摘要
为了实现异育银鲫Carassius auratus gibelio肿瘤坏死因子α(Tumor necrosis factor,TNF-α)在大肠杆菌表达系统的高效表达,并研究其在促鲫鱼细胞凋亡中的作用,采用PCR的方法从体质量(15±1)g异育银鲫脾脏组织中扩增TNF-α基因,构建pET28a-TNF-α重组质粒并转化至大肠杆菌BL21(DE3)中,经IPTG诱导表达后,通过Ni亲和层析柱纯化并采用尿素浓度梯度透析法进行蛋白复性,最后用rTNF-α蛋白处理鲫鱼鳍条细胞,通过Hoechst 33258染色法和荧光定量PCR技术,分析异育银鲫经rTNF-α蛋白处理后鲫鱼细胞的凋亡情况及凋亡相关基因Caspase 3和Caspase 8的表达变化。结果表明:诱导后获得了相对分子质量约为23000的蛋白条带,且重组蛋白以包涵体形式存在;Western-blot显示,纯化产物与抗His单抗在相对分子量约23000处出现了明显的反应条带,表明成功地实现了异育银鲫TNF-α的重组表达;进一步采用Hoechst 33258染色后发现,与正常细胞相比,经rTNF-α蛋白处理后的细胞核染色加深,呈碎块状浓染现象;荧光定量PCR显示,rTNF-α蛋白处理能使凋亡相关基因Caspase 3和Caspase 8 mRNA表达水平呈明显的上调趋势。研究表明,本文成功实现了异育银鲫TNF-α蛋白在大肠杆菌中的高效表达,且rTNF-α蛋白可诱导鲫鱼鳍条细胞发生凋亡,该工作将为后续研究TNF-α蛋白的抗病毒作用奠定基础。
In order to realize the high-efficiency expression of TNF-a protein in allogynogenetic silver crucian carp in the expression system of E.coli and to investigate its role in promoting cell apoptosis,tumor necrosis factor-α(TNF-α)gene was amplified from spleen of allogynogenetic silver crucian carp Carassius auratus gibelio with body weight of(15±1)g by PCR,and the recombinant plasmid pET28a-TNF-αwas constructed and then transformed into Escherichia coli,BL21(DE3).After induction with isopropylthio-β-galactoside(IPTG),the protein was purified by Ni affinity chromatography and protein renaturation was performed by urea concentration gradient dialysis.Finally,rTNF-αprotein was used to treat fin cells(CAF cell)of allogynogenetic silver crucian carp,the apoptosis of which and the expression of related genes after treatment with rTNF-αprotein were analyzed by Hoechst 33258 staining and qPCR.The results showed that the protein band with relative molecular weight of about 23000 was obtained after induction,and the recombinant protein was found in the form of inclusion body.Western-blot revealed that an obvious band at about 23000 appeared with the purified protein and anti-His monoclonal antibody,with successful recombinant expression of TNF-αprotein.Furthermore,Hoechst 33258 staining showed that the nucleus of GCF cells treated by rTNF-αprotein was more deeply and densely stained as fragmented,compared with the normal cells.The qPCR showed that the rTNF-αprotein led to significantly increase in the mRNA expression levels of Caspase 3 and Caspase 8.The findings reveal that TNF-αprotein of allogynogenetic silver crucian carp was successfully and efficiently expressed in E.coli,and could induce the apoptosis of GCF cells,which will lay the foundation for the subsequent research on the antiviral effect of TNF-αprotein.
作者
崔正一
胡光耀
姜新宇
王佳
于新然
乔帼
李振
李强
张家林
CUI Zhengyi;HU Guangyao;JIANG Xinyu;WANG Jia;YU Xinran;QIAO Guo;LI Zhen;LI Qiang;ZHANG Jialin(Key Laboratory of Mariculture&Stock Enhancement in North China's Sea,Ministry of Agriculture and Rural Affairs,Dalian Ocean University,Dalian 116023,China;College of Marine and Biology Engineering,Yancheng Institute of Technology,Yancheng 224051,China)
出处
《大连海洋大学学报》
CAS
CSCD
北大核心
2020年第2期247-252,共6页
Journal of Dalian Ocean University
基金
江苏省自然科学基金青年基金资助项目(BK20181053,BK20191044)
江苏省农业科技自主创新资金项目(CX(18)3028)
盐城工学院人才引进项目(xjr2019044)。
关键词
肿瘤坏死因子Α
异育银鲫
原核表达
蛋白纯化
细胞凋亡
TNF-α
Carassius auratus gibelio
prokaryotic expression
protein purification
apoptosis
