CN114540392A - 一种碱性成纤维细胞生长因子制备方法及其应用 - Google Patents
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Abstract
本发明公开了一种碱性成纤维细胞生长因子制备方法,包括以下步骤:融合质粒构建;工程菌构建;将上述工程菌培养于培养基,当大肠杆菌的OD值达到2值,温度调为32℃诱导16小时,收集菌体,破碎,离心取上清;将上清37℃下水浴加热,37℃离心,取沉淀部分;向沉淀部分加入预冷的PBS后负40度冷冻2小时,超声溶解,低温离心取溶解部分;溶解部分加入带自备的His‑Tag的EK酶,冰水中酶切12小时;酶切完成后,蛋白质溶液过Ni‑NTA层析柱,ELP‑IDP标签以及带His‑Tag的EK酶将结合至层析柱上,收集流穿部分。bFGF在促进细胞生长增殖方面应用。本发明所制备bFGF的可溶性得到显著提升;同时大幅度简化纯化步骤,具有良好的经济价值。
Description
技术领域
本发明属于蛋白质技术领域,具体涉及一种碱性成纤维细胞生长因子制备方法及其应用。
背景技术
碱性成纤维细胞生长因子(Basic fbroblast growth factor,bFGF,或者FGF-2)是FGF家族的重要成员。作为单链蛋白,bFGF具有146个氨基酸和pI为9.6。研究报道显示bFGF在不同的细胞和器官系统中具有多效性。bFGF可以刺激平滑肌细胞生长,伤口愈合,以及损伤组织修复。此外,bFGF对肺,生殖系统,神经系统,皮肤,眼睛,造血系统,肌肉,骨骼和消化系统的分化起重要作用。相关研究表明bFGF可以预防大鼠脑缺血再灌注损伤。
基于bFGF重要作用,医药工业上对特别是在无血清细胞培养基添加剂对bFGF有巨量需求。从动物器官和组织(包括腺瘤,脑,下丘脑,胸腺和肾)提取的bFGF,由于bFGF在组织中的含量非常低,因此几乎不可能从动物组织中大量获取bFGF。1986年研究者克隆bFGF的人类基因并进行外源表达,在随后的几十年中,bFGF在各类表达系统进行重组表达。常规的表达系统如大肠杆菌表达的bFGF,基本以包涵体的形式存在,包涵体难以复性成有生物活性的bFGF,研究显示,bFGF在各类菌株中的直接表达,每升培养基只能产生约1-10mg可溶性蛋白质。为了克服这一缺陷,有研究报道采用融合表达方式进行重组表达可提高可溶性,如maltose,binding protein (MBP)、谷胱甘肽S-转移酶(GST)以及硫氧还蛋白(Trx)。这些融合表达纯化方法只能部分解决可溶性表达问题,未见有大幅度提高bFGF可溶性表达研究报道。加之采用常规的色谱纯化程序较为复杂,bFGF的产率严重偏低,难以产生经济效益。
之前有研究者与我们课题组将目的蛋白与弹性蛋白(elastin-likepolypeptides,ELP)融合后,可提高目的蛋白可溶性;但采用使用可逆相变循环方式进行纯化时,目的蛋白损失量较大,会导致相应制备工艺所产生的经济效益欠佳。
据此,有效提升bFGF可溶性表达,快速且简易纯化bFGF蛋白,并提高bFGF蛋白纯化得率,是本发明要解决的问题。
发明内容
本发明的目的在于提供一种碱性成纤维细胞生长因子制备方法及其应用,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:一种碱性成纤维细胞生长因子制备方法,包括以下步骤:
S1:ELP-IDP-bFGF质粒的构建:其中ELP-IDP-bFGF质粒的基因序列如SEQ ID NO1所示;
S2:大肠杆菌工程菌构建:将上述带有ELP-IDP-bFGF质粒转入BL21(DE3)大肠杆菌中,得到工程菌;
S3:将上述工程菌培养于LB培养基,当大肠杆菌的OD值达到2值,温度调为32℃诱导16小时,收集菌体,破碎,4℃离心取上清;
S4:将上述步骤得到的上清37℃下水浴加热,37℃离心,取沉淀部分;
S5:向沉淀部分加入预冷的PBS后负40度冷冻2小时,超声溶解,低温离心取溶解部分;
S6:溶解部分加入带自备的His-Tag的EK酶,冰水中酶切12小时;
S7:酶切完成后,蛋白质溶液过Ni-NTA层析柱,ELP-IDP标签以及带His-Tag的EK酶将结合至层析柱上,收集流穿部分,流穿部分即为bFGF。
优选的是,在步骤S1中,ELP-IDP-bFGF质粒构建步骤如下:
(1):合成ELP-IDP基因,将ELP-IDP克隆至pET-26b的NdeI与HindIII酶切位点;
(2):合成带EK酶蛋白酶切位点的ELP-IDP基因序列,借助无缝连接酶将含有EK酶切位点的bFGF基因序列连接至pET-26b-ELP的HindIII酶切位点,得到ELP-IDP-bFGF质粒。
上述任一方案中优选的是,在步骤S3中,LB培养基包括蛋白胨10g/L、酵母提取物5g/L、NaCl为10g/L。
上述任一方案中优选的是,在步骤S6中,所述EK酶的添加量为1000mg蛋白加入1mg的EK酶。
一种碱性成纤维细胞生长因子在促进细胞生长增殖方面应用。
本发明的技术效果和优点:1、bFGF与特定的ELP整合表达之后,约80%以上的重组蛋白可以得到可溶性表达,bFGF的可溶性得到显著提升;
2、本发明通过纯化标签的氨基酸序列组成,一步法去除酶,纯化标签以及尚未被切断的ELP-IDP-bFGF,简化纯化步骤;
3、本发明采用冻融-离心法可以使90%以上重组蛋白得到回收,得率有较大幅度改善,适用于工业规模制备。
附图说明
图1为ELP-IDP-bFGF可溶性表达以及比较可逆相变纯化与冻融-离心法蛋白得率;
图2纯后ELP-IDP-bFGF酶切、色谱法去除纯化标签和EK酶;
图3为本发明的bFGF促进小鼠成纤维细胞生长(CCK-8)结果;
图4为本发明的bFGF促进小鼠成纤维细胞生长细胞结果图。
具体实施方式
下面结合附图对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
在本发明的描述中,需要理解的是,术语“中心”、“纵向”、“横向”、“长度”、“宽度”、“厚度”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”“内”、“外”、“顺时针”、“逆时针”、“轴向”、“径向”、“周向”等指示的方位或位置关系为基于附图所示的方位或位置关系,仅是为了便于描述本发明和简化描述,而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理解为对本发明的限制。
此外,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。
一种碱性成纤维细胞生长因子制备方法,包括以下步骤:
S1:ELP-IDP-bFGF质粒的构建:其中ELP-IDP-bFGF质粒的基因序列如SEQ ID NO1所示;
SEQ ID NO1;
5’-ATGAGTACCGGTTCAAAACAGCGTTCACAGAATCGCAGCAAAACCCCGAAAAATCAGGAAGCACTGCGCATGGCAAATGTTGCAGAAGCAAATGTGGCAGAAAATAGCAGCAGCGATCAGCGTCAGGCATGTAAAAAGCATGAACTGTATGTTAGCTTTCGCGATCTGGGTTGGCAGGATTGGATTATTGCACCGGAAGGCTATGCAGCCTATTATTGTGAAGGTGAATGTGCATTTCCGCTGAATAGTTATATGAATGCAACCAATCATGCAATTGTTCAGACTCTGGTTCATTTTATTAACCCGGAAACCGTGCCGAAACCATGTTGTGCCCCGACACAGCTGAATGCAATTAGCGTTCTGTATTTTGATGATAGTAGCAACGTTATTCTGAAAAAGTATCGTAATATGGTGGTGCGCGCCTGTGGCTGTCATGGTGGCGGTAGCGGTGGCCAGGCAAAGCATAAACAGCGTAAACGTCTGAAAAGCAGCTGTAAACGTCATCCGCTGTATGTGGATTTTAGCGATGTTGGTTGGAACGATTGGATTGTTGCCCCGCCGGGTTATCATGCATTTTATTGTCATGGTGAATGTCCGTTTCCGCTGGCAGATCATCTGAATTCAACCAACCATGCGATTGTTCAGACCCTGGTTAATAGTGTTAACTCTAAAATTCCTAAAGCCTGTTGTGTTCCGACCGAACTGAGCGCAATTTCTATGCTGTATCTGGATGAAAACGAAAAAGTGGTTCTGAAAAATTATCAGGATATGGTTGTTGAAGGCTGTGGTTGTCGTGGTGGTGGATCCGAAAACCTGTATTTTCAGGGCAGCCACGGCGTGCACGGCGTGGGTGTTCCGGGCCACGGTGTCCCAGGTCACGGCGTACCGGGCCACGGTGTTCCTGGTCACGGCGTGCCGGGCGCGGCCGCAGCTGCGGCGGCAGCCGCGGCTGCCGCGGCTGCAGCGGCAGCCGCGGCTGCGGCAGCCGCAGCTGCGGCGGCCGCAGCTGCGGCGGCAGCCGCGGCTGCCGCGGCTGCAGCGGCAGCCGCGGCTGCGGCAGCCGCAGCTGCAAGCTTATGACGATGACAAAGCGGCCGGTAGCATTACCACGCTGCCGGCGCTGCCGGAAGATGGCGGTAGTGGCGCGTTTCCTCCGGGTCATTTTAAAGATCCGAAACGTCTGTATTGTAAAAATGGCGGGTTCTTTCTGCGCATTCATCCGGATGGCCGTGTTGACGGTGTTCGCGAAAAGTCTGATCCGCATATTAAACTGCAGCTGCAAGCCGAAGAGCGCGGCGTTGTGAGCATTAAAGGTGTTTGTGCCAATCGTTATCTGGCAATGAAAGAAGATGGCCGTCTGTTAGCAAGCAAATGTGTTACCGACGAATGCTTCTTTTTCGAGCGTCTGGAAAGTAATAACTATAATACGTACCGTAGCCGCAAATATACCTCTTGGTACGTGGCCCTGAAACGTACCGGTCAGTATAAACTGGGCAGCAAAACCGGTCCGGGCCAGAAAGCAATTCTGTTTCTGCCGATGAGCGCGAAAAGCTAA;
ELP-IDP-bFGF融合蛋白序列为:
SKGHGVGGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGVGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGVPGVGVPGHGVPGHGVPGHGVPGHGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGLDDDDKAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS;
S2:大肠杆菌工程菌构建:将上述带有ELP-IDP-bFGF质粒转入BL21(DE3)大肠杆菌中,得到工程菌;
S3:将上述工程菌培养于LB培养基,当大肠杆菌的OD值达到2值,温度调为32℃诱导16小时,收集菌体,破碎,4℃离心取上清;
S4:将上述步骤得到的上清37℃下水浴加热,37℃离心,取沉淀部分;
S5:向沉淀部分加入预冷的PBS后负40度冷冻2小时,超声溶解,低温离心取溶解部分(冻融-离心法);
S6:溶解部分加入带自备的His-Tag的肠激酶(Enterokinase,EK酶),冰水中酶切12小时;
S7:酶切完成后,蛋白质溶液过Ni-NTA层析柱,ELP-IDP标签以及带His-Tag的EK酶将结合至层析柱上,收集流穿部分,流穿部分即为bFGF。
结果如附件图1所示:ELP-IDP-bFGF在E.coli可溶性可以达到80%以上(图1a的Lane 1与Lane 2),经冻融法纯化之后,ELP-IDP-bFGF可以产率可以达到100mg/L。
纯化后ELP-IDP-bFGF用聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,ELP-IDP-bFGF纯化可以达到90%以上,经EK酶切后,产生两条带(分别对应为ELP-IDP与bFGF,图2的泳道1),其所对应的分子量与理论值一致;采用Ni-NTA亲和色谱去除标签ELP-IDP,可获取bFGF蛋白(图2的泳道2)。
具体的,在步骤S1中,ELP-IDP-bFGF质粒构建步骤如下:
(1):合成ELP-IDP基因,将ELP-IDP克隆至pET-26b的NdeI与HindIII酶切位点;
(2):合成带EK酶蛋白酶切位点的ELP-IDP基因序列,借助无缝连接酶将含有EK酶切位点的bFGF基因序列连接至pET-26b-ELP的HindIII酶切位点,得到ELP-IDP-bFGF质粒。
具体的,在步骤S3中,LB培养基包括蛋白胨10g/L、酵母提取物5g/L、NaCl为10g/L。
具体的,在步骤S6中,所述EK酶的添加量为1000mg蛋白加入1mg的EK酶。
一种碱性成纤维细胞生长因子在促进细胞生长增殖方面应用,具体在小鼠胚胎成纤维细胞(NIH3T3)生长增殖。
bFGF促进细胞增殖检测:用NIH3T3细胞测试所纯化bFGF活性。NIH3T3培养于含有10%胎牛血清DMEM培养基(加入链霉素/青霉素)中,待细胞长至60%后,消化细胞,铺至96孔板中,每孔5000个细胞,培养12后小时,培养基换成含0.4%FBS的DMEM培养基,再培养12小时。加入一系列浓度梯度的bFGF孵育48小时后,加入CCK-8试剂,按CCK-8试剂说明书测试96孔板的吸光度。
综上,本发明是将富含组氨酸的ELP序列(ELP的五肽(VPGXG)单元的第四个可变氨基酸为缬氨酸与组氨酸)以及五肽重复次数差异,会引发ELP性质的千差万别。为了克服ELP不可逆相变现象,本发明引入无序蛋白序列,纯化过程采取冻融方式有效解决了重组蛋白回收率低的问题。ELP-IDP-bFGF在大肠杆菌中主要以可溶形式表达。采用过柱方式进行一步色谱法纯化,简化纯化程序。纯化后的bFGF生物活性(EC50=10.6)接近于Abcam公司的bFGF。本发明所述的融合表达方式有助于提升bFGF的可溶性,缩短制备时间,压缩纯化步骤,利于工业规模放大生产。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (5)
1.一种碱性成纤维细胞生长因子制备方法,其特征在于:包括以下步骤:
S1:ELP-IDP-bFGF质粒的构建:其中ELP-IDP-bFGF质粒的基因序列如SEQ ID NO1所示;
S2:大肠杆菌工程菌构建:将上述带有ELP-IDP-bFGF质粒转入BL21大肠杆菌中,得到工程菌;
S3:将上述工程菌培养于LB培养基,当大肠杆菌的OD值达到2值,温度调为32℃诱导16小时,收集菌体,破碎,4℃离心取上清;
S4:将上述步骤得到的上清37℃下水浴加热,37℃离心,取沉淀部分;
S5:向沉淀部分加入预冷的PBS后负40度冷冻2小时,超声溶解,低温离心取溶解部分;
S6:溶解部分加入带自备的His-Tag的EK酶,冰水中酶切12小时;
S7:酶切完成后,蛋白质溶液过Ni-NTA层析柱,ELP-IDP标签以及带His-Tag的EK酶将结合至层析柱上,收集流穿部分,流穿部分即为bFGF。
2.根据权利要求1所述的一种碱性成纤维细胞生长因子制备方法,其特征在于:在步骤S1中,ELP-IDP-bFGF质粒构建步骤如下:
(1):合成ELP-IDP基因,将ELP-IDP克隆至pET-26b的NdeI与HindIII酶切位点;
(2):合成带EK酶蛋白酶切位点的ELP-IDP基因序列,借助无缝连接酶将含有EK酶切位点的bFGF基因序列连接至pET-26b-ELP的HindIII酶切位点,得到ELP-IDP-bFGF质粒。
3.根据权利要求1所述的一种碱性成纤维细胞生长因子制备方法,其特征在于:在步骤S3中,LB培养基包括蛋白胨10g/L、酵母提取物5g/L、NaCl为10g/L。
4.根据权利要求1所述的一种碱性成纤维细胞生长因子制备方法,其特征在于:在步骤S6中,所述EK酶的添加量为1000mg蛋白加入1mg的EK酶。
5.根据权利要求1-4任一所述的一种碱性成纤维细胞生长因子在促进细胞生长增殖方面应用。
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