REGULATION OF HSP70 GENE BY NOTCH1A AND NOTCH1B IN ZEBRAFISH (DANIO RERIO)
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摘要:
研究旨在通过双荧光素酶报告基因实验探究斑马鱼notch1a和notch1b基因对hsp70的调控作用。通过NCBI数据库检索获取了斑马鱼notch1a和notch1b基因的CDS序列, 对其胞内段(Notch1a/Notch1b intracellular domain, N1aICD/N1bICD)进行克隆并构建pCMV-N1aICD和pCMV-N1bICD真核表达载体, 在人胚肾细胞(HEK293T)中用Western Blot和亚细胞定位检测N1aICD和N1bICD的表达。通过生信分析并克隆斑马鱼hsp70启动子序列, 构建pGL3-hsp70-pro报告基因, 并在HEK293T中检测该报告基因的双荧光素酶活性。之后在HEK293T细胞中过表达notch1a和notch1b基因, 通过双荧光素酶报告基因实验检测pGL3-hsp70-pro的活性变化, 以此探究notch1a和notch1b对hsp70基因的调控作用。实验结果显示真核表达载体pCMV-N1aICD和pCMV-N1bICD构建成功, Western Blot显示pCMV-N1aICD和pCMV-N1bICD可正常表达, 亚细胞定位显示N1aICD和N1bICD蛋白表达在HEK293T的细胞核中; 双荧光素酶实验显示pGL3-hsp70-pro报告基因在HEK293T细胞中具有活性, 是阴性对照质粒的3.7倍; 在HEK293T细胞中pCMV-N1aICD和pCMV-N1bICD真核表达载体能够显著增强pGL3-hsp70-pro的活性, 分别为对照组的4.9倍和5.1倍。实验结果表明斑马鱼 notch1a和notch1b基因可显著增强hsp70基因的表达, 为进一步研究Notch分子通过Hsp70进行抗感染免疫的分子机制提供了实验材料并奠定了理论基础。
Abstract:The aim of this study was to investigate the regulatory role of zebrafish (Danio rerio) notch1a and notch1b genes on hsp70 by using a dual luciferase reporter gene assay. The CDS sequences of zebrafish notch1a and notch1b genes were obtained through NCBI database search, and their intracellular domains (Notch1a/Notch1b intracellular domain, N1aICD/N1bICD) were cloned. Eukaryotic expression vectors, pCMV-N1aICD and pCMV-N1bICD, were constructed. The expressions of N1aICD and N1bICD were detected in human embryonic kidney cells (HEK293T) through Western Blot and subcellular localization. The zebrafish hsp70 promoter sequence was analyzed, cloned, and incorporated into the pGL3-hsp70-pro reporter gene, which was constructed and assayed for dual luciferase activity in HEK293T. Overexpression of notch1a and notch1b genes in HEK293T cells was performed, and changes in pGL3-hsp70-pro activity were detected using a dual-luciferase reporter gene assay. The results showed that the successful construction of eukaryotic expression vectors pCMV-N1aICD and pCMV-N1bICD. Western Blot analysis confirmed the normal expression of pCMV-N1aICD and pCMV-N1bICD, while the subcellular localization assay showed the normal expression in the nucleus of HEK293T cells. The dual-luciferase reporter gene showed that the pGL3-hsp70-pro reporter gene was active in HEK293T cells, exhibiting activity 3.7 times higher than that of the empty plasmid. Additionally, the pCMV-N1aICD and pCMV-N1bICD eukaryotic expression vectors significantly enhanced the activity of pGL3-hsp70-pro in HEK293T cells, showing increases of 4.9-fold and 5.1-fold compared to the control, respectively. The results indicated that zebrafish notch1a and notch1b genes play a substantial role in enhancing the expression of hsp70 gene. This provides experimental materials and a theoretical foundation for further investigation into the immune mechanism of Notch molecular in defense against infection and apoptosis through Hsp70.
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Keywords:
- notch1a /
- notch1b /
- hsp70 /
- Luciferase /
- Apoptosis /
- Danio rerio
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图 1 斑马鱼Notch1a和Notch1b结构示意图
NECD. 胞外结构域; TM. 跨膜域; NICD. 胞内段
Figure 1. Structure diagram of zebrafish Notch1a and Notch1b
NECD. Notch extracellular domain; TM. Transmembrane domain; NICD. Notch intracellular domain
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图 3 pCMV-N1aICD和pCMV-N1bICD真核表达载体的构建及验证
M1. DL5000 DNA Marker; 1. notch1a基因胞内段; 2. notch1b基因胞内段; M2. 蛋白分子量标准; 3. pCMV-Tag2B; 4. pCMV-N1aICD; 5. pCMV-N1bICD
Figure 3. Construction and validation of pCMV-N1aICD and pCMV-N1bICD eukaryotic expression vectors
M1. DL5000 DNA Marker; 1. N1aICD; 2. N1bICD; M2. protein marker; 3. pCMV-Tag2B; 4. pCMV-N1aICD; 5. pCMV-N1bICD
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图 4 N1aICD和N1bICD在HEK293T细胞中的定位
Figure 4. Localization of N1aICD and N1bICD in HEK293T cells
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图 5 斑马鱼hsp70启动子CpG岛和转录因子结合位点的预测
Figure 5. Prediction of CpG island and transcription factor binding sites in zebrafish hsp70 promote
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图 6 pGL3-hsp70-pro报告基因的构建及活性检测
M. DL5000 DNA Marker; 1. hsp70启动子的扩增; **表示与对照组有极显著性差异(P<0.01)
Figure 6. Construction and activity detection of pGL3-hsp70-pro reporter gene
M. DL5000 DNA Marker; 1. cloning of hsp70 promoter; ** means very significant difference compare with the control (P<0.01)
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图 7 验证N1aICD和N1bICD 对pGL3-hsp70-pro转录活性的影响
**表示与对照组有极显著性差异(P<0.01); 1. pCMV-Tag2B+pGL3-hsp70-pro; 2. pCMV-N1aICD+pGL3-hsp70-pro; 3. pCMV-N1bICD+ pGL3-hsp70-pro
Figure 7. Verification the effects of N1aICD and N1bICD on pGL3-hsp70-pro transcriptional activity
**means very significant difference compare with the control (P<0.01); 1. pCMV-Tag2B+pGL3-hsp70-pro; 2. pCMV-N1aICD+pGL3-hsp70-pro; 3. pCMV-N1bICD+pGL3-hsp70-pro
表 1 实验所用引物序列
Table 1 Primer sequence used in the experiment
引物名称
Primer name序列
Sequence (5′—3′)用途
Usagenotch1a-F1 GGCCTCCAGTGGAAACCT CDS 序列扩增
Amplification of CDS sequencesnotch1a-R1 CTACTTGAAGGCTTCTGGAATATG notch1b-F1 CAAAGGCTCCGTGGTTTAT notch1b-R1 AAGCAGGCTTATGGAATGTG notch1a-F2 CGCGGATCCTCCAGGAAGAGGAAGCGGG 表达载体构建
Construction of expression vectornotch1a-R2 CCCAAGCTTCTTGAAGGCTTCTGGAATATGGTTCATC notch1b-F2 CGCGGATCCTCCCGTAAACGGCGCCG notch1b-R2 CCCAAGCTTCTTGAATTGCTCGGGCATGTG hsp70-F CGAGCTCTTACGCGTGCTAGCTGAAGTGGACGGATTGAGTGA 报告基因载体构建
Construction of reporter gene vectorhsp70-R ACTTAGATCGCAGATCTCGAGAAACACCTCGTCGGGGAAAA 
下载: 导出CSV
表 2 hsp70报告基因活性检测转染体系
Table 2 Transfection system for detecting hsp70 promoter reporter gene activity
组别
GrouppGL3-
Enhancer (ng)pGL3-hsp70-
pro (ng)pRL-
TK (ng)1 200 0 20 2 0 200 20
下载: 导出CSV
表 3 转染复合物配制表
Table 3 Formulation table of transfected compound
组别
GrouppCMV-
Tag2B (ng)N1aICD
(ng)N1bICD
(ng)pGL3-hsp70-
pro (ng)pRL-TK
(ng)1 200 0 0 100 10 2 0 200 0 100 10 3 0 0 200 100 10
下载: 导出CSV
表 4 斑马鱼Notch1a、Notch1b和人、小鼠Notch1氨基酸相似性分析(%)
Table 4 Amino acid similarity analysis of zebrafish Notch1a, Notch1b, human and mouse Notch1 (%)
物种
Species斑马鱼
Danio rerio人
Homo sapiens小鼠
Mus musculusNotch1a Notch1b Notch1 Notch1 斑马鱼
Danio
rerioNotch1a 100.00 68.72 66.86 64.41 Notch1b 68.72 100.00 67.67 67.72
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