CN114507291A - 一种重组人免疫球蛋白ε和γ的Fc融合蛋白的表达方法 - Google Patents
一种重组人免疫球蛋白ε和γ的Fc融合蛋白的表达方法 Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种重组人免疫球蛋白ε和γ的Fc融合蛋白(抗过敏融合蛋白)的表达方法及用途。所述的重组人免疫球蛋白ε和γ的Fc融合蛋白,其能够结合于肥大细胞和嗜碱粒细胞表面介导过敏反应的Fcε受体I(FcεRI)和抑制性Fcγ受体IIb(FcγRIIb)。本发明提供的重组人免疫球蛋白ε和γ的Fc融合蛋白能够用于过敏疾病,如过敏哮喘、鼻炎等。
Description
技术领域
本发明属于生物医药领域,涉及一种重组人免疫球蛋白ε和γ的Fc融合蛋白(抗过敏融合蛋白)的表达方法及操作。
背景技术
过敏是人体接触环境中的过敏原后,所引发的一系列过度反应的现象,包含过敏性鼻炎、食物过敏、荨麻疹、哮喘等。
即发性过敏反应(又称I型超敏反应)是IgE介导的免疫反应,是一类常见的过敏类型,主要可分为全身性过敏反应和异位性过敏反应两大类。
过敏产生的原理是:当过敏原(抗原)第一次接触免疫B细胞后,B细胞和T细胞接触而分化成浆细胞并产生大量抗体IgE,这些IgE会与肥大细胞等免疫细胞结合,当下一次接触过敏原时,IgE结合的免疫细胞被激活,免疫细胞释放组胺等物质,引起过敏症状并影响免疫反应。
1995年,法国的科研小组Daeron等首次报道了介导肥大细胞活化的IgE高亲和力受体(FcεRI)受到低亲和力IgG受体(FcγRII)的负调控,同一多价配体交联肥大细胞表面的FcεRI和FcγRII可以抑制细胞的活化,阻止介体的释放[Daeron M,Malbec O,Latour S,Arock M,Fridman W.Regulation of high-affinity IgE receptor-mediated mast cellactivation by murine low-affinity IgG receptors.J Clin Invest,95:577-585,1995.],在当时的文献中,并未关注不同种类的FcγRII存在区别。这一区别在之后的研究中被发现。
WuJ.等人的研究表明,FcγRII一族单抗在过敏反应中扮演着重要角色,FcγRII可进一步细分为 FcγRIIa,FcγRIIb,FcγRIIc。其中对IgE有负调控作用的受体是FcγRIIb,FcγRIIa与特异性反应呈现正相关,其他的受体与特异性反应及哮喘没有明显关联[Wu J,LinR,Huang J,Guan W,Oetting WS,et al.(2014) Functional Fcgamma ReceptorPolymorphisms Are Associated with Human Allergy.PLoS ONE9(2):e89196. doi:10.1371/journal.pone.0089196.]。其中FcγRIIa为人类独有。在FcγRII一族中,FcγRIIb与IgG的亲和力较FcγRIIa及FcγRIIc为低。
随着研究的深入进行,人们发现不同种类的FcγRII受体其在体内作用存在的差异相当明显。Rosales C. 等人的研究进一步证实了不同受体的调控作用截然相反。[RosalesC.FcγReceptor Heterogeneity in Leukocyte Functional Responses.FrontImmunol.2017;8:280.Published 2017Mar 20. doi:10.3389/fimmu.2017.00280.]在人体中,FcγRIIb受体可以激活胞内链上的免疫受体酪氨酸抑制基序 (ImmunoreceptorTyrosine-based Inhibition Motif,ITIM),它也是唯一能激活ITIM的受体,而FCγRIIa与FCγRIIc则会激活胞内链上的免疫受体酪氨酸活化基序(Immunoreceptor Tyrosine-basedActivation Motif, ITAM),这与IgE相同的活化效果将会导致SYK(蛋白脾酪氨酸激酶)活性化,加重哮喘的症状。
王静娴的研究证实[王静娴.鼠源AAFP(Anti-allergy fusionprotein,简称AAFP)蛋白对IgE介导小鼠过敏反应的干预作用及机制研究[D].贵阳:贵阳医学院,2014.],通过交联FcεRI和FcγRIIb受体,激活 FcγRIIb受体胞内链上的ITIM抑制基序,进一步激活5'-肌醇磷酸酶(SHIP)的磷酸化,SHIP磷酸化使得 FcεRI信号途径中的关键信号蛋白脾酪氨酸激酶(Syk)脱磷酸化,阻断Syk启动的细胞脱颗粒作用。AAFP 蛋白作用于过敏细胞的活化和炎性介质的释放,以此来抑制IgE介导过敏反应的发生。
抗过敏融合蛋白AAFP由人源Fcε和Fcγ构成,可以交联肥大细胞和嗜碱粒细胞表面介导过敏反应的 Fcε受体I(FcεRI)和抑制性Fcγ受体IIb(FcγRIIb),抑制肥大细胞和嗜碱粒细胞的活化及过敏炎性介质的释放,从而有效阻断过敏的发生。
近年来,市场上对于IgE介导过敏性疾病生物药物的研究开发主要集中于抗IgE或抗FcεRI单克隆抗体和抑制其信号传导两大类。不过临床研究发现,体内IgE水平的降低会导致肿瘤发生率的增加。而AAFP 并不是单纯地与IgE抗体结合,因此AAFP没有减少体内IgE抗体的含量,不会因为IgE抗体在体内含量的降低而引起不良后果,相较于抗IgE单克隆抗体相对安全,为IgE介导过敏性疾病的治疗开辟了新的途径。
生物学中,表达载体就是在克隆载体基本骨架的基础上增加表达元件(如启动子、增强子、终止子等),使目的基因能够表达的载体。表达载体主要分为四部分:目的基因、启动子、终止子、标记基因。
表达载体常用细菌质粒构建,构建过程中运用限制性核酸内切酶切割出与目的基因相合的末端(多为黏性末端,也有平末端),再采用DNA连接酶连接,导入生物体实现表达。标记基因可帮助识别质粒并检测是否成功整合到染色体DNA中。
CHO细胞(Chinese Hamster Ovary,中国仓鼠卵巢细胞)是目前重组蛋白药物的生产的常用体系,与其他表达***相比具有许多优点:(1)具有产物胞外分泌功能,便于下游产物分离纯化;(2)具有重组基因的高效扩增和表达能力;(3)具有贴壁生长特性,且有较高的耐受剪切力和渗透压能力,也可进行悬浮培养,表达水平较高;(4)CHO细胞属于成纤维细胞,很少分泌自身的内源蛋白,利于外源蛋白的启分离; (5)CHO细胞既可贴壁生长,也可悬浮生长,容易发生基因突变与基因转染;(6)在无血清及化学限定培养基中快速稳定地悬浮成长;(7)具有准确的转录后修饰功能,表达的蛋白在分子结构、理化特性和生物学功能接近天然蛋白分子;(8)具有重组基因的高效扩增和表达能力,能稳定融合外源蛋白。
选择标记和基因扩增CHO细胞表达载体主要有两类选择标记。一类是neo(新霉素抗性基因)等非扩增基因,它对目的基因的拷贝数没有影响,用于构建瞬时表达载体。另一类具有基因扩增的功能,也称共扩增基因,如二氢叶酸还原酶基因(Dihydrofolatereductase,DHFR)、谷氨酰胺合成酶基因 (Glutaminesynthetase,GS)。
稳定细胞株使用DHFR基因筛选周期长,超过半年时间,产量较低,已较少使用。而稳定细胞株使用 GS基因筛选周期短,一般少于3个月,产量较稳定,能利用生物代谢产生的氨等,可避免或减少细胞代谢过程中谷氨酰胺降解积累的氨对细胞的损伤、抑制作用。
将装载好的pGS质粒转入大肠杆菌感受态细胞DH5α中,进行菌落PCR,由于该质粒带有氨苄青霉素抗性基因,所以含有该质粒的大肠杆菌会在含有氨苄青霉素的培养基上生长,而不含该质粒的大肠杆菌则会死亡,因此可用于筛选未导入该质粒的菌落。
另外,pGS质粒带有GS标记(即带有谷氨酰胺合成酶基因),而CHO-K1细胞为GS缺陷型细胞,不能合成谷氨酰胺,在谷氨酰胺类似物MSX(蛋氨酸亚氨基代砜)的筛选下,未转入质粒的CHO-K1细胞会因为无法合成谷氨酰胺而死亡,存活的细胞即为含有质粒的阳性细胞,因此使用该质粒还可用于筛选未导入该质粒的细胞。因此在使用含有GS标记的载体时,选择GS缺陷型细胞作为宿主细胞,使用MSX作为筛选阳性克隆的条件。
脂质体转染法可用于DNA的瞬时转染及稳定转染。中性脂质体利用脂质膜包裹DNA,借助脂质膜将 DNA导入细胞膜内。对于带正电的阳离子脂质体,带负电的DNA磷酸基团自动结合到带正电的脂质体上,形成DNA-阳离子脂质体复合物,从而吸附到带负电的细胞膜表面,经过内吞被导入细胞。此法使得转染的效率、稳定性,以及可重复性大大提高。
因此选用脂质体转染的方法将线性化的pGS-AAFP质粒导入CHO-K1细胞,可得到能稳定表达AAFP 基因的CHO细胞株。
对于确诊为IgE(免疫球蛋白E)介导的哮喘患者,经吸入型糖皮质激素和长效吸入型β2-肾上腺素受体激动剂治疗后,仍不能有效控制症状的中至重度持续性过敏性哮喘,AAFP可交联嗜碱性粒细胞或肥大细胞表面的受体FcγRIIb和FcεRI,其中AAFP上的IgE Fc端可以与细胞表面的FcεRI结合,IgG Fc端可以与FcγRIIb结合。一旦发生交联后,FcγRIIb的信号通路即被激活而抑制FcεRI的传导,从而阻止细胞释放炎性介质,有效降低患者的哮喘发生率。
发明内容
本发明提供了一种抗过敏融合蛋白AAFP及其表达。
第一方面,本发明提供了一种编码重组人免疫球蛋白ε和γ的Fc融合蛋白的核苷酸序列。
具体地,AAFP基因片段由IgE Fc与IgG Fc片段连接而成,所述基因的核苷酸序列如SEQ ID NO.1 所示:
其中1~63为Igκ链的分泌信号肽基因序列,64~1192位为IgE Fc的基因,1193~2106位为IgG Fc的基因。
为去除pSecTaq-AAFP载体中AAFP基因序列中所掺杂的非人源序列,利用OverlapPCR方法扩增所述AAFP基因;首先使用PCR从pSecTaq-AAFP质粒上获取IgE Fc与IgGFc基因片段,之后将两个片段以overlapPCR方法连接。
第二方面,本发明提供了一种由上述基因序列表达的重组人免疫球蛋白ε和γ的Fc融合蛋白,所述重组人免疫球蛋白ε和γ的Fc融合蛋白的氨基酸序列如SEQ ID NO.2所示:
其中,1~21为Igκ链的分泌信号肽序列;22~25、346和347为载体序列编码的氨基酸残基;26~345为人IgE Fc的氨基酸序列(CH2-CH3-CH4);346~579为人IgG Fc的氨基酸序列(Hinge-CH2-CH3)。
第三方面,本发明提供了一种表达载体,所述表达载体包含如专利要求1所述的核苷酸序列。具体操作步骤如下:
(1)设计overlap PCR引物,通过第一轮overlap PCR从pSecTaq-AAFP质粒上获取IgE Fc与IgG Fc基因片段,通过第二轮overlap PCR将两个片段连接,获得如上文SEQ IDNO.1所示的序列。overlap PCR需要5 条引物,forward1如SEQ ID NO.3所示,forward2如SEQ ID NO.4所示,forward3如SEQ ID NO.5所示,reverse1 如SEQ ID NO.6所示,reverse2如SEQ ID NO.7所示:
(2)利用XhoI酶和MluI酶对PCR产物和pGS质粒进行双酶切,之后在T4连接酶的作用下,将AAFP 基因片段与pGS的载体片段整合成闭合的重组质粒;
(3)验证质粒是否装载目的基因的方法为将质粒转入大肠杆菌感受态细胞DH5α中,进行菌落PCR,使用含氨苄青霉素的培养基培养菌落,挑取阳性菌落进行菌落PCR,验证阳性菌落是否包含目标载体;
(4)对阳性克隆抽提质粒,经MluI酶及XhoI酶双酶切鉴定载体是否已成功构建,构建的载体命名为pGS-AAFP。
第四方面,本发明提供了一种可生产重组人免疫球蛋白ε和γ的Fc融合蛋白的CHO-K1细胞株,细胞株通过如下的步骤构建:
(1)用BcgI酶切pGS-AAFP载体使其线性化;
(2)利用脂质体Lipofectamine(Invitrogen)转染方法将线性化的pGS-AAFP载体转染CHO-K1宿主细胞。
(3)对转染后的CHO-K1细胞用MSX进行加压筛选,再利用ELISA方法对阳性克隆的表达量进行筛选,最终得到权利要求4所述CHO-K1细胞。
第五方面,本发明提供了AAFP基因片段的细胞株的放大培养实施条件。
具体地,将含有AAFP基因片段的细胞株复苏后,经过三级种子培养后,接入生产培养反应器进行培养。
本发明所指的CHO细胞包括CHO-K1,CHO-DG44等商业化的CHO细胞株。
本发明使用的表达载体可以选用各种已知的载体,如商业化载体:pCDNA3.1、pSecTag等等。将重组表达载体通过电转染或化学转染的方法转入宿主细胞,利用合适的条件筛选稳定的重组细胞,作为制备重组人免疫球蛋白ε和γ的Fc融合蛋白的工程细胞株。
附图说明
图1为本发明建议的对CHO-K1细胞株的加压筛选流程图。
图2为本发明建议的含有AAFP基因片段的细胞培养工艺流程图。
图3为重组质粒转化大肠杆菌DH5α菌落PCR电泳结果,图示结果表明挑取的DH5α阳性克隆均带有目的基因,pGS-AAFP质粒构建完成。
具体实施方式
实施例
1.含有AAFP基因片段的CHO-K1细胞株的构建及筛选
1.1AAFP基因的扩增
为了去除pSecTaq-AAFP载体中AAFP基因序列中所掺杂的非人源序列,我们设计了3对引物:
利用OverlapPCR方法扩增我们所需要的AAFP的基因。第一轮PCR中,包括了两个PCR反应,第一个反应以pSecTag-AAFP为模板,扩增IgE Fc片段序列。其中F1与IgE Fc的5’端完全配对,R1的3’端与IgE Fc反义链的5’端配对,而R1的5’端部分与IgG Fc的5’端配对。第二个反应也是以pSecTag-AAFP 为模板,扩增IgG Fc片段序列。其中F2的3’端部分与IgGFc的5’端配对,而5’端部分则与R1的5’端部分配对,R2的3’端与IgG Fc反义链的5’端配对,其5’端部分为MluI的酶切序列和保护碱基。第一轮所涉及的两个PCR的反应体系如下:
PCR条件为94℃预变性2min,1个热循环;98℃热变性10s,55℃退火5s,72℃延伸80s,30个热循环;72℃复性10min;4℃保存。
第一轮反应之后,用PCR产物抽提试剂盒分离纯化出PCR产物,并以其作为模板进行第二轮PCR。第二轮PCR的正向F3引物依次由保护性碱基、XhoI酶切序列、Kozak序列、鼠Kappa链分泌信号肽序列和IgE Fc的5’端序列构成,反向引物为R2,模板是第一轮PCR的抽提所得到的产物IgE Fc和IgG Fc。
第二轮反应体系如下:
IgE Fc PCR产物(稀释100倍后) | 0.5μL |
IgG Fc PCR产物(稀释100倍后) | 0.5μL |
引物F3工作液 | 1μL |
引物R2工作液 | 1μL |
5×PS buffer | 5μL |
dNTP | 2μL |
PrimeStar酶 | 0.25μL |
水 | 14.75μL |
PCR条件为94℃预变性2min,1个热循环;98℃热变性10s,55℃退火5s,72℃延伸2min,35个热循环;72℃复性10min;4℃保存。
1.2PCR产物双酶切
利用XhoI和MluI对1.1中的PCR产物进行双酶切,回收目标片段,双酶切反应体系如下:
37℃酶切2hr,酶切后,经1.0%凝胶电泳分离和胶回收,获得AAFP基因。
1.3pGS质粒双酶切
利用XhoI和MluI对pGS质粒进行双酶切,回收目标片段,双酶切反应体系如下:
Buffer3(NEB) | 5μL |
MluI | 2μL |
XhoI | 2μL |
pGS质粒 | 2μL |
水 | 39μL |
37℃酶切2hr,酶切后,经1.0%凝胶电泳分离和胶回收,获得pGS载体片段。
1.4pGS-AAFP载体的连接
将1.2和1.3中获得的AAFP基因片段与pGS的载体片段,在T4连接酶的作用下,整合成闭合的重组质粒。DNA的连接反应体系为:
T4 ligase | 1μL |
Buffer | 1μL |
AAFP基因片段 | 7μL |
pGS的载体片段 | 1μL |
4℃反应过夜。
1.5菌落PCR
用连接反应产物转化感受态大肠杆菌DH5α,在含有Amp的LB平板上筛选阳性克隆。挑取阳性克隆接种于0.5ml的LB培养基中发酵4小时,取菌液作为模板以F3、R2为引物进行克隆PCR反应,反应体系为:
克隆发酵液 | 1μL |
引物F3工作液 | 1μL |
引物R2工作液 | 1μL |
2×Taq PCR MasterMix | 10μL |
灭菌去离子水 | 7μL |
PCR条件为95℃预变性5min,1个热循环;94℃热变性30s,55℃退火30s,72℃延伸2min,25个热循环;72℃复性7min;4℃保存。
用琼脂糖凝胶电泳对PCR产物进行鉴定,有2KB大小的片段为阳性克隆,进一步对阳性克隆进行 MluI+XhoI双酶切鉴定及基因测序,确认与目标序列一致,命名所得质粒为pGS-AAFP。
1.6pGS-AAFP质粒的线性化
通过分析pGS-AAFP质粒的基因序列,发现BcgI可以单酶切pGS-AAFP,并且不会影响到pGS-AAFP 质粒上的功能单位。因此在转染CHO-K1S细胞之前,首先应用BcgI酶切pGS-AAFP,并用PCR抽提试剂盒回收线性化质粒。
1.7脂质体转染及细胞株筛选
利用脂质体Lipofectamine(Invitrogen)转染方法将线性化的pGS-AAFP质粒转染CHO-K1宿主细胞。转染后,转入6孔板中培养,并加入25MMSX进行筛选,未被质粒成功转染的细胞将被杀死。待细胞适应后,限定稀释法,以每孔1个细胞接种96孔细胞培养板进行单克隆筛选,并在显微镜下,挑选标记出只含有单个细胞的孔。当单个细胞长出克隆后,利用ELISA方法比较各个单克隆的AAFP表达水平,选取前96个克隆,移种接入1块96孔板中,继续培养,当细胞长满时,再次利用ELISA方法对这96个克隆进行表达量的比较,取表达量最高的24个克隆,并放大到24孔板中继续培养,细胞长满后,再次进行ELISA 比较各孔之间的表达量,选取表达量最大的5个孔的细胞接种入6孔板,放大培养,然后接种入125mL 的摇瓶中培养比较细胞的生长和AAFP的表达水平,选取表达水平最高的细胞株作为生产细胞株。
由此,得到目标AAFP细胞株。
具体构建步骤详见说明书附图1。
2.含有AAFP基因片段的细胞培养
2.1培养基的确定
Invitrogen公司的CD CHO培养基能够支持CHO-K1细胞的快速生长,但是重组蛋白的表达量较低,而CHO-K1细胞在Hyclone公司的PFCHO培养基中生长速度相对较慢,但是蛋白表达量较高。故选用 Invitrogen公司的CD CHO培养基与Hyclone公司的PFCHO培养基,按1:1混合,添加GSSupplement、 F-68、NaHCO3等作为细胞培养的基础培养基,应用于种子培养阶段。另一方面,在构建基因工程细胞中,我们采用了GS表达***,因此,细胞株可以在无谷氨酰胺的培养基中生长。在1:1CD/PF的无血清培养基中,不另添加谷氨酰胺而是补充1*GS Supplement添加剂。在生产流加培养中,为了支持细胞在反应器培养中达到更高的细胞密度,维持更长的生产时间,获得更高的产物浓度,培养中期开始流加营养富集的补料培养基。补料培养基的基本成分是在无盐、无谷氨酰胺、无糖的DMEM/F12(BDF培养基)的基础上,添加高浓度的葡萄糖、氨基酸、维生素和核苷酸等营养成分。所用基础培养基配方如下:
CD CHO AGT | 121.3g |
PF-CHO part1 | 30g |
PF-CHO part2 | 52g |
F-68 | 5g |
50*GS supplement | 200ml |
NaHCO<sub>3</sub> | 10g |
超纯水H<sub>2</sub>O | 10L |
2.2I级和II级种子培养(摇瓶培养)
按常规细胞复苏方法,从工作细胞库中取一支冻存细胞迅速投入到37℃温水中解冻。解冻完全后,无菌操作将冻存管中的细胞转入10ml的离心管中,并加入5ml37℃预热培养基,1000rpm离心5min,弃上清,再加入5ml37℃预热培养基重新悬浮细胞。将细胞悬浮液转入125ml摇瓶,并补加25ml的培养基后,置于5%CO2、37℃恒温培养箱中培养。摇床转速设定为120rpm。每天进行细胞密度和活性的监测,养4天后,细胞密度约为3.0×106cells/ml时进行传代转入1000ml摇瓶,补加37℃预热的新鲜培养基至 270ml。再培养3天后,细胞密度约为3×106cells/mL,接种入5L反应器开始III级种子培养。
2.3III级种子培养
将摇瓶中约300ml细胞液转移至含有2.7L1:1CD/PF+1*GS Supplement培养基的NBS7.5L或BBraun 5L生物反应器中,培养体积约3L,温度36.8℃,溶氧50%,pH7.1,搅拌转速设定为80rpm,培养3 天,细胞密度约为3.0×106cells/ml,接种入生产罐中进行生产。
2.4生产培养
中试生产在B Braun的C30罐中进行,反应器的总体积为42L,工作体积为30L。接种时,将7.5L 或5L反应器中约3L细胞液转移至含有20L基础培养基(1:1CD/PF+1*GSSupplement)的C30生物反应器中,起初的培养温度设定为36.8℃,溶氧50%,pH 7.1±0.05,搅拌转速60rpm。当培养基中残留糖浓度下降到1.5g/L左右时,启动流加培养,连续流加优化确定的补料培养基10×BDF+5×GS+45g/LGlc。为了尽可能地延长生产周期,当细胞密度达到1×107cells/ml时,降低培养温度至31℃。根据每天取样的测糖结果,控制培养液中葡萄糖含量在1~2g/L间,当细胞活力下降到80%以下时结束培养。
具体步骤详见说明书附图2。
序列表
<110> 上海景峰制药有限公司
<120> 一种重组人免疫球蛋白ε和γ的Fc融合蛋白的表达方法
<141> 2020-11-09
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2106
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60
gacttcaccc cgcccaccgt gaagatctta cagtcgtcct gcgacggcgg cgggcacttc 120
cccccgacca tccagctcct gtgcctcgtc tctgggtaca ccccagggac tatcaacatc 180
acctggctgg aggacgggca ggtcatggac gtggacttgt ccaccgcctc taccacgcag 240
gagggtgagc tggcctccac acaaagcgag ctcaccctca gccagaagca ctggctgtca 300
gaccgcacct acacctgcca ggtcacctat caaggtcaca cctttgagga cagcaccaag 360
aagtgtgcag gtacgttccc acctgccctg gtggccgcca cggaggccag agaagagggg 420
cgggtgggcc tcacacagcc ctccggtgta ccacagattc caacccgaga ggggtgagcg 480
cctacctaag ccggcccagc ccgttcgacc tgttcatccg caagtcgccc acgatcacct 540
gtctggtggt ggacctggca cccagcaagg ggaccgtgaa cctgacctgg tcccgggcca 600
gtgggaagcc tgtgaaccac tccaccagaa aggaggagaa gcagcgcaat ggcacgttaa 660
ccgtcacgtc caccctgccg gtgggcaccc gagactggat cgagggggag acctaccagt 720
gcagggtgac ccacccccac ctgcccaggg ccctcatgcg gtccacgacc aagaccagcg 780
gtgagccatg ggcaggccgg ggtcgtgggg gaagggaggg agcgagtgag cggggcccgg 840
gctgacccca cgtctggcca caggcccgcg tgctgccccg gaagtctatg cgtttgcgac 900
gccggagtgg ccggggagcc gggacaagcg caccctcgcc tgcctgatcc agaacttcat 960
gcctgaggac atctcggtgc agtggctgca caacgaggtg cagctcccgg acgcccggca 1020
cagcacgacg cagccccgca agaccaaggg ctccggcttc ttcgtcttca gccgcctgga 1080
ggtgaccagg gccgaatggg agcagaaaga tgagttcatc tgccgtgcag tccatgaggc 1140
agctagcccc tcacagaccg tccagcgagc ggtgtctgta aatcccggta aagagcccaa 1200
atcttgtgac aaaactcaca catgcccacc gtgcccaggt aagccagccc aggcctcgcc 1260
ctccagctca aggcgggaca ggtgccctag agtagcctgc atccagggac aggccccagc 1320
cgggtgctga cacgtccacc tccatctctt cctcagcacc tgaactcctg gggggaccgt 1380
cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg acccctgagg 1440
tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc aactggtacg 1500
tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag tacaacagca 1560
cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt 1620
acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc atctccaaag 1680
ccaaaggtgg gacccgtggg gtgcgagggc cacatggaca gaggccggct cggcccaccc 1740
tctgccctga gagtgaccgc tgtaccaacc tctgtcccta cagggcagcc ccgagaacca 1800
caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 1860
tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1920
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1980
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 2040
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 2100
aaatga 2106
<210> 2
<211> 579
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ala Ala Gln Pro Phe Thr Pro Pro Thr Val Lys
20 25 30
Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile
35 40 45
Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile
50 55 60
Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala
65 70 75 80
Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr
85 90 95
Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val
100 105 110
Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp
115 120 125
Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe
130 135 140
Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp
145 150 155 160
Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala Ser
165 170 175
Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn
180 185 190
Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp
195 200 205
Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro His Leu Pro
210 215 220
Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala
225 230 235 240
Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp
245 250 255
Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile
260 265 270
Ser Val Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His
275 280 285
Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe
290 295 300
Ser Arg Leu Glu Val Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe
305 310 315 320
Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln
325 330 335
Arg Ala Val Ser Val Asn Pro Gly Lys Gly Ser Glu Pro Lys Ser Cys
340 345 350
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
355 360 365
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
370 375 380
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
385 390 395 400
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
405 410 415
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
420 425 430
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
435 440 445
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
450 455 460
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
465 470 475 480
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
485 490 495
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
500 505 510
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
515 520 525
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
530 535 540
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
545 550 555 560
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
565 570 575
Pro Gly Lys
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ttcaccccgc ccaccgtgaa g 21
<210> 4
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tctgtaaatc ccggtaaaga gcccaaatct tgtgac 36
<210> 5
<211> 99
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaatctcgag gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt 60
tccaggttcc actggtgact tcaccccgcc caccgtgaa 99
<210> 6
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtcacaagat ttgggctctt taccgggatt tacaga 36
<210> 7
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtcacgcgtt catttacccg gagacag 27
Claims (6)
1.一种编码重组人免疫球蛋白ε和γ的Fc融合蛋白的核苷酸序列,其特征在于,核苷酸序列如SEQ ID NO.1所示。
2.一种重组人免疫球蛋白ε和γ的Fc融合蛋白,其特征在于,重组人免疫球蛋白ε和γ的Fc融合蛋白的氨基酸序列如SEQ ID NO.2所示,由权利要求1所述的核苷酸序列编码表达获得。
3.一种权利要求2所述融合蛋白的表达方法,包括如下步骤:构建重组质粒,导入CHO细胞中,经筛选获得阳性克隆,表达融合蛋白。
4.如权利要求2所述融合蛋白在抗过敏药物中的应用。
5.一种CHO宿主细胞株,其特征在于含有权利要求3中所述的表达载体。
6.一种表达载体,其特征在于,表达载体包含权利要求1所述的核苷酸序列并通过基因工程手段构建获得;构建方法为overlap PCR;构建时所用的PCR引物如SEQ ID NO.3,SEQID NO.4,SEQ ID NO.5,SEQ ID NO.6,SEQ ID NO.7所示。
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