摘要
为研究麻鸭(Anas platyrhynchas)热休克蛋白HSP90α结合肌内磷脂及抑制磷脂水解的作用机制,采用反转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)技术,以麻鸭骨骼肌细胞cDNA为模板扩增HSP90α基因,构建重组表达质粒在大肠杆菌中诱导表达,镍柱亲和层析和凝胶层析纯化可溶性重组蛋白。结果表明,获得的HSP90α开放阅读框全长为2 187 bp,编码728个氨基酸,预测的理论等电点约为5。构建的原核表达载体pCold1-HSP90α在大肠杆菌中成功可溶性表达了麻鸭HSP90α,并获得了高纯度的重组HSP90α蛋白。运用薄层层析证明该重组蛋白具有稳定结合磷脂酰胆碱的活性,并且该蛋白能显著削弱磷脂酶A2对磷脂的水解作用。该研究成功克隆并建立了麻鸭HSP90α基因的原核表达体系,制备了具有磷脂结合活性的重组蛋白,为进一步研究HSP90α与磷脂的相互作用及对肉品加工中磷脂的保护效应提供了理论支持。
The present study was conducted to express and purify the heat shock protein 90 alpha（HSP90α） of Anas platyrhynchas in prokaryotic expression system for further study of the mechanism of interaction between HSP90α and phospholipids and inhibition of phospholipid hydrolysis by HSP90α.The HSP90α gene was cloned from A.platyrhynchas skeletal muscle c DNA by RT-PCR.The gene sequence and its amino acid sequence were analyzed with bioinformatic tools.An inducible expression vector was constructed by enzyme digestion-ligation reactions and transformed into Escherichia coli for expression using isopropyl β-D-1-thiogalactopyranoside（IPTG） as an inducer.The recombinant protein was purified by Ni-NTA affinity chromatography and gel chromatography.As results,the open reading frame of HSP90α was 2 187 bp in length,and the deduced protein was composed of 728 amino acids with 5 glycosylation sites and 67 phosphorylation sites; its predicted isoelectric point was about 5.The E.coli vector pCold1-HSP90α successfully expressed the recombinant HSP90α protein in the supernatant of bacterial lysate.Thin-layer chromatography demonstrated that the recombinant HSP90α could stably bind to phosphatidylcholine.Lipolysis assay showed that HSP90α significantly restrained the hydrolysis of phospholipid.In conclusion,this study may provide a foundation for further study of the interaction between HSP90α and phospholipids and the potential of HSP90α to protect phospholipids in processed meat.
作者
张玉梅
李鹏鹏
张牧焓
王晶晶
王道营
徐为民
ZHANG Yumei;LI Pengpeng;ZHANG Muhan;WANG Jingjing;WANG Daoying;XU Weimin(Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, China;Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, Nanjing 210095, China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2018年第2期99-104,共6页
Food Science
基金
国家自然科学基金青年科学基金项目(31701532
31401560)
江苏省自然科学基金面上项目(BK20161378)
江苏省农业科学院农产品加工研究所基金项目(JG201702)
关键词
麻鸭
HSP90Α
原核表达
纯化
磷脂结合
Anas platyrhynchas
HSP90α
prokaryotic expression
purification
phospholipid binding
