摘要
目的建立并优化重组小鼠鞘磷脂脂蛋白(PLP)免疫原性多肽片段(PLP139-208)的原核表达方法.方法构建重组质粒PLP-pET-32a(+),利用基因工程技术将质粒转化到BL21(DE3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,进行PLP139-208的原核表达.为提高多肽片段的获得量,从诱导时间、诱导温度2个方面进行原核表达条件优化.诱导表达的蛋白多肽经Ni-NTA树脂纯化后,用Weestern blot法进行蛋白鉴定.结果表达条件优化、并经Western blot法蛋白鉴定后,确定诱导温度23℃、诱导时间20 h、0.5 mmol/L IPTG作为PLP多肽的最适原核表达条件.结论建立小鼠重组PLP139-208蛋白原核表达方法,为改良EAE动物模型的制备方法及研究PLP诱导自身免疫性脱髓鞠病变的机制奠定了实验基础.
Objective To establish and optimize the prokaryotic expression method for the recombinant mouse myelin proteolipid protein(PLP,139-208 aa)which is a critical immunogenic polypeptide of PLP.Methods The sequence coding for PLP139-208 polypeptide was cloned into pET-32a(+)vector.Afterwards,the expression vector prepared in this research was transformed into E.coli BL21,and the recombinant PLP polypeptide was induced to express by isopropyl-p-D-thiogalactoside(IPTG).Two key prokaryotic expression conditions,IPTG’s induction length and temperature,were analyzed for further optimization.The recombinant PLP polypeptide was induced to express by the expression method under the optimal expression conditions,and then was purified by Ni-NTA agarose and amylose resin.Finally,the gain of PLP139-208 polypeptide was verified by Western blot analysis.Results The results in the combinatorial optimization revealed that the expression of PLP139-208 was obtained at a satisfactory level when it was incubated at 23℃ for 20 hours with the IPTG concentration of 0.5 mmol/L.Conclusion The optimized prokaryotic expression method for the recombinant mouse PLP139-208 was successfully established and effectively performed.This will shed light on the further researches on the improved preparation for experimental autoimmune encephalitis(EAE,an animal model of multiple sclerosis)and the underlying mechanism underlying PLP-induced autoimmune demyelination.
作者
白晔
董荔红
周远红
兰风华
BAI Ye;DONG Lihong;ZHOU Yuanhong;LAN Fenghua(Department of Clinical Genetics and Experimental Medicine,900th Hospital of PLA Joint Service Support Force,Fuzhou 350025;Department of Rehabilitation Sciences,Wuyi University,Wuyishang 354300;Clinical Medical College,Fuzhou General Hospital,Fujian Medical University,Fuzhou 350025,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2019年第10期886-891,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
福建省自然科学基金(13161509)。
关键词
鞘磷脂脂蛋白(PLP)
原核表达
蛋白纯化
自身免疫性脱髓鞘
myelin proteolipid protein(PLP)
prokaryotic expression
protein purification
demyelinating autoimmune diseases
作者简介
第一作者:白晔(1984-),女,福建建阳人,讲师,博士,Tel:0599-22859103;E-mail:ellenpai@wuyiu.edu.cn;第一作者:董荔红;通讯作者:兰风华,E-mail:fhlan@xmu.edu.cn。
