大鼠His-Akt1重组蛋白的真核表达、蛋白纯化及活性鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-0497

摘要/Abstract

摘要：

构建His-Akt1-pcDNA3.1真核表达载体,于HEK293细胞进行表达,Ni-NTA亲和纯化His-Akt1,并对Akt1进行活性鉴定。以Akt1（全长）-pcDNA3.1重组体为模板,采用PCR扩增目的基因,然后克隆入真核表达载体pcDNA3.1;将His-Akt1-pcDNA3.1转染HEK293细胞表达,Ni-NTA纯化His-Akt1,用SDS-PAGE、考马斯亮蓝染色及免疫印迹鉴定蛋白纯度;蛋白经透析后,免疫印迹检测Akt1 Ser473和Thr308磷酸化水平。His-Akt1- pcDNA3.1真核表达载体构建成功,在HEK293细胞中高效表达;考马斯亮蓝染色和免疫印迹结果显示,取2 mg过表达His-Akt1的蛋白混合液经 Ni-NTA纯化后,加入含100 mmol/L咪唑的洗脱液洗脱,可获得高纯度His-Akt1重组蛋白。上述His-Akt1重组蛋白经透析后,免疫印迹结果显示His-Akt1 Ser473和Thr308磷酸化水平（活化水平）均呈高水平。重组His-Akt1-pcDNA3.1真核表达载体成功构建,且蛋白高表达,His-Akt1经提取纯化后具有较高的酶活性。

关键词: Akt1, 真核表达载体, 蛋白纯化, 活性鉴定

Abstract:

To obtain rat His-Akt1 recombinant protein with high activity,His-Akt1-pcDNA3.1 eukaryotic expression vector was constructed firstly,and then transfected into HEK293 cells. Next,the His-Akt1 was purified by Ni column affinity chromatography（Ni-NTA）,and its activity was then identified. His-Akt1 cDNAs were amplified from the full-length recombinant Akt1-pcDNA3.1 by PCR and cloned into eukaryotic expression vector pcDNA3.1. The HEK293 cells transfected with His-Akt1-pcDNA3.1 plasmid were collected and sent to Ni-NTA. His-Akt1 purity was identified by SDS-PAGE,Coomassie blue staining and Western blot. Dialytically purified His-Akt1 finally was subjected to Western blot analysis to detect Ser473 and Thr308 phosphorylation levels. His-Akt1-pcDNA3.1 eukaryotic expression vector was constructed successfully and efficiently expressed in HEK293 cells. The Coomassie blue staining and Western blot results showed that high-purity His-Akt1 recombinant protein was obtained by purifying 2 mg overexpressing His-Akt1 protein samples with Ni-NTA and eluting with 100 mmol/L imidazole. In addition,the Ser473 and Thr308 phosphorylation（i.e. activation）of the His-Akt1 after dialysis presented both high levels. The recombinant His-Akt1-pcDNA3.1 eukaryotic expression vector is constructed successfully,and the protein highly expressed,and the purified His-Akt1 recombinant protein demonstrates high enzymatic activity.

Key words: Akt1, eukaryotic expression vector, protein purification, activity identification

孟利, 杜彩萍. 大鼠His-Akt1重组蛋白的真核表达、蛋白纯化及活性鉴定[J]. 生物技术通报, 2020, 36(12): 98-103.

MENG Li, DU Cai-ping. Eukaryotic Expression,Purification and Activity Identification of Rat His-Akt1 Recombinant Protein[J]. Biotechnology Bulletin, 2020, 36(12): 98-103.

图/表 6

表1 试验所用引物

引物名称	引物序列（5'-3'）	酶切位点
His-Akt1上游	CCCAAGCTTGGGATGCATCATCATCAT- CATCACATGAACGACGTAGCCATTGT	Hind III
His-Akt1下游	GAATTCTCAGGCTGTGCCACTGGCT- GAGTC	EcoR I

图1 重组质粒His-Akt1-pcDNA3.1的构建与鉴定 A：PCR产物鉴定;B：His-Akt1与真核表达载体 pcDNA3.1连接产物的鉴定;C：His-Akt1-pcDNA3.1经Hind III和EcoR I酶切鉴定;M：DNA marker

图2 His-Akt1重组蛋白高表达将His-Akt1-pcDNA3.1转入HEK293细胞中,用anti-Akt1 antibody免疫印迹检测蛋白的表达。-：空细胞对照

图3 His-Akt1重组蛋白纯度初步鉴定 A：考马斯亮蓝染色鉴定经 Ni-NTA纯化的His-Akt1重组蛋白纯度;-,HEK293空细胞;B：免疫印迹鉴定经 Ni-NTA纯化的His-Akt1重组蛋白纯度

图4 优化纯化条件后His-Akt1重组蛋白纯度初步鉴定 A：考马斯亮蓝染色鉴定His-Akt1重组蛋白纯度;-,HEK293空细胞;B：免疫印迹鉴定His-Akt1重组蛋白纯度;M ：protein ladder

图5 免疫印迹检测His-Akt1 Ser473（S473）和Thr308（T308）磷酸化水平 V：pcDNA3.1空载体

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