摘要
为获得可溶的鲤鱼IL-17N重组蛋白,构建原核重组表达质粒GST-17N、SUMO-GST-17N和MBP-17N,确定IL-17N的最适促溶标签,并优化其诱导表达条件(诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)的浓度、诱导时间和诱导温度)。结果:成功构建了鲤鱼IL-17N原核重组表达载体GST-17N、SUMO-GST-17N和MBP-17N;融合标签MBP、SUMO-GST和GST的促溶效果依次降低,SUMO-GST-17N、MBP-17N上清蛋白占总蛋白比例分别为47.4%、87.0%;MBP-17N的最佳表达条件为0.5 mmol/L IPTG,25℃诱导8~12 h;经过镍柱纯化,获得可溶MBP-17N蛋白,每克总菌约获得0.09 mg MBP-17N蛋白。
To obtain soluble recombinant Cyprinus carpio IL-17N protein,recombinant prokaryotic expression plasmids(GST-17N,SUMO-GST-17N,MBP-17N)were constructed,the optimal fusion tag(GST,GST-SUMO and MBP)was determined,and the expression conditions of recombinant Cyprinus carpio IL-17N protein(the content of IPTG,time and temperature)were optimized.Results:the recombinant Cyprinus carpio IL-17N prokaryotic expression plasmids(GST-17N,SUMO-GST-17N,MBP-17N)were successfully constructed.The soluble expression of rccIL-17N protein from high to low were MBP-17N,SUMO-GST-17N and GST-17N.The soluble protein of SUMO-GST-17N and MBP-17N accounted for 47.4%and 87.0%of the total protein,respectively.The optimal induction condition for MBP-17N was incubate the transformed Escherichia coil for 8-12 h under 25℃with 0.5 mmol/L IPTG induction.The prokaryotic recombinant protein was purified by Ni-NTA and 0.09 mg protein could be obtained by 1 g total bacterial.
作者
张磊
唐永凯
李红霞
徐逾鑫
李迎宾
俞菊华
ZHANG Lei;TANG Yongkai;LI Hongxia;XU Yuxin;LI Yingbin;YU Juhua(Wuxi Fisheries College,Nanjing Agricultural University,Wuxi,Jiangsu 214128,China;Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi,Jiangsu 214128,China)
出处
《湖南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2020年第3期364-369,共6页
Journal of Hunan Agricultural University(Natural Sciences)
基金
中央级基本科研业务费(2018HY-ZD02)。
关键词
鲤鱼
白细胞介素17N
原核表达
重组蛋白
融合标签
蛋白纯化
Cyprinus carpio
interleukin-17N
prokaryotic expression
recombinant protein
fusion tag
protein purification
