使用,並且涵蓋各種抗體結構,只要它們展現出期望的抗原結合活性,所述抗體包括但不限於單克隆抗體、多克隆抗體、多特異性抗體(例如雙特異性抗體)和抗原結合片段。抗體可以包括鼠源抗體、人抗體、人源化抗體、嵌合抗體、駱駝抗體。例示性的,抗體可以是免疫球蛋白,是由兩條相同的重鏈和兩條相同的輕鏈通過鏈間二硫鍵連接而成的四肽鏈結構。免疫球蛋白重鏈恒定區的氨基酸組成和排列順序不同,故其抗原性也不同。據此,可將免疫球蛋白分為五類,或稱為免疫球蛋白的同種型,即IgM、IgD、IgG、IgA和IgE,其相應的重鏈分別為μ鏈、δ鏈、γ鏈、α鏈和ε鏈。同一類Ig根據其鉸鏈區氨基酸組成和重鏈二硫鍵的數目和位置的差別,又可分為不同的亞類,如IgG可分為IgG1、IgG2、IgG3、IgG4。輕鏈通過恒定區的不同分為κ鏈或λ鏈。五類Ig中第每類Ig都可以有κ鏈或λ鏈。本發明所述“抗體”還包括scFv,sdFv,Fab,Fab’,F(ab’)
2和Fv。
本發明術語“分離的”指將材料由其原始或天然環境(例如其天然存在的自然環境)中取出。因此,存在於活體動物中的天然多核苷酸或多肽不是分離的,但通過人為干預由天然系統中某些或全部共存材料中分離的相同多核苷酸或多肽是分離的。
“分離的核酸片段”是RNA或DNA聚合物,其為單鏈或雙鏈,任選含合成的、非天然或改變的核苷酸堿基。DNA聚合物形式的分離核酸片段可由一個或多個cDNA、基因組DNA或合成DNA片段組成。本發明所述“核酸片段”可為載體的一部分,在異源位點整合入宿主細胞染色體。本發明所述“核酸片段”可為組合物的一部分。由於這種載體或組合物不是其天然存在環境的一部分,所以仍然是分離的。
本發明術語“載體”包括核酸載體,例如DNA載體(如質粒),RNA載體,病毒或其他適合的複製子(例如病毒載體)。已經開發了多種載體用於將編碼外源蛋白質的多核苷酸遞送到原核或真核細胞中。本發明所述“載體”可含有用於表達蛋白質和/或將這些多核苷酸序列整合到哺乳動物細胞基因組中的附加序列元件、用於指導基因轉錄的調控序列(如啟動子和增強子區域)或用於增強基因的翻譯速率或改善由基因轉錄產生的mRNA的穩定性或核輸出的序列。這些序列元件包括例如5’和3’非翻譯區、內部核糖體進入位點(IRES)和聚腺苷酸化信號位元點,以便指導表達載體上攜帶的基因的有效轉錄。本發明所述“載體”還可以含有以下多核苷酸,該多核苷酸編碼用於選擇含有這種載體的細胞的標記。適合的標記的實例包括編碼抗生素(如氨苄青黴素、氯黴素、卡那黴素或諾爾絲菌素)抗性的基因。
本發明術語“宿主細胞”是指已引入外源核酸的細胞,包括這類細胞的後代。所述“後代”在核酸內容物上可與親本完全相同,也可含有突變,與親本細胞不完全相同。所述“後代”包括具有與原始轉化細胞中所篩選或選擇的功能或生物學活性相同的功能或生物學活性的突變後代。
本發明術語“藥物組合物”是指含有一種或多種本發明所述IL-2突變體、融合蛋白、核酸片段、載體或宿主細胞的混合物,所述混合物還含有其他組分,所述其他組分包括但不限於其在藥學上可接受的運載體、稀釋劑或助劑。本發明所述藥物組合物的目的是促進對生物體的給藥,利於活性成分的吸收進而發揮生物活性。
本發明術語“治療”是指外科手術或藥物處理(surgical or therapeutic treatment),其目的是預防、減緩(減少)治療物件中不希望的生理變化或病變,如細胞增殖性病症(如癌症或傳染性疾病),如自身免疫性疾病(如系統性紅斑狼瘡)的進展。有益的或所希望的臨床結果包括但不限於症狀的減輕、疾病程度減弱、疾病狀態穩定(即,未惡化)、疾病進展的延遲或減慢、疾病狀態的改善或緩和、以及緩解(無論是部分緩解或完全緩解),無論是可檢測的或不可檢測的。需要治療的物件包括已患有病症或疾病的物件以及易於患上病症或疾病的物件或打算預防病症或疾病的對象。當提到減緩、減輕、減弱、緩和、緩解等術語時,其含義也包括消除、消失、不發生等情況。
本發明術語“受試者”是指接受對如本發明所述的特定疾病或病症(如癌症或傳染性疾病或自身免疫性疾病)的治療的生物體。物件和患者的實例包括接受疾病或病症治療的哺乳動物,如人、靈長類動物、豬、山羊、兔、倉鼠、貓、狗、豚鼠、牛或其他牛科家族成員、綿羊和馬等。
本發明術語“有效量”指單獨給予或與另一治療劑組合給予細胞、組織或物件時能有效防止或緩解疾病病症或該疾病進展的治療劑用量。“有效量”還指足以緩解症狀,例如治療、治癒、防止或緩解相關醫學病症,或治療、治癒、防止或緩解這些病症的速度增加的化合物用量。當將活性成分單獨給予個體時,治療有效劑量單指該成分。當應用某一組合時,治療有效劑量指產生治療作用的活性成分的組合用量,而無論是組合、連續或同時給予。
本發明術語“自身免疫病”指特徵是物件對其自身的細胞、組織和/或器官產生免疫反應而引起的細胞、組織和/或器官損傷的物件病症。示例性地,自身免疫病包括但不限於類風濕關節炎、強直性脊柱炎、系統性紅斑狼瘡、皮膚紅斑狼瘡、狼瘡性腎炎、IgA腎病、乾燥綜合征、多肌炎、皮肌炎、硬皮病、銀屑病、斑塊狀銀屑病、斑禿、多發性硬化症、肌萎縮側索硬化症、炎性腸病、潰瘍性結腸炎、克羅恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、濕疹或哮喘。
本發明術語“增生性疾病”指細胞或組織的生長不受調節和/或異常生長,可導致發展不需要的病況或疾病的病況,可為或可不為癌性的,其包括但不限於贅生物、實體瘤、血液瘤、惡性腹水或惡性胸水。本發明“實體瘤”可為良性(benign)或惡性(maligant),原發性(Primary)或轉移性(metastatic);惡性實體瘤可為癌(carcinomas)或肉瘤(sarcomas)。示例性地,本發明“實體瘤”包括但不限於上皮細胞癌、內皮細胞癌、鱗狀細胞癌、畸胎瘤、肺部腫瘤、乳頭瘤病毒引起的癌、腺癌、癌腫、黑色素瘤、血管肉瘤、神經母細胞瘤、轉移性肺癌、非小細胞肺癌、小細胞肺癌、乳腺癌、Merkel細胞癌、卵巢癌、腎細胞癌、轉移性腎癌、頭頸癌、膀胱癌、非肌層浸潤性膀胱癌。示例性地,本發明“血液瘤”包括但不限於白血病、淋巴瘤、多發性骨髓瘤,例如B細胞淋巴瘤、T細胞淋巴瘤、皮膚T細胞淋巴瘤、T細胞大顆粒淋巴細胞白血病。
本發明術語“IL-2受體α亞基”(IL-2Rα),又稱“CD25”,指來自任何脊椎動物來源,包括哺乳動物如靈長類(例如人)和齧齒動物(例如小鼠和大鼠)的任何天然IL-2受體α亞基或其突變體,包括“全長”的未加工的IL-2受體α亞基以及源自細胞中的加工的任何形式的IL-2受體α亞基,還包括天然存在的IL-2受體α亞基變體,例如剪接變體或等位變體,還包括在天然IL-2受體α亞基基礎上經過人工改造的突變體。在某些實施方案中,IL-2受體α亞基是人IL-2受體α亞基,例示性序列如SEQ ID NO:57所示。
本發明術語“IL-2受體β亞基”(IL-2Rβ),又稱“CD122”,指來自任何脊椎動物來源,包括哺乳動物如靈長類(例如人)和齧齒動物(例如小鼠和大鼠)的任何天然IL-2受體β亞基或其突變體,包括“全長”的未加工的IL-2受體β亞基以及源自細胞中的加工的任何形式的IL-2受體β亞基,還包括天然存在的IL-2受體β亞基變體,例如剪接變體或等位變體,還包括在天然IL-2受體β亞基基礎上經過人工改造的突變體。在某些實施方案中,IL-2受體β亞基是人IL-2受體β亞基,例示性序列如SEQ ID NO:58所示。
本發明術語“IL-2受體γ亞基”(IL-2Rγ),又稱“CD132”,指來自任何脊椎動物來源,包括哺乳動物如靈長類(例如人)和齧齒動物(例如小鼠和大鼠)的任何天然IL-2受體γ亞基或其突變體,包括“全長”的未加工的IL-2受體γ亞基以及源自細胞中的加工的任何形式的IL-2受體γ亞基,還包括天然存在的IL-2受體γ亞基變體,例如剪接變體或等位變體,還包括在天然IL-2受體γ亞基基礎上經過人工改造的突變體。在某些實施方案中,IL-2受體γ亞基是人IL-2受體γ亞基,例示性序列如SEQ ID NO:59所示。
本發明術語“Treg”,又稱“調節性T細胞”或“T
調節細胞”,是指一種能抑制其它T細胞的應答的特殊化CD4
+T細胞類型。Treg的特徵在於表達IL-2受體α亞基(CD25)和轉錄因數叉頭框P3(Forkhead box protein P3,FOXP3),並在誘導和維持對抗原的外周自體耐受中起著關鍵作用。Treg需要IL-2來實現其功能和發育以及其抑制性特徵的誘導。
本發明術語“結合力”是指成對的分子之間表現出的相互間的結合或相互作用。常見的成對分子包括:配體與受體、抗原與抗體、酶和底物等,更具體地,例如本發明所示IL-2與IL-2βγ亞基複合物,或IL-2與IL-2αβγ亞基複合物。本發明所述“結合力”可通過本領域已經的常規方法進行檢測,包括但不限於ELISA或FACS。
下面結合具體實施例來進一步描述本發明,本發明的優點和特點將會隨著描述而更為清楚。實施例中未注明具體條件者,按照常規條件或製造商建議的條件進行。所用試劑或儀器未注明生產廠商者,均為可以通過市售購買獲得的常規產品。
本發明實施例僅是範例性的,並不對本發明的範圍構成任何限制。本領域技術人員應該理解的是,在不偏離本發明的精神和範圍下可以對本發明技術方案的細節和形式進行修改或替換,但這些修改和替換均落入本發明的保護範圍內。
實施例
1 IL-2
熱穩定性突變的設計與表達質粒構建
使用多種演算法獲得熱穩定性提高的IL-2突變,設計合成相應序列,將編碼野生型IL2和前述突變型IL2的核酸片段克隆至帶有Fc標籤的pTT5載體上,並按已建立的標準分子生物學方法製備質粒,所述質粒編碼以下融合蛋白:IL2-linker2-hFc、mut0.08-linker2-hFc、mut0.31-linker2-hFc、mut0.36-linker2-hFc、mut0.39-linker2-hFc、mut0.46-linker2-hFc、mut0.57-linker2-hFc、mut0.66-linker2-hFc和mut0.68-linker2-hFc。
前述融合蛋白及各元件的具體序列資訊如表1所示,其中,“IL2”表示野生型IL2,“mutXX”表示與野生型相比,發生突變的突變型IL2。
表1 IL-2穩定性突變序列
突變號 | 序列編號 | 序列資訊 |
IL2 | SEQ ID NO:1 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut0.08 (Y31V/A73L/H79Q) | SEQ ID NO:2 | APTSSSTKKTQLQLEHLLLDLQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut0.31 (Q13L) | SEQ ID NO:3 | APTSSSTKKTQL L LEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut0.36 (R120F) | SEQ ID NO:4 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT |
mut0.39 (L18I/V91A/F117W) | SEQ ID NO:5 | APTSSSTKKTQLQLEHL I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN A IVLELKGSETTFMCEYADETATIVE W LNRWITFAQSIISTLT |
mut0.46 (L18I/I89L/V93I) | SEQ ID NO:6 | APTSSSTKKTQLQLEHL I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN L NVI I LELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut0.57 (G27W/R120F) | SEQ ID NO:7 | APTSSSTKKTQLQLEHLLLDLQMILN W INNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT |
mut0.66 (P82L/R120F) | SEQ ID NO:8 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR L RDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT |
mut0.68 (N90Y/R120F) | SEQ ID NO:9 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI Y VIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT |
linker2 | SEQ ID NO:10 | GGGGSGGGGSGGGGS |
Fc (C220S/N297G) | SEQ ID NO:11 | EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
IL2-linker2-hFc,又稱WT IL-2-linker2-hFc(C220S/N297G) | SEQ ID NO:12 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT G GGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut0.08-linker2-hFc (Y31V/A73L/H79Q) | SEQ ID NO:13 | APTSSSTKKTQLQLEHLLLDLQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut0.31-linker2-hFc (Q13L) | SEQ ID NO:14 | APTSSSTKKTQL L LEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut0.36-linker2-hFc (R120F) | SEQ ID NO:15 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut0.39-linker2-hFc (L18I/V91A/F117W) | SEQ ID NO:16 | APTSSSTKKTQLQLEHL I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN A IVLELKGSETTFMCEYADETATIVE W LNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut0.46-linker2-hFc (L18I/I89L/V93I) | SEQ ID NO:17 | APTSSSTKKTQLQLEHL I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN L NVI I LELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTG GGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut0.57-linker2-hFc (G27W/R120F) | SEQ ID NO:18 | APTSSSTKKTQLQLEHLLLDLQMILN W INNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut0.66-linker2-hFc (P82L/R120F) | SEQ ID NO:19 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR L RDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut0.68-linker2-hFc (N90Y/R120F) | SEQ ID NO:20 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI Y VIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
實施例
2 IL-2
熱穩定性突變的生產與純化
對HEK293細胞(購自中國科學院細胞庫)暫態轉染實施例1構建的質粒(PEI,Polysciences)並使用FreeStyle TM 293 Expression Medium(購自Gibco)在37℃下進行擴大培養。7天后收集細胞培養液,離心去除細胞成分,獲得含IL-2-hFc融合蛋白的培養上清液。
用10 mL蛋白A柱(購自柏格隆)純化細胞培養上清液中的融合蛋白。蛋白A柱先用3~5倍柱體積的平衡緩衝液(PBS磷酸緩衝液,pH7.4)平衡,然後將澄清的培養上清液上樣到蛋白A柱,控制流速在10 mL/分鐘。上樣完畢後用平衡緩衝液清洗蛋白A柱,平衡緩衝液的體積為蛋白A柱柱床體積的3~5倍。用洗脫液(0.02 M檸檬酸緩衝液,0.1M甘氨酸,0.1M氯化鈉,pH3.0)洗脫結合在蛋白A柱上的蛋白,用核酸蛋白檢測儀監測洗脫情況(A280紫外吸收峰)。收集洗脫的蛋白,加入1M精氨酸,0.4M丁二酸,pH9.0的緩衝液中和pH。然後過分子篩(購自柏格隆),緩衝體系為20mM PB,200mM 氯化鈉,pH6.0-6.5,收集目的蛋白。用0.22 μm的濾器進行無菌過濾,無菌保存,即得純化的IL-2突變融合蛋白。
將純化的IL-2突變融合蛋白進行蛋白產量、蛋白濃度(A280/1.4)、SEC純度等檢測分析,純化後的IL-2穩定性突變融合蛋白的品質合格,同時IL-2穩定性突變(mutXX-linker2-hFc)的產量也較野生型IL-2(IL2-linker2-hFc)有顯著提高。其中關於蛋白產量、蛋白濃度和純度的檢測資訊參見表2。
表2 IL-2穩定性突變融合蛋白的檢測分析
突變號 | 蛋白產量 (mg/L) | 蛋白SEC純度 | 蛋白濃度 (mg/mL) |
IL2-linker2-hFc | 0.95 | 97.25% | 1.11 |
mut0.08-linker2-hFc | 8.95 | 99.85% | 1.79 |
mut0.31-linker2-hFc | 1.54 | 98.49% | 2.20 |
mut0.36-linker2-hFc | 2.98 | 99.99% | 1.49 |
mut0.39-linker2-hFc | 43.10 | 99.58% | 2.13 |
mut0.46-linker2-hFc | 13.82 | 99.92% | 1.22 |
mut0.57-linker2-hFc | 1.25 | 99.91% | 0.89 |
mut0.66-linker2-hFc | 7.39 | 99.90% | 1.12 |
mut0.68-linker2-hFc | 11.90 | 99.99% | 1.19 |
實施例
3 IL-2
熱穩定性突變
DSF
檢測
將Protein Thermal Shift Dye Kit(購自Applied Biosystems,貨號4461146)中的緩衝液稀釋成50×,稀釋通過實施例2所述方法純化所得IL-2突變蛋白至0.5mg/mL,染料2×濃度,補充至20μL反應體系。混勻後加入到8連管中,每個樣品2個複孔,蓋上管蓋,離心5-10秒,放置到檢測儀器(Applied Biosystems 7500)檢測,Boltzmann分析法分析熔解曲線,獲得Tm值。結果如表3所示,穩定性IL-2突變(mutXX-linker2-hFc)與野生型IL-2(IL2-linker2-hFc)相比,Tm提高3℃以上,熱穩定性有顯著提高。
表3 IL-2穩定性突變DSF分析
突變號 | Tm (℃) |
IL2-linker2-hFc | 46.74 |
mut0.08-linker2-hFc | 54.91 |
mut0.31-linker2-hFc | 57.53 |
mut0.36-linker2-hFc | 56.59 |
mut0.39-linker2-hFc | 55.93 |
mut0.46-linker2-hFc | 53.83 |
mut0.57-linker2-hFc | 57.98 |
mut0.66-linker2-hFc | 57.24 |
mut0.68-linker2-hFc | 54.77 |
實施例 4 IL-2 突變(與βγ
亞基相互作用減弱的 IL2 突變及在此基礎上組合熱穩定性突變)的設計與表達質粒構建
使用包括MOE軟體在內的多種演算法類比人的IL-2與相應的受體α、β、γ亞基的相互作用介面,獲得與βγ亞基相互作用減弱的突變位點。設計合成與βγ亞基相互作用減弱的IL-2突變體序列及其與熱穩定突變組合後的IL-2突變體序列。將編碼野生型IL-2和前述IL-2突變體的核酸片段克隆到帶有Fc標籤的pTT5載體上,並按已建立的標準分子生物學方法製備質粒,所述質粒編碼以下融合蛋白:IL2-linker2-hFc、mut7-linker2-hFc、mut7.08-linker2-hFc 、mut7.36-linker2-hFc、mut7.39-linker2-hFc、mut7.46-linker2-hFc、mut7.57-linker2-hFc 、mut7.66-linker2-hFc、mut8-linker2-hFc、mut8.08-linker2-hFc、mut8.36-linker2-hFc、mut11-linker2-hFc、 mut11.08-linker2-hFc、mut11.31-linker2-hFc、mut11.36-linker2-hFc 、mut11.46-linker2-hFc、mut61-linker2-hFc、mut61.08-linker2-hFc和mut61.46-linker2-hFc。所述融合蛋白及其組成元件的具體序列資訊如表4所示,其中,“IL2”表示野生型IL2,“mutXX”表示與野生型相比,發生突變的突變型IL2。
表4 IL-2突變序列
突變號 | 序列編號 | 序列 |
IL2 | SEQ ID NO:1 | 參見表1 |
mut7 (H16E) | SEQ ID NO:21 | APTSSSTKKTQLQLE E LLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut7.08 (H16E/Y31V/A73L/H79Q) | SEQ ID NO:22 | APTSSSTKKTQLQLE E LLLDLQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut7.36 (H16E/R120F) | SEQ ID NO:23 | APTSSSTKKTQLQLE E LLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT |
mut7.39 (H16E/L18I/V91A/F117W) | SEQ ID NO:24 | APTSSSTKKTQLQLE E L I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN A IVLELKGSETTFMCEYADETATIVE W LNRWITFAQSIISTLT |
mut7.46 (H16E/L18I/I89L/V93I) | SEQ ID NO:25 | APTSSSTKKTQLQLE E L I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN L NVI I LELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut7.57 (H16E/G27W/R120F) | SEQ ID NO:26 | APTSSSTKKTQLQLE E LLLDLQMILN W INNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT |
mut7.66 (H16E/P82L/R120F) | SEQ ID NO:27 | APTSSSTKKTQLQLE E LLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR L RDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT |
mut8 (D20A) | SEQ ID NO:28 | APTSSSTKKTQLQLEHLLL A LQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut8.08 (D20A/Y31V/A73L/H79Q) | SEQ ID NO:29 | APTSSSTKKTQLQLEHLLL A LQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut8.36 (D20A/R120F) | SEQ ID NO:30 | APTSSSTKKTQLQLEHLLL A LQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT |
mut11 (V91R) | SEQ ID NO:31 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut11.08 (V91R/Y31V/A73L/H79Q) | SEQ ID NO:32 | APTSSSTKKTQLQLEHLLLDLQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut11.31 (V91R/Q13L) | SEQ ID NO:33 | APTSSSTKKTQL L LEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut11.36 (V91R/R120F) | SEQ ID NO:34 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT |
mut11.46 (V91R/L18I/I89L/V93I) | SEQ ID NO:35 | APTSSSTKKTQLQLEHL I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN L N R I I LELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut61 (H16E/V91R) | SEQ ID NO:36 | APTSSSTKKTQLQLE E LLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut61.08 (H16E/V91R/Y31V/A73L/H79Q) | SEQ ID NO:37 | APTSSSTKKTQLQLE E LLLDLQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
mut61.46 (H16E/V91R/L18I/I89L/V93I) | SEQ ID NO:38 | APTSSSTKKTQLQLE E L I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN L N R I I LELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT |
linker2 | SEQ ID NO:10 | 參見表1 |
Fc (C220S/N297G) | SEQ ID NO:11 | 參見表1 |
IL2-linker2-hFc | SEQ ID NO:12 | 參見表1 |
mut7-linker2-hFc (H16E) | SEQ ID NO:39 | APTSSSTKKTQLQLE E LLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut7.08-linker2-hFc (H16E/Y31V/A73L/H79Q) | SEQ ID NO:40 | APTSSSTKKTQLQLE E LLLDLQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut7.36-linker2-hFc (H16E/R120F) | SEQ ID NO:41 | APTSSSTKKTQLQLE E LLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLTG GGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut7.39-linker2-hFc (H16E/L18I/V91A/F117W) | SEQ ID NO:42 | APTSSSTKKTQLQLE E L I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN A IVLELKGSETTFMCEYADETATIVE W LNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut7.46-linker2-hFc (H16E/L18I/I89L/V93I) | SEQ ID NO:43 | APTSSSTKKTQLQLE E L I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN L NV I ILELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut7.57-linker2-hFc (H16E/G27W/R120F) | SEQ ID NO:44 | APTSSSTKKTQLQLE E LLLDLQMILN W INNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut7.66-linker2-hFc (H16E/P82L/R120F) | SEQ ID NO:45 | APTSSSTKKTQLQLE E LLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR L RDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut8-linker2-hFc (D20A) | SEQ ID NO:46 | APTSSSTKKTQLQLEHLLL A LQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut8.08-linker2-hFc (D20A/Y31V/A73L/H79Q) | SEQ ID NO:47 | APTSSSTKKTQLQLEHLLL A LQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut8.36-linker2-hFc (D20A/R120F) | SEQ ID NO:48 | APTSSSTKKTQLQLEHLLL A LQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut11-linker2-hFc (V91R) | SEQ ID NO:49 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut11.08-linker2-hFc (V91R/Y31V/A73L/H79Q) | SEQ ID NO:50 | APTSSSTKKTQLQLEHLLLDLQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut11.31-linker2-hFc (V91R/Q13L) | SEQ ID NO:51 | APTSSSTKKTQL L LEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut11.36-linker2-hFc (V91R/R120F) | SEQ ID NO:52 | APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLN F WITFAQSIISTLT GGGGSGGGGSGGGGSE PKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut11.46-linker2-hFc(V91R/L18I/I89L/V93I) | SEQ ID NO:53 | APTSSSTKKTQLQLEHL ILDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN L N R I I LELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut61-linker2-hFc (H16E/V91R) | SEQ ID NO:54 | APTSSSTKKTQLQLE E LLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut61.08-linker2-hFc (H16E/V91R/Y31V/A73L/H79Q) | SEQ ID NO:55 | APTSSSTKKTQLQLE E LLLDLQMILNGINN V KNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL L QSKNF Q LRPRDLISNIN R IVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
mut61.46-linker2-hFc (H16E/V91R/L18I/I89L/V93I) | SEQ ID NO:56 | APTSSSTKKTQLQLE E L I LDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN L N R I I LELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGS EPKS S DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY G STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK |
實施例
5 IL-2
突變的生產與純化
對HEK293細胞(購自中國科學院細胞庫)暫態轉染(PEI,Polysciences)實施例4構建的質粒並使用FreeStyle TM 293 Expression Medium(購自Gibco)在37℃下進行擴大培養。7天后收集細胞培養液,離心去除細胞成分,獲得含IL-2-hFc融合蛋白的培養上清液。
用10 mL蛋白A柱(購自柏格隆)純化細胞培養上清液中的融合蛋白。蛋白A柱先用3~5倍柱體積的平衡緩衝液(PBS磷酸緩衝液,pH7.4)平衡,然後將澄清的培養上清液上樣到蛋白A柱,控制流速在10 mL/分鐘。上樣完畢後用平衡緩衝液清洗蛋白A柱,平衡緩衝液的體積為蛋白A柱柱床體積的3~5倍。用洗脫液(0.02 M檸檬酸緩衝液,0.1M甘氨酸,0.1M氯化鈉,pH3.0)洗脫結合在蛋白A柱上的蛋白,用核酸蛋白檢測儀監測洗脫情況(A280紫外吸收峰)。收集洗脫的蛋白,加入1M精氨酸,0.4M丁二酸,pH9.0的緩衝液中和pH。然後過分子篩(購自柏格隆),緩衝體系為20mM PB,200mM 氯化鈉,pH6.0-6.5,收集目的蛋白。用0.22 μm的濾器進行無菌過濾,無菌保存,即得純化的IL-2突變融合蛋白。
對純化的IL-2突變融合蛋白(mutXX-linker2-hFc)進行蛋白濃度(A280/1.4)、SEC純度等檢測分析,純化後的IL-2突變融合蛋白的品質合格。其中,關於蛋白純度和濃度的檢測資訊參見表5。
表5 IL-2突變融合蛋白的檢測分析
突變號 | 蛋白SEC純度 | 蛋白濃度 (mg/mL) |
IL2-linker2-hFc | 97.25% | 1.11 |
mut7-linker2-hFc | 100% | 0.97 |
mut7.08-linker2-hFc | 99.84% | 1.73 |
mut7.36-linker2-hFc | 99.94% | 2.40 |
mut7.39-linker2-hFc | 99.77% | 1.16 |
mut7.46-linker2-hFc | 99.90% | 1.28 |
mut7.57-linker2-hFc | 99.89% | 1.37 |
mut7.66-linker2-hFc | 99.89% | 1.92 |
mut8-linker2-hFc | 97.19% | 0.98 |
mut8.08-linker2-hFc | - | 0.82 |
mut8.36-linker2-hFc | - | 1.65 |
mut11-linker2-hFc | 99.13% | 1.65 |
mut11.08-linker2-hFc | 99.88% | 1.29 |
mut11.31-linker2-hFc | 100.00% | 1.37 |
mut11.36-linker2-hFc | 99.97% | 1.15 |
mut11.46-linker2-hFc | 99.90% | 1.52 |
mut61-linker2-hFc | 99.93% | 1.36 |
mut61.08-linker2-hFc | 99.99% | 1.15 |
mut61.46-linker2-hFc | 99.99% | 1.30 |
實施例
6 IL-2
突變
DSF
檢測
將Protein Thermal Shift Dye Kit(購自Applied Biosystems,貨號4461146)中的緩衝液稀釋成50×,稀釋實施例5純化的IL-2突變蛋白至0.5mg/mL,染料2×濃度,補充至20μL反應體系。混勻後加入到8連管中,每個樣品2個複孔,蓋上管蓋,離心5-10秒,放置到檢測儀器(Applied Biosystems 7500)檢測,Boltzmann分析法分析熔解曲線,獲得Tm值。具體Tm值如表6所示。
根據表6所示結果可知,實施例4-5所示IL-2突變體(mutXX-linker2-hFc)與野生型IL-2(IL2-linker2-hFc)相比,Tm均提高5℃以上,其中部分IL-2突變體的Tm提高9℃以上,所有突變體的熱穩定性均有顯著提高。
令人意外,與βγ亞基相互作用減弱的IL-2突變體,較之野生型IL-2,其Tm值升高,熱穩定性得到提升;在此基礎上,進一步組合熱穩定性突變的組合型突變體,均保持高Tm值,其中部分組合了熱穩定突變的組合突變,與僅發生βγ亞基相互作用減弱的突變相比,Tm仍提高2℃以上,最高可以提高6℃以上,熱穩定性獲得進一步的提高。
表6 IL-2突變DSF分析
突變號 | Tm (℃) |
IL2-linker2-hFc | 45.95 |
mut7-linker2-hFc | 51.08 |
mut7.08-linker2-hFc | 54.70 |
mut7.36-linker2-hFc | 56.95 |
mut7.57-linker2-hFc | 57.54 |
mut7.66-linker2-hFc | 56.64 |
mut8-linker2-hFc | 55.24 |
mut8.08-linker2-hFc | 55.32 |
mut8.36-linker2-hFc | 56.84 |
mut11-linker2-hFc | 56.63 |
mut11.08-linker2-hFc | 58.80 |
mut11.31-linker2-hFc | 59.37 |
mut11.36-linker2-hFc | 57.56 |
IL2-linker2-hFc | 46.74 |
mut7-linker2-hFc | 51.05 |
mut7.39-linker2-hFc | 56.30 |
mut7.46-linker2-hFc | 54.84 |
mut11-linker2-hFc | 57.17 |
mut11.46-linker2-hFc | 57.02 |
IL-2-linker2-hFc | 46.86 |
mut61-linker2-hFc | 56.84 |
mut61.08-linker2-hFc | 57.71 |
mut61.46-linker2-hFc | 56.84 |
實施例
7
過表達人或小鼠
IL-2
受體的穩轉細胞株的構建
A、過表達人源IL-2受體α的穩轉細胞株構建
將人的IL-2受體α亞基的氨基酸序列(NCBI中的基因登陸號為P01589,具體序列如SEQ ID NO:57所示)克隆到pLVX-IRES-Puro載體(購自優寶生物,貨號:VT1464)中,進行慢病毒包裝,隨後感染CHO-K1細胞系(購自中國科學院細胞庫)。病毒感染CHO-K1細胞72個小時後,用已知的IL-2受體α亞基抗體(貨號302606,購自BioLegend)經流式細胞分析法進行檢測。當確定轉染後的細胞開始表達人IL-2受體α亞基,加入Puromycin(購自Gibco)進行篩選,待細胞恢復後用有限稀釋法在96孔培養板中進行亞克隆,並置於37℃、5%(v/v)CO
2條件下培養,大約2周後選擇部分單克隆孔擴增到6孔板中。對擴增後的克隆再用已知的IL-2受體α亞基抗體經流式細胞分析法進行篩選。選擇長勢較好、螢光強度較高、單克隆的細胞系繼續擴大培養,用流式細胞分析法再次複測後並液氮凍存,即獲得表達人IL-2受體α亞基的穩轉細胞株。具體選擇結果如表7和圖1所示,表7中陽性細胞(%)指陽性細胞占總細胞數目的百分比。表7說明,已經製得一系列IL-2受體α亞基過表達的CHO-K1細胞系。
表7 表達人源IL-2受體α的CHO-K1細胞FACS篩選檢測結果
序號 | 轉染細胞克隆號 | IL-2受體α抗體 | IgG亞型對照 | ||
陽性細胞(%) | 平均螢光強度 | 陽性細胞(%) | 平均螢光強度 | ||
1 | CHO-K1 hIL-2Rα 1A6 | 100.00 | 38168 | - | - |
2 | CHO-K1 hIL-2Rα 2D2 | 100.00 | 21817 | - | - |
3 | CHO-K1 hIL-2Rα 2D5 | 100.00 | 18111 | - | - |
4 | CHO-K1 hIL-2Rα 2B8 | 99.80 | 19018 | 0.04 | 80.70 |
B、過表達人源IL-2受體β的穩轉細胞株構建
將人的IL-2受體β亞基的氨基酸序列(NCBI中的基因登陸號為P14784,具體序列如SEQ ID NO:58所示)克隆到pLVX-IRES-Puro載體(購自優寶生物,貨號:VT1464)中,進行慢病毒包裝,隨後感染CHO-K1細胞系(購自中國科學院細胞庫)。病毒感染CHO-K1細胞72個小時後,用已知的IL-2受體β亞基抗體(貨號339010,購自BioLegend)經流式細胞分析法進行檢測。當確定轉染後的細胞開始表達人IL-2受體β亞基,加入Puromycin(購自Gibco)進行篩選,待細胞恢復後用有限稀釋法在96孔培養板中進行亞克隆,並置於37℃、5%(v/v)CO
2條件下培養,大約2周後選擇部分單克隆孔擴增到6孔板中。對擴增後的克隆再用已知的IL-2受體β亞基抗體經流式細胞分析法進行篩選。選擇長勢較好、螢光強度較高、單克隆的細胞系繼續擴大培養,用流式細胞分析法再次複測後並液氮凍存,即獲得表達人IL-2受體β亞基的穩轉細胞株。具體選擇結果如表8和圖2所示,表8中陽性細胞(%)指陽性細胞占總細胞數目的百分比。表8說明,已經製得一系列IL-2受體β亞基過表達的CHO-K1細胞系。
表8 表達人源IL-2受體β的CHO-K1細胞FACS篩選檢測結果
序號 | 轉染細胞克隆號 | IL-2受體β抗體 | IgG亞型對照 | ||
陽性細胞(%) | 平均螢光強度 | 陽性細胞(%) | 平均螢光強度 | ||
1 | CHO-K1 hIL-2Rβ 2A5 | 100.00 | 7905 | - | - |
2 | CHO-K1 hIL-2Rβ 2B3 | 99.40 | 5081 | 0.03 | 41.50 |
3 | CHO-K1 hIL-2Rβ 2H4 | 99.40 | 1226 | - | - |
C、過表達人源IL-2受體βγ的穩轉細胞株構建
將人的IL-2受體γ亞基的氨基酸序列(NCBI中的基因登陸號為P31785,具體序列如SEQ ID NO:59所示)克隆到pLVX-IRES-Hygro載體中,進行慢病毒包裝,隨後感染CHO-K1 IL2受體β亞基細胞系過表達細胞系(CHO-K1 hIL-2R β 2A5)。病毒感染CHO-K1細胞72個小時後,用已知的IL-2受體β、γ亞基抗體(貨號339010、338608,購自BioLegend)經流式細胞分析法進行檢測。當確定轉染後的細胞開始表達人IL-2受體βγ亞基,加入Puromycin、Hygromycin(購自Gibco、Thermo)進行篩選,待細胞恢復後用有限稀釋法在96孔培養板中進行亞克隆,並置於37℃、5%(v/v)CO
2條件下培養,大約2周後選擇部分單克隆孔擴增到6孔板中。對擴增後的克隆再用已知的IL-2受體β、γ亞基抗體經流式細胞分析法進行篩選。選擇長勢較好、螢光強度較高、單克隆的細胞系繼續擴大培養,用流式細胞分析法再次複測後並液氮凍存,即獲得表達人IL-2受體βγ亞基的穩轉細胞株。具體選擇結果如表9和圖3A-圖3B所示,表9中陽性細胞(%)指陽性細胞占總細胞數目的百分比。表9說明,已經製得一系列IL-2受體βγ亞基過表達的CHO-K1細胞系。
表9 表達人源IL-2受體βγ的CHO-K1細胞FACS篩選檢測結果
序號 | 轉染細胞克隆號 | IL-2受體β抗體 | IgG亞型對照 | IL-2受體γ抗體 | IgG亞型對照 | ||||
陽性細胞(%) | 平均螢光強度 | 陽性細胞(%) | 平均螢光強度 | 陽性細胞(%) | 平均螢光強度 | 陽性細胞(%) | 平均螢光強度 | ||
1 | CHO-K1 IL-2Rβγ 2D3 | 100.00 | 4569 | 0.65 | 115 | 98.90 | 8932 | 23.30 | 1070 |
2 | CHO-K1 hIL-2Rβγ 2C11 | 99.70 | 2111 | - | - | 99.00 | 31664 | - | - |
3 | CHO-K1 IL-2Rβγ 2E6 | 100.00 | 2909 | - | - | 98.80 | 11764 | - | - |
D、過表達人源IL-2受體 αβγ的穩轉細胞株構建
將人IL-2受體α亞基的氨基酸序列克隆到pLVX-IRES-zsGreen載體中,進行慢病毒包裝,隨後感染IL-2受體βγ亞基過表達的CHO-K1細胞系。病毒感染CHO-K1細胞72個小時後,用已知的IL-2受體α、β、γ亞基抗體(同上,購自BioLegend)經流式細胞分析法進行檢測。當確定轉染後的細胞開始表達人IL-2受體αβγ亞基,加入Puromycin、Hygromycin(購自Gibco、Thermo)、GFP螢光表達進行篩選,待細胞恢復後用有限稀釋法在96孔培養板中進行亞克隆,並置於37℃、5%(v/v)CO
2條件下培養,大約2周後選擇部分單克隆孔擴增到6孔板中。對擴增後的克隆再用已知的IL-2受體α、β、γ亞基抗體經流式細胞分析法進行篩選。選擇長勢較好、螢光強度較高、單克隆的細胞系繼續擴大培養,用流式細胞分析法再次複測後並液氮凍存,即獲得表達人IL-2受體αβγ亞基的穩轉細胞株。具體選擇結果如表10和圖4A-圖4C所示,表10中陽性細胞(%)指陽性細胞占總細胞數目的百分比。表10說明,已經製得一系列IL-2受體αβγ亞基過表達的CHO-K1細胞系。
表10 表達人源IL-2受體αβγ的CHO-K1細胞FACS篩選檢測結果
序號 | 轉染細胞克隆號 | IL-2受體α抗體 | IL-2受體β抗體 | IL-2受體γ抗體 | |||
陽性細胞(%) | 平均螢光強度 | 陽性細胞(%) | 平均螢光強度 | 陽性細胞(%) | 平均螢光強度 | ||
1 | CHO-K1 hIL-2R αβγ 1E3 | 99.90 | 10667 | 100.00 | 3002 | 99.90 | 14863 |
2 | CHO-K1 hIL-2R αβγ 2D6 | 99.90 | 5265 | 99.90 | 1324 | 99.80 | 2575 |
3 | CHO-K1 hIL-2R αβγ 1A10 | 100.00 | 12255 | 88.60 | 465 | 99.60 | 4106 |
表11 實施例7 A-D構建穩轉細胞株使用的基因名稱及其編碼蛋白的序列資訊
基因名稱 | 序列編號 | 序列資訊 |
hIL2Rα | SEQ ID NO:57 | MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI |
hIL2Rβ | SEQ ID NO:58 | MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV |
hIL2Rγ | SEQ ID NO:59 | MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET |
實施例
8
流式細胞實驗(
FACS
)檢測
IL-2
突變與受體表達細胞的結合
將CHO-K1 IL2受體αβγ或CHO-K1 IL2受體βγ細胞(CHO-K1 hIL-2R αβγ 2D6、CHO-K1 hIL-2R βγ 2E6)在T-75細胞培養瓶中擴大培養至90%匯合度。移除培養基,用PBS緩衝液(購自Hyclone,貨號SH30256.01)洗滌一次,然後用2mL含有0.25% EDTA的胰酶(購自Invitrogen,貨號25200072)處理細胞2-3分鐘,加入8mL含有10% (w/w)胎牛血清(購自Gibco,貨號10099-141C)的DMEM/F-12(購自Gibco,貨號12634-010)進行中和,用移液器吹打3-4次,將吹散後的細胞收集到15毫升離心管中,進行細胞計數後,室溫條件下1000rpm離心5分鐘,棄去培養基。將細胞用含有2%(w/w)胎牛血清的RPMI-1640(購自Gibco,貨號A10491-01)重懸並稀釋至1.43×10
6個細胞每毫升,按每孔70μL加入到U型底96孔FACS 反應板中,放在4℃或者冰上備用。用含有2%(w/w)胎牛血清的RPMI-1640稀釋待測IL-2突變,按每孔30μL加入細胞中混勻,冰上孵育1小時。用FACS緩衝液[含有2%(w/w)牛血清蛋白的PBS緩衝液]離心洗滌2次,加入每孔100μL螢光標記的二抗(購自Biolegend,貨號409306),冰上孵育30分鐘。用FACS緩衝液離心洗滌2次。用FACS(FACS Canto II,購自BD公司)檢測和分析結果。或者用100μL 含有2%(w/w)多聚甲醛(購自DingGuo,貨號AR-0211)的FACS緩衝液懸浮細胞,放於4℃保存至上機檢測,檢測前每孔加入100μL PBS緩衝液,用FACS檢測和分析結果。結果如圖5A-圖5D和表12-13示。結果顯示,IL-2突變可結合細胞表面的人源IL2受體αβγ三聚體。IL-2突變與細胞表面的人源IL2受體βγ二聚體的結合活性與野生型相比較弱,其中,mut61-linker2-hFc、mut61.08-linker2-hFc和mut61.46-linker2-hFc與CHO-K1 IL2Rβγ二聚體的結合活性受到極大抑制。表中資料MFI為所測細胞群的平均螢光強度值。
表12 FACS檢測IL-2突變與CHO-K1 IL2受體αβγ重組細胞(CHO-K1 IL2R αβγ)的結合活性
蛋白濃度(nM) | 23.529 | 4.7059 | 0.9412 | 0.1882 | 0.0376 | 0.0075 | 0.0015 | 0.0003 |
IL2-linker2-hFc(MFI) | 25072 | 22623 | 22295 | 9026 | 1728 | 457 | 161 | 251 |
mut7.36-linker2-hFc(MFI) | 23892 | 19565 | 18132 | 9608 | 2416 | 570 | 272 | 171 |
mut11.08-linker2-hFc(MFI) | 22230 | 17370 | 14857 | 8713 | 2736 | 621 | 268 | 251 |
mut61-linker2-hFc(MFI) | 23858 | 18259 | 16403 | 9188 | 2307 | 544 | 224 | 194 |
mut61.08-linker2-hFc(MFI) | 22558 | 19652 | 18992 | 9557 | 2131 | 575 | 303 | 190 |
mut61.46-linker2-hFc(MFI) | 24244 | 19701 | 19386 | 10124 | 2384 | 521 | 280 | 186 |
蛋白濃度(nM) | 26.667 | 5.3333 | 1.0667 | 0.2133 | 0.0427 | 0.0085 | 0.0017 | 0.0003 |
人IgG對照 | 118 | 124 | 157 | 181 | 274 | 119 | 120 | 146 |
蛋白濃度(nM) | 23.529 | 4.7059 | 0.9412 | 0.1882 | 0.0376 | 0.0075 | 0.0015 | 0.0003 |
IL2-linker2-hFc(MFI) | 17021 | 16362 | 15503 | 9402 | 1983 | 423 | 188 | 156 |
mut7.66-linker2-hFc(MFI) | 15503 | 11604 | 9659 | 7683 | 2485 | 568 | 227 | 177 |
mut11.31-linker2-hFc(MFI) | 13449 | 10906 | 9502 | 7521 | 2181 | 553 | 214 | 172 |
蛋白濃度(nM) | 26.667 | 5.3333 | 1.0667 | 0.2133 | 0.0427 | 0.0085 | 0.0017 | 0.0003 |
人IgG對照(MFI) | 187 | 136 | 138 | 139 | 144 | 145 | 140 | 146 |
表13 FACS檢測IL-2突變與CHO-K1 IL2受體βγ重組細胞(CHO-K1 IL2Rβγ)的結合活性
蛋白濃度(nM) | 117.65 | 39.216 | 13.072 | 4.357 | 1.452 | 0.484 | 0.161 | 0.054 |
IL2-linker2-hFc(MFI) | 1210 | 1226 | 1162 | 980 | 904 | 603 | 325 | 191 |
mut7.36-linker2-hFc(MFI) | 615 | 438 | 318 | 250 | 210 | 165 | 156 | 153 |
mut11.08-linker2-hFc(MFI) | 854 | 645 | 447 | 342 | 232 | 178 | 160 | 144 |
mut61-linker2-hFc(MFI) | 169 | 155 | 142 | 142 | 145 | 146 | 148 | 142 |
mut61.08-linker2-hFc(MFI) | 164 | 163 | 144 | 144 | 157 | 144 | 146 | 141 |
mut61.46-linker2-hFc(MFI) | 148 | 159 | 132 | 131 | 134 | 133 | 136 | 134 |
蛋白濃度(nM) | 133.33 | 44.444 | 14.815 | 4.938 | 1.646 | 0.549 | 0.183 | 0.061 |
人IgG對照(MFI) | 130 | 130 | 136 | 134 | 139 | 141 | 141 | 146 |
蛋白濃度(nM) | 117.65 | 39.216 | 13.072 | 4.357 | 1.452 | 0.484 | 0.161 | 0.054 |
IL2-linker2-hFc(MFI) | 2694 | 2703 | 2518 | 2170 | 1729 | 831 | 363 | 177 |
mut7.66-linker2-hFc(MFI) | 905 | 597 | 357 | 216 | 157 | 136 | 124 | 118 |
mut11.31-linker2-hFc(MFI) | 1320 | 950 | 560 | 285 | 180 | 134 | 121 | 115 |
蛋白濃度(nM) | 133.33 | 44.444 | 14.815 | 4.938 | 1.646 | 0.549 | 0.183 | 0.061 |
人IgG對照(MFI) | 259 | 100 | 101 | 107 | 101 | 102 | 104 | 109 |
實施例
9 STAT5
磷酸化實驗檢測
IL-2
突變對不同細胞的信號通路啟動作用
將冷凍的PBMC(購自Allcells)復蘇,每孔加入50μl的5x10
5個細胞PBMC,以及50μl IL-2突變蛋白,二氧化碳培養箱中反應15分鐘。反應結束後,每孔加100μl預冷的DPBS終止反應,離心後分別進行Livedead Violet 染色(Invitrogen-L34964),Fix I(BD-557870)37℃固定10分鐘,PermIII(BD-558050)冰上破膜30分鐘。用CD3-AF700(BD-557943),CD4-PerCP cy5.5(BD-560650),CD8-FTIC(BD-555366),CD25-PE(BD-557138),FoxP3-AF647(BD-560045),pSTAT5-PE Cy7(Invitrogen-25-9010-42)室溫染色1小時,洗滌兩遍後上機檢測。結果如圖6A-圖6I和表14-16所示。結果顯示,與野生型IL-2相比,IL-2突變啟動Treg細胞內STAT5磷酸化的水準相近,但是啟動CD4
+CD25
-FoxP3
-T細胞或者CD8
+T細胞內的STAT5磷酸化水準顯著降低。其中表中的資料MFI為所測細胞群內STAT5磷酸化的平均螢光強度值。
表14 FACS檢測IL-2突變啟動Treg STAT5磷酸化信號
蛋白濃度(pM) | 10000 | 1000 | 100 | 10 | 1 | 0.1 | 0.01 |
IL2-linker2-hFc(MFI) | 3706 | 3498 | 3607 | 3440 | 2554 | 680 | 662 |
mut7.36-linker2-hFc(MFI) | 3463 | 3498 | 3498 | 3139 | 1250 | 735 | 522 |
IL2-linker2-hFc(MFI) | 3371 | 3451 | 3382 | 3619 | 1909 | 464 | 415 |
mut11.08-linker2-hFc(MFI) | 3451 | 3440 | 3668 | 3558 | 1365 | 462 | 366 |
IL2-linker2-hFc(MFI) | 3807 | 3731 | 3706 | 3833 | 1955 | 561 | 467 |
mut7.66-linker2-hFc(MFI) | 3718 | 3743 | 3280 | 2323 | 668 | 544 | 419 |
mut11.31-linker2-hFc(MFI) | 3570 | 3522 | 3428 | 2149 | 680 | 465 | 473 |
表15 FACS檢測IL-2突變啟動CD4+CD25-FoxP3- T細胞STAT5磷酸化信號
蛋白濃度(pM) | 10000 | 1000 | 100 | 10 | 1 | 0.1 |
IL2-linker2-hFc(MFI) | 1601 | 1370 | 1130 | 612 | 377 | 333 |
mut7.36-linker2-hFc(MFI) | 527 | 473 | 388 | 371 | 368 | 336 |
IL2-linker2-hFc(MFI) | 1111 | 960 | 833 | 485 | 362 | 370 |
mut11.08-linker2-hFc(MFI) | 718 | 584 | 496 | 382 | 355 | 354 |
IL2-linker2-hFc(MFI) | 1532 | 1361 | 1205 | 789 | 529 | 501 |
mut7.66-linker2-hFc(MFI) | 592 | 564 | 481 | 496 | 496 | 501 |
mut11.31-linker2-hFc(MFI) | 612 | 582 | 557 | 481 | 481 | 467 |
表16 FACS檢測IL-2突變啟動CD8+ T細胞STAT5磷酸化信號
蛋白濃度(pM) | 10000 | 1000 | 100 | 10 | 1 | 0.1 |
IL2-linker2-hFc(MFI) | 973 | 413 | 274 | 221 | 233 | 239 |
mut7.36-linker2-hFc(MFI) | 270 | 264 | 245 | 247 | 259 | 236 |
IL2-linker2-hFc(MFI) | 758 | 381 | 321 | 260 | 270 | 288 |
mut11.08-linker2-hFc(MFI) | 336 | 295 | 296 | 281 | 286 | 294 |
IL2-linker2-hFc(MFI) | 910 | 481 | 372 | 361 | 344 | 339 |
mut7.66-linker2-hFc(MFI) | 326 | 344 | 319 | 331 | 343 | 346 |
mut11.31-linker2-hFc(MFI) | 311 | 324 | 346 | 327 | 326 | 317 |
實施例
10 IL-2
突變對
T
細胞增殖水準的調節作用
將冷凍的PBMC復蘇,細胞重懸於RPMI-1640(購自Gibco,貨號A10491-01)含10% FBS(購自Gibco,貨號10099-141C)中,加入預包被有100ng/ml的CD3抗體(購自BD,貨號566685)的六孔板中培養兩天。收集細胞,用PBS緩衝液(購自Hyclone,貨號SH30256.01)洗滌三次,重懸在RPMI-1640加10%FBS的培養基中,在六孔板中培養五天。收集細胞,用PBS緩衝液洗滌一次,用Celltrace Violet(購自Invitrogen,貨號C34557)染色,加培養基洗滌一次,用培養基重懸,24孔板每孔加入900ul細胞,加100μl的IL2突變蛋白樣品,培養七天。收集細胞,用含1%BSA(購自生工,貨號A500023-0100)的PBS(購自生工,貨號B548117-0500)重懸,細胞加入96孔板中。
用BV605-CD8(購自Biolengend,貨號344742)室溫染色30分鐘,含1%BSA的PBS洗滌。每孔加入200μl固定液(購自eBioscience,貨號00-5523-00),4℃固定半小時,用含1%BSA的PBS洗滌,每孔加入200μl破膜液液(購自eBioscience,貨號00-5523-00),4℃破膜半小時,1%BSA的PBS洗滌。加入抗體APC-CY7-CD3(購自Biolengend,貨號344818),CD25抗體(購自Biolengend,貨號302606),Foxp3抗體(購自ThermoFisher#17-4777-42),室溫染色半小時。用含1%BSA的PBS洗滌,將樣品重懸在200μl的含1%BSA的PBS中,FACS(FACS Canto II,購自BD公司)檢測和分析結果。結果如圖7A-7F和表17-19所示。結果顯示:與野生型IL-2相比,IL-2突變對Treg細胞的增殖水準影響相近,但是對CD4
+CD25
-FoxP3
-T細胞或者CD8
+CD25
-T細胞細胞的增殖水準影響略微降低。其中表中的資料MFI為所測細胞群Celltrace Violet的平均螢光強度值,增殖後細胞內Celltrace Violet的平均螢光強度值降低,即,同樣的檢測時間內,細胞增殖越快,倍增數越多時,檢測到的螢光強度平均值越低。
表17 FACS檢測IL-2突變啟動Treg細胞增殖
蛋白濃度(nM) | 10 | 1 | 0.1 | 0.01 | 0.001 | 0.0001 | 0.00001 |
IL2-linker2-hFc(MFI) | 1995 | 1670 | 2857 | 3362 | 3624 | 3863 | 4518 |
mut7.36-linker2-hFc(MFI) | 1823 | 1407 | 2070 | 3162 | 3555 | 4077 | 4389 |
mut7.66-linker2-hFc(MFI) | 1583 | 1437 | 2068 | 3384 | 4392 | 4027 | 4887 |
IL2-linker2-hFc(MFI) | 1807 | 1838 | 2651 | 3319 | 3459 | 3691 | 4758 |
mut11.08-linker2-hFc(MFI) | 1633 | 1536 | 1884 | 2371 | 2930 | 3369 | 3953 |
mut11.31-linker2-hFc(MFI) | 1735 | 1855 | 1985 | 2669 | 2626 | 3379 | 4323 |
表18 FACS檢測IL-2突變啟動CD4+CD25-FoxP3-T細胞增殖
蛋白濃度(nM) | 10 | 1 | 0.1 | 0.01 | 0.001 | 0.0001 | 0.00001 |
IL2-linker2-hFc(MFI) | 1653 | 2058 | 2216 | 3198 | 4578 | 5274 | 5602 |
mut7.36-linker2-hFc(MFI) | 2361 | 2372 | 3317 | 4169 | 4971 | 5319 | 5815 |
mut7.66-linker2-hFc(MFI) | 2258 | 2524 | 3160 | 4633 | 5374 | 5495 | 6022 |
IL2-linker2-hFc(MFI) | 1679 | 1838 | 2055 | 2728 | 3997 | 4826 | 5314 |
mut11.08-linker2-hFc(MFI) | 2254 | 2166 | 2538 | 3372 | 4154 | 5072 | 5391 |
mut11.31-linker2-hFc(MFI) | 2332 | 2559 | 2885 | 3878 | 4779 | 5284 | 5509 |
表19 FACS檢測IL-2突變啟動CD8+CD25-T細胞增殖
蛋白濃度(nM) | 10 | 1 | 0.1 | 0.01 | 0.001 | 0.0001 | 0.00001 |
IL2-linker2-hFc(MFI) | 1128 | 1422 | 1696 | 3303 | 4907 | 5767 | 6303 |
mut7.36-linker2-hFc(MFI) | 1955 | 1921 | 2640 | 3732 | 4917 | 5562 | 6179 |
mut7.66-linker2-hFc(MFI) | 1820 | 1983 | 2683 | 4038 | 5539 | 5553 | 6617 |
IL2-linker2-hFc(MFI) | 1064 | 1236 | 1435 | 2497 | 4087 | 5010 | 5715 |
mut11.08-linker2-hFc(MFI) | 1876 | 1682 | 1956 | 2965 | 4124 | 5195 | 5567 |
mut11.31-linker2-hFc(MFI) | 1750 | 1945 | 2252 | 3463 | 4610 | 5406 | 5583 |
實施例
11 IL-2
突變對
NK
細胞增殖水準的調節作用
用NK分選試劑盒(購自Miltenyi Biotec,貨號130-092-657)將NK細胞分選並計數,重懸在MEM培養基(購自Gibco,貨號12634-010),培養基中含25%牛血清(購自Gibco,貨號10099-141C)、0.2mM肌醇(購自Sigma Aldrich,貨號I7508-50G)、0.1mM β-巰基乙醇(購自Sigma Aldrich,貨號M3148-100ML)、0.02mM葉酸(購自Sigma Aldrich,貨號F8758-5G)。將細胞種入96孔板,每孔加入Fc blocker(購自Biolengend,貨號422302),再加入IL2突變蛋白培養三天。在最後18小時加入BrdU(購自Biolengend,貨號423401)。收集細胞,用1%BSA的PBS洗滌並重懸,用等體積4%多聚甲醛(購自DingGuo,貨號AR-0211)室溫固定30分鐘,用1%BSA的PBS洗滌,用0.5% Triton-X 100(購自ThermoFisher,貨號HFH10)室溫破膜15分鐘,用1%BSA的PBS洗滌,加入DnaseI(購自Sigma Aldrich,貨號D4513-1VL)37°C消化一小時。用1%BSA的PBS洗滌並重懸,加入APC anti-BrdU 抗體(購自Biolengend,貨號339808)室溫染色二十分鐘,用1%BSA的PBS洗滌,將樣品重懸在200μl 的1%BSA的PBS中,FACS(FACS Canto II,購自BD公司)檢測和分析結果。結果如圖8和表20所示。結果顯示:與野生型IL-2相比,IL-2突變對NK細胞的增殖水準顯著降低。其中表中的資料為所測NK細胞群BrdU陽性細胞比例。
表20 FACS檢測IL-2突變啟動NK細胞增殖
蛋白濃度(nM) | 100.00 | 33.33 | 11.11 | 3.70 | 1.23 | 0.41 | 0.14 | 0.05 |
IL2-linker2-hFc(APC+%) | 13.9 | 12.0 | 12.9 | 14.3 | 11.4 | 8.5 | 9.1 | 4.0 |
mut7.36-linker2-hFc(APC+%) | 7.7 | 5.6 | 7.0 | 4.6 | 4.8 | 3.2 | 3.5 | 3.6 |
mut7.66-linker2-hFc(APC+%) | 2.6 | 5.3 | 3.9 | 5.6 | 5.6 | 3.8 | 4.3 | 3.8 |
mut11.08-linker2-hFc(APC+%) | 9.1 | 12.3 | 7.2 | 5.3 | 6.3 | 4.8 | 4.2 | 3.2 |
實施例
12
野生型小鼠皮下給藥的
PD
(藥效動力學)結果
採用Balb/c小鼠,雌性,6-8周齡,購自維通利華。Fc-linker-IL2_V91K、mut11.08-linker2-hFc蛋白,PBS稀釋,小鼠背部皮下給藥,給藥體積為200μL/只,之後在不同時間點收取小鼠全血及脾臟樣本,用於FACS檢測。Fc-linker-IL2_V91K序列如SEQ ID NO:60所示,純化方法參照實施例2。
PKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
GGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO:60 )。
2 mL EDTA/2K抗凝管 (新康醫療,貨號X424)中收集小鼠全血,上下混勻血樣及抗凝劑,使其充分接觸。將300 μL全血轉入FACS管中,每管加入染色抗體的混合液,室溫、避光孵育20 min。隨後在管中加入紅細胞裂解液1 mL/樣品 (Hybri-Max,貨號R7757-100mL),室溫、避光,靜置5 min,見絮狀物質,20℃,400 g,離心6 min,棄去上清,打散細胞。重複進行紅細胞裂解。洗細胞:PBS 4 mL/樣品重懸細胞,4℃,500 g,離心6 min,棄去上清,打散細胞。加固定液,500μL/管,一滴滴加入,(點震)每次加入後間歇震動FACS管,於4℃固定一小時或者固定過夜。
取小鼠脾臟,70 μm細胞濾網上碾磨 (Falcon Corning,貨號352350),500 g離心,每個脾臟加入3 mL紅細胞裂解液,裂解5 min,加入20 mL PBS終止裂解,500 g離心5 min,加入5 mL PBS重懸細胞,過70 μm細胞篩。細胞計數,每個FACS管加入1×10
6個細胞。加Fc blocker 5 μL/管(Biolengend,貨號156603),渦旋振盪器上震盪,4℃孵育20 min,每10 min震盪一次。加4 mL/管FACS洗液(PBS+1% BSA),4℃,400 g,離心6 min。倒掉上清,用吸水紙吸幹管口。加入Anti-Mouse CD3e (BD Bioscience,貨號740014)、Anti-Mouse CD4 (BD Bioscience,貨號553407)、anti-mouse CD25 (Biolengend,貨號102008),震盪一次,4℃孵育20 min,每10 min震盪一次。加4 mL/管FACS洗液,4℃,400 g,離心6 min。倒掉上清,用吸水紙吸幹管口,渦旋振盪器上震盪一次。加固定液,500 μL/管,一滴滴加入,(點震)每次加入後間歇震動FACS管,於4℃固定一小時或者固定過夜。
固定液:Set3901 (eBioscience,貨號00-5523-00)中Fixation/Permeabilization Concentrate 一份,Fixation/Permeabilization Diluent 三份,1:3混合配置成固定液。
Permeabilization buffer1份+9份dd H
2O配製成穿核液,震盪混勻。2 mL/管,4℃,500 g,離心6 min,倒掉上清,吸幹管口。重複,加穿核液3mL/管。加Foxp3抗體(eBioscience,貨號25-5773-82),10 μL/管,4℃,40 min,每20 min震盪一次。加4 mL/管FACS洗液,4℃,500 g,離心6 min。倒掉上清,用吸水紙吸幹管口,渦旋振盪器上震盪一次。加FACS洗液100μL/管,重懸上機。各組動物Treg(CD4
+CD25
+Foxp3
+)、CD4
+CD25
-Foxp3
-、CD3
+CD4
-T細胞占比均用平均值±標準差(Mean ± SEM)表示,並用Graphpad Prism 5軟體作圖分析。
實驗結果如圖9A-圖9C、圖10A-10C所示,1 mpk單次皮下給藥,mut11.08-linker2-hFc顯著提高小鼠脾臟及外周血Treg占比,CD4
+CD25
-Foxp3
-T細胞占比降低,CD3
+CD4
-T細胞變化不顯著。mut11.08-linker2-hFc表現優於Fc-linker-IL2_V91K。
實施例
13
野生型小鼠遲髮型超敏反應(
delayed-type hypersensitivity
,
DTH
)模型
致敏階段3 mg/mL KLH (Sigma,貨號H7017-50 mg):IFA (Sigma,貨號F5506-10 mL):CFA (Sigma,貨號F5581-10 mL)體積比為1:1:1,用連通管注射器法將抗原乳化,大約1 h左右,可使抗原充分乳化形成粘稠的乳劑。每只小鼠100μL乳化劑,即注射100μg KLH。每只小鼠肩胛骨中間部位分兩點皮下注射乳化的KLH,每點注射50 μL。同時將WT IL-2-linker2-hFc(簡稱WT IL-2,參見SEQ ID NO:12)、Fc-linker-IL2_V91K、mut11.08-linker2-hFc蛋白給與皮下注射,劑量為1 mpk,200 μL/只鼠,每3天一次,作為WT IL-2組、Fc-linker-IL2_V91K組、mut11.08-linker-hFc組;將環孢菌素(Cyclosporin A,CsA, Sigma,貨號F5581-10 mL)給與腹腔注射,劑量為10 mpk,200 μL/只鼠,每天一次,作為CsA組;腹腔注射PBS,作為vehicle組。
致敏後第五天進行刺激。10 mg/mL KLH用PBS 10倍稀釋為1μg/μL,每只小鼠右耳皮內注射10 μL,即注射10μg KLH,左耳皮內注射10μL PBS作為對照。
致敏前,螺旋測微器 (0-25 mm,精度0.001,購自南京蘇測計量儀器有限公司) 測量每只小鼠左右耳片厚度,做好記錄。刺激前,螺旋測微器測量每只小鼠左右耳片厚度,作為本底值。刺激後,在24 h、48 h、72 h、96 h測定耳片厚度一次。各組動物體重及耳厚變化均用平均值±標準差(Mean ± SEM)表示,並用Graphpad Prism 5軟體作圖分析。
實驗結果如圖11A所示,1 mpk單次皮下給藥,與vehicle組相比,WT IL-2 給藥組小鼠Δ耳厚沒有明顯變化,而mut11.08-linker2-hFc給藥組小鼠Δ耳厚變小,表現出抑炎作用,且抑炎作用優於Fc-linker-IL2_V91K及CsA。如圖11B所示,各組動物體重沒有明顯變化。
第二輪實驗結果如圖12A所示,Fc-linker-IL2_V91K 1 mpk單次皮下給藥,mut11.08-linker2-hFc使用0.04、0.2、1、5 mpk四個劑量進行單次皮下給藥。mut11.08-linker2-hFc的抑炎作用呈現劑量依賴;mut11.08-linker2-hFc 0.2 mpk與Fc-linker-IL2_V91K 1 mpk作用相當,該條件下體重均無異常;mut11.08-linker2-hFc 5 mpk時小鼠體重出現波動及下降,其他組體重無異常,詳見圖12B。
實施例
14 PBMC
小鼠皮下給藥的
PD
結果
採購NOG小鼠,雌性,11-12周齡,購自維通利華。在Day-1復蘇PBMC並添加CD3(購自eBioscience,貨號16-0037-85/2106800,終濃度為12.5 ng/mL)和CD28(購自eBioscience,貨號16-0289-85/2073954,終濃度為25 ng/mL)活化,置於37℃ 5% CO
2培養箱中過夜培養16 h。在Day0收集PBMC(20×10
6個/鼠,400μL)尾靜脈接種於NOG小鼠體內,然後根據體重隨機分為對照組PBS,mut11.08-linker2-hFc組(0.3 mpk,1 mpk), Fc-linker-IL2_V91K組(0.3 mpk,1 mpk)共5組,每組3只,並開始(Day0)頸部皮下注射藥物,單次給藥。在給藥後Day3,安樂死小鼠取脾,用於流式檢測,並記錄資料。各組動物Treg(CD4
+CD25
+Foxp3
+)、Tcon(CD4
+CD25
-)、CD8
+T細胞的數目及變化倍數用Graphpad Prism 8軟體作圖分析。
實驗結果如圖13A-13B、圖14A-14C所示,1 mpk單次皮下給藥,mut11.08-linker2-hFc在給藥後3天表現出提高Treg/Tcon、Treg/CD8
+T比例,並且呈劑量依賴,提高占比優於Fc-linker-IL2_V91K。給藥後3天除Treg數量顯著變化外,Tcon有增加趨勢但不顯著,CD8
+T細胞數量沒有顯著變化。
實施例
15 PBMC
小鼠植物抗宿主病
(graft-versus-host disease
,
GVHD)
模型
採購NOG小鼠,雌性,13-14周齡,購自維通利華。操作方式同實施例14,根據體重將NOG小鼠隨機分為G1組(PBS,未接種活化的PBMC,3只),G2 組(PBS,10只),G3組( 按0.2mpk給藥mut11.08-linker2-hFc,10只),共3組,其中G2組和G3組參照實施例14接種活化的PBMC。開始(Day0)頸部皮下注射藥物,單次給藥。每週2次稱重,待出現GVHD特徵後開始評分(評分體系:體重減少(0分 <10%,1分 10%-20%,2分 >20%,3分 >30%);貧血(0分 紅色或粉紅色的尾;1分 白色的尾);姿勢(0分 正常,1分 駝背);一般活動(0分 正常,1分 受限);脫毛(0分 無脫毛,1分 脫毛)和黃疸(0分 白色或紅色的尾,1分 黃色的尾);最大疾病嚴重程度或死亡對應於8分)並記錄資料。注:1. 如小鼠屬於最大疾病嚴重程度評分其他症狀將不評分;2.死亡小鼠死亡後一直評分到實驗結束。
結果如圖15A-15B所示,PBMC接種Day13後 G2組體重下降,GVHD症狀在該組提前發生;PBMC接種Day17後G3組體重下降,整體下降幅度不及G1,且發生時間較G2組晚,符合預期,mut11.08-linker2-hFc有效抑制小鼠發生GVHD。通過體重評估可見mut11.08-linker2-hFc在0.2 mpk給藥劑量無毒副作用,且表現出較好的抗GVHD能力。同時G3組動物死亡率和GVHD評分都低於G2組,有顯著差異。
實施例
16
小鼠藥代動力學
本實施例血藥濃度檢測方法如下所示:以1μg/mL hIL2R alpha (百普賽斯,貨號ILA-H52H9-100μg) 蛋白包被,在空白小鼠血清中以藥物配製標準曲線,濃度範圍為500-3.90625 ng/mL, 同時配製高中低三個濃度的質控品,所有待測樣品、標準品及質控品用稀釋液稀釋40倍後以100μL複孔入板(待測樣品根據實際情況可進行額外稀釋),再加入10000倍稀釋的檢測抗體Peroxidase AffiniPure Mouse Anti-Human IgG, Fc fragment specific (Jackson,貨號209-035-098) ,最後加入100μL TMB顯色液顯色,50μL 1 M硫酸終止讀數。
野生型小鼠皮下給藥,劑量為1 mpk,檢測小鼠血藥濃度。兩次結果如圖16A-16B和表21-22所示,mut11.08-linker2-hFc暴露量均高於對照Fc-linker-IL2_V91K (4-6倍),Tmax較Fc-linker-IL2_V91K延後。1 mpk單次皮下給藥,mut11.08-linker2-hFc暴露量高於mut11-linker2-hFc。
表21 野生型小鼠單次皮下給藥後的藥代動力學參數
Mean | T1/2(h) | Tmax(h) | Cmax(ng/mL) | AUClast(h*ng/mL) |
Fc-linker-IL2_V91K | NA | 6.00 | 1430.94 | 28208.77 |
mut11-linker2-hFc | NA | 12.00 | 1843.32 | 43254.14 |
mut11.08-linker2-hFc | NA | 24.00 | 4433.17 | 139652.97 |
表22 野生型小鼠單次皮下給藥後的藥代動力學參數
Mean | T1/2(h) | Tmax(h) | Cmax(ng/mL) | AUClast(h*ng/mL) |
Fc-linker-IL2_V91K | NA | 6.00 | 1628.30 | 42874.91 |
mut11.08-linker2-hFc | 11.51 | 24.00 | 5391.62 | 177902.19 |
野生型小鼠靜脈給藥,劑量為1 mpk,檢測血藥濃度。結果如圖17和表23所示,mut11.08-linker2-hFc暴露量高於對照Fc-linker-IL2_V91K,但與皮下注射相比,差異變小(Cmax 3倍→1.3倍,AUC 4倍→2.4倍)。mut11.08-linker2-hFc暴露量高於mut11-linker2-hFc,但與皮下注射相比,差異變小(Cmax 2.4倍→1.2倍,AUC 3.2倍→1.6倍)。
因此,皮下給藥mut11.08-linker2-hFc的生物利用度優於Fc-linker-IL2_V91K及mut11-linker2-hFc。穩定性突變提高藥物的暴露量以及生物利用度。
表23 野生型小鼠單次靜脈給藥後的藥代動力學參數
Mean | T1/2(h) | Tmax(h) | Cmax(ng/mL) | AUClast(h*ng/mL) |
Fc-linker-IL2_V91K | 7.35 | 0.08 | 14518.61 | 92522.67 |
mut11-linker2-hFc | 5.27 | 0.08 | 15450.84 | 138450.96 |
mut11.08-linker2-hFc | 8.41 | 0.08 | 18761.22 | 217851.01 |
PBMC接種小鼠,方法如實施例14,皮下給藥,劑量為1 mpk,檢測小鼠血藥濃度。結果如圖18和表24所示,PBMC小鼠中1 mpk單次皮下給藥,mut11.08-linker2-hFc的暴露量具有明顯優勢,為Fc-linker-IL2_V91K暴露量的5-8倍,Tmax較Fc-linker-IL2_V91K延後。
表24 PBMC小鼠單次皮下給藥後的藥代動力學參數
Mean | T1/2(h) | Tmax(h) | Cmax(ng/mL) | AUClast(h*ng/mL) |
Fc-linker-IL2_V91K | 12.29 | 12.00 | 2338.82 | 99848.69 |
mut11.08-linker2-hFc | 43.06 | 24.00 | 7405.26 | 546719.71 |
實施例
17
食蟹猴藥代動力學及
PD
結果
本實施例食蟹猴血藥濃度檢測方法如下所示:1μg/mL包被hIL2R alpha 蛋白 (百普賽斯,貨號ILA-H52H9-100μg) ,在空白食蟹猴血清中以藥物配製標準曲線,濃度範圍為15-0.11718 ng/mL, 同時配製高中低三個濃度的質控品,所有待測樣品、標準品及質控品用稀釋液稀釋5倍後以100μL複孔入板(待測樣品根據實際情況可進行額外稀釋),後加入1000倍稀釋的檢測抗體Goat Anti-Human IgG, Monkey ads-BIOT (SoutherBiotech,貨號2049-08),再加入5000倍稀釋的Streptavidin-HRP (Thermo,貨號21126) ,最後加入100μL TMB顯色液顯色,50μL 1 M硫酸終止讀數。食蟹猴空白血清:上海禾開生物製藥有限公司。
4只食蟹猴,按0.05mpk的劑量皮下給藥IL-2,其中1只給藥WT IL-2-linker2-hFc(簡稱WT IL-2,參見SEQ ID NO:12),1只給藥Fc-linker-IL2_V91K,2只給藥mut11.08-linker2-hFc(分別簡稱為Mut11.08-1和Mut11.08-2),並檢測給藥後的血藥濃度。結果如圖19和表25所示,0.05 mpk劑量下,終末半衰期WT IL-2分子較長,Fc-linker-IL2_V91K和mut11.08-linker2-hFc近似。Cmax和AUC 排序為mut11.08-linker2-hFc>Fc-linker-IL2_V91K>WT IL-2。
表25 食蟹猴單次皮下給藥後的藥代動力學參數
mean | T1/2(h) | Tmax(h) | Cmax(ng/mL) | AUClast(h*ng/mL) |
WT IL-2 | 204.97 | 4.00 | 57.70 | 1564.86 |
Fc-linker-IL2_V91K | 13.20 | 24.00 | 125.07 | 4669.24 |
mut11.08-1 | 11.05 | 24.00 | 185.40 | 8165.21 |
mut11.08-2 | 11.80 | 24.00 | 355.50 | 15033.23 |
為檢測mut11.08-linker2-hFc單次皮下給藥對食蟹猴Treg細胞的擴增作用,共取4只食蟹猴。1只給藥WT IL-2-linker2-hFc,1只給藥Fc-linker-IL2_V91K,另外2只給藥mut11.08-linker2-hFc。劑量為0.05 mpk,一次皮下給藥。在給藥前以及給藥後Day1、3、5、7、10、14取食蟹猴外周血。不同時間點取2 mL食蟹猴血液,從中分離出PBMC並凍存。各時間點分別取一支凍存的PBMC,一同進行復蘇檢測。將復蘇的PBMC用1 mL染色液Staining Buffer(含2% FBS的DPBS緩衝液)重懸後,各取200 μL轉移至兩塊96孔V底板中,標記為Panel 1和Panel2。兩塊板用DPBS洗一遍後,加入Live/Dead Fixable Near-IR染色(Thermo,貨號L34976)20分鐘。染色結束用Staining Buffer終止後,加入Human TruStain FcX(Biolegend,貨號422302)孵育20分鐘。隨後兩塊板分別加入不同的螢光抗體混合液進行染色30 min,Panel 1為BV605 Mouse Anti-Human CD3(BD,貨號562994)、PerCP-Cy5.5 Mouse Anti-Human CD4(BD,貨號552838)、FITC Mouse Anti-Human CD8(Biolegend,貨號301050)、BV421 Mouse Anti-Human CD25(Biolegend,貨號302630)稀釋至Brilliant stain buffer(BD,貨號563794),Panel 2為BV605 Mouse Anti-Human CD3、PerCP-Cy5.5 Mouse Anti-Human CD4、FITC Mouse Anti-Human CD8、Brilliant Violet 421 anti-human CD16(Biolegend,貨號302038)稀釋至Brilliant stain buffer。染色結束用Staining Buffer洗一遍後,用Foxp3 / Transcription Factor Staining Buffer Set試劑(eBioscience,貨號00-5523-00)盒進行固定破膜。然後兩塊板分別加入不同的螢光抗體混合液進行染色45 min,Panel 1為PE anti-human FOXP3(Biolegend,貨號320208)和Ki67 Monoclonal Antibody APC (eBioscience,貨號17-5698-82)稀釋至Permeabilization Buffer,Panel 2為Ki67 Monoclonal Antibody APC 稀釋至Permeabilization Buffer。染色結束用Permeabilization Buffer洗一遍後,用400 μL Staining Buffer將細胞重懸,用流式定量收取200μl樣品進行檢測。各組動物Treg、CD4+Foxp3-、CD8+T、NK細胞的數目及變化倍數用Graphpad Prism 9軟體作圖分析。
實驗結果如圖20A-20F所示,0.05 mpk單次皮下給藥,mut11.08-linker2-hFc顯著提高食蟹猴外周血Treg數目及占比。根據Treg/CD4
+T比例,mut11.08-linker2-hFc具有Treg偏向性,且Treg占比約為Fc-linker-IL2_V91K的2倍。mut11.08-linker2-hFc對比Fc-linker-IL2_V91K對Treg的增殖能力更高(11.08 vs Fc-linker-IL2_V91K= 79/55 vs 18),Fc-linker-IL2_V91K優於WT(18 vs 9.5)。從Treg活化標誌物分析,其中Ki67+ Treg%不同分子給藥後區分度不大。Treg活化標誌Foxp3及CD25的表達量顯著增加,與Treg增殖水準保持正相關。
實驗結果如圖21A-21F所示,0.05 mpk單次皮下給藥,Fc-linker-IL2_V91K給藥後FoxP3
-CD4
+T變化倍數最大值在2倍左右,CD8
+T變化倍數最大值略高在4倍左右。mut11.08-linker2-hFc對FoxP3
-CD4
+T的擴增強於Fc-linker-IL2_V91K約2-3倍;對CD8
+T 的擴增稍弱于或類似於Fc-linker-IL2_V91K,強於WT約1.5-2倍。Fc-linker-IL2_V91K給藥後NK細胞數目變化不明顯,mut11.08-linker2-hFc對NK的擴增水準類似於Fc-linker-IL2_V91K。
無。
圖1為流式細胞分析(FACS)方法檢測CHO-K1 hIL2-Rα 1A6重組細胞系人源IL-2受體α蛋白表達水準的結果圖,其中IL-2受體α抗體購自BioLegend;陰性對照指同型抗體對照;
圖2為流式細胞分析(FACS)方法檢測CHO-K1 hIL-2Rβ 2A5重組細胞系人源IL-2受體β蛋白表達水準的結果圖,其中IL-2受體β抗體購自BioLegend;陰性對照指同型抗體對照;
圖3A與圖3B為流式細胞分析(FACS)方法檢測CHO-K1 hIL-2Rβγ 2E6重組細胞系人源IL-2受體βγ蛋白表達水準的結果圖,IL-2受體β、γ抗體購自BioLegend;陰性對照指同型抗體對照(圖3A為IL2受體β蛋白表達水準結果圖;圖3B為IL2受體γ蛋白表達水準的結果圖);
圖4A至圖4C為流式細胞分析(FACS)方法檢測CHO-K1 hIL-2Rαβγ 2D6重組細胞系人源IL-2受體αβγ蛋白表達水準的結果圖,IL-2受體α、β、γ抗體購自BioLegend;對照為CHO-K1空細胞對應受體表達水準(圖4A為IL2受體α蛋白表達水準結果圖;圖4B為IL2受體β蛋白表達水準結果圖;圖4C為IL2受體γ蛋白表達水準的結果圖);
圖5A至圖5D為流式細胞分析(FACS)方法檢測IL-2突變蛋白與CHO-K1 IL-2受體αβγ、IL-2受體βγ重組細胞結合活性的結果圖
(圖5A為mut7.36-linker2-hFc、mut11.08-linker2-hFc、mut61-linker2-hFc、mut61.08-linker2-hFc或mut61.46-linker2-hFc與CHO-K1 IL2受體αβγ重組細胞結合活性的結果圖;
圖5B為mut11.31-linker2-hFc、mut7.66-linker2-hFc與CHO-K1 IL2受體αβγ重組細胞結合活性的結果圖;
圖5C為mut7.36-linker2-hFc、mut11.08-linker2-hFc、mut61-linker2-hFc、mut61.08-linker2-hFc或mut61.46-linker2-hFc與CHO-K1 IL2受體βγ重組細胞結合活性的結果圖;
圖5D為mut11.31-linker2-hFc、mut7.66-linker2-hFc與CHO-K1 IL2受體βγ重組細胞結合活性的結果圖);
圖6A至圖6I分別為檢測IL-2突變蛋白對Treg(圖6A-圖6C)、CD4
+CD25
-FoxP3
-T細胞(圖6D-圖6F)、CD8
+T細胞(圖6G-圖6I)STAT5磷酸化水準的影響;
(圖6A為mut7.36-linker2-hFc對Treg STAT5磷酸化水準的影響;
圖6B為mut11.08-linker2-hFc對Treg STAT5磷酸化水準的影響;
圖6C為mut11.31-linker2-hFc、mut7.66-linker2-hFc對Treg STAT5磷酸化水準的影響;
圖6D為mut7.36-linker2-hFc對CD4
+CD25
-FoxP3
-T細胞磷酸化水準的影響;
圖6E為mut11.08-linker2-hFc對CD4
+CD25
-FoxP3
-T細胞磷酸化水準的影響;
圖6F為mut11.31-linker2-hFc、mut7.66-linker2-hFc對CD4
+CD25
-FoxP3
-T細胞磷酸化水準的影響;
圖6G為mut7.36-linker2-hFc對CD8
+T細胞磷酸化水準的影響;
圖6H為mut11.08-linker2-hFc對CD8
+T細胞磷酸化水準的影響;
圖6I為mut11.31-linker2-hFc、mut7.66-linker2-hFc對CD8
+T細胞磷酸化水準的影響);
圖7A至圖7F分別為檢測IL-2突變蛋白對Treg(圖7A-圖7B)、CD4
+CD25
-FoxP3
-T細胞(圖7C-圖7D)、CD8
+CD25
-T細胞(圖7E-圖7F)增殖水準的影響;
(圖7A為mut7.36-linker2-hFc、mut7.66-linker2-hFc對Treg增殖水準的影響;
圖7B為mut11.08-linker2-hFc、mut11.31-linker2-hFc對Treg增殖水準的影響;
圖7C為mut7.36-linker2-hFc、mut7.66-linker2-hFc對CD4
+CD25
-FoxP3
-T細胞增殖水準的影響;
圖7D為mut11.08-linker2-hFc、mut11.31-linker2-hFc對CD4
+CD25
-FoxP3
-T細胞增殖水準的影響;
圖7E為mut7.36-linker2-hFc、mut7.66-linker2-hFc對CD8
+CD25
-T細胞增殖水準的影響;
圖7F為mut11.08-linker2-hFc、mut11.31-linker2-hFc對CD8
+CD25
-T細胞增殖水準的影響);
圖8為檢測IL-2突變蛋白對NK細胞增殖水準的影響;
圖9A為脾臟中Treg細胞在CD4
+T細胞中的占比;
圖9B為脾臟中CD4
+CD25
-Foxp3
-細胞在CD4
+細胞中的占比;
圖9C為脾臟中CD3
+CD4
-細胞在CD3
+細胞中的占比;
圖10A為外周血中Treg細胞在CD4
+細胞中的占比;
圖10B為外周血中CD4
+CD25
-Foxp3
-細胞在CD4
+細胞中的占比;
圖10C為外周血中CD3
+CD4
-細胞在CD3
+細胞中的占比;
圖11A為野生型小鼠DTH模型不同組別的Δ耳厚變化,Δ耳厚是指刺激前後,右耳片厚度變化;
圖11B為野生型小鼠DTH模型不同組別的體重變化;
圖12A為野生型小鼠DTH模型不同組別的Δ耳厚變化,Δ耳厚是指刺激前後,右耳片厚度變化;
圖12B為野生型小鼠DTH模型不同組別的體重變化;
圖13A為NOG小鼠不同組別的Treg/Tcon;
圖13B為NOG小鼠不同組別的Treg/CD8+;
圖14A為NOG小鼠不同組別的Treg細胞數目;
圖14B為NOG小鼠不同組別的Tcon細胞數目;
圖14C為NOG小鼠不同組別的CD8+細胞數目;
圖15A為NOG小鼠不同組別的體重變化;
圖15B為NOG小鼠不同組別的GVHD評分;
圖16A為野生型小鼠皮下給藥後血藥濃度;
圖16B為野生型小鼠皮下給藥後血藥濃度;
圖17為野生型小鼠靜脈給藥後血藥濃度;
圖18為PBMC接種小鼠皮下給藥後血藥濃度;
圖19為食蟹猴皮下給藥後血藥濃度;
圖20A為食蟹猴皮下給藥後Treg數目;
圖20B為食蟹猴皮下給藥後Treg數目變化倍數;
圖20C為食蟹猴皮下給藥後Treg占CD4
+T細胞比例;
圖20D為食蟹猴皮下給藥後Treg Ki67
+細胞比例;
圖20E為食蟹猴皮下給藥後FoxP3平均螢光強度變化倍數;
圖20F為食蟹猴皮下給藥後CD25平均螢光強度變化倍數;
圖21A為食蟹猴皮下給藥後FoxP3
-CD4
+細胞數目;
圖21B為食蟹猴皮下給藥後FoxP3
-CD4
+細胞數目變化倍數;
圖21C為食蟹猴皮下給藥後CD8
+T細胞數目;
圖21D為食蟹猴皮下給藥後CD8
+T細胞數目變化倍數;
圖21E為食蟹猴皮下給藥後NK細胞數目;
圖21F為食蟹猴皮下給藥後NK細胞數目變化倍數。
序列表 <![CDATA[<110> 江蘇先聲藥業有限公司]]> <![CDATA[<120> IL-2突變體及其應用]]> <![CDATA[<130> 114871-TW-PA ]]> <![CDATA[<140> TW 110132852]]> <![CDATA[<141> 2021-09-03]]> <![CDATA[<150> CN 202010918842.0]]> <![CDATA[<151> 2020-09-04 ]]> <![CDATA[<150> CN 202110932286.7]]> <![CDATA[<151> 2021-08-13 ]]> <![CDATA[<160> 60]]> <![CDATA[<170> SIPOSequenceListing 1.0]]> <![CDATA[<210> 1]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人類(Homo sapiens)]]> <![CDATA[<400> 1]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 2]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 2]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 3]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 3]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Leu Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 4]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 4]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 5]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 5]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Ala Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Trp Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 6]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 6]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Leu Asn Val Ile Ile Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 7]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 7]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Trp Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 8]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 8]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Leu Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 9]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 9]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Tyr Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 10]]> <![CDATA[<211> 15]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 10]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <![CDATA[<210> 11]]> <![CDATA[<211> 232]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 11]]> Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[<210> 12]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 12]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 13]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 13]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 14]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 14]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Leu Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 15]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 15]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 16]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 16]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Ala Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Trp Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 17]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 17]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Leu Asn Val Ile Ile Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 18]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 18]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Trp Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 19]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 19]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Leu Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 20]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 20]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Tyr Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 21]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 21]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 22]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 22]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 23]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 23]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 24]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 24]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Ala Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Trp Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 25]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 25]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Leu Asn Val Ile Ile Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 26]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 26]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Trp Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 27]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 27]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Leu Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 28]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 28]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ala Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 29]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 29]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ala Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 30]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 30]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ala Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 31]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 31]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 32]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 32]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 33]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 33]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Leu Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 34]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 34]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 35]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 35]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Leu Asn Arg Ile Ile Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 36]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 36]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 37]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 37]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 38]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 38]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Leu Asn Arg Ile Ile Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <![CDATA[<210> 39]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 39]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 40]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 40]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 41]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 41]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 42]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 42]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Ala Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Trp Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 43]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 43]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Leu Asn Val Ile Ile Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 44]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 44]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Trp Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 45]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 45]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Leu Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 46]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 46]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ala Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 47]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 47]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ala Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 48]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 48]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Ala Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 49]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 49]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 50]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 50]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 51]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 51]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Leu Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 52]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 52]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Phe Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 53]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 53]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Leu Asn Arg Ile Ile Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 54]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 54]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 55]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 55]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Val Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Leu Gln Ser Lys Asn Phe Gln Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Arg Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 56]]> <![CDATA[<211> 380]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 56]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu Glu 1 5 10 15 Leu Ile Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Leu Asn Arg Ile Ile Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <![CDATA[<210> 57]]> <![CDATA[<211> 272]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人類(Homo sapiens)]]> <![CDATA[<400> 57]]> Met Asp Ser Tyr Leu Leu Met Trp Gly Leu Leu Thr Phe Ile Met Val 1 5 10 15 Pro Gly Cys Gln Ala Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro 20 25 30 His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn 35 40 45 Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr 50 55 60 Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys 65 70 75 80 Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro 85 90 95 Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro 100 105 110 Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro 115 120 125 Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val 130 135 140 Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His 145 150 155 160 Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg 165 170 175 Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln 180 185 190 Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu 195 200 205 Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr 210 215 220 Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln 225 230 235 240 Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu 245 250 255 Ser Gly Leu Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile 260 265 270 <![CDATA[<210> 58]]> <![CDATA[<211> 551]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人類(Homo sapiens)]]> <![CDATA[<400> 58]]> Met Ala Ala Pro Ala Leu Ser Trp Arg Leu Pro Leu Leu Ile Leu Leu 1 5 10 15 Leu Pro Leu Ala Thr Ser Trp Ala Ser Ala Ala Val Asn Gly Thr Ser 20 25 30 Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp 35 40 45 Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp 50 55 60 Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser 65 70 75 80 Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln 85 90 95 Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu 100 105 110 Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu 115 120 125 Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu 130 135 140 Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr 145 150 155 160 Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His 165 170 175 Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp 180 185 190 Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val 195 200 205 Arg Val Lys Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser 210 215 220 Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr 225 230 235 240 Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly 245 250 255 Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro 260 265 270 Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe 275 280 285 Phe Ser Gln Leu Ser Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu 290 295 300 Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro 305 310 315 320 Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu 325 330 335 Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ala Ser Leu Ser Ser Asn 340 345 350 His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe Phe Phe His 355 360 365 Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr Phe Thr Tyr 370 375 380 Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val Ala Gly Ala Pro 385 390 395 400 Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro Leu Ser Gly Glu Asp Asp 405 410 415 Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp Leu Leu Leu Phe Ser Pro 420 425 430 Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser 435 440 445 Gly Ala Gly Glu Glu Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro 450 455 460 Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro 465 470 475 480 Asp Leu Val Asp Phe Gln Pro Pro Pro Glu Leu Val Leu Arg Glu Ala 485 490 495 Gly Glu Glu Val Pro Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro 500 505 510 Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg 515 520 525 Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly 530 535 540 Gln Asp Pro Thr His Leu Val 545 550 <![CDATA[<210> 59]]> <![CDATA[<211> 369]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人類(Homo sapiens)]]> <![CDATA[<400> 59]]> Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu 1 5 10 15 Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly 20 25 30 Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp 35 40 45 Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val 50 55 60 Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro 65 70 75 80 Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn 85 90 95 Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr 100 105 110 Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe 115 120 125 Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln 130 135 140 Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu 145 150 155 160 Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn 165 170 175 Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp 180 185 190 Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe 195 200 205 Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg 210 215 220 Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp 225 230 235 240 Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe 245 250 255 Leu Phe Ala Leu Glu Ala Val Val Ile Ser Val Gly Ser Met Gly Leu 260 265 270 Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro 275 280 285 Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His 290 295 300 Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser 305 310 315 320 Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro 325 330 335 Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn 340 345 350 Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu 355 360 365 Thr <![CDATA[<210> 60]]> <![CDATA[<211> 133]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工序列(Artificial Sequence)]]> <![CDATA[<400> 60]]> Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Xaa Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130
Claims (30)
- 一種IL-2突變體,其中,與野生型IL-2相比,所述IL-2突變體包括發生在Q13、L18、G27、Y31、A73、H79、P82、I89、N90、V91、V93、F117或R120的一個或多個突變。
- 如請求項1所述的IL-2突變體,其中,所述IL-2突變體包括Q13L、L18I、G27W、Y31V、A73L、H79Q、P82L、I89L、N90Y、V91A、V93I、F117W或R120F的一個或多個突變。
- 如請求項1所述的IL-2突變體,其中,所述IL-2突變體包括(a)-(h)組中至少一組突變: (a)、Y31/A73/H79突變;優選地,Y31V/A73L/H79Q突變; (b)、Q13突變;優選地,Q13L突變; (c)、R120突變,優選地,R120F突變; (d)、L18/V91/F117突變;優選地,L18I/V91A/F117W突變; (e)、L18/I89/V93突變;優選地,L18I/I89L/V93I突變; (f)、G27/R120突變;優選地,G27W/R120F突變; (g)、P82/R120突變;優選地,P82L/R120F突變; (h)、N90/R120突變;優選地,N90Y/R120F突變。
- 如請求項3所述的IL-2突變體,其中,所述IL-2突變體具有如SEQ ID NO:2-9任一項所示氨基酸序列。
- 如請求項1-4任一項所述的IL-2突變體,其中,所述IL-2突變體的Tm值高於所述野生型IL-2。
- 如請求項1-3、5任一項所述的IL-2突變體,其中,所述IL-2突變體還包括選自下組的一個或多個突變:H16、D20、N88、V91或Q126突變,例如H16E突變,D20A、D20H或D20Y突變,N88A、N88I、N88G、N88R或N88D突變,V91R或V91K突變,Q126L或Q126F突變; 優選地,所述IL-2突變體還包括選自(ⅰ)-(ⅳ)中至少一組的突變: (ⅰ)、H16突變,優選H16E突變; (ⅱ)、D20突變,優選D20A突變; (ⅲ)、V 91突變,優選V91R突變; (ⅳ)、H16/V91突變,優選H16E/V91R突變。
- 如請求項6所述的IL-2突變體,其中,所述IL-2突變體包括(a)-(n)組中至少一組突變: (a)、H16/Y31/A73/H79突變;優選為H16E/Y31V/A73L/H79Q突變; (b)、H16/R120突變;優選H16E/R120F突變; (c)、H16/L18/V91/F117突變;優選H16E/L18I/V91A/F117W突變; (d)、H16/L18/I89/V93突變;優選H16E/L18I/I89L/V93I突變; (e)、H16/G27/R120突變;優選H16E/G27W/R120F突變; (f)、H16/P82/R120突變;優選H16E/P82L/R120F突變; (g)、D20/Y31/A73/H79突變;優選D20A/Y31V/A73L/H79Q突變; (h)、D20/R120突變;優選D20A/R120F突變; (i)、V91/Y31/A73/H79突變;優選V91R/Y31V/A73L/H79Q突變; (j)、V91/Q13突變;優選V91R/Q13L突變; (k)、V91/R120突變;優選V91R/R120F突變; (l)、V91/L18/I89/V93突變;優選V91R/L18I/I89L/V93I突變; (m)、H16/V91/Y31/A73/H79突變;優選H16E/V91R/Y31V/ A73L/H79Q突變; (n)、H16/V91/L18/I89/V93突變;優選H16E/V91R/L18I/ I89L/V93I突變。
- 如請求項7所述的IL-2突變體,其中,所述IL-2突變體具有如SEQ ID NO:22-27、29-30、32-35或37-38所示的氨基酸序列。
- 如請求項1-3、5任一項所述的IL-2突變體,其中,所述IL-2突變體還包括選自下組的一個或多個突變:F42、Y45或L72的突變;優選F42A、Y45A或L72G突變。
- 一種IL-2突變體,其中,與野生型IL-2相比,所述IL-2突變體包括發生在H16、D20或V91中的一個或多個突變;優選地,所述IL-2突變體包括選自下組的突變: (ⅰ)、H16突變,優選H16E突變; (ⅱ)、D20突變,優選D20A突變; (ⅲ)、V 91突變,優選V91R突變; (ⅳ)、H16/V91突變,優選H16E/V91R突變。
- 如請求項10所述的IL-2突變體,其中,所述IL-2突變體具有如SEQ ID NO:21、28、31或36所示的氨基酸序列。
- 如請求項1-3、5-7、9-10任一項所述的IL-2突變體,其中,所述IL-2突變體還包括選自下組的一個或多個突變:N26、N29、N30、N71、Q11、L132、L70、P82、G27或F28突變; 優選地,所述IL-2突變體還包括選自下組的一個或多個突變:N26Q、N29S、N30S、N71Q、Q11C、L132C、L70C、P82C、G27C或F78C突變; 更優選地,所述IL-2突變體還包括(a)-(g)組中的至少一組突變: (a)、N26Q突變; (b)、N29S突變; (c)、N30S突變; (d)、N71Q突變; (e)、Q11C/L132C突變; (f)、L70C/P82C突變; (g)、G27C/F78C突變。
- 如請求項6-8、10-12任一項所述的IL-2突變體,其中,與所述野生型IL-2相比,所述IL-2突變體與IL-2Rβγ亞基複合物的結合能力下降;優選地,結合力 IL-2Rβγ 亞基複合物/結合力 IL-2αβγ 亞基複合物下降。
- 如請求項13所述的IL-2突變體,其中,與野生型IL-2相比,所述IL-2突變體對非調節性T細胞或NK(自然殺傷)細胞的刺激能力下降,所述刺激可選自:胞內STAT5磷酸化或細胞增殖; 或者,與非調節性T細胞或NK(自然殺傷)細胞相比,所述IL-2突變體優先刺激外周血或T細胞群體中的調節性T細胞(Treg);所述優先刺激可選自:刺激調節性T細胞內STAT5磷酸化、刺激調節性T細胞增殖、提高調節性T細胞與非調節性T細胞的比率,或提高調節性T細胞與NK細胞的比率。
- 如請求項1-14任一項所述的IL-2突變體,其中,所述突變包括缺失、插入或替換,優選為替換。
- 如請求項1-15任一項所述的IL-2突變體,其中,所述野生型IL-2具有如SEQ ID NO:60或SEQ ID NO:1所示的氨基酸序列。
- 一種融合蛋白,其中,所述融合蛋白包括第一多肽和第二多肽,其中,所述第一多肽為如請求項1-16所述的任一項所述的IL-2突變體,所述第二多肽為非IL-2多肽。
- 如請求項17所述的融合蛋白,其中,所述第二多肽為Fc、腫瘤抗原結合分子或IL-2受體亞基; 可選地,所述Fc為人IgG Fc,例如人IgG1 Fc; 優選地,所述人IgG1 Fc包括選自(a)-(i)組中的至少一組突變: (a)、C220S; (b)、N297G; (c)、C220S和N297G; (d)、A327Q; (e)、L234A和L235A; (f)、A287C和L306C; (g)、A259C和V302C; (h)、R292C和V306C; (i)、V323C和I332C; 更優選地,所述人Ig G1 Fc具有如SEQ ID NO:11所示的氨基酸序列; 可選地,所述腫瘤抗原包括:EDB-FN(extra domain of fibronectin)、Muc1、p53、FAP、GD2、EpCAM、tenascin-C、CD20、CEA、MAdCAM-1或WT1(Wilms tumor protein1);可選地,所述腫瘤抗原結合分子為抗體,例如scFv,sdFv、Fab、Fab’、F(ab’)2或Fv; 可選地,所述IL-2受體亞基為IL-2受體α亞基。
- 如請求項17-18任一項所述的融合蛋白,其中,所述第一多肽的C端通過或不通過連接肽與所述第二多肽的N端連接;或所述第一多肽的N端通過或不通過連接肽與所述第二多肽的C端連接; 所述連接肽優選選自:(G 4S) n、(GGNGT) n或(YGNGT) n,所述n選自1、2、3、4或5; 更優選地,所述第一多肽的C端與所述第二多肽的N端通過連接肽(G4S) 3連接。
- 如請求項17所述的融合蛋白,其中,所述融合蛋白包括SEQ ID NO:13-20或SEQ ID NO:39-56任一項所述的氨基酸序列。
- 一種綴合物,其中,所述綴合物包括如請求項1-16任一項所述的IL-2突變體或如請求項17-20任一項所述的融合蛋白,還包括綴合至所述突變體或融合蛋白的穩定劑、藥物或者示蹤分子,其中所述穩定劑可選自聚乙二醇,例如單甲氧基聚乙二醇。
- 一種分離的核酸片段,其中,所述核酸片段編碼如請求項1-16任一項所述的IL-2突變體或如請求項17-20任一項所述的融合蛋白。
- 一種載體,其中,所述載體包括如請求項22所述的核酸片段。
- 一種宿主細胞,其中,所述宿主細胞包含如請求項23所述的載體;可選地,所述宿主細胞是原核細胞或真核細胞;所述原核細胞或所述真核細胞可選自大腸桿菌、酵母、昆蟲細胞或哺乳動物細胞,所述哺乳動物細胞可選自CHO細胞系或HEK293細胞系。
- 一種製備如請求項1-16任一項所述的突變體或如請求項17-20任一項所述的融合蛋白的方法,其中,所述方法包括培養如請求項24所述的宿主細胞,以及分離所述宿主細胞表達的所述IL-2突變體或所述融合蛋白。
- 如請求項25所述的方法製備而成的IL-2突變體或融合蛋白。
- 一種藥物組合物,其中,所述藥物組合物包括如請求項1-16任一項所述的所述突變體、如請求項17-20任一項所述的融合蛋白、如請求項21所述的綴合物、如請求項22所述的核酸片段、如請求項23所述的載體、如請求項24所述的宿主細胞或如請求項26所述的突變體或融合蛋白;以及藥學上可接受的運載體、稀釋劑或助劑; 優選地,所述藥物組合物為可注射藥物組合物,例如靜脈注射藥物組合物或皮下注射藥物組合物;更優選地,每劑所述藥物組合物包含能夠給與受試者有效量的融合蛋白;最優選地,所述有效量為0.001-10mpk,例如0.001mpk、0.002mpk、0.003mpk、0.004mpk、0.005mpk、0.006mpk、0.007mpk、0.008mpk、0.009mpk、0.01mpk、0.02mpk、0.03mpk、0.04mpk、0.05mpk、0.06mpk、0.07mpk、0.08mpk、0.09mpk、0.1 mpk、0.2mpk、0.3mpk、0.4mpk、0.5mpk、0.6mpk、0.7mpk、0.8mpk、0.9mpk、1mpk、2mpk、3mpk、4mpk、5mpk、6mpk、7mpk、8mpk、9mpk或10mpk。
- 如請求項1-16任一項所述的突變體、如請求項17-20任一項所述的融合蛋白、如請求項21所述的綴合物、如請求項22所述的核酸片段、如請求項23所述的載體、如請求項24所述的宿主細胞或如請求項26所述的突變體或融合蛋白在製備藥物中的用途; 優選地,所述藥物為注射藥物,例如靜脈注射藥物或皮下注射藥物; 優選地,每劑所述藥物包含能夠給與受試者有效量的融合蛋白;最優選地,所述有效量為0.001-10mpk,例如0.001mpk、0.002mpk、0.003mpk、0.004mpk、0.005mpk、0.006mpk、0.007mpk、0.008mpk、0.009mpk、0.01mpk、0.02mpk、0.03mpk、0.04mpk、0.05mpk、0.06mpk、0.07mpk、0.08mpk、0.09mpk、0.1 mpk、0.2mpk、0.3mpk、0.4mpk、0.5mpk、0.6mpk、0.7mpk、0.8mpk、0.9mpk、1mpk、2mpk、3mpk、4mpk、5mpk、6mpk、7mpk、8mpk、9mpk或10mpk; 優選地,所述藥物用於治療自身免疫性疾病,或增生性疾病,或病毒感染; 更優選地,所述自身免疫性疾病包括類風濕關節炎、強直性脊柱炎、系統性紅斑狼瘡、皮膚紅斑狼瘡、狼瘡性腎炎、IgA腎病、乾燥綜合征、多肌炎、皮肌炎、硬皮病、銀屑病、斑塊狀銀屑病、斑禿、多發性硬化症、肌萎縮側索硬化症、炎性腸病、潰瘍性結腸炎、克羅恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、濕疹或哮喘; 更優選地,所述增生性疾病包括贅生物、實體瘤、血液瘤、惡性腹水或惡性胸水;其中,所述實體瘤可為良性或惡性,原發性或轉移性,所述惡性實體瘤可為癌或肉瘤,例如,上皮細胞癌、內皮細胞癌、鱗狀細胞癌、畸胎瘤、肺部腫瘤、乳頭瘤病毒引起的癌、腺癌、癌腫、黑色素瘤、血管肉瘤、神經母細胞瘤、轉移性肺癌、非小細胞肺癌、小細胞肺癌、乳腺癌、Merkel細胞癌、卵巢癌、腎細胞癌、轉移性腎癌、頭頸癌、膀胱癌、非肌層浸潤性膀胱癌;所述血液瘤可選自白血病、淋巴瘤、多發性骨髓瘤,例如B細胞淋巴瘤、T細胞淋巴瘤、皮膚T細胞淋巴瘤、T細胞大顆粒淋巴細胞白血病; 更優選地,所述病毒感染選自HIV感染、新型冠狀病毒感染或HPV病毒感染。
- 一種治療自身免疫性疾病,或增生性疾病,或病毒感染的方法,其中,所述方法包括向受試者施用有效量的如請求項1-16任一項所述的IL-2突變體或如請求項17-20任一項所述的融合蛋白,或如請求項21所述的綴合物,或如請求項22所述的核酸片段,或如請求項23所述的載體,或如請求項24所述的宿主細胞,或如請求項26所述的突變體或融合蛋白,或如請求項27所述的藥物組合物; 優選地,施用方式為注射,例如靜脈注射或者皮下注射; 優選地,所述有效量為0.001-10mpk,例如0.001mpk、0.002mpk、0.003mpk、0.004mpk、0.005mpk、0.006mpk、0.007mpk、0.008mpk、0.009mpk、0.01mpk、0.02mpk、0.03mpk、0.04mpk、0.05mpk、0.06mpk、0.07mpk、0.08mpk、0.09mpk、0.1mpk、0.2mpk、0.3mpk、0.4mpk、0.5mpk、0.6mpk、0.7mpk、0.8mpk、0.9mpk、1mpk、2mpk、3mpk、4mpk、5mpk、6mpk、7mpk、8mpk、9mpk或10mpk; 優選地,所述自身免疫性疾病自身免疫性疾病包括類風濕關節炎、強直性脊柱炎、系統性紅斑狼瘡、皮膚紅斑狼瘡、狼瘡性腎炎、IgA腎病、乾燥綜合征、多肌炎、皮肌炎、硬皮病、銀屑病、斑塊狀銀屑病、斑禿、多發性硬化症、肌萎縮側索硬化症、炎性腸病、潰瘍性結腸炎、克羅恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、濕疹或哮喘; 優選地,所述增生性疾病包括贅生物、實體瘤、血液瘤、惡性腹水或惡性胸水;其中,所述實體瘤可為良性或惡性,原發性或轉移性,惡性實體瘤可為癌或肉瘤,例如,上皮細胞癌、內皮細胞癌、鱗狀細胞癌、畸胎瘤、肺部腫瘤、乳頭瘤病毒引起的癌、腺癌、癌腫、黑色素瘤、血管肉瘤、神經母細胞瘤、轉移性肺癌、非小細胞肺癌、小細胞肺癌、乳腺癌、Merkel細胞癌、卵巢癌、腎細胞癌、轉移性腎癌、頭頸癌、膀胱癌、非肌層浸潤性膀胱癌;所述血液瘤可選自白血病、淋巴瘤、多發性骨髓瘤,例如B細胞淋巴瘤、T細胞淋巴瘤、皮膚T細胞淋巴瘤、T細胞大顆粒淋巴細胞白血病。
- 一種優先刺激T細胞群體或外周血中調節性T細胞的方法,其中,所述方法包括將所述T細胞群體或外周血與如請求項1-16任一項所述的IL-2突變體或如請求項17-20任一項所述的融合蛋白,或如請求項21所述的綴合物,或如請求項22所述的核酸片段,或如請求項23所述的載體,或如請求項24所述的宿主細胞,或如請求項26所述的突變體或融合蛋白,或如請求項27所述的藥物組合物接觸;優選地,所述優先刺激包括: (a)與非調節性T細胞或NK細胞相比,優先刺激調節性T細胞的STAT5磷酸化; (b)與非調節性T細胞或NK細胞相比,優先刺激調節性T細胞增殖; 和/或(c)提高調節性T細胞與非調節性T細胞的比率,或提高調節性T細胞與NK細胞的比率。
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US20230265148A1 (en) | 2023-08-24 |
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