Prokaryotic expression, purification and pro-apoptotic activity analysis of TNF-α in allogynogenetic silver crucian carp Carassius auratus gibelio
CUI Zhengyi1,2, HU Guangyao2, JIANG Xinyu2, WANG Jia2, YU Xinran1, QIAO Guo2, LI Zhen2, LI Qiang2, ZHANG Jialin2*
1.Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs, Dalian Ocean University, Dalian; 2.College of Marine and Biology Engineering, Yancheng Institute of Technology, Yancheng
Abstract: 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 Carassiusauratusgibelio with body weight of(15±1)g by PCR, and the recombinant plasmid pET28a-TNF-α was constructed and then transformed into Escherichiacoli, 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-α protein 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-α protein were analyzed by Hoechst 33258 staining and qPCR.The results showed that the protein band with relative molecular weight of about 23 000 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 23 000 appeared with the purified protein and anti-His monoclonal antibody, with successful recombinant expression of TNF-α protein.Furthermore, Hoechst 33 258 staining showed that the nucleus of GCF cells treated by rTNF-α protein was more deeply and densely stained as fragmented, compared with the normal cells.The qPCR showed that the rTNF-α protein led to significantly increase in the mRNA expression levels of Caspase 3 and Caspase 8.The findings reveal that TNF-α protein 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-α protein.
2020, Vol. 35 
