鸡TIGAR基因真核表达质粒的构建及其抗凋亡功能评价

doi:10.3864/j.issn.0578-1752.2019.06.013

摘要/Abstract

摘要：

【背景】 TP53诱导的糖酵解和凋亡调节因子（TP53-induced glycolysis and apoptosis regulator,TIGAR）是p53下游的靶基因,具有调节糖酵解水平和去除活性氧（Reactive oxygen species,ROS）并降低由活性氧诱发的细胞凋亡水平。【目的】构建鸡源TIGAR基因的真核表达质粒,并检测TIGAR基因在DF1细胞中的抗凋亡作用,为建立稳定表达鸡TIGAR基因的细胞系做准备。【方法】根据GenBank（登录号：XM_417232.6）中预测基因设计引物,利用RT-PCR的方法从SPF鸡脾脏中扩增鸡TIGAR基因,将扩增产物克隆至载体（Flag-CMV14）后送公司测序验证;随后构建进化树对鸡TIGAR基因与其他哺乳动物及水生动物的TIGAR基因进行同源性分析。将重组质粒（Flag-TIGAR）转染入DF1细胞24 h后,使用新城疫病毒诱导细胞凋亡,利用Western Blot检测重组质粒表达情况以及Poly ADP-Ribose Polymerase（PARP）裂解情况。此外还将重组质粒（Flag-TIGAR）转染入DF1细胞,并于收样前2 h使用Staurosporine刺激细胞发生凋亡,分别在转染后24、48 h收集样品,并用流式细胞仪检测细胞凋亡情况。【结果】 RT-PCR扩增TIGAR基因,在843 bp处出现目的条带与预测相符,构建的TIGAR真核表达质粒（Flag-TIGAR）经测序,结果显示其序列与GenBank上预测的基本一致。Western Blot结果显示在30 h、36 h收集的样品中PARP均被裂解且转染重组质粒（Flag-TIGAR）的实验组与未转染质粒（MOCK）组或转染空载体（Flag-CMV14）组的样品相比,裂解的PARP表达量明显降低,且差异极显著（P<0.01）。流式结果显示：24 h 检测转染Flag-CMV14后细胞总凋亡率为11%（早期凋亡7.8%,晚期凋亡3.2%）,而转染Flag-TIGAR后细胞总凋亡率仅为4%（早期凋亡3.7%,晚期凋亡0.3%）,转染Flag-CMV14的早期凋亡率和晚期凋亡率均高于转染Flag-TIGAR组,且差异显著（P<0.05）。48 h转染Flag-CMV14后细胞总凋亡率为20.3%（早期凋亡14.3%,晚期凋亡6.0%）,而转染Flag-TIGAR后细胞总凋亡率为6.4%（早期凋亡4.8%,晚期凋亡1.6%）,转染Flag-CMV14组的细胞早期凋亡和晚期凋亡率均高于转染Flag-TIGAR的组,且差异极显著（P<0.01）。【结论】成功扩增出鸡TIGAR基因,并构建了其真核表达质粒,通过Western Blot和流式实验均证实过表达TIGAR后可降低细胞的凋亡程度并有利于细胞存活。

关键词: TIGAR, 真核表达质粒, 新城疫病毒, 抗凋亡, 鸡

Abstract:

【Background】TP53-induced glycolysis and apoptosis regulator (TIGAR) is a target gene downstream of p53, which can regulate glycolysis level, remove reactive oxygen species (ROS) and reduce apoptosis induced by reactive oxygen species (ROS). 【Objective】The objective of this study was to construct the eukaryotic expression plasmid contain of chicken TIGAR gene, and to evaluate its resistance apoptosis effect in DF1 cells, in order to establish the cell line with stable expression of the chicken TIGAR gene. 【Method】According to design primers based on the predicted TIGAR gene published in Genbank (accession number: XM_417232.6), the TIGAR gene from SPF chicken spleen was amplified by RT-PCR, and then amplified products were cloned into expression vector (Flag-CMV14), finally the positive clone was verified by DNA sequencing. Then the phylogenetic tree of chicken TIGAR genes with other mammals and aquatic animals TIGAR gene was constructed to do homology analysis. The recombinant plasmid was transfected into DF1 cells, and infected with Newcastle disease virus to induce apoptosis. The expression of the TIGAR gene and the cleavage level of PARP protein were detected by Western Blot. At the same time the recombinant plasmid (Flag-TIGAR) was transfected into DF1 cells and incubated with apoptosis inducer staurosporine for 2 hours before sample collection, then cells were collected at 24 hours and 48 hours after transfection to evaluate apoptosis level by flow cytometry. 【Result】The TIGAR gene was amplified by RT-PCR, and a band appeared at 843 bp, which was consistent with the prediction. The constructed TIGAR eukaryotic expression plasmid (Flag-TIGAR) was sequenced, and the result showed that the sequence of amplified TIGAR gene were almost consistent with the predicted sequence published on GenBank. Western Blot results showed: cleavaged PARP bands were existed, and the expression level in transfected recombinant plasmid (Flag-TIGAR) group was significantly different (P<0.05) from that in non-transfected plasmid (MOCK) group and transfected empty vector (Flag-CMV14) group. Flow cytometry results showed that the total apoptotic rate was 11% (early apoptosis 7.8%, late apoptosis 3.2%) after transfection with Flag-CMV14 for 24 h, while the total apoptotic rate was decreased to 4% (early apoptosis 3.7%, late apoptosis 0.3%) in Flag-TIGAR transfected group. The early apoptotic rate of Flag-CMV14 transfected group was much higher than that of Flag-TIGAR transfection group, and the difference was very significant (P<0.01), while the late apoptotic rate was also higher than that of Flag-TIGAR transfected group and the difference was significant (P<0.05). The total apoptotic rate was 20.3% (early apoptosis 14.3%, late apoptosis 6.0%) after transfection with Flag-CMV14 for 48 h, while the total apoptotic rate was only 6.4% (early apoptosis 4.8%, late apoptosis 1.6%) after transfection with Flag-TIGAR for 48 h. Both the early apoptotic rate and late apoptotic rate of Flag-CMV14 transfected group were much higher than that of Flag-TIGAR transfected group and the difference was very significant (P<0.01). 【Conclusion】The chicken TIGAR gene was amplified and the eukaryotic expression plasmid was constructed successfully. It was also confirmed that overexpression of chicken TIGAR could obviously reduce the degree of apoptosis and increase cell survival rate.

Key words: TIGAR, Eukaryotic expression plasmid, Newcastle disease virus, Anti-apoptotic function

栗永华, 车路平, 仇旭升, 谭磊, 孙英杰, 刘炜炜, 宋翠萍, 廖瑛, 丁铲, 王金泉, 孟春春. 鸡TIGAR基因真核表达质粒的构建及其抗凋亡功能评价[J]. 中国农业科学, 2019, 52(6): 1102-1109.

LI YongHua, CHE LuPing, QIU XuSheng, TAN Lei, SUN YingJie, LIU WeiWei, SONG CuiPing, LIAO Ying, DING Chan, WANG JinQuan, MENG ChunChun. Construction of Chicken TIGAR Gene Eukaryotic Expression Plasmid and Evaluation of Its Anti-Apoptotic Function[J]. Scientia Agricultura Sinica, 2019, 52(6): 1102-1109.

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