CN117003892A - 一种含有PADRE和Fc的gE融合蛋白及其制备方法和应用 - Google Patents
一种含有PADRE和Fc的gE融合蛋白及其制备方法和应用 Download PDFInfo
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- CN117003892A CN117003892A CN202310999189.9A CN202310999189A CN117003892A CN 117003892 A CN117003892 A CN 117003892A CN 202310999189 A CN202310999189 A CN 202310999189A CN 117003892 A CN117003892 A CN 117003892A
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Abstract
本发明公开了一种含有PADRE和Fc的gE融合蛋白及其制备方法和应用,gE融合蛋白包括水痘‑带状疱疹病毒(VZV)糖蛋白E(gE)胞外区段、通用DR Th表位序列(PADRE)和人源IgG1抗体的Fc序列(Fc)。本发明利用哺乳动物细胞表达载体pXNM3.0将所述融合蛋白在CHOK1细胞中表达,采用亲和层析Protein A、疏水色谱Capto Phenyl ImpRes、分子筛色谱Sephacryl S‑300 High Resolution纯化后,得到融合蛋白。本发明中的gE融合蛋白具有较好的免疫原性,同单纯gE蛋白相比,能诱导更强的体液免疫和细胞免疫水平。
Description
技术领域
本发明涉及生物医学领域,具体涉及一种含有PADRE和Fc的gE融合蛋白及其制备方法和应用。
背景技术
水痘-带状疱疹病毒(varicella zoster virus,VZV)又称3型人疱疹病毒,是一种人类α疱疹病毒。水痘-带状疱疹病毒在正常人群中的原发性感染,会在儿童时期导致水痘。感染VZV后,病毒会于人体神经节内潜伏,当各种原因导致的免疫力下降时,病毒会再度被激活并大量复制,会导致带状疱疹(herpes zoster,HZ),并常常伴随有带状疱疹后遗神经痛(Post-herpetic neuralgia,PHN)。在免疫功能受损的人群中VZV感染可能伴有髓炎、脑髓炎等严重的并发症。VZV的基因组大小约为125kb,编码约69种蛋白,编码的糖蛋白有8个,包括gB、gC、gE、gH、gI、gK、gL、gM。其中糖蛋白E(glycoprotein E,gE)是宿主免疫系统识别的主要糖蛋白,也是病毒包膜和感染细胞膜上抗原性最强、含量最丰富的糖蛋白,同时它还广泛存在于在VZV颗粒的表面和宿主细胞的细胞膜和胞质中,能诱导细胞免疫和体液免疫。
水痘-带状疱疹病毒的传染性及强,主要通过空气飞沫和直接接触传播,据报道全世界50岁以上成人中约有90%的血清VZV检测呈阳性。儿童感染VZV后会引起发热,并出现周身性红色斑丘疹、疱疹和痂疹,有自限性。随着年龄增长,机体免疫力下降,带状疱疹的发病率呈逐渐增加,随着现在生活节奏加快,生活压力增加,带状疱疹的发病呈现年轻化的趋势。水痘-带状疱疹的发病严重影响人们的生活质量,特别是PHN的存在。PHN是带状疱疹最常见的后遗症,在带状疱疹中的发生率为10%~30%,疼痛可持续数月至数年,最长可达10年。
默沙东公司高剂量(约20000PFU)减毒活疫苗产品Zostavax,2005年被批准在50岁以上成人中用于预防带状疱疹,疫苗的不良反应主要以接种部位的疼痛、红斑和肿胀为主,大规模临床实验证明,在50-59岁年龄阶段人群中,疫苗的保护率为69.8%,大于60岁以上人群中保护率为51%,随年龄的增加保护率逐渐降低,≥80岁以上人群中保护率只有18%。在预防PHN方面,对≥60岁以上人群,疫苗保护率为39%。有文献报道Zostavax疫苗接种5~8年后疫苗效力持续下降,超过8年后统计学上不在显著。国内目前有长春百克的带状疱疹减毒活疫苗于2023年02月获批上市。
葛兰素史克公司的佐剂带状疱疹亚单位疫苗Shingrix于2017年经FDA批准上市,用于在50岁以上成人中预防带状疱疹。该疫苗有两部分组成,一是由CHO细胞表达的截短VZV糖蛋白E,一是AS01B佐剂系统。相对于减毒活疫苗Zostavax,佐剂亚单位疫苗Shingrix的副反应更严重,疫苗接种后7天内,试验组和安慰剂组受试者至少1种征集性症状的发生率分别为84.5%和33.7%,至少1种3级征集性AE的发生率分别为16.0%和2.5%。临床实验证明,该疫苗在≥50岁的受试者中,对HZ的总体保护率为97.16%。按50-59岁、60-69岁、≥70岁分层分析,本品对HZ的保护率在各年龄层相当,其中50-59岁为96.57%,60-69岁为97.36%,≥70岁为97.93%。在预防PHN反面,在≥50岁正常人群中,Shingrix可使PHN的发病率降低91.2%。在≥70岁正常人群中,降低88.8%。对疫苗效力的持续研究表明,疫苗的有效性可持续10年以上。AS01B佐剂系统含有3D-MPL、QS-21以及磷脂酰胆碱、胆固醇等成分,能显著提高疫苗的效力,但它有个极大的缺点是副反应程度和发生率都高。
公布号为CN110343722的专利公布了一种重组表达水痘-带状疱疹病毒v-Oka株截短型糖蛋白E的方法,该方法将截短的gE蛋白基因导入杆状病毒中,利用重构的杆状病毒感染昆虫细胞表达可溶性的gE蛋白。该方法易于筛选,批间稳定,但用昆虫细胞表达的蛋白相较于哺乳动物细胞糖基化方面有较大差异,且单独的蛋白并不能有效激活人体的体液及细胞免疫功能。
公布号为CN112870344专利公开了一种重组水痘带状疱疹疫苗的制备方法,该方法在CHO细胞中表达截短gE和IgG抗体Fc段的融合蛋白gE-Fc。重组蛋白经一系列色谱纯化,并经过病毒灭活后,得到了高度纯化的融合蛋白质。融合蛋白配合铝佐剂免疫动物可以产生高效价的血清中和抗体。有大量研究证明,带状疱疹疫苗发挥做用的主要是其细胞免疫功能决定的,虽然融合蛋白中的Fc可提高一定水平的细胞免疫功能,但该发明中使用佐剂为铝佐剂,单独的融合蛋白配合铝佐剂,可能不能高效激活人体的细胞免疫功能。
目前被批准用于预防VZV感染的疫苗有两种,一种是VZV减毒活疫苗(Oka株),低剂量用于预防儿童水痘,高剂量用于预防带状疱疹;一种是配合佐剂系统使用的基因工程重组亚单位疫苗。临床实验证明减毒活疫苗的副反应较小,但是保护效率及保护持久性较低,疫苗生产工艺复杂,放大生产受限;配合佐剂系统的重组亚单位疫苗,保护效率好持久性高,但疫苗副反应及强,佐剂系统中3D-MPL来源于沙门氏菌的脂多糖的脱毒产物,产量低,疫苗成本高。大量研究证明,细胞免疫功能在VZV感染中发挥着极其重要的作用,文献报道的其他基因工程重组亚单位疫苗,要么因抗原设计原因导致的抗原免疫原性较弱,要么是因为使用的佐剂系统为传统铝佐剂,细胞免疫功能弱。因此在预防水痘带状疱疹发病方面较弱。
发明内容
本发明的目的在于提供一种含有PADRE和Fc的gE融合蛋白及其制备方法和应用,以解决现有技术中的抗原免疫原性低,疫苗生产工艺复杂,疫苗副反应强和疫苗成本高的技术问题。
为实现上述目的,本发明提供了以下技术方案:
本发明提供的一种含有PADRE和Fc的gE融合蛋白,所述融合蛋白包括水痘-带状疱疹病毒(VZV)糖蛋白E(gE)胞外区段、通用DR Th表位序列(PADRE);人源IgG1抗体的Fc序列(Fc),编码gE、PADRE、Fc的基因序列能够在基因工程化细胞中表达含有PADRE和Fc的gE融合蛋白。
进一步的,所述含有PADRE和Fc的gE融合蛋白其氨基酸序列组合方式包括PADRE-gE-Fc、gE-PADRE-Fc、gE-Fc-PADRE、Fc-gE-PADRE、Fc-PADRE-gE、PADRE-Fc-gE;所述gE、PADRE、Fc之间通过连接肽连接,所述连接肽为GGS和/或GGGS和/或GGGGS和/或GSGSG连接肽。
进一步的,所述含有PADRE和Fc的gE融合蛋白其氨基酸序列组合方式优选为PADRE-gE-Fc,所述PADRE-gE-Fc的氨基酸序列如SEQ ID NO.7所示。
进一步的,所述的含有PADRE和Fc的gE融合蛋白在制备水痘-带状疱疹疫苗中的应用。
本发明提供的一种水痘-带状疱疹疫苗,所述疫苗由含有PADRE和Fc的gE融合蛋白和佐剂组合而成。
进一步的,所述佐剂为氢氧化铝佐剂、磷酸铝佐剂、含有皂素的中性脂质体佐剂、含有皂素的阳离子脂质体佐剂、含有皂素的阴离子脂质体佐剂,CpG佐剂、纳米乳剂、含有3D-MPL的佐剂中的任意一种或任意多种组合使用。
进一步的,所述水痘-带状疱疹病毒的疫苗的每个剂量单位中含有融合蛋白5~200μg,优选的,每个剂量单位中含有融合蛋白10~100μg,更优选的,每个剂量单位中含有融合蛋白20~80μg。
本发明提供的一种融合蛋白的制备方法,包括下述步骤:
S1、将经由密码子优化后的融合蛋白基因序列进行全基因合成,得到融合蛋白基因;
S2、将所述融合蛋白基因连接到哺乳动物表达载体pXNM3.0中,得到重组表达质粒;
S3、将所述重组表达质粒转染至CHO细胞,通过迷你细胞群的筛选和单克隆筛选,获得稳定表达重组蛋白的细胞株;
S4、培养所述的稳定表达重组蛋白的细胞株,收集发酵物上清进行纯化处理,得到融合蛋白。
进一步的,步骤S2中,所述的表达载体为携带GS筛选系统和/或携带博莱霉素抗性基因的质粒表达载体。
进一步的,步骤S3中,所述CHO细胞优选为CHO-K1细胞。
基于上述技术方案,本发明实施例至少可以产生如下技术效果:
抗原为融合蛋白,在其中融合表达了提高CD4+T活化增强抗体及杀伤T细胞表达的通用T细胞抗原表位肽和增强抗原稳定性,提高抗原递呈效率及表达量的Fc。小鼠试验证明其诱导的体液免疫和细胞免疫效果高于单纯的gE蛋白。同时有临床试验证明融合表达的元件安全性高,无毒副作用。
融合蛋白与含有QS-21的中性脂质体佐剂组成的疫苗,在佐剂成分减少的情况下,能诱导比上市更强的T细胞免疫反应和体液免疫反应。
附图说明
图1是本发明实施例表达载体PXNM3.0的结构图;
图2是本发明实施例融合蛋白(PADRE-gE-Fc)表达载体结构图;
图3是本发明实施例两次免疫gE蛋白特异性血清GMT效价图;
图4是本发明实施例gE特异性的CD4+T细胞反应图。
具体实施方式
下面将结合本发明实施例中的附图;对本发明实施例中的技术方案进行清楚、完整地描述;显然,所描述的实施例仅仅是本发明一部分实施例;而不是全部的实施例,基于本发明中的实施例;本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例都属于本发明保护的范围。
一种含有PADRE和Fc的gE融合蛋白,所述融合蛋白包括水痘-带状疱疹病毒(VZV)糖蛋白E(gE)胞外区段、通用DR Th表位序列(PADRE);人源IgG1抗体的Fc序列(Fc),编码gE、PADRE、Fc的基因序列能够在基因工程化细胞中表达含有PADRE和Fc的gE融合蛋白。
所述含有PADRE和Fc的gE融合蛋白其氨基酸序列组合方式包括PADRE-gE-Fc、gE-PADRE-Fc、gE-Fc-PADRE、Fc-gE-PADRE、Fc-PADRE-gE、PADRE-Fc-gE;所述gE、PADRE、Fc之间通过连接肽连接,所述连接肽为GGS和/或GGGS和/或GGGGS和/或GSGSG连接肽。
所述含有PADRE和Fc的gE融合蛋白其氨基酸序列组合方式优选为PADRE-gE-Fc,所述PADRE-gE-Fc的氨基酸序列如SEQ ID NO.7所示。
涉及到的序列如下:
编码水痘-带状疱疹病毒糖蛋白E胞外区的基因序列记为gE的核酸序列,如序列表SEQ ID NO.1所示;
编码通用DR Th表位序列记为PADRE的核酸序列,如序列表SEQ ID NO.2所示;
编码人源IgG1抗体的Fc序列记为Fc的核酸序列,如序列表SEQ ID NO.3所示;
gE的氨基酸序列如序列表SEQ ID NO.4所示;
PADRE的氨基酸序列如序列表SEQ ID NO.5所示;
Fc的氨基酸序列如序列表SEQ ID NO.6所示;
S1、融合蛋白密码子优化与全基因合成
对融合蛋白的基因进行密码子优化:避开常用的酶切位点;根据CHO细胞中密码子的偏好性,用频率高的密码子替代频率低的同义密码子,控制稀有密码子;控制序列中的GC含量在40%~60%,以提高mRNA的转录效率,同时避免高GC含量影响mRNA二级结构,进而影响翻译效率。在优化后的序列前面加上信号肽序列;序列的上游引入HindⅢ酶切位点,序列下游加入终止密码子及BamHⅠ酶切位点,进行核苷酸序列的全基因组合成。
S2、融合蛋白表达质粒的构建
含有全基因合成序列的克隆载体转化DH5α感受态细菌,然后大量扩增,提质粒后将克隆载体用限制性内切酶HindⅢ和BamHⅠ双酶切,同时用限制性内切酶HindⅢ和BamHⅠ双酶切表达载体pXNM3.0(如图1所示)。双酶切的克隆载体切胶回收融合蛋白基因部分,表达载体切胶回收骨架部分。二者用T4酶连接后转化DH5α感受态细菌,涂布含有氨苄抗性的平板进行筛选,阳性菌落挑斑扩增提质粒,然后用HindⅢ和BamHⅠ双酶切鉴定,正确的重组表达载体测序验证。
S3、稳转细胞株构建
鉴定正确的重组表达载体,增菌后大量提质粒,用Pvu I单酶切重组表达载体,切胶回收酶切线性化的载体,过滤除菌后待用。CHO-K1细胞复苏后连续传代两次以上,细胞活率大于95%时用于电转化。在洁净工作台中,向4mm电转杯内加入0.6ml的细胞悬浮液(约1×107个细胞),200ul线性化后的重组表达载体(约50μg),设置电转条件为电压为300V,电容900μF。电转结束后将细胞转移至含有30ml CD CHO培养基的细胞摇瓶中,37℃,5%二氧化碳浓度,125rpm条件下培养24小时。100g离心电转后细胞悬液10分钟,弃去上清,用含25μM MSX和200μg/ml博来霉素的CD CHO培养基重悬细胞,然后接种至24空板中,培养3周后用ELISA方法检测表达量,表达量高的3个细胞孔混合后用有限稀释法接种96孔板进行单克隆筛选,用单细胞成像设备在第0、1、2、3、7、15天拍照。15天后用ELISA方法检测表达量,表达量高的3株细胞进行冻存。稳定性研究后确定用于疫苗制备的细胞株,然后建立两级细胞库。
S4、目的产物表达
用OPM-CHO CDP9培养基复苏一只冻存的工作种子,在摇瓶中逐级放大,最后转移进5L的生物反应器内进行培养,接种密度为0.8×106cells/ml,设定培养参数为温度37℃、pH7.0、转速150r/min、溶解氧浓度40%。每日取样检测细胞活率、密度、乳酸含量、葡萄糖含量,培养至第3天,活细胞密度达到3×106cells/ml时加入补料培养基CDF18和CDF26,分别接入250ml和25ml。然后每隔1天加入相同体积的补料培养基。维持培养液中的葡萄糖含量在2g/L以上,当低于此浓度时补加葡萄糖浓度至4g/L。培养15天左右,当细胞活率低至70%时,终止培养。深层滤器过滤去除细胞及细胞碎片,收集细胞培养物上清液。
S5、融合蛋白纯化
细胞培养物上清调节pH至7.5,用pH7.5含150mM氯化钠的40mM PB缓冲液平衡亲和层析protein A至紫外吸收基线水平,pH稳定,然后将细胞上清液过柱,用相同平衡缓冲液平衡至紫外吸收基线水平后,pH3.0~4.0的乙酸-乙酸钠缓冲液,洗脱目的物。纯化产物低pH灭活(pH 3.0~4.0,18~25℃放置60min),灭活后产物加入1M硫酸铵并调节pH至7.5,用pH7.5 50mM PB缓冲液+1M硫酸铵缓冲液平衡疏水层析柱Capto Phenyl ImpRes至紫外吸收基线水平,pH稳定,然后将灭活液过柱,相同缓冲液平衡层析柱,最后用pH7.550mM PB缓冲液线性洗脱目的物。疏水纯化产物经分子筛色谱Sephacryl S-300High Resolution分子筛色谱纯化并换液,获得纯化后的蛋白。纯化后蛋白经15nm滤器纳滤及0.22μm滤膜除菌过滤后获得疫苗原液。
S6、疫苗配制
(1)脂质体的制备(乙醇注入法;100ml体积,DOPC和胆固醇浓度分别为4mg/ml和1mg/ml):分别称取400mgDOPC和100mg胆固醇;然后将DOPC和胆固醇完全溶于10ml无水乙醇,并混合均匀,即为有机相;将10ml有机相注入90ml的10mM PBS缓冲盐溶液(pH为7.0)中,制成脂质体初乳;再使用高压微射流均质机整粒至脂质体粒径为100nm左右;然后,通过透析方法去除乙醇;最后使用0.22μm除菌滤器过滤除菌,即为脂质体成品;
(2)佐剂的配制:取5ml脂质体,加入1ml皂苷QS-21溶液(浓度为1mg/ml),并搅拌均匀,即为佐剂;
(3)疫苗的配制(10ml体积;gE融合蛋白浓度为100μg/ml;免疫增强剂皂苷QS-21为100μg/ml;脂质体成分:DOPC和胆固醇分别为2mg/ml和0.5mg/ml)
向佐剂中加入gE融合蛋白溶液,再使用10mM PBS缓冲盐溶液(pH为7.0)补足总体积至10ml,并搅拌均匀,即为gE融合蛋白佐剂疫苗;加入gE融合蛋白溶液体积(ml)计算公式为:加入gE融合蛋白的质量(μg)/gE融合蛋白浓度(μg/ml)=gE融合蛋白理论分子量*疫苗中gE浓度(μg/ml)*疫苗体积(ml)/[gE理论分子量*gE融合蛋白浓度(μg/ml)]=gE融合蛋白理论分子量*100*10/(gE融合蛋白理论分子量*gE融合蛋白浓度)。
PADRE-gE-Fc、gE-PADRE-Fc、gE-Fc-PADRE、Fc-gE-PADRE、Fc-PADRE-gE、PADRE-Fc-gE;均按照S1-S6制得疫苗。
PADRE-gE-Fc的氨基酸序列如序列表SEQ ID NO.7所示。
S7、疫苗免疫动物
实验动物为6-8周龄的C57BL/6雌性小鼠;动物免疫及饲养在无特定病原体条件下进行。为模拟在自然环境中水痘-带状疱疹病毒的感染,在进行候选疫苗免疫前35天,用水痘-带状疱疹病毒(包含不少于3.3lg PFU活病毒,0.5ml)颈部皮下预敏免疫小鼠。在第0天和28天,腿部肌肉注射候选疫苗,注射剂量为每只小鼠50ul。在二免后28天采用眼球取血法采血,采血后处死小鼠。采集好的全血2~8℃下静置过夜,第二天3000rpm离心30min,吸取顶部血清。用间接ELISA法测小鼠血清效价,并计算GMT值。小鼠脾脏分离脾脏细胞,用胞内细胞因子染色法检测gE特异性分泌INF-γ和IL-2的CD4+T细胞数量,评价细胞免疫水平。
疫苗免疫原性分析
(1)血清中gE蛋白结合抗体检测
利用间接ELISA的方式检测所有个体小鼠二免后28天血清样本中的总gE特异性抗体。流程为用碳酸盐缓冲液将gE蛋白包被至96孔板中,包被量为500ng/孔,4℃包被过夜,用含BSA的TPBS溶液封闭,TPBS溶液洗板3次,然后所有个体小鼠血清按不同稀释度稀释后,加入孔板中,37℃孵育1小时,TPBS溶液洗板3次,然后用HRP标记的羊抗鼠二抗37℃孵育1小时,TPBS溶液洗板3次后用TMB显色液显色10min,加入0.2M硫酸终止反应,酶标仪读取OD450处数值。以一免前血清混合样品的读数的3倍作为Cut-Off值,判定免疫后血清效价,一免和二免后gE结合抗体比较如图3所示。
(2)胞内细胞因子流式细胞仪检测
二免后28天,试验小鼠取脾脏,制备脾单细胞悬液,调整细胞浓度后用裂红液裂解单细胞悬液中的红细胞,用特异性肽池刺激脾脏细胞,使其分泌细胞因子,加入ContainingBrefeldin A分泌阻断剂后阻断分泌,然后经过细胞活死染色,表面受体FcR阻断,CD3、CD45、CD4表面染色,固定破膜和IL-2、IFN-γ胞内染色等步骤后,利用流式细胞仪检测分泌IL-2、IFN-γ的CD4+T细胞的数量,检测数量如图4所示。
Claims (10)
1. 一种含有PADRE和Fc的gE融合蛋白,其特征在于,所述融合蛋白包括水痘-带状疱疹病毒(VZV)糖蛋白E(gE)胞外区段、通用DR Th表位序列(PADRE);人源IgG1抗体的Fc序列(Fc),编码gE、PADRE、Fc的基因序列能够在基因工程化细胞中表达含有PADRE和Fc的gE融合蛋白。
2. 根据权利要求1所述的一种含有PADRE和Fc的gE融合蛋白,其特征在于,所述含有PADRE和Fc的gE融合蛋白其氨基酸序列组合方式包括PADRE-gE-Fc、gE-PADRE-Fc、gE-Fc-PADRE、Fc-gE-PADRE、Fc-PADRE-gE、PADRE -Fc-gE;所述gE、PADRE、Fc之间通过连接肽连接,所述连接肽为GGS和/或GGGS和/或GGGGS和/或GSGSG连接肽。
3. 根据权利要求2所述的一种含有PADRE和Fc的gE融合蛋白,其特征在于,所述含有PADRE和Fc的gE融合蛋白其基酸序列组合方式优选为PADRE-gE-Fc,所述PADRE-gE-Fc的氨基酸序列如SEQ ID NO.7所示。
4.根据权利要求1-3任意一项所述的含有PADRE和Fc的gE融合蛋白在制备水痘-带状疱疹疫苗中的应用。
5.一种水痘-带状疱疹疫苗,其特征在于:所述疫苗由权利要求1~3任意一项所述的含有PADRE和Fc的gE融合蛋白和佐剂组合而成。
6.根据权利要求5所述的水痘-带状疱疹疫苗,其特征在于:所述佐剂为氢氧化铝佐剂、磷酸铝佐剂、含有皂素的中性脂质体佐剂、含有皂素的阳离子脂质体佐剂、含有皂素的阴离子脂质体佐剂,CpG佐剂、纳米乳剂、含有3D-MPL的佐剂中的任意一种或任意多种组合使用。
7.根据权利要求5所述的水痘-带状疱疹疫苗,其特征在于:所述水痘-带状疱疹疫苗的每个剂量单位中含有融合蛋白5~200μg,优选的,每个剂量单位中含有融合蛋白10~100μg,更优选的,每个剂量单位中含有融合蛋白20~80μg。
8.一种融合蛋白的制备方法,用于制备权利要求1~3任意一项所述的含有PADRE和Fc的gE融合蛋白,其特征在于,包括下述步骤:
S1、将经由密码子优化后的融合蛋白基因序列进行全基因合成,得到融合蛋白基因;
S2、将所述融合蛋白基因连接到哺乳动物表达载体pXNM3.0中,得到重组表达质粒;
S3、将所述重组表达质粒转染至CHO细胞,通过迷你细胞群的筛选和单克隆筛选,获得稳定表达重组蛋白的细胞株;
S4、培养所述的稳定表达重组蛋白的细胞株,收集发酵物上清进行纯化处理,得到融合蛋白。
9.根据权利要求8所述的融合蛋白的制备方法,其特征在于:步骤S2中,所述的表达载体为携带GS筛选系统和/或携带博莱霉素抗性基因的质粒表达载体。
10.根据权利要求8所述的融合蛋白的制备方法,其特征在于:步骤S3中,所述CHO细胞优选为CHO-K1细胞。
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