CN117327681A - 促进哺乳动物细胞表达蛋白正确折叠的分子伴侣质粒及其应用 - Google Patents
促进哺乳动物细胞表达蛋白正确折叠的分子伴侣质粒及其应用 Download PDFInfo
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Abstract
本发明公开了一种促进哺乳动物细胞表达蛋白正确折叠的分子伴侣质粒,其特征在于含有P4HB‑IRES‑HSPA8融合基因,其中P4HB基因的核酸序列如SEQ ID No.1所示,HSPA8基因的核酸序列如SEQ ID No.3所示,IRES的核酸序列如SEQ ID No.5所示。还公开了其在促进哺乳动物细胞表达蛋白正确折叠中的应用。本发明还公开了P4HB蛋白与HSPA8蛋白联合在促进哺乳动物细胞表达蛋白正确折叠中的应用。本发明的分子伴侣质粒可以有效帮助FAP蛋白实现正确折叠。
Description
技术领域
本发明涉及一种促进哺乳动物细胞表达蛋白正确折叠的分子伴侣质粒及其应用,属于蛋白表达技术领域。
背景技术
重组蛋白表达技术是指将编码目标蛋白的外源基因通过分子克隆的方法插入表达载体,随后使用表达载体侵染宿主细胞,通过宿主细胞自身的转录和翻译机制进行蛋白表达生产,再通过分离纯化获得目标蛋白的方法,至今已有50多年的历史。常用的表达用的宿主细胞包括大肠杆菌、酵母、昆虫和哺乳动物细胞等。
驯化后的哺乳动物细胞是现代重组蛋白生物技术的重要组成部分,常用的此类表达系统包括HEK293和CHO。由于哺乳动物细胞具有高度复杂的蛋白折叠和翻译后修饰的机制,通过其表达的重组蛋白具备正确折叠和修饰,以及最接近体内的天然形式等特点。哺乳动物细胞通常用于生产分泌蛋白、跨膜蛋白胞外区、重组抗体等,为现代生物医药的发展奠定了基础。然而对于某些结构极其复杂的蛋白,哺乳动物细胞在蛋白大量表达的模式下也无法完成全部目标蛋白的正确折叠,造成纯化后获得构象不正确的蛋白,影响蛋白的收率和后期的使用。
分子伴侣是一类可促进蛋白正确折叠的辅助蛋白,以热休克蛋白和二硫键异构酶为主,在原核系统中,通常使用共转分子伴侣的方法提高蛋白的正确折叠的概率,因此使用较为频繁。对于哺乳动物细胞,现阶段暂时未有相关的辅助蛋白折叠的分子伴侣。本发明通过构建一个含两个分子伴侣蛋白的辅助质粒与目标蛋白质粒的共转,以实现协助所表达的目标蛋白的正确折叠。
发明内容
本发明的目的是提供一种促进哺乳动物细胞表达蛋白正确折叠的分子伴侣,能协助所表达的目标蛋白的正确折叠。
本发明采用的技术方案为:一种促进哺乳动物细胞表达蛋白正确折叠的分子伴侣质粒,其特征在于含有P4HB-IRES-HSPA8融合基因,其中P4HB基因的核酸序列如SEQ IDNo.1所示,HSPA8基因的核酸序列如SEQ ID No.3所示,IRES的核酸序列如SEQ ID No.5所示。
优选的,所述质粒的载体为pCMV。
本发明还公开了上述的分子伴侣质粒在促进哺乳动物细胞表达蛋白正确折叠中的应用。
优选的,所述表达的蛋白为FAP蛋白。
本发明还公开了P4HB蛋白与HSPA8蛋白联合在促进哺乳动物细胞表达蛋白正确折叠中的应用,所述P4HB蛋白的氨基酸序列如SEQ ID No.2所示,所述HSPA8蛋白的氨基酸序列如SEQ ID No.4所示。
优选的,所述表达的蛋白为FAP蛋白。
其中IRES=internalribosomeentrysite,是一个质粒上的元件,其作用是在一个启动子调控下同时表达两个不同的蛋白,该序列本身不会被翻译。
本发明使用脯氨酰内肽酶(Prolylendopeptidase,FAP)作为验证蛋白。FAP蛋白是一种II型跨膜丝氨酸蛋白酶,FAP在肿瘤组织和伤口愈合中的反应性基质成纤维细胞和类风湿性关节炎中的滑膜细胞上表达,参与细胞外基质的降解,参与许多细胞过程,包括组织重塑、纤维化、伤口愈合、炎症和肿瘤生长,是重要的癌症治疗药物靶点之一。此外,由于与脯氨酸相连的肽键对广谱性肽酶具有相对抗性,这表明脯氨酸内肽酶在含有脯氨酸的生物活性肽的代谢中起着重要的作用。该酶能水解许多含有脯氨酸的生物活性肽,如催产素、促甲状腺素释放激素、促黄体生成素释放激素、血管紧张素II、缓激肽、P物质、神经紧张素和抗利尿激素。因此,研究FAP具有重要的生物学意义。
FAP的胞外催化区段可通过蛋白重组表达技术进行生产,现有重组表达技术主要使用哺乳动物细胞系HEK293细胞来表达生产FAP蛋白。FAP蛋白需要形成同源二聚体以获得正确的酶切活性,然而通过HEK293细胞重组表达获得的蛋白多呈现为单体,需通过孵育后方可形成少量二聚体,影响蛋白的活性和后期应用。通过FAP质粒与本发明的分子伴侣辅助质粒的共转,有效形成了二聚化的FAP蛋白。
附图说明
图1:Helper-5分子伴侣质粒构建方式。
图2:FAP蛋白活性计算公式。
图3:使用Helper-5分子伴侣质粒前后重组FAP蛋白二聚程度。左:SE-HPLC测定表达后以及孵育后FAP的分子量,判定二聚体情况,从上到下:单转FAP,纯化后孵育0小时;单转FAP,纯化后孵育48小时;FAP和Hepler-5共转,纯化后孵育0小时。右:SDS-PAGE测试FAP蛋白纯度,单转表达和共转表达的FAP蛋白纯化后纯度为95%,且在整个孵育过程中蛋白纯度不变。
具体实施方式
下面结合实施例对本发明作进一步的说明,但实施例的描述不对本发明的保护范围产生任何限制。
除非另有定义,本文所使用的所有的技术术语和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同,本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
下列实施例中所用的物质或仪器,如果未进行特殊说明的话,均可以从常规的商用渠道获取。
实施例1分子伴侣辅助质粒的构建
先分别扩增P4HB、IRES、HSPA8三个基因片段,通过OverlapPCR得到全长4056bp的P4HB-IRES-HSPA8片段,然后通过同源重组方式使用HindIII+XbaI内切酶组合插入表达质粒pCMV(图谱https://www.sinobiological.com/cdna-clone/contol-vector-pcmv-untagged-cv001)中,获得Helper-5分子伴侣质粒。扩增模板和引物分别为:
P4HB扩增模板:P4HB质粒(义翘神州货号HG10827-CM),引物对为1022Helper-5-F1+1022Helper-5-R1;
HSPA8扩增模板:HSPA8质粒(义翘神州货号HG11329-CM),引物对为1022Helper-5-F3+1022Helper-5-R3;
IRES扩增模板:pLenti-CD86-P2A-BLAST-IRES-CD19-XH9-2质粒,引物对为1022Helper-5-F2+1022Helper-5-R2;
引物对序列见表1,同源重组后获得的P4HB-IRES-HSPA8串联片段核苷酸序列见图1。编码P4HB的蛋白的核苷酸序列见SEQ ID No.1,氨基酸序列见SEQ ID No.2。编码HSPA8的蛋白的核苷酸序列见SEQ ID No.3,氨基酸序列见SEQ ID No.4。编码IRES的核苷酸序列见SEQ ID No.5。
表1引物对序列
名称 | 序列 |
1022Helper-5-F1 | gtcaccgtcctgacacgaagcttgccgccaccatgctgcgccgcgctctg |
1022Helper-5-R1 | ttacagttcatctttcacagctttctga |
1022Helper-5-F2 | agctgtgaaagatgaactgtaagcccctctccctcccccccccctaacg |
1022Helper-5-R2 | caactgcaggtcccttggacatggttgtggccatattatcatcgtgt |
1022Helper-5-F3 | atgtccaagggacctgcagttggtattgatc |
1022Helper-5-R3 | actatagaatagggccctctagattaatcaacctcttcaatggtgggccctgag |
实施例2FAP蛋白表达载体构建
使用重组DNA技术分别将FAP蛋白的基因整合进入哺乳动物细胞表达载体pCMV中,后通过测序的方法验证插入基因的序列正确性。编码FAP蛋白的cDNA序列通过全基因合成方法制备得到,并通过XbaI和BshTI酶切位点整合至pCDNA3.4表达载体。编码FAP蛋白的cDNA序列见SEQ ID No.6,翻译后得到的氨基酸序列见SEQ ID No.7。
实施例3重组FAP蛋白的表达
用无血清CD培养基(义翘神州,货号SMM293-TI)传代培养HEK293细胞,单转FAP蛋白表达质粒时,调节细胞密度至1~2.5*106时将FAP蛋白表达质粒与转染试剂TF1(义翘神州内部专用试剂)混合,随后加入细胞中进行转染。转染后第1、3、5天添加293无血清加料液(义翘神州,货号M293-SUPI-100)。细胞培养7天后进行蛋白纯化。使用分子伴侣载体时,将FAP蛋白表达质粒与分子伴侣质粒以质量比1:1混合后与转染试剂TF1混合后转染细胞,后使用相同工艺加料和培养。
实施例4重组FAP蛋白的纯化
培养7天后收集培养液上清,过滤后加样至镍离子亲和层析柱,随后使用含10mM咪唑的Tris缓冲液(20mMTris,pH7.4,150mMNaCl,10mM咪唑)进行平衡。平衡完成后使用线性咪唑浓度梯度洗脱,收集含有目标蛋白的洗脱峰。洗脱缓冲液为20mMTris,pH7.4,150mMNaCl,1M咪唑。使用还原和非还原SDS-PAGE对纯化得到的FAP蛋白的纯度进行表征,使用SEC-HPLC表征重组FAP蛋白的聚集形式,结果如图3所示,可以看出,未添加Helper-5分子伴侣辅助质粒进行生产的FAP蛋白二聚体占比较低,孵育48小时后才达到25%左右。使用Helper-5分子伴侣辅助质粒共转表达,纯化得到的FAP蛋白二聚体比例占比达95%以上(图3),酶活性较单独表达FAP蛋白活性提高5倍(表2),证明含分子伴侣的Helper-5辅助质粒可以有效帮助FAP蛋白实现正确折叠。
实施例5
重组FAP蛋白酶活性测定:
试剂和材料:
测试缓冲液=50mMTris,1MNaCl,1mg/mLBSA,pH7.5
测试底物=Z-GlyPro-AMC(Bachem货号M-1300),溶解于DMSO后配置成10mM母液材料=96孔Maxisorp板(Costar货号3915),读板器(SpectraMaxPlus,MolecularDevices)。
测试方法:
1、将重组FAP蛋白使用测试缓冲液稀释至0.2μg/mL;
2、将底物使用测试缓冲液稀释至100μM;
3、在孔板的一个样品孔中添加50μL的0.2μg/mL重组FAP蛋白,并加入50μL的100μM底物溶液,底物加入时间点为反应开始时间。在孔板的另一个样品中添加50μL的100μM底物溶液和50μL的测试缓冲液,作为对照;
4、底物加入后反应5分钟,使用读板器的动力学模式在激发波长380nm和吸收波长460nm持续读取荧光信号变化;
5、酶活性计算:公式见图2,其中conversionfactor=71.2pmole/RFU(根据AMC底物的荧光信号和量之间关系做标曲测定),Vmax=(RFUFAP-RFUBlank)/5。
对于单转FAP的条件,由于蛋白二聚体形成较少,因此需要通过孵育的方式获得一定量的二聚体,孵育的时间是0hr,即不孵育直接测试,以及48hr,即4℃下孵育48小时待一定程度的二聚体出现后再进行酶活和SEC的测定。共转Hepler-5的条件下获得的FAP蛋白不需要孵育大多数情况下就是二聚体,因此为不孵育的0hr的条件。
表2.重组FAP蛋白的二聚体程度和对应的活性测试结果
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (6)
1.P4HB蛋白与HSPA8蛋白联合在促进哺乳动物细胞表达蛋白正确折叠中的应用,其特征在于所述P4HB蛋白的氨基酸序列如SEQ ID No.2所示,所述HSPA8蛋白的氨基酸序列如SEQ ID No.4所示。
2.根据权利要求1所述的应用,其特征在于:所述表达的蛋白为FAP蛋白。
3.一种促进哺乳动物细胞表达蛋白正确折叠的分子伴侣质粒,其特征在于含有P4HB-IRES-HSPA8融合基因,其中P4HB基因的核酸序列如SEQ ID No.1所示,HSPA8基因的核酸序列如SEQ ID No.3所示,IRES的核酸序列如SEQ ID No.5所示。
4.根据权利要求3所述的分子伴侣质粒,其特征在于所述质粒的载体为pCMV。
5.权利要求3或4所述的分子伴侣质粒在促进哺乳动物细胞表达蛋白正确折叠中的应用。
6.根据权利要求1所述的应用,其特征在于:所述表达的蛋白为FAP蛋白。
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