斑马鱼notch3基因真核表达载体的构建及其表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0289

摘要/Abstract

摘要：

为了进一步研究notch3基因在斑马鱼中的功能,构建了斑马鱼notch3真核表达载体并在真核细胞中成功表达。其中斑马鱼notch3基因编码序列（coding sequence,CDS）从NCBI的在线数据库中获得,根据序列克隆其胞内段（notch intracellular domain,NICD）,接着利用同源重组技术构建pCMV-N3ICD表达载体。再通过双酶切和测序筛选阳性重组载体,借由脂质体法转染至HEK293T细胞中,最后经细胞免疫荧光技术和蛋白质印迹法验证N3ICD的表达。结果显示,重组质粒经酶切电泳片段及测序比对均与预期结果一致。在蛋白质印迹实验中N3ICD蛋白可正常表达,且大小与预测结果一致。细胞免疫荧光显示重组质粒可以在HEK293T细胞的细胞核中表达。同时,构建的N3ICD真核表达载体能够明显增强NF-κB（nuclear factor-кB）家族中rela和nfκb1启动子的活性。成功构建了pCMV-N3ICD真核表达载体。

关键词: 斑马鱼, notch3, N3ICD, 免疫荧光, 真核表达载体, NF-κB

Abstract:

In order to further study the function of notch3 in Danio rerio,the eukaryotic expression vector of notch3 was constructed and it successfully expressed in eukaryotic cells. The coding sequence（CDS）of notch3 gene in D. rerio was obtained from NCBI online database. According to the sequence,Notch intracellular domain（NICD）was cloned,and then the expression vector pCMV-N3ICD was constructed by homologous recombination. The positive recombinant vector was screened by double enzyme digestion and sequencing,and then transfected into HEK293T cells by liposome method. Finally,the expression of N3ICD was verified by immunofluorescence and Western blot. The results showed that the recombinant plasmids were in accordance with the expected results via enzymatic digestion electrophoresis fragment and sequencing comparison. In Western blot,N3ICD protein expressed normally,and the size was consistent with the predicted one. Cell immunofluorescence showed that the recombinant plasmid expressed in the nucleus of the HEK293T cells. Concurrently,the constructed eukaryotic expression vector N3ICD significantly enhanced the activities of rela and nfkb1 promoters in NF-κB family（nuclear factor-κB）.

Key words: Danio rerio, notch3, N3ICD, immunofluorescence, eukaryotic expression vector, NF-κB

吴坤坤, 徐行, 季策, 任建峰, 李伟明, 张庆华. 斑马鱼notch3基因真核表达载体的构建及其表达分析[J]. 生物技术通报, 2022, 38(1): 179-186.

WU Kun-kun, XU Xing, JI Ce, REN Jian-feng, LI Wei-ming, ZHANG Qing-hua. Eukaryotic Expression Vector Construction of Danio rerio notch3 Gene and Its Expression Analysis[J]. Biotechnology Bulletin, 2022, 38(1): 179-186.

图/表 6

表1 N3ICD引物扩增序列

Table 1 N3ICD primer amplification sequence

长度 Length/bp	引物序列 Sequence of primer	酶切位点选择 Selection of enzyme cutting sites
2 487	TGGGCTAGGCTACTGCTAGTAGG	Pst I和BamH I
2 487	TCAAGCAAACACCTGCATCTTC	Pst I和BamH I

图1 NJ法构建Notch3进化树

Fig. 1 Construction of Notch3 evolutionary tree by NJ method

图2 notch3基因胞内段的克隆（A）及重组质粒的构建（B） M：分子量标准;1：notch3基因胞内段片段;2、3：阳性克隆

Fig. 2 Cloning of notch3 gene Intracellular domain（A）and construction of recombinant plasmid（B） M：Molecular weight standard. 1：Intracellular segment of notch3 gene. 2, 3：Positive clone

图3 pCMV-N3ICD在HEK293T细胞中的定位

Fig. 3 Localization of pCMV-N3ICD in HEK293T cells

图4 Western blot检测N3ICD蛋白的表达（通过Flag标签显示）

Fig. 4 Expression of N3ICD protein was detected by Western blot（shown by Flag tag）

图5 过表达斑马鱼notch3基因 t test,n=3,ns P>0.05,* P<0.05,**P<0.01 pGL3-rela-promoter-Enhancer（A）和pGL3-nfκb1-Enhancer（B）的转录活性

Fig. 5 Overexpression of notch3 gene Transcriptional activity of pGL3-rela-promoter-enhancer（A）and pGL3-nfκb1-enhancer（B）

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