CN116790670A - Ythdf3过表达载体的应用、cho细胞系的应用、重组蛋白表达系统 - Google Patents
Ythdf3过表达载体的应用、cho细胞系的应用、重组蛋白表达系统 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,具体涉及YTHDF3过表达载体的应用、CHO细胞系的应用、重组蛋白表达系统。本发明将YTHDF3过表达载体转染至CHO细胞构建过表达YTHDF3的CHO细胞系,该CHO细胞系能够显著提高YTHDF3的mRNA和蛋白表达水平,同时该CHO细胞系作为重组蛋白表达用宿主细胞,相比野生型CHO细胞系,提升重组蛋白表达量,有效克服了目前CHO细胞表达系统存在的表达水平较低的问题;进一步的,本发明以含转座子元件的出发载体,构建YTHDF3过表达载体,并且通过PBase持续高效催化YTHDF3过表达载体整合到CHO细胞的基因中,实现CHO细胞中YTHDF3的稳定过表达。
Description
技术领域
本发明属于基因工程技术领域,具体涉及YTHDF3过表达载体的应用、CHO细胞系的应用、重组蛋白表达系统。
背景技术
中国仓鼠卵巢(Chinesehamsterovary,CHO)细胞是目前应用最为广泛的哺乳动物细胞表达系统,但仍突出存在重组蛋白表达量较低和异质性等问题,无法满足用于疫苗开发、临床治疗药物和基因疗法等方面日益增长的需求。因此,利用基因和细胞工程的方法进行CHO细胞重组表达研究,对于提高其转基因表达水平和表达稳定性、建立高产稳产的重组蛋白CHO细胞表达系统具有十分重要的意义和应用价值。
对于大多数细胞的mRNA来说mRNA的翻译在人类细胞中发现,m6A控制RBM(第一个抑癌基因)的RNA结构依赖的可及性,从而影响RNA-蛋白质的相互作用以进行生物调节。m6A是真核mRNA中最丰富、具有普遍特征的内部修饰,与RNA代谢的许多基本方面如翻译、剪接、稳定性和衰减有关。在m6A中被称为擦除器去甲基化酶(ALKBH5),其包含一些位于细胞质的阅读器YTHDF1、YTHDF2、YTHDF3等的m6A已被发现,这些基因能够识别RNA甲基化修饰的信息,并参与下游RNA的翻译、降解等过程。其中YTHDF3可通过核糖体蛋白在翻译过程中发挥重要作用。细胞质m6A阅读器YTHDF3(Cytoplasmic m6A reader YTHDF3)可与40S/60S亚基的核糖体蛋白结合进而调控基因表达,主要参与染色质修饰、细胞周期、染色体组织、翻译延伸等,以维持染色质的结构或控制特定基因的表达,这提示YTHDF3在调控基因表达调控方面具有重要作用。但以YTHDF3过表达为途径,提高CHO细胞系重组蛋白表达的技术及应用未见报道。
发明内容
为了克服现有技术的缺陷,本发明的目的之一在于提供YTHDF3过表达载体在构建重组蛋白表达系统方面的应用,能够提高重组蛋白表达量。
本发明的目的之二在于过表达YTHDF3的CHO细胞系在作为重组蛋白表达用宿主细胞方面的应用,能够提升重组蛋白表达量。
同时,本发明的目的之三在于提供一种重组蛋白表达系统,以本发明提供的过表达YTHDF3的CHO细胞系为宿主细胞,提升重组蛋白表达量。
为了实现上述发明目的,本发明采用的技术方案如下:
YTHDF3过表达载体在构建重组蛋白表达系统方面的应用。
在本发明的具体实施例中,所述YTHDF3过表达载体是通过将YTHDF3编码基因插入出发载体构建而成。
具体的,所述YTHDF3编码基因根据YTHDF3的CDS序列设计,其核酸序列如SEQ IDNO.1所示。
可选的,所述出发载体为piggyBac。
可选的,所述YTHDF3过表达载体是通过将YTHDF3编码基因插入LAC启动子下游构建而成。进一步的,所述YTHDF3过表达载体是通过将YTHDF3编码基因片段、抗性筛选片段依次插入到出发载体的LAC启动子下游构建而成。
一种过表达YTHDF3的CHO细胞系作为重组蛋白表达用宿主细胞方面的应用。
在本发明的具体实施例中,所述CHO细胞系由将带有转座子元件的YTHDF3过表达载体在转座酶作用下转染CHO细胞构建而成。
一种重组蛋白表达系统,由CHO细胞系宿主细胞、带有转座子元件的YTHDF3过表达载体、转座酶、包含重组蛋白编码基因的表达载体构建而成。
可选的,所述重组表达系统包括将带有转座子元件的YTHDF3过表达载体、转座酶表达质粒共转染CHO细胞系宿主细胞构建过表达YTHDF3的CHO细胞系,然后将包含重组蛋白编码基因的表达载体转染至过表达YTHDF3的CHO细胞系构建而成;
其中带有转座子元件的YTHDF3过表达载体为将如SEQ ID NO.1所示核苷酸序列插入piggyBac载体的LAC启动子下游构建而成。
本发明有益效果:
(1)本发明创造性的将YTHDF3过表达载体应用于构建重组蛋白表达系统,提高重组蛋白表达量。具体的,将YTHDF3过表达载体转染至CHO细胞构建过表达YTHDF3的CHO细胞系,该CHO细胞系能够显著提高YTHDF3的mRNA和蛋白表达水平,同时该CHO细胞系作为重组蛋白表达用宿主细胞,相比野生型CHO细胞系,提升重组蛋白表达量有效克服了目前CHO细胞表达系统存在的表达水平较低的问题;
(2)进一步的,本发明根据中国仓鼠的目的基因设计合成YTHDF3的编码基因核酸序列,如SEQ ID NO.1所示,整合至具有转座子元件的出发载体,构建YTHDF3过表达载体,并且通过PBase持续高效催化YTHDF3过表达载体整合到CHO细胞的基因中,实现CHO细胞中YTHDF3的稳定过表达。
附图说明
图1为本发明实施例1构建的YTHDF3过表达载体示意图。
图2为本发明实施例3中qPCR法检测CHO细胞中过表达YTHDF3 mRNA相对表达量结果图;其中Control:正常CHO细胞对照组,OE YTHDF3:过表达YTHDF3的CHO细胞组;
图3为本发明实施例5Western blot法检测CHO细胞中重组阿达木单抗的相对表达量结果图;Control:正常CHO细胞对照组,OE YTHDF3:过表达YTHDF3的CHO细胞组。
具体实施方式
下面结合具体实施方式对本发明作进一步描述,但本发明的保护范围并不仅限于此;
实施例1构建YTHDF3过表达载体
本实施提供YTHDF3过表达载体,其构建方法为:
1)按照NCBI GenBank中国仓鼠YTHDF3的序列(编号:NW003614179),根据表达载体的设计原则,设计编码YTHDF3的核酸片段序列为SEQ ID NO.1所示;
2)以piggyBac载体(购自海星生物科技有限公司)为出发载体,通过酶切、连接的方式将步骤1)合成的核酸片段插入出发载体的LAC启动子的下游,构建获得表达YTHDF3的转座子载体,其结构示意图如图1所示。
实施例2构建过表达YTHDF3的CHO细胞系
本实施例提供过表达YTHDF3的CHO细胞系,其构建过程为:
1)准备用于转染的细胞:将CHO-S细胞在含10%灭活胎牛血清的DMEM/F12培养基中于37℃和5%CO2条件下进行生长培养。转染前将2.0×105个CHO-S细胞接种于24孔培养板中,铺板培养24h后当细胞融合度达到约85%时用于细胞转染;
2)转染:转染准备前换液成为0.5mL无血清培养基,加入50μL DMEM/F12培养基,采用脂质体转染法将实施例1构建的表达YTHDF3的转座子载体(2.5μg/孔)在转座酶的作用下转染至按照步骤1)准备的细胞,构建获得过表达YTHDF3的CHO细胞系,作为overexpression(OE)组;
采用脂质体转染法仅将出发载体piggyBac在转座酶作用下转染至在按照步骤1)制备的细胞,作为对照组(control);
脂质体转染法具体为:
(1)以24孔板为例,取一个1.5ml离心管标记载体名称,将50μL的基础DMEM/F12培养基加入管内,再吸取0.6μg的YTHDF3质粒(实施例1构建的表达YTHDF3的转座子载体)与0.2μg的转座酶,分别加入到管内,充分混匀,室温静置五分钟;
(2)再取一离心管,标记为转染试剂管,将50μL基础DMEM/F12培养基加入管内,再添加2μL转染试剂Lipofectamine 2000于管内,充分混匀,室温静置五分钟;
(3)将步骤(2)中的混合液滴加于步骤1中的质粒管中,充分混匀,室温静置25min。
(4)将前一日铺板用于转染的细胞从培养箱取出,吸弃旧培养基,每孔用1×PBS缓冲液洗涤两遍,随后加入500μL的新的不含双抗的DMEM/F12的减血清培养基;
(5)将步骤(3)中混合液滴加至孔中,轻轻晃动培养板使混合液分布均匀后放置于培养箱中培养。孵育5h时后,取出培养板,更换新鲜完全培养基继续培养;
3)筛选:转染48h后,使用潮霉素(hygro)筛选,细胞汇合度达到80%后,按照1:2~1:3传代,继续药物筛选培养获得转染后稳定表达的细胞系。
实施例3过表达效率检测
分别收集转染后筛选的稳定表达的正常CHO细胞对照组(Control)和过表达YTHDF3的CHO细胞组(OE),采用qPCR实验在mRNA水平检测各组YTHDF3的表达水平,具体实验方法如下:
1)qPCR实验
分别设计并合成靶基因YTHDF3和内参基因β-actin进行qPCR检测的引物,收集培养的Control组和OE组CHO细胞系,分别用Trizol提取各组细胞的总RNA,然后用All-in-one1st Strand cDNA Synthesis SuperMix试剂盒(购自近岸蛋白质科技股份有限公司)逆转录总RNA为cDNA。cDNA以1∶4的比例稀释后作为模板;使用20.0μL反应体系:分别加入SYBRGreen Mix 10.0μL,稀释后的模板2.0μL,引物(F/R)各1.0μL,无酶水6.0μL。反应条件为:95℃预变性30s,扩增阶段(95℃10s、60℃30s,45个循环反应)。融解曲线阶段为95℃15s,60~95℃1min。最后,根据2-△△CT算法计算YTHDF3mRNA的相对表达量。结果表明(图2),OE组CHO细胞系中YTHDF3的mRNA表达量显著提高至control组CHO细胞的8.84±1.22倍。
2)Western blot实验
采用Western blot检测分析YTHDF3蛋白在OE组和control组CHO细胞系中的表达。具体地,用冰冷的PBS洗涤细胞两次,并用RIPA缓冲液裂解细胞,将两组CHO细胞培养上清液加入5×SDS样品缓冲液煮水浴煮10min充分变性。分别取10μL蛋白样品经10%SDS-PAGE凝胶电泳分离后,湿转法转移至硝酸纤维素膜。5%脱脂牛奶的TBST缓冲液中封闭膜2h,并分别与Anti-YTHDF3(1:1000)、Anti-β-actin(1:1000)孵育过夜,然后与Anti-Rabbit IgG(1:10000)孵育2h。TBST洗膜三次后加化学发光显色液显影,在凝胶成像仪上观察并采集图像结果,分析待测蛋白的表达水平。结果表明,与Control组CHO细胞相比,OE组CHO细胞中YTHDF3蛋白的表达量显著提高。
实施例4过表达YTHDF3的CHO细胞的生物学特性验证
通过检测细胞增殖等细胞生物学特性验证YTHDF3基因过表达CHO细胞系能否进行正常的生长和传代培养:
以野生型CHO-S细胞作为对照(Control组),对获得的YTHDF3基因过表达CHO细胞(OE组)进行细胞生长特性验证,包括细胞形态和生长状态观察、CCK-8法检测细胞增殖以及悬浮计数检测活细胞密度(viablecelldensity,VCD)。采用CCK-8试剂盒(CellCountingKit-8试剂盒,碧云天Beyotime生物公司)检测细胞增殖;将野生型和YTHDF3基因过表达的CHO细胞以5×105/mL悬浮7天,每天收集细胞通过BioTech细胞计数仪(上海睿钰生物科技有限公司)检测VCD。结果表明,获得的过表达YTHDF3的CHO细胞株细胞生长状态、形态、细胞增殖、活细胞密度等生物学特性与野生型CHO细胞无显著性差别。这些结果提示,YTHDF3过表达的CHO细胞系能够正常进行生长增殖和传代培养。
实施例5重组蛋白表达
本实施例将实施例2筛选验证的过表达YTHDF3的CHO细胞系应用于重组蛋白表达,包括构建重组蛋白表达系统,具体构建过程为:
1)构建重组阿达木单抗(adalimumab)真核表达载体
构建真核表达载体所需的元件序列包括CMV启动子、SV40polyA、IRES、EF-1α启动子、阿达木单抗的HC链和LC链、杀稻瘟菌素(Blasticidin,BSD)抗性基因、嘌呤霉素(puromycin,Puro)抗性基因序列由通用生物(安徽)股份有限公司合成。首先,将LC和HC序列分别克隆至pCHO1.0表达载体(赛默飞世尔科技公司)的启动子EF2/CMV和CMV/EF1之后,获得对照真核表达载体pCHO-HL。随后,用CMV启动子和BSD抗性基因序列分别替换pCHO-HL表达载体中pEF2/CMV-LC-polyA-pCMV/EF1和Puro序列,构建载体pHC-BSD。然后,用LC和Puro抗性基因序列分别替换pHC-BSD载体中HC、BSD抗性基因序列,构建pLC-Puro载体。最后将LC-polyA-EF-1α表达盒克隆至pHC-BSD载体,构建成表达阿达木单抗的pLC-pHC双启动子真核表达载体;
2)转染和重组蛋白表达检测
将步骤1)构建的真核表达载体分别转染OE组和Control组CHO细胞,转染的细胞在含有杀稻温菌素(1mg/mL)的培养基中培养15d以筛选稳定转染的重组细胞池(cellpool)。随后将重组CHO细胞在六孔板中用3mL无蛋白、无血清、化学成分确定的CD CHO培养基(河南普诺易生物制品研究院有限公司)培养6d至细胞数达到1×107,每天收集细胞通过BioTech细胞计数仪(上海睿钰生物科技有限公司)检测细胞密度和活力,第7d离心收集上清液,以相同细胞数定量,用于重组阿达木单抗表达量检测分析。采用Westernblot检测分析阿达木单抗在OE组和Control组CHO细胞中的表达。如图3所示,结果表明,YTHDF3过表达的CHO细胞可明显提高重组阿达木单抗的表达水平,表达水平增加1.47±0.11倍。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.YTHDF3过表达载体在构建重组蛋白表达系统方面的应用。
2.如权利要求1所述的应用,其特征在于,所述YTHDF3过表达载体是通过将YTHDF3编码基因插入出发载体构建而成。
3.如权利要求2所述的应用,其特征在于,所述YTHDF3编码基因的核酸序列如SEQ IDNO.1所示。
4.如权利要求3所述的应用,其特征在于,所述出发载体为piggyBac。
5.如权利要求4所述的应用,其特征在于,所述YTHDF3过表达载体是通过将YTHDF3编码基因片段插入出发载体的LAC启动子下游构建而成;
进一步的,所述YTHDF3过表达载体是通过将YTHDF3编码基因片段、抗性筛选片段依次插入到出发载体的LAC启动子下游构建而成。
6.如权利要求5所述的应用,其特征在于,包括首先将YTHDF3过表达载体在转座酶的作用下转染CHO细胞,构建过表达YTHDF3的CHO细胞,然后将重组蛋白表达质粒转染过表达YTHDF3的CHO细胞。
7.一种过表达YTHDF3的CHO细胞系作为重组蛋白表达用宿主细胞方面的应用。
8.如权利要求7所述的应用,其特征在于,所述CHO细胞系由将带有转座子元件的YTHDF3过表达载体在转座酶的作用下转染CHO细胞构建而成。
9.一种重组蛋白表达系统,其特征在于,由CHO细胞系宿主细胞、带有转座子元件的YTHDF3过表达载体、转座酶、包含重组蛋白编码基因的表达载体构建而成。
10.如权利要求10所述的重组蛋白表达系统,其特征在于,包括将带有转座子元件的YTHDF3过表达载体在转座酶作用下转染CHO细胞系宿主细胞构建过表达YTHDF3的CHO细胞系,然后将包含重组蛋白编码基因的表达载体转染至过表达YTHDF3的CHO细胞系构建而成;
其中带有转座子元件的YTHDF3过表达载体为将如SEQ ID NO.1所示核苷酸序列插入piggyBac载体的LAC启动子下游构建而成。
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