CN113292647B - 一种igf-1的低成本制备方法及其应用 - Google Patents
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Abstract
本发明公开一种IGF‑1的低成本制备方法,包括:S1:ELP‑IGF‑1质粒的构建;S2:工程菌的构建;S3:将上述工程菌培养于TB培养基,过夜诱导,收集菌体,超声破碎,低温离心取上清;S4:将上述步骤得到的上清于水浴加热,离心取沉淀部分;S5:沉淀部分用预冷Tris‑HCl缓冲液溶解,离心取上清;S6:过夜酶切;S7:酶切完成后,蛋白质溶液置于30℃下水浴加热,30℃离心后取上清,上清部分用磁珠结合TEV酶,离心回收磁珠,上清经冷冻干燥后即为IGF‑1。IGF‑1应用于促进细胞生长增殖。本发明提供了全新、无需任何色谱填料制备方法,所制备蛋白应于刺激含IGF‑1受体的细胞生长增殖。
Description
技术领域
本发明属于蛋白质工程技术领域,具体涉及一种IGF-1的低成本制备方法及其应用。
背景技术
胰岛素样生长因子-1(insulin like growth factor,IGF-1)是生长激素IGF轴上最重要的生长因子之一。IGF-1是由70个氨基酸组成的单一多肽蛋白,分子量为7649 Da。IGF-1在肝脏中产生的合成代谢激素,能刺激多种细胞类型的增殖和分化,并在组织更新和修复中发挥重要作用。IGF-1可对体细胞生长的调节,研究表明它参与哺乳动物的各种生理进程。此外相关研究也表明IGF-1在细胞能量代谢以及生长和发育中起着至关重要作用。基于IGF-1参与细胞各类重要代谢进程,IGF-1是细胞培养基不可或缺的组分。相关市场调研表明,在细胞培养基市场中IGF-1需求量巨大。
商品化的IGF-1来源途径主要有真核表达系统以及原核表达系统。有研究显示采用真核表达系统的动物细胞可表达出有生物活性的IGF-1;而细胞表达系统复杂,培养成本高昂。常规原核表达系统如大肠杆菌(E.coli),因含70个氨基酸的IGF-1有6个半胱氨酸,以及大肠杆菌内部的氧化环境,6个半胱氨酸极易发生二硫键错配而形成包涵体,IGF-1包涵体复性难以获取有活性的蛋白。
已有研究报道中,IGF-1表达后,均采用色谱分离进行纯化IGF-1。色谱分离方法需经上样,结合,洗涤,洗脱等多重步骤,耗时耗力,加之色谱分离方式在工业应用时难以实现规模放大,色谱分离法所需要填料价格高企。基于经济效益,特别是考虑工业级别的制备成本,优化来自大肠杆菌的IGF-1可溶性表达以及采用非色谱法纯化可能是最佳的选择。
综上,提高IGF-1可溶性表达,快速且简易纯化蛋白,并产生的有活性的IGF-1,大幅度削减制备成本,是本发明要解决的主要问题。
发明内容
本发明的目的在于提供一种IGF-1的低成本制备方法及其应用,以解决上述技术背景中所提出的问题。
为实现上述目的,本发明提供以下技术方案:一种IGF-1的低成本制备方法,按照先后顺序包括以下步骤:
S1:ELP-IGF-1质粒构建:构建ELP-IGF-1质粒,其中ELP-IGF-1基因序列如SEQ IDNO:1所示;
S2:工程菌的构建:将上述ELP-IGF-1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TB培养基,当OD值达到0.5值,温度调为23℃过夜诱导,收集菌体,超声破碎,4℃离心取上清;
S4:将上述步骤得到的上清30℃下水浴加热,30℃离心,取沉淀部分;
S5:将沉淀部分用预冷的Tris-HCl缓冲液溶解,4℃离心取上清;
S6:上清加入带His-Tag的TEV酶,4℃过夜酶切;
S7:酶切完成后,蛋白质溶液置于30℃下水浴加热,30℃离心后取上清,上清部分用带镍的磁珠结合游离的TEV酶,离心回收磁珠,上清经冷冻干燥后即为IGF-1。
在步骤S1中,所述ELP-IGF-1质粒构建步骤如下:
(1):合成ELP基因,将ELP克隆至pET-23a的NdeI与HindIII酶切位点;
(2):合成带TEV(Tobacco Etch Virus)蛋白酶切位点Glu-Asn-Leu-Tyr-Phe-Gln-Gly的IGF基因序列,使用无缝连接酶将TEV酶切位点的IGF基因序列连接至pET-23a-ELP的HindIII酶切位点,得到ELP-IGF-1质粒。
上述任一方案中优选的是,在步骤S5中,所述Tris-HCl缓冲液pH值为9.0。
上述任一方案中优选的是,在步骤S6中,所述TEV酶的添加量为1mg蛋白加入25UTEV酶。
上述制备方法获取的IGF-1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、该IGF-1的低成本制备方法通过离心的方法纯化了IGF-1,极大简化制备程序;经体外细胞实验证实所纯化的IGF-1可促进细胞增长,其生物活性略高于商品化的IGF-1;
2、提供了一种新的IGF-1融合办法,实现了高效可溶性表达;
3、提供了全新的、无需任何色谱填料的、低廉的IGF-1制备方案。
附图说明
图1为本发明ELP-IGF-1的电泳图;
图2为本发明的纯化后的IGF-1(IC50=6.4ng/ml)与派普泰克公司的商品化IGF-1(IC50=6.5ng/ml)活性比较图。
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。
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。在本发明的描述中,“多个”的含义是至少两个,例如两个,三个等,除非另有明确具体的限定。
一种IGF-1的低成本制备方法,按照先后顺序包括以下步骤:
S1:ELP-IGF-1质粒的构建:构建ELP-IGF-1质粒,其中ELP-IGF-1序列如SEQ IDNO1所示;
SEQ ID NO1:5’-ATGAGCAAAGGCCACGGCGTGGGTGTTCCGGGCCACGGTGTCCCAGGTCACGGCGTACCGGGCCACGGTGTTCCTGGTCACGGCGTGCCGGGCGTGGGTGTTCCGGGCCACGGTGTCCCAGGTCACGGCGTACCGGGCCACGGTGTTCCTGGTCACGGCGTGCCGGGCGTGGGTGTTCCGGGCCACGGTGTCCCAGGTCACGGCGTACCGGGCCACGGTGTTCCTGGTCACGGCGTGCCGGGCGTGGGTGTTCCGGGCCACGGTGTCCCAGGTCACGGCGTACCGGGCCACGGTGTTCCTGGTCACGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCCACGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCCATGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCCACGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCCATGGTGTTCCTGGTGTCGGCGTGCCGGGCGGGCTGTAAAAGCTTGAAAACCTGTATTTTCAGGGTCCTGAAACACTGTGTGGTGCAGAACTGGTTGATGCACTGCAGTTTGTTTGTGGTGATCGTGGTTTTTATTTTAATAAACCGACAGGTTATGGCAGTAGTAGTCGTCGTGCGCCGCAGACCGGTATTGTTGATGAATGTTGTTTTCGTAGCTGTGATCTGCGTCGTCTGGAAATGTATTGTGCCCCGCTGAAACCGGCAAAAAGCGCGTAAGCTT-3’;
融合蛋白序列如下:SKHGVGVPGVGVPGVGVPGHGVPGVGVPGVGVPGVGVPGVGVPGHGVPGVGVPGVGVPGVGVPGVGVPGHGVPGVGVPGVGVPGVGVPGVGVPGHGVPGVGVPGVGVPGVGVPGVGVPGHGVPGVGVPGVGVPGVGVPGVGVPGHGVPGVGVPGVGVPGVGVPGVGVPGHGVPGVGVPGVGVPGVGVPGVGVPGHGVPGVGVPGGSKLENLYFQGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA。
S2:工程菌的构建:将上述ELP-IGF-1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TB培养基,当OD值达到0.5值,温度调为23℃过夜诱导,收集菌体,超声破碎,4℃离心取上清;
S4:将上述步骤得到的上清30℃下水浴加热,30℃离心,取沉淀部分;
S5:将沉淀部分用预冷的Tris-HCl缓冲液溶解,4℃离心取上清;
S6:上清加入带His-Tag的TEV酶,4℃过夜酶切;
S7:酶切完成后,蛋白质溶液置于30℃下水浴加热,30℃离心后取上清,上清部分用带镍的磁珠结合游离的TEV酶,离心回收磁珠,上清经冷冻干燥后即为IGF-1。
在这个过程中用SDS-PAGE鉴定ELP-IGF-1在E.coli可溶性表达情况、蛋白纯度以及酶切产生的IGF-1。
结果如图1所示:ELP-IGF-1在E.coli主要以可溶的形式进行表达(图1的泳道1与2),经本工艺纯化之后,得率为50 mg/L,纯化后ELP-IGF-1用聚丙烯酰胺凝胶电泳分析,纯后的ELP-IGF-1呈单一条带(图1泳道3),经TEV酶切后,产生两条带(分别对应为ELP与IGF-1,图1的泳道4),其所对应的分子量与理论值一致;经可逆相变后可有效去除ELP,进而获得纯的IGF-1(图1泳道5)。
其中图1中泳道1:含ELP-IGF-1质粒的工程菌经诱导后的破碎液;泳道2:破碎液经低温离心后上清部分,对比于泳道1可获知ELP-IGF-1主要以可溶性质表达;泳道3:离心纯化后的ELP-IGF-1;泳道4:经TEV酶切分别产生两种蛋白质分子,相对应分别为ELP与IGF-1;泳道5:采用可逆相变方式去除ELP,溶液即为IGF-1。
申请者对于本工艺的所纯化的IGF-1进行成本测算,每制备1克的IGF-1,其成本可以控制在8000元以内,大幅度低于市场价(上海近岸科技有限公司以及派普泰克生物市场报价均为4-5万元/克),显示出本制备工艺所生产的IGF-1具有很强市场竞争力。
具体的,在步骤S1中,ELP-IGF-1质粒构建步骤如下:
(1):合成ELP基因,将ELP克隆至pET-23a的NdeI与HindIII酶切位点;
(2):合成带TEV(Tobacco Etch Virus)蛋白酶切位点Glu-Asn-Leu-Tyr-Phe-Gln-Gly的IGF基因序列,使用无缝连接酶将TEV酶切位点的IGF基因序列连接至pET-23a-ELP的HindIII酶切位点,得到ELP-IGF-1质粒。
具体的,在步骤S3中,TB培养基包括蛋白胨12g/L、酵母提取物24g/L、甘油4ml/L,17mM的KH2PO4与72 mM的K2HPO4。
具体的,在步骤S6中,TEV酶的添加量为1mg蛋白加入25U TEV酶。
上述制备方法获取的IGF-1在促进细胞生长增殖方面应用。
IGF-1促进细胞增殖检测:用乳腺癌细胞(MCF-7)测试所纯化IGF-1活性。MCF-7培养于含有10%牛血清DMEM培养基中,待细胞长至70%后,消化细胞,铺至96孔板中(每个孔为4000个细胞),培养4小时,加入一系列浓度梯度的IGF-1,孵育24小时后,加入CCK8(碧云天公司),按CCK8试剂说明书测试96孔板的吸光度。
结果如图2所示,本发明所制备的IGF-1的IC50值4ng/ml),其生物活性略高于派普泰克公司的商品化IGF-1(IC50=6.5ng/ml)。
综上,本发明是将特定的ELP序列(ELP的五肽(VPGXG)单元的第四个可变氨基酸(X)以及五肽重复次数差异,会引发ELP性质的千差万别;本发明所选取的ELP序列组成是经多次试验后挑选出来的)与IGF-1进行选择性融合并表达,ELP-IGF-1在大肠杆菌中主要以可溶形式表达,经过简易离心后,可获取高纯度的目的蛋白IGF-1。纯化后的IGF-1的生物活性与商品化的IGF-1基本一致。本发明所述的离心纯化方式简易,制备成本尤为低廉。
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。
序列表
<110> 南京谷浦生物科技有限公司
<120> 一种IGF-1的极低成本制备方法
<141> 2021-07-22
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 868
<212> DNA
<213> 人工合成(ArtificialSequence)
<400> 2
atgagcaaag gccacggcgt gggtgttccg ggccacggtg tcccaggtca cggcgtaccg 60
ggccacggtg ttcctggtca cggcgtgccg ggcgtgggtg ttccgggcca cggtgtccca 120
ggtcacggcg taccgggcca cggtgttcct ggtcacggcg tgccgggcgt gggtgttccg 180
ggccacggtg tcccaggtca cggcgtaccg ggccacggtg ttcctggtca cggcgtgccg 240
ggcgtgggtg ttccgggcca cggtgtccca ggtcacggcg taccgggcca cggtgttcct 300
ggtcacggcg tgccgggcgt gggtgttccg ggcgtaggtg tcccaggtgt gggcgtaccg 360
ggccacggtg ttcctggtgt cggcgtgccg ggcgtgggtg ttccgggcgt aggtgtccca 420
ggtgtgggcg taccgggcca tggtgttcct ggtgtcggcg tgccgggcgt gggtgttccg 480
ggcgtaggtg tcccaggtgt gggcgtaccg ggccacggtg ttcctggtgt cggcgtgccg 540
ggcgtgggtg ttccgggcgt aggtgtccca ggtgtgggcg taccgggcca tggtgttcct 600
ggtgtcggcg tgccgggcgg gctgtaaaag cttgaaaacc tgtattttca gggtcctgaa 660
acactgtgtg gtgcagaact ggttgatgca ctgcagtttg tttgtggtga tcgtggtttt 720
tattttaata aaccgacagg ttatggcagt agtagtcgtc gtgcgccgca gaccggtatt 780
gttgatgaat gttgttttcg tagctgtgat ctgcgtcgtc tggaaatgta ttgtgccccg 840
ctgaaaccgg caaaaagcgc gtaagctt 868
Claims (6)
1.一种IGF-1的低成本制备方法,其特征在于:按照先后顺序包括以下步骤:
S1:ELP-IGF-1质粒的构建:构建ELP-IGF-1质粒,其中ELP-IGF-1基因序列如SEQ IDNO:1所示;
S2:工程菌的构建:将上述ELP-IGF-1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TB培养基,当OD值达到0.5值,温度调为23℃过夜诱导,收集菌体,超声破碎,离心取上清;
S4:将上述步骤得到的上清30℃下水浴加热,30℃离心,取沉淀部分;
S5:将沉淀部分用预冷的Tris-HCl缓冲液溶解,4℃离心取上清;
S6:上清加入带His-Tag的TEV酶,4℃过夜酶切;
S7:酶切完成后,蛋白质溶液置于30℃下水浴加热,30℃离心后取上清,上清部分用带镍的磁珠结合游离的TEV酶,离心回收磁珠,上清经冷冻干燥后即为IGF-1。
2.根据权利要求1所述的一种IGF-1的低成本制备方法,其特征在于:在步骤S1中,所述ELP-IGF-1质粒构建步骤如下:
(1):合成ELP基因,将ELP克隆至pET-23a的NdeI与HindIII酶切位点;
(2):合成带TEV蛋白酶切位点Glu-Asn-Leu-Tyr-Phe-Gln-Gly的IGF基因序列,使用无缝连接酶将TEV酶切位点的IGF基因序列连接至pET-23a-ELP的HindIII酶切位点,得到ELP-IGF-1质粒。
3.根据权利要求1所述的一种IGF-1的低成本制备方法,其特征在于:在步骤S3中,所述TB培养基包括蛋白胨12g/L、酵母提取物24g/L、甘油4ml/L,17mM的KH2PO4与72 mM的K2HPO4。
4.根据权利要求1所述的一种IGF-1的低成本制备方法,其特征在于:在步骤S3中,所述离心温度为4℃。
5.根据权利要求1所述的一种IGF-1的低成本制备方法,其特征在于:所述Tris-HCl缓冲液pH值为9.0。
6.根据权利要求1所述的一种IGF-1的低成本制备方法,其特征在于:在步骤S6中,所述TEV酶的添加量为1mg蛋白加入25U TEV酶。
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