IgE Fc受体的α亚基的胞外域、包含其的药物组合物及其制备方法
阅读说明:本技术 IgE Fc受体的α亚基的胞外域、包含其的药物组合物及其制备方法 (Extracellular domain of alpha subunit of IgE Fc receptor, pharmaceutical composition comprising the same, and preparation method thereof ) 是由 成永哲 梁祯允 于 2019-01-08 设计创作,主要内容包括:本发明提供了一种包含两个单体的多肽二聚体蛋白,每个单体包含IgE Fc受体的α亚基的胞外域(FcεRIa-ECD)。与包含抗IgE抗体的常规治疗剂相比,本发明的二聚体蛋白显示出具有对IgE优异结合能力的优点,并且由于其缺乏ADCC和CDC功能而表现出较少的其他副作用。因此,该二聚体蛋白质可以应用于治疗或预防IgE介导的过敏性疾病的医疗产品。(The present invention provides a polypeptide dimeric protein comprising two monomers, each monomer comprising the extracellular domain of the alpha subunit of the IgE Fc receptor (FcRIa-ECD). The dimeric protein of the present invention shows the advantage of having excellent binding ability to IgE and shows less other side effects due to its lack of ADCC and CDC functions, compared to conventional therapeutic agents comprising anti-IgE antibodies. Therefore, the dimeric protein may be applied to medical products for treating or preventing IgE-mediated allergic diseases.)
具体实施方式
在下文中,将参考以下实施例更详细地描述本发明。然而,以下实施例仅旨在解释本发明,并且本发明的范围不仅限于此。
实施例1.含有FcεRIα-ECD和经修饰的Fc区的多肽的制备
根据美国专利号7,867,491中公开的方法,制备IgE Fc受体的α亚基的胞外域(FcεRIα-ECD)的C末端经修饰的多肽。
首先,为了表达如SEQ ID NO:1所示氨基酸序列的FcεRI的α链的胞外域和如SEQID NO:2所示的经修饰的免疫球蛋白Fc分别通过如SEQ ID NO:19所示的铰链、如SEQ IDNO:3所示的铰链、如SEQ ID NO:4所示的铰链连接所得到的蛋白质(FcεRIαECD-Fc1)、蛋白质(FcεRIαECD-Fc2)和蛋白质(FcεR1αECD-Fc3),将通过连接编码每种蛋白的基因获得的表达盒克隆到pAD15载体(Genexin,Inc.)中,以构建FcεRIαECD-Fc蛋白表达载体。然后,将每种表达载体转导至CHO DG44细胞(来自美国哥伦比亚大学的Chasin博士)。
其中,在转导至细胞系中时,同时转导通过将α-2,6--唾液酸转移酶的基因克隆至pCI Hygro载体(Invitrogen)中而获得的表达载体,以分别制备能够表达添加了唾液酸的FcεRIαECD-Fc2ST和FcεRIαECD-Fc3ST蛋白。
作为初步筛选程序,使用无5-羟色胺(HT)的10%dFBS培养基(Gibco,USA,30067-334)、MEMα培养基(Gibco,12561,USA,目录号12561-049)和HT+培养基(USA,Gibco,11067-030)进行HT筛选。然后,使用HT筛选出的克隆进行甲氨蝶呤(MTX)扩增,以使用二氢叶酸还原酶(DHFR)系统扩增生产力。
在MTX扩增完成后,为了评估生产力,进行约1-5次传代培养以使细胞稳定。之后,进行MTX扩增细胞的单位生产力评价。结果显示在下表1中。
表1
如表1所示,在2μM的甲氨蝶呤扩增后,FcεRIαECD-Fc3细胞系显示出16.9μg/106个细胞的生产力。另一方面,在1μM的甲氨蝶呤扩增后,与2,6-唾液酸转移酶共转导的FcεRIαECD-Fc3细胞系(FcεRIαECD-Fc3ST)的生产力为17.5μg/106个细胞。此外,在0.5μM的甲氨蝶呤扩增后,FcεRIαECD-Fc2细胞系显示出20.9μg/106个细胞的生产力。另外,在0.1μM的甲氨蝶呤扩增后,与2,6-唾液酸转移酶共转导的FcεRIαECD-Fc2细胞系(FcεRIαECD-Fc2ST)显示出25.1μg/106个细胞的生产力。即可以确认的是,在0.1μM的氨甲蝶呤扩增条件后,与2,6-唾液酸转移酶共转染的FcεRIαECD-Fc2细胞系显示出最优异的生产力。
实施例2.FcεRIαECD融合蛋白的纯化及其纯度的鉴定
在以上实施例1中筛选的细胞系中,i)FcεRIαECD-Fc3,ii)FcεRIαECD-Fc3ST,和iii)FcεRIαECD-Fc2ST,以60ml规模通过分批培养方法进行培养。使用Protein-A亲和柱纯化所得的培养物,然后对纯化的蛋白进行SDS-PAGE和尺寸排阻HPLC(SE-HPLC)以鉴定蛋白的纯度。
如图1所示,通过SE-HPLC法纯化的所有蛋白质的纯度均为93%以上。另外,SDS-PAGE分析的结果鉴定出,在非还原条件和还原条件下分别检测到具有约150kDa和约75kDa大小的蛋白质(图1,泳道1至6)。由此发现,结合Fc的FcεRIαECD形成二聚体。另外,在SDS-PAGE结果中没有观察到诸如截短形式的杂质。特别地,即使在解冻/冷冻过程之后(图1,泳道7至8),也已经确定所有蛋白质具有93%或更高的纯度,并且没有杂质。由此发现,相对于应用了野生型IgD铰链的FcεRIαECD-Fc1,截短的蛋白质形式的产生减少了。
其中,在以下测试条件下进行Gel-IEF以鉴定在引入唾液酸转移酶之后蛋白质中唾液酸含量的程度。由此可知,由于唾液酸含量的增加,酸性蛋白质的含量有所增加。
表2
为了鉴定纯化产量的再现性,将FcεRIαECD-Fc2ST细胞系在1L烧瓶中以250mL的规模分批培养,并使用Protein-A亲和柱纯化。随后,在Tris-甘氨酸SDS(TGS)缓冲液和200V的条件下,将培养上清液和纯化的产物在4%至15%的TGXTM凝胶(Bio-Rad Laboratories,Inc.)上运行30分钟,然后进行SDS PAGE分析。结果显示,不仅通过仅第一步纯化就纯化出了纯度很高(98%以上)的蛋白质,而且即使在培养上清液中也表达了很高纯度的蛋白质。这表明,可以将在相关细胞系中表达的FcεRIαECD-Fc蛋白开发为医疗产品,从而简化过程开发步骤,因而医疗产品的开发成本极有可能显著下降。
实施例3.鉴定FcεRIαECD融合蛋白与IgE的结合能力
比较地检测了以下四种蛋白质对IgE的结合能力:通过上述实施例2的方法纯化的i)FcεRIαECD-Fc2,ii)FcεRIαECD-Fc2ST,iii)FcεRIαECD-Fc3和iv)FcεRIαECD-Fc3ST,以及市售的抗IgE抗体奥马珠单抗(omalizumab,商品名:Xolair)。具体而言,通过在蛋白GLC传感器芯片(Bio-Rad Laboratories,Inc.,Cat#176-5011)的通道上包被IgE,并使得不同浓度的奥马珠单抗或每种FceR1αECD-Fc蛋白以每分钟30μl的速度流动,来测量与IgE的结合能力。
通过使用25mM NaOH作为再生缓冲液鉴定出零基线,然后重复上述步骤进行实验。之后,使用蛋白质结合分析仪(ProteOn XPR36,Bio-Rad Laboratories,Inc.,USA)鉴定结合曲线。结果示于表3、以及图3和4。
表3
如表3所示,测量的本发明的一个实施方案的多肽二聚体的结合速率(ka)的值比奥马珠单抗低1.5-2.0倍。即,发现与奥马珠单抗相比,其与除IgE以外的物质的结合能力低1.5-2.0倍。另外,测量的本发明的一个实施方案的多肽二聚体的解离速率(kd)的值比奥马珠单抗高40至106倍。另外,如图3和图4所示,在结合一定的时间后,奥马珠单抗无法与IgE结合,而本发明的FcεRIαECD D融合蛋白的多肽二聚体结合至IgE,就不会与IgE分离。
即,可以看出本发明的多肽二聚体不易与IgE分离,并且与奥马珠单抗相比具有更好的维持其结合状态的能力。从结果可以看出,本发明的一个实施方案的多肽二聚体的平衡解离常数(KD<kd/ka>)的值比奥马珠单抗高22-69倍。由此可知,与奥马珠单抗相比,本发明的FcεRIαECD融合蛋白与IgE的结合能力显著提高。特别地,已确定添加了唾液酸的FcεRIαECD-Fc2(FcεRIαECD-Fc2ST)表现出最强的IgE结合能力,其比奥马珠单抗高69倍。
实施例4.鉴定FcεRIαECD融合蛋白与IgG受体的结合能力
使用Octet RED384系统(ForteBio,CA,USA)测定IgETRAP和奥马珠单抗与IgG受体的结合程度。将FcγRI、FcγRIIA、FcγRIIB、FcγRIIIA和FcγRIIIB重组蛋白(R&DSystems Inc.,5μg/ml)固定在活化的AR2G生物传感器上的300mM乙酸盐缓冲液(pH5)中。使用含有0.1%Tween-20和1%牛血清的PBS作为运行缓冲液。在30℃下用速率为1,000rpm的样品平板振荡器进行所有实验。结果如图5A至5E所示,奥马珠单抗和IgETRAP与IgG受体的结合能力的定量检测显示在图6中。
实施例5.通过β-己糖胺酶测定法鉴定在小鼠骨髓来源的肥大细胞中的FcεRIαECD融合蛋白的活性
进行β-己糖胺酶测定以用于本发明的FcεRIαECD融合蛋白的体外活性分析。具体地,将本发明的一个实施方案的FcεRIαECD-Fc2蛋白在不同浓度下与小鼠IgE(1μg/mL)混合,并在室温(20℃)下孵育30分钟以准备样品。用汉克平衡盐溶液(Hank's balanced saltsolution,HBSS)缓冲液洗涤培养物中的小鼠骨髓肥大细胞,以除去培养基,从而活化肥大细胞,同时测量细胞数。然后调整细胞数,将5×105细胞注入40μL HBSS缓冲液中。
然后,将事先进行孵育制得的50μL样品溶液加入到活化的肥大细胞中。然后,将所得产物在5%CO2培养箱中于37℃孵育30分钟。随后,在加入10μL DNP(2,4-二硝基苯酚,100ng/mL)(一种外源抗原)后,再次在37℃的5%CO2中孵育30分钟,然后分离出30μL的上清液。充分混合30μL分离的上清液和30μL底物(4-硝基苯基N-乙酰基-β-D-氨基葡萄糖苷,5.84mM),然后在5%CO2中于37℃孵育20分钟。接着,加入140μL作为终止溶液的0.1M碳酸钠缓冲液(pH 10)以终止反应。此后,测量在405nm处的吸光度,以鉴定在活化的肥大细胞中由外源抗原分泌的α-己糖胺酶的分泌量。结果示于图7。
如图7所示,本发明的一个实施方案的多肽二聚体在浓度为小鼠IgE的一半(0.5μg/mL)的情况下,显示出对肥大细胞的抑制率约为49.4%,并且其浓度与小鼠IgE相同(1μg/mL)的情况下,显示出对肥大细胞的抑制率约为99.4%。即可以看出,本发明的FceRIa-ECD多肽二聚体极大地抑制了IgE诱导的骨髓来源的肥大细胞的活性。
实施例6.在表达人FcεRI的骨髓来源的肥大细胞中使用β-己糖胺酶测定法比较FcεRIαECD融合蛋白和抗人IgE抗体的活性
进行β-己糖胺酶测定,以通过体外活性分析鉴定FcεRIαECD融合蛋白相对于Xolair的优越性。制备不同浓度的相应药物FcεRIαECD-Fc2ST(IgETRAP)和Xolair,然后与人IgE(1μg/mL)混合。接着在室温下进行30分钟的孵育。在药物的预孵育过程中,导入人FcεRI基因,并制备从已去除FcεRI gene基因的小鼠的骨髓衍生并分化出的肥大细胞。制备的肥大细胞用HBSS缓冲液洗涤,然后将5×105细胞注入60μL HBSS缓冲液中。将20μL预孵育的样品添加到制备的肥大细胞中,然后在5%CO2培养箱中于37℃孵育30分钟。
随后,在加入20μL抗人IgE抗体(BioLegend,目录号325502,0.5μg/mL)后,将所得产物再次在5%CO2培养箱中于37℃孵育30分钟。随后,在4℃以1,500rpm离心后,分离出30μL的上清液。将30μL分离的上清液和30μL底物(4-硝基苯基N-乙酰基-β-氨基葡萄糖,5.84mM)充分混合,然后在5%CO2培养箱中于37℃孵育25分钟。然后,加入140μL的0.1M碳酸钠缓冲液(pH 10)以终止反应。
接着,检测在405nm处的吸光度以比较分泌的β-己糖胺酶的相对量,并且鉴定出基于每种药物浓度的肥大细胞的抑制作用。结果示于图8。如图8所示,测得FcεRIαECD融合蛋白的IC50约为11.16ng/mL,Xolair蛋白的IC50约为649.8ng/mL。因此,已确定FcεRIαECD融合蛋白对肥大细胞活性的抑制能力比Xolair高58倍。
实施例7.FcεRIαECD融合蛋白的体内测定(食物过敏模型)
以14天的间隔向Balb/c小鼠(Orientbio Inc.)腹膜内施用两次50μg卵清蛋白(OVA)和1mg明矾以引起过敏。此后,在第28、30、32、34和36天总共口服五次共50mg OVA,以诱发肠道食物过敏。
在口服OVA两次后,即在第31天,将小鼠分为三组,每组包含7只小鼠。三组分别为:第一组接受高浓度(200μg)的FcεRIαECD-Fc2ST融合蛋白,第二组接受低浓度的FcεRIαECD-Fc2ST融合蛋白(20μg),第三组未接受任何药物。口服OVA时,测定是否由于食物过敏引起了腹泻。在第37天处死小鼠,分析每个组的小鼠中小肠肥大细胞数量、血液中IgE的浓度和血液中肥大细胞脱粒的酶(肥大细胞蛋白酶-1(MCPT-1))浓度。
如图9可知,经证明与未接受任何药物的小鼠组相比,接受多肽二聚体FcεRIαECD-Fc2ST的小鼠组在高浓度时显示出缓解食物过敏的效果,并且呈浓度依赖性。
序列表
SEQ ID NO:1
SEQ ID NO:2
SEQ ID NO:3
RNTGRGGEEK KGSKEKEEQE ERETKTPECP
SEQ ID NO:4
AQPQAEGSLA KATTAPATTR NTGRGGEEKK GSKEKEEQEE RETKTPECP
SEQ ID NO:5
SEQ ID NO:6
SEQ ID NO:7
SEQ ID NO:8
SEQ ID NO:9
MDAMLRGLCC VLLLCGAVFV SPSHA
SEQ ID NO:10
SEQ ID NO:11
SEQ ID NO:12
SEQ ID NO:13
SEQ ID NO:14
SEQ ID NO:15
SEQ ID NO:16
SEQ ID NO:17
RNTGRGGEEK KXXKEKEEQE ERETKTPECP
SEQ ID NO:18
AQPQAEGSLA KATTAPATTR NTGRGGEEKK XXKEKEEQEE RETKTPECP
SEQ ID NO:19
RNTGRGGEEK KKEKEKEEQE ERETKTPECP
SEQ ID NO:20
SEQ ID NO:21
SEQ ID NO:22
SEQUENCE LISTING
<110> GI 医诺微新
<120> IgE FC受体的α亚基的胞外域、包含其的药物组合物及其制备方法
<130> P20113066WP
<150> KR 10-2018-0002248
<151> 2018-01-08
<160> 22
<170> PatentIn version 3.5
<210> 1
<211> 180
<212> PRT
<213> Artificial Sequence
<220>
<223> FCeRI1 ECD
<400> 1
Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile
1 5 10 15
Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe
20 25 30
Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu
35 40 45
Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly
50 55 60
Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr
65 70 75 80
Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val
85 90 95
Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn
100 105 110
Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys
115 120 125
Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu
130 135 140
Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr
145 150 155 160
Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys
165 170 175
Tyr Trp Leu Gln
180
<210> 2
<211> 215
<212> PRT
<213> Artificial Sequence
<220>
<223> 经修饰的Fc
<400> 2
Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys
1 5 10 15
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
20 25 30
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
35 40 45
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
50 55 60
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
65 70 75 80
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
85 90 95
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
100 105 110
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
115 120 125
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
130 135 140
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
145 150 155 160
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
165 170 175
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
180 185 190
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
195 200 205
Leu Ser Leu Ser Leu Gly Lys
210 215
<210> 3
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> IgD铰链变体
<400> 3
Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys
1 5 10 15
Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro
20 25 30
<210> 4
<211> 49
<212> PRT
<213> Artificial Sequence
<220>
<223> IgD铰链变体
<400> 4
Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro
1 5 10 15
Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser
20 25 30
Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys
35 40 45
Pro
<210> 5
<211> 540
<212> DNA
<213> Artificial Sequence
<220>
<223> FCeRI1 ECD的核苷酸序列
<400> 5
gtgccccaga agcccaaggt gagcctgaac cctccctgga acagaatctt caagggcgag 60
aacgtgaccc tgacctgcaa cggcaacaac ttcttcgagg tgagcagcac caagtggttc 120
cacaatggca gcctgagcga ggagaccaac agctccctga acatcgtgaa cgccaagttc 180
gaggacagcg gcgagtacaa gtgccagcac cagcaggtga acgagagcga gcccgtgtac 240
ctggaggtgt tcagcgactg gctgctgctg caggccagcg ccgaggtggt gatggagggc 300
cagcccctgt tcctgagatg ccacggctgg agaaactggg acgtgtacaa ggtgatctac 360
tacaaggatg gcgaggccct gaagtactgg tacgagaacc acaacatctc catcaccaac 420
gccaccgtgg aggacagcgg cacctactac tgcacaggca aggtgtggca gctggactac 480
gagagcgagc ccctgaacat caccgtgatc aaggctccca gagagaagta ctggctgcag 540
<210> 6
<211> 561
<212> DNA
<213> Artificial Sequence
<220>
<223> 经修饰的Fc的核苷酸序列
<400> 6
tgcgtggtcg tggatgtgag ccaggaagat cccgaagtgc agttcaactg gtacgtggat 60
ggcgtggaag tgcacaacgc caagaccaag cccagagaag agcagttcaa ctccacctac 120
agagtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 180
tgcaaggtgt ccaacaaagg cctgcccagc tccatcgaga agaccatcag caaagccaaa 240
ggccagccca gagaacccca ggtgtacacc ctgcctccca gccaggaaga gatgaccaag 300
aaccaggtgt ccctgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgtggag 360
tgggaaagca acggccagcc cgagaacaat tacaagacaa cccctcccgt gctggatagc 420
gatggcagct tctttctgta cagcagactg accgtggaca agagcagatg gcaggaaggc 480
aacgtgttca gctgcagcgt gatgcacgaa gccctgcaca accactacac ccagaagagc 540
ctgtccctga gcctgggcaa g 561
<210> 7
<211> 174
<212> DNA
<213> Artificial Sequence
<220>
<223> IgD铰链变体的核苷酸序列
<400> 7
aggaacaccg gcagaggagg cgaggaaaag aaaggaagca aggagaagga ggagcaggag 60
gaaagagaaa ccaagacccc cgagtgcccc agccacaccc agcccctggg cgtgttcctg 120
ttccccccca agcccaagga caccctgatg atcagcagaa cccccgaggt gacc 174
<210> 8
<211> 231
<212> DNA
<213> Artificial Sequence
<220>
<223> IgD铰链变体的核苷酸序列
<400> 8
gcccagcccc aggccgaggg cagcctggct aaggccacca cagctcccgc caccaccagg 60
aacaccggca gaggaggcga ggaaaagaaa ggaagcaagg agaaggagga gcaggaggaa 120
agagaaacca agacccccga gtgccccagc cacacccagc ccctgggcgt gttcctgttc 180
ccccccaagc ccaaggacac cctgatgatc agcagaaccc ccgaggtgac c 231
<210> 9
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> 信号肽
<400> 9
Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His Ala
20 25
<210> 10
<211> 75
<212> DNA
<213> Artificial Sequence
<220>
<223> 信号肽核苷酸
<400> 10
atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg 60
tcccctagcc acgcc 75
<210> 11
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> FceRIa ECD-铰链-Fc2
<400> 11
Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His Ala Val Pro Gln Lys Pro Lys Val
20 25 30
Ser Leu Asn Pro Pro Trp Asn Arg Ile Phe Lys Gly Glu Asn Val Thr
35 40 45
Leu Thr Cys Asn Gly Asn Asn Phe Phe Glu Val Ser Ser Thr Lys Trp
50 55 60
Phe His Asn Gly Ser Leu Ser Glu Glu Thr Asn Ser Ser Leu Asn Ile
65 70 75 80
Val Asn Ala Lys Phe Glu Asp Ser Gly Glu Tyr Lys Cys Gln His Gln
85 90 95
Gln Val Asn Glu Ser Glu Pro Val Tyr Leu Glu Val Phe Ser Asp Trp
100 105 110
Leu Leu Leu Gln Ala Ser Ala Glu Val Val Met Glu Gly Gln Pro Leu
115 120 125
Phe Leu Arg Cys His Gly Trp Arg Asn Trp Asp Val Tyr Lys Val Ile
130 135 140
Tyr Tyr Lys Asp Gly Glu Ala Leu Lys Tyr Trp Tyr Glu Asn His Asn
145 150 155 160
Ile Ser Ile Thr Asn Ala Thr Val Glu Asp Ser Gly Thr Tyr Tyr Cys
165 170 175
Thr Gly Lys Val Trp Gln Leu Asp Tyr Glu Ser Glu Pro Leu Asn Ile
180 185 190
Thr Val Ile Lys Ala Pro Arg Glu Lys Tyr Trp Leu Gln Arg Asn Thr
195 200 205
Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys Glu Glu Gln
210 215 220
Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro
225 230 235 240
Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
260 265 270
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
435 440 445
Gly Lys
450
<210> 12
<211> 1350
<212> DNA
<213> Artificial Sequence
<220>
<223> FceRIa ECD-铰链-Fc2的核苷酸序列
<400> 12
atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg 60
tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga 120
atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc 180
agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc 240
gtgaacgcca agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag 300
agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag 360
gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg 420
tacaaggtga tctactacaa ggatggcgag gccctgaagt actggtacga gaaccacaac 480
atctccatca ccaacgccac cgtggaggac agcggcacct actactgcac aggcaaggtg 540
tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag 600
aagtactggc tgcagaggaa caccggcaga ggaggcgagg aaaagaaagg aagcaaggag 660
aaggaggagc aggaggaaag agaaaccaag acccccgagt gccccagcca cacccagccc 720
ctgggcgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag cagaaccccc 780
gaggtgacct gcgtggtcgt ggatgtgagc caggaagatc ccgaagtgca gttcaactgg 840
tacgtggatg gcgtggaagt gcacaacgcc aagaccaagc ccagagaaga gcagttcaac 900
tccacctaca gagtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgtc caacaaaggc ctgcccagct ccatcgagaa gaccatcagc 1020
aaagccaaag gccagcccag agaaccccag gtgtacaccc tgcctcccag ccaggaagag 1080
atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc cagcgacatc 1140
gccgtggagt gggaaagcaa cggccagccc gagaacaatt acaagacaac ccctcccgtg 1200
ctggatagcg atggcagctt ctttctgtac agcagactga ccgtggacaa gagcagatgg 1260
caggaaggca acgtgttcag ctgcagcgtg atgcacgaag ccctgcacaa ccactacacc 1320
cagaagagcc tgtccctgag cctgggcaag 1350
<210> 13
<211> 469
<212> PRT
<213> Artificial Sequence
<220>
<223> FceRIa ECD-铰链-Fc3
<400> 13
Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His Ala Val Pro Gln Lys Pro Lys Val
20 25 30
Ser Leu Asn Pro Pro Trp Asn Arg Ile Phe Lys Gly Glu Asn Val Thr
35 40 45
Leu Thr Cys Asn Gly Asn Asn Phe Phe Glu Val Ser Ser Thr Lys Trp
50 55 60
Phe His Asn Gly Ser Leu Ser Glu Glu Thr Asn Ser Ser Leu Asn Ile
65 70 75 80
Val Asn Ala Lys Phe Glu Asp Ser Gly Glu Tyr Lys Cys Gln His Gln
85 90 95
Gln Val Asn Glu Ser Glu Pro Val Tyr Leu Glu Val Phe Ser Asp Trp
100 105 110
Leu Leu Leu Gln Ala Ser Ala Glu Val Val Met Glu Gly Gln Pro Leu
115 120 125
Phe Leu Arg Cys His Gly Trp Arg Asn Trp Asp Val Tyr Lys Val Ile
130 135 140
Tyr Tyr Lys Asp Gly Glu Ala Leu Lys Tyr Trp Tyr Glu Asn His Asn
145 150 155 160
Ile Ser Ile Thr Asn Ala Thr Val Glu Asp Ser Gly Thr Tyr Tyr Cys
165 170 175
Thr Gly Lys Val Trp Gln Leu Asp Tyr Glu Ser Glu Pro Leu Asn Ile
180 185 190
Thr Val Ile Lys Ala Pro Arg Glu Lys Tyr Trp Leu Gln Ala Gln Pro
195 200 205
Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr
210 215 220
Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys
225 230 235 240
Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His
245 250 255
Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
340 345 350
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Leu Gly Lys
465
<210> 14
<211> 1407
<212> DNA
<213> Artificial Sequence
<220>
<223> FceRIa ECD-铰链-Fc3的核苷酸序列
<400> 14
atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg 60
tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga 120
atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc 180
agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc 240
gtgaacgcca agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag 300
agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag 360
gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg 420
tacaaggtga tctactacaa ggatggcgag gccctgaagt actggtacga gaaccacaac 480
atctccatca ccaacgccac cgtggaggac agcggcacct actactgcac aggcaaggtg 540
tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag 600
aagtactggc tgcaggccca gccccaggcc gagggcagcc tggctaaggc caccacagct 660
cccgccacca ccaggaacac cggcagagga ggcgaggaaa agaaaggaag caaggagaag 720
gaggagcagg aggaaagaga aaccaagacc cccgagtgcc ccagccacac ccagcccctg 780
ggcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 840
gtgacctgcg tggtcgtgga tgtgagccag gaagatcccg aagtgcagtt caactggtac 900
gtggatggcg tggaagtgca caacgccaag accaagccca gagaagagca gttcaactcc 960
acctacagag tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1020
tacaagtgca aggtgtccaa caaaggcctg cccagctcca tcgagaagac catcagcaaa 1080
gccaaaggcc agcccagaga accccaggtg tacaccctgc ctcccagcca ggaagagatg 1140
accaagaacc aggtgtccct gacctgcctg gtgaaaggct tctaccccag cgacatcgcc 1200
gtggagtggg aaagcaacgg ccagcccgag aacaattaca agacaacccc tcccgtgctg 1260
gatagcgatg gcagcttctt tctgtacagc agactgaccg tggacaagag cagatggcag 1320
gaaggcaacg tgttcagctg cagcgtgatg cacgaagccc tgcacaacca ctacacccag 1380
aagagcctgt ccctgagcct gggcaag 1407
<210> 15
<211> 406
<212> PRT
<213> Artificial Sequence
<220>
<223> 人a-2,6-唾液酸转移酶
<400> 15
Met Ile His Thr Asn Leu Lys Lys Lys Phe Ser Cys Cys Val Leu Val
1 5 10 15
Phe Leu Leu Phe Ala Val Ile Cys Val Trp Lys Glu Lys Lys Lys Gly
20 25 30
Ser Tyr Tyr Asp Ser Phe Lys Leu Gln Thr Lys Glu Phe Gln Val Leu
35 40 45
Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp Ser Gln Ser Val Ser
50 55 60
Ser Ser Ser Thr Gln Asp Pro His Arg Gly Arg Gln Thr Leu Gly Ser
65 70 75 80
Leu Arg Gly Leu Ala Lys Ala Lys Pro Glu Ala Ser Phe Gln Val Trp
85 90 95
Asn Lys Asp Ser Ser Ser Lys Asn Leu Ile Pro Arg Leu Gln Lys Ile
100 105 110
Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser Tyr Lys Gly
115 120 125
Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu Ala Leu Arg Cys His Leu
130 135 140
Arg Asp His Val Asn Val Ser Met Val Glu Val Thr Asp Phe Pro Phe
145 150 155 160
Asn Thr Ser Glu Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile Arg Thr
165 170 175
Lys Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser Ser Ala Gly Ser
180 185 190
Leu Lys Ser Ser Gln Leu Gly Arg Glu Ile Asp Asp His Asp Ala Val
195 200 205
Leu Arg Phe Asn Gly Ala Pro Thr Ala Asn Phe Gln Gln Asp Val Gly
210 215 220
Thr Lys Thr Thr Ile Arg Leu Met Asn Ser Gln Leu Val Thr Thr Glu
225 230 235 240
Lys Arg Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile Leu Ile Val
245 250 255
Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys Trp Tyr Gln Asn
260 265 270
Pro Asp Tyr Asn Phe Phe Asn Asn Tyr Lys Thr Tyr Arg Lys Leu His
275 280 285
Pro Asn Gln Pro Phe Tyr Ile Leu Lys Pro Gln Met Pro Trp Glu Leu
290 295 300
Trp Asp Ile Leu Gln Glu Ile Ser Pro Glu Glu Ile Gln Pro Asn Pro
305 310 315 320
Pro Ser Ser Gly Met Leu Gly Ile Ile Ile Met Met Thr Leu Cys Asp
325 330 335
Gln Val Asp Ile Tyr Glu Phe Leu Pro Ser Lys Arg Lys Thr Asp Val
340 345 350
Cys Tyr Tyr Tyr Gln Lys Phe Phe Asp Ser Ala Cys Thr Met Gly Ala
355 360 365
Tyr His Pro Leu Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn Gln
370 375 380
Gly Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala Thr Leu Pro Gly
385 390 395 400
Phe Arg Thr Ile His Cys
405
<210> 16
<211> 1218
<212> DNA
<213> Artificial Sequence
<220>
<223> 人a-2,6-唾液酸转移酶的核苷酸序列
<400> 16
atgatccaca ccaacctgaa gaagaagttc agctgctgcg tgctggtgtt cctgctgttc 60
gccgtgatct gcgtgtggaa ggagaagaag aaaggcagct actacgacag cttcaagctg 120
cagaccaagg agttccaggt gctgaagagc ctgggcaagc tggccatggg cagcgacagc 180
cagagcgtgt ccagctcctc cacccaggat ccccacagag gcagacagac cctgggcagc 240
ctgagaggcc tggccaaggc caagcccgag gccagcttcc aggtgtggaa caaggacagc 300
agcagcaaga acctgatccc cagactgcag aagatctgga agaactacct gagcatgaac 360
aagtacaagg tgagctacaa aggacccgga cccggcatca agttcagcgc cgaggccctg 420
aggtgccacc tgagagacca cgtgaacgtg agcatggtgg aagtgaccga cttccccttc 480
aacaccagcg agtgggaagg ctacctgccc aaggagagca tcaggaccaa ggctggcccc 540
tggggcagat gcgccgtggt gagcagcgct ggcagcctga agagctccca gctgggcaga 600
gagatcgacg accacgatgc cgtgctgagg ttcaatggcg ctcccaccgc caacttccag 660
caggacgtgg gcaccaagac cacaatccgg ctgatgaaca gccagctggt gacaaccgag 720
aagcggttcc tgaaggacag cctgtacaac gagggcatcc tgatcgtgtg ggatcccagc 780
gtgtaccaca gcgacatccc caagtggtac cagaatcccg actacaactt cttcaacaac 840
tacaagacct atagaaagct gcaccccaac cagcccttct acatcctgaa gccccagatg 900
ccctgggagc tgtgggacat cctgcaggag atcagccctg aagagatcca gcccaaccct 960
ccctccagcg gcatgctggg cattatcatc atgatgaccc tgtgcgacca ggtggacatc 1020
tacgagttcc tgcccagcaa gagaaagacc gacgtgtgct actactatca gaagttcttc 1080
gacagcgcct gcaccatggg cgcctaccac cccctgctgt acgagaagaa cctggtgaag 1140
cacctgaacc agggcaccga cgaggacatc tacctgctgg gcaaagccac cctgcccggc 1200
ttcagaacca tccactgc 1218
<210> 17
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> IgD铰链变体
<220>
<221> misc_feature
<222> (12)
<223> Xaa is Lys or Gly
<220>
<221> misc_feature
<222> (13)
<223> Xaa is Glu, Gly or Ser
<400> 17
Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa Xaa Lys Glu Lys
1 5 10 15
Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro
20 25 30
<210> 18
<211> 49
<212> PRT
<213> Artificial Sequence
<220>
<223> IgD铰链变体
<220>
<221> misc_feature
<222> (31)
<223> Xaa is Lys or Gly
<220>
<221> misc_feature
<222> (32)
<223> Xaa is Glu, Gly or Ser
<400> 18
Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro
1 5 10 15
Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa Xaa
20 25 30
Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys
35 40 45
Pro
<210> 19
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> IgD铰链
<400> 19
Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys
1 5 10 15
Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro
20 25 30
<210> 20
<211> 425
<212> PRT
<213> Artificial Sequence
<220>
<223> FceRIa ECD-铰链-Fc1
<400> 20
Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile
1 5 10 15
Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe
20 25 30
Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu
35 40 45
Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly
50 55 60
Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr
65 70 75 80
Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val
85 90 95
Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn
100 105 110
Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys
115 120 125
Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu
130 135 140
Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr
145 150 155 160
Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys
165 170 175
Tyr Trp Leu Gln Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys
180 185 190
Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu
195 200 205
Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys
210 215 220
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
225 230 235 240
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
245 250 255
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
260 265 270
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
275 280 285
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
290 295 300
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
305 310 315 320
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
325 330 335
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
340 345 350
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
355 360 365
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
370 375 380
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
385 390 395 400
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
405 410 415
Lys Ser Leu Ser Leu Ser Leu Gly Lys
420 425
<210> 21
<211> 425
<212> PRT
<213> Artificial Sequence
<220>
<223> FceRIa ECD-铰链-Fc2
<400> 21
Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile
1 5 10 15
Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe
20 25 30
Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu
35 40 45
Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly
50 55 60
Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr
65 70 75 80
Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val
85 90 95
Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn
100 105 110
Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys
115 120 125
Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu
130 135 140
Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr
145 150 155 160
Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys
165 170 175
Tyr Trp Leu Gln Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly
180 185 190
Ser Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu
195 200 205
Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys
210 215 220
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
225 230 235 240
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
245 250 255
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
260 265 270
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
275 280 285
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
290 295 300
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
305 310 315 320
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
325 330 335
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
340 345 350
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
355 360 365
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
370 375 380
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
385 390 395 400
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
405 410 415
Lys Ser Leu Ser Leu Ser Leu Gly Lys
420 425
<210> 22
<211> 444
<212> PRT
<213> Artificial Sequence
<220>
<223> FceRIa ECD-铰链-Fc3
<400> 22
Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile
1 5 10 15
Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe
20 25 30
Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu
35 40 45
Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly
50 55 60
Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr
65 70 75 80
Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val
85 90 95
Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn
100 105 110
Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys
115 120 125
Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu
130 135 140
Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr
145 150 155 160
Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys
165 170 175
Tyr Trp Leu Gln Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala
180 185 190
Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu
195 200 205
Lys Lys Gly Ser Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys
210 215 220
Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
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