C_2H_2型锌指蛋白--ZNFD的生物学功能初探
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摘要
C2H2型锌指基因是高等动物基因组中最大且最复杂的基因超家族,在人与小鼠基因组中拥有数百个成员。本实验室使用生物信息学分析,并结合NCBI数据库筛选得到一人源锌指基因ZNFD,基因号为NM_182761,对ZNFD基因进行生物信息学方法分析,结果表明ZNFD为C2H2型锌指蛋白的一员,该锌指基因尚未见相关研究,故本实验室对该基因展开了以下几个方面的初步研究。
首先,通过RT-PCR的方法从睾丸组织cDNA中克隆获得ZNFD基因,经测序鉴定ZNFD基因序列的正确性。利用生物信息学方法对ZNFD基因和蛋白进行分析,表明ZNFD基因位于第5号染色体,定位在5q23.1。ZNFD cDNA由5个外显子和4个内含子组成,含有990bp的开放阅读框,编码一个由329个氨基酸组成,分子量为37kDa的蛋白。对ZNFD基因进行组织表达谱分析证实ZNFD基因在前列腺、睾丸、脑、胰、子宫中表达,且睾丸和脑组织中表达量较高。通过生物信息学软件CLUSTAL W分析7种物种的ZNFD基因的同源性,分析表明目的蛋白的17-260aa有较高的保守性。
通过构建真核表达重组质粒pEGFP-C1 -ZNFD,细胞亚定位分析结果显示ZNFD蛋白定位于细胞核。同时为了鉴定ZNFD蛋白的N端氨基酸序列与锌指模序对ZNFD蛋白细胞核定位的影响,分别构建了一系列截短型ZNFD与绿色荧光蛋白表达载体(pEGFP-C1)的重组质粒,转染Hela细胞进行细胞亚定位分析,证明了ZNF结构域对于ZNFD蛋白的核定位不是必须的。
大量研究证明ZNF蛋白多为转录因子,因此,本文进一步对ZNFD在特定的信号通路中是否有转录调控作用进行了分析。运用Dual-荧光酶报道基因分析系统来检测ZNFD是否对AP1(PMA), GRE, SRE, p53, CRE, HSE, NF-κB和AP1的信号路径有转录调控作用,且ZNFD蛋白对AP1(PMA)有剂量依耐性激活。数据表明ZNFD蛋白导致AP1(PMA)转录活性的增强,pAP1(PMA)-TA-luc质粒含有AP1元件,该质粒专门用来监控蛋白激酶C转导路径的转录调控作用。这说明ZNFD可能作为蛋白激酶C信号途径的转录激活因子来介导调控细胞的一系列功能。另外,串联亲和纯化(TAP)为一种两步亲和纯化方法,该方法适用于分离类似生理条件下的蛋白互作复合体,因此我们构建了TAP-ZNFD真核表达载体,为后续寻找ZNFD互作蛋白奠定基础。
C2H2 zinc finger (C2H2-ZNF) genes are one of the largest and most complex gene super-families in metazoan genomes, with hundreds of members in the human and mouse genome. We got a novel human zinc finger gene ZNFD by bioinformatics analyse and NCBI database screening. The gene ID is NM_182761. The bioinformatics character suggested that ZNFD is a member of C2H2 type zinc finger protein, and this gene has not been investigated. The main complishments are as follows.
Firstly, we cloned the ZNFD gene from testis cDNA by RT-PCR, then identified by sequencing. Bioinformatics databases were used to analyze and predict its function. We confirmed that ZNFD gene has a 990bp full open reading frame (ORF), which was localized on chromosome 5q23.1. It was composed of five exons and four introns. The ZNFD gene encoded a protein with 329 amino acids. The molecular weight was predicted to be 37kDa. RT–PCR was used to determine the expression pattern of ZNFD in human multiple tissue. The tissue distribution pattern of ZNFD mRNA showed that the band was detected in the prostate, testis, brain, spleen, pancreas, and uterus. ZNFD gene is strongly expressed in adult testis and brain.
Multiple alignment analysis was performed by ClustalW software in 7 ZNFD orthologs. ZNFD was highly conservative in 17 to 260aa in different species. To verify the subcelluar location of ZNFD protein, we contruct the plasmid pEGFP-C1-ZNFD which express the fused protein EGFP-ZNFD, then transfected it into Hela cells. The result indicated that ZNFD is a nuclear protein. To verify the subcelluar location and the function of N-terminal residues and ZNF motif in location of our interested protein, pEGFP-C1-ZNFD, pEGFP-C1-D-ZNF, and pEGFP-C1-ZNF were transfected into Hela cells. These results indicate that ZNFD is a nuclear protein and the ZNF domain is not required for nuclear localization of the ZNFD protein in Hela cells.
Many papers showed that many ZNF proteins function as transcription factors. Accordingly, it was reasonable to postulate that ZNFD possibly possesses transcription function. Here we performed Dual-luciferase reporter assay system to investigate the potentially role of ZNFD on signaling pathway including AP1(PMA), GRE, SRE, p53, CRE, HSE, NF-κB and AP1. The data indicate that ZNFD protein induces the activation of AP1(PMA) transcription, and ZNFD induced a dose- dependent activation of the AP1(PMA) reporter gene. The pAP1(PMA)-TA-luc plasmid contains the AP1(Activator of protein 1) elements, which are designed for monitoring the induction of the PKC signal transduction pathway. So it suggests that the ZNFD may act as a transcriptional activator in PKC signal pathway to mediate cellular functions. Addition, tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions. So we construct the plasmid which expresses the fusion TAP-ZNFD protein to analysis of protein-protein interaction.
首先,通过RT-PCR的方法从睾丸组织cDNA中克隆获得ZNFD基因,经测序鉴定ZNFD基因序列的正确性。利用生物信息学方法对ZNFD基因和蛋白进行分析,表明ZNFD基因位于第5号染色体,定位在5q23.1。ZNFD cDNA由5个外显子和4个内含子组成,含有990bp的开放阅读框,编码一个由329个氨基酸组成,分子量为37kDa的蛋白。对ZNFD基因进行组织表达谱分析证实ZNFD基因在前列腺、睾丸、脑、胰、子宫中表达,且睾丸和脑组织中表达量较高。通过生物信息学软件CLUSTAL W分析7种物种的ZNFD基因的同源性,分析表明目的蛋白的17-260aa有较高的保守性。
通过构建真核表达重组质粒pEGFP-C1 -ZNFD,细胞亚定位分析结果显示ZNFD蛋白定位于细胞核。同时为了鉴定ZNFD蛋白的N端氨基酸序列与锌指模序对ZNFD蛋白细胞核定位的影响,分别构建了一系列截短型ZNFD与绿色荧光蛋白表达载体(pEGFP-C1)的重组质粒,转染Hela细胞进行细胞亚定位分析,证明了ZNF结构域对于ZNFD蛋白的核定位不是必须的。
大量研究证明ZNF蛋白多为转录因子,因此,本文进一步对ZNFD在特定的信号通路中是否有转录调控作用进行了分析。运用Dual-荧光酶报道基因分析系统来检测ZNFD是否对AP1(PMA), GRE, SRE, p53, CRE, HSE, NF-κB和AP1的信号路径有转录调控作用,且ZNFD蛋白对AP1(PMA)有剂量依耐性激活。数据表明ZNFD蛋白导致AP1(PMA)转录活性的增强,pAP1(PMA)-TA-luc质粒含有AP1元件,该质粒专门用来监控蛋白激酶C转导路径的转录调控作用。这说明ZNFD可能作为蛋白激酶C信号途径的转录激活因子来介导调控细胞的一系列功能。另外,串联亲和纯化(TAP)为一种两步亲和纯化方法,该方法适用于分离类似生理条件下的蛋白互作复合体,因此我们构建了TAP-ZNFD真核表达载体,为后续寻找ZNFD互作蛋白奠定基础。
C2H2 zinc finger (C2H2-ZNF) genes are one of the largest and most complex gene super-families in metazoan genomes, with hundreds of members in the human and mouse genome. We got a novel human zinc finger gene ZNFD by bioinformatics analyse and NCBI database screening. The gene ID is NM_182761. The bioinformatics character suggested that ZNFD is a member of C2H2 type zinc finger protein, and this gene has not been investigated. The main complishments are as follows.
Firstly, we cloned the ZNFD gene from testis cDNA by RT-PCR, then identified by sequencing. Bioinformatics databases were used to analyze and predict its function. We confirmed that ZNFD gene has a 990bp full open reading frame (ORF), which was localized on chromosome 5q23.1. It was composed of five exons and four introns. The ZNFD gene encoded a protein with 329 amino acids. The molecular weight was predicted to be 37kDa. RT–PCR was used to determine the expression pattern of ZNFD in human multiple tissue. The tissue distribution pattern of ZNFD mRNA showed that the band was detected in the prostate, testis, brain, spleen, pancreas, and uterus. ZNFD gene is strongly expressed in adult testis and brain.
Multiple alignment analysis was performed by ClustalW software in 7 ZNFD orthologs. ZNFD was highly conservative in 17 to 260aa in different species. To verify the subcelluar location of ZNFD protein, we contruct the plasmid pEGFP-C1-ZNFD which express the fused protein EGFP-ZNFD, then transfected it into Hela cells. The result indicated that ZNFD is a nuclear protein. To verify the subcelluar location and the function of N-terminal residues and ZNF motif in location of our interested protein, pEGFP-C1-ZNFD, pEGFP-C1-D-ZNF, and pEGFP-C1-ZNF were transfected into Hela cells. These results indicate that ZNFD is a nuclear protein and the ZNF domain is not required for nuclear localization of the ZNFD protein in Hela cells.
Many papers showed that many ZNF proteins function as transcription factors. Accordingly, it was reasonable to postulate that ZNFD possibly possesses transcription function. Here we performed Dual-luciferase reporter assay system to investigate the potentially role of ZNFD on signaling pathway including AP1(PMA), GRE, SRE, p53, CRE, HSE, NF-κB and AP1. The data indicate that ZNFD protein induces the activation of AP1(PMA) transcription, and ZNFD induced a dose- dependent activation of the AP1(PMA) reporter gene. The pAP1(PMA)-TA-luc plasmid contains the AP1(Activator of protein 1) elements, which are designed for monitoring the induction of the PKC signal transduction pathway. So it suggests that the ZNFD may act as a transcriptional activator in PKC signal pathway to mediate cellular functions. Addition, tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions. So we construct the plasmid which expresses the fusion TAP-ZNFD protein to analysis of protein-protein interaction.
引文
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