Alu串联序列抑制GFP报告基因表达
发布时间：2018-08-02 21:40
【摘要】： 目的:基因组学的研究表明,98%的基因组DNA属于非编码DNA,在非编码DNA中重复序列的含量最高,约占50%,对于这样的事实一直不能很好的解释。近年来的研究表明重复序列与多种生物学现象有关:基因转座,基因突变,肿瘤发生,自身免疫病,生物进化等。Alu元件属于重复序列中的短散布核元件(Short Interspersed Nuclear Elements),约占人类基因组的10%,在人类基因组中约有1,000,000个拷贝。近年来越来越多的证据表明Alu序列对人类基因组的结构、功能以及进化等方面有重要的影响。虽然对重复序列的研究取得了一定的进展但是对于其功能及机理性的研究还需要进一步的探讨。本实验是在pEGFP-C1质粒的报告基因GFP的下游依次装入1, 2, 4,8,14个首尾串联的Alu序列,然后在GFP基因和Alu14(14个串联Alu)之间加入SV40早期mRNA加尾信号SV40-polyA反序(240bp),共构建出6个重组质粒。将这些质粒转染入HeLa细胞中,观察GFP绿色荧光蛋白的表达,研究基因的下游不同长度的Alu序列对上游基因表达的影响,为短散布核元件的功能研究积累实验资料。 方法:1串联表达载体构建 用基因分析软件找出pEGFP-C1(pEGFP)质粒多克隆位点具有(Fig.1 Fig.2)但是待插入序列不具有的酶切位点。经分析Alu序列不含有EcoRⅠ, HindⅢ, NheⅠ, KpnⅠ和XbaⅠ酶切位点。XbaⅠ和NheⅠ两种核酸内切酶切出的粘性末端可以用T4DNA连接酶连接,但是连接以后则对XbaⅠ和NheⅠ两种核酸内切酶均不敏感。利用这种特性可以制备插有不同个数Alu串联重复序列的质粒。设计引物上游带有EcoRⅠ, XbaⅠ酶切位点,下游带有KpnⅠ,NheⅠ酶切位点,用RP11-29107克隆作为模板,扩增Alu序列。EcoRⅠ和KpnⅠ作为Alu插入的位点,制备pEGFP-Alu1(以下简称p-Alu1)。pEGFP-Alu2的制备过程为:用HindⅢ和XbaⅠ酶切构建好的pEGFP-Alu1质粒,胶回收大片段,HindⅢ和NheⅠ酶切,胶回收小片段(Fig.3),再将大、小片断用T4DNA连接酶连接,转化DH5a感受态菌,PCR筛选含有目的序列的阳性菌,进一步用酶切和测序鉴定。反复重复上述步骤制备pEGFP-Alu4、pEGFP-Alu8。酶切和连接pEGFP-Alu4(以下简称p-Alu4)和pEGFP-Alu2(以下简称p-Alu2)质粒获得pEGFP-Alu6(以下简称p-Alu6),再用pEGFP-Alu6和pEGFP-Alu8(以下简称p-Alu8)制备pEGFP-Alu14(以下简称p-Alu14)质粒。 2 pEGFP-polyAas-Alu14(以下简称p-polyAas-Alu14)构建在p-Alu14质粒GFP基因下游插入SV40早期mRNA加尾信号SV40-polyA反序(240bp),称为p-polyAas-Alu14。 3细胞转染和荧光计数 将构建好的六种质粒和pEGFP-C1以质脂体法分别转入HeLa细胞,培养24小时后在荧光显微镜下观察。在×100倍视野白光下计数细胞总数,同样视野紫兰光下计数荧光阳性细胞数并在荧光和白光下拍下细胞照片。计数细胞总数至少500个,按以下公式计算荧光细胞阳性率:荧光细胞阳性率=荧光阳性细胞数/同样视野细胞总数×100%。实验数据用均数±标准差( x±SD)表示。 结果:1构建出的p-Alu1,p-Alu2,p-Alu4,p-Alu8,p-Alu14质粒及p-polyAas-Alu14的鉴定。 (1) p-Alu1,p-Alu2,p-Alu4,p-Alu8,p-Alu14质粒酶切鉴定图谱(Fig.4 Fig.5)。 (2) p-polyAas-Alu14鉴定的序列和测序(Fig.6Fig.7)。 2 p-Alu1、p-Alu2、p-Alu4、p-Alu8、p-Alu14、p-polyAas-Alu14和pEGFP-C1七种质粒瞬时转染HeLa细胞,24小时后荧光照片及荧光计数结果(Table 1 Fig.8 Fig.9)各质粒转染后荧光细胞阳性率均值分别为:p- Alu1 17.4±0.7%、p-Alu2 13.7±1.31%、p- Alu4 10.2±0.41%、p-Alu8 5.6±0.27%、p-Alu14 0.07±0.16%、p-polyAasAlu14 10.0±0.26%和pEGFP-C1 35.3±2.66%。 结论:1成功的在pEGFP-C1质粒中插入了不同串联数目(1、2、4、8、14个)Alu序列。 2在pEGFP基因的下游插入不同长度的序列可以抑制荧光报告基因的表达,并且随着长度的增加这种抑制作用增强。 3上述的这种抑制作用不是由于pEGFP基因下游插入基因的增多而导致的质粒增大引起的。
[Abstract]:Objective: genomics studies have shown that 98% of the genomic DNA is a non coded DNA, and the repeat sequence in the non coded DNA is the highest, accounting for about 50%. The fact that this fact is not well explained. Recent studies have shown that the repeat sequences are related to a variety of biological phenomena: gene transposition, gene mutation, oncology, autoimmune disease, .Alu components, such as biological evolution, belong to the Short Interspersed Nuclear Elements in the repetitive sequence, accounting for about 10% of the human genome, and about 1000000 copies in the human genome. In recent years, more and more evidence shows that Alu sequences have important effects on the structure, function and evolution of the human genome. Although some progress has been made in the study of repeat sequences, the study of its function and rationality needs to be further explored. This experiment is in the lower reaches of the pEGFP-C1 plasmid reporting gene GFP in sequence of 1, 2, 4,8,14 first and tail series of Alu sequences, and then adding early mRN to SV40 between the GFP base and Alu14 (14 series Alu). 6 recombinant plasmids were constructed with A plus tail signal SV40-polyA reverse order (240bp). These plasmids were transfected into HeLa cells, and the expression of GFP green fluorescent protein was observed. The effect of Alu sequence on the upstream gene expression in the downstream length of the gene was studied, and the experimental data for the function of short scattered nuclear elements were accumulated.
Method: 1 construction of tandem expression vector
The pEGFP-C1 (pEGFP) plasmid polyclonal site was found to have (Fig.1 Fig.2) but the inserted sequence did not have the enzyme tangent site. After analysis, the Alu sequence did not contain EcoR I, Hind III, Nhe I, Kpn I and Xba I enzyme tangent site.Xba I and Nhe I two kinds of nucleic acid endonucleases can be linked with the ligase. It is not sensitive to two kinds of endonucleases of Xba I and Nhe I after connection. Using this characteristic, we can prepare plasmids with different number of Alu series repeats. The primers are designed to carry the EcoR I, Xba I enzyme cut site upstream, the downstream with Kpn I, Nhe I enzyme cut site, RP11-29107 clones as templates, Alu sequence.EcoR I and Kp N I as the insertion site of Alu, the preparation process of pEGFP-Alu1 (hereinafter referred to as p-Alu1).PEGFP-Alu2 was prepared by the pEGFP-Alu1 plasmids constructed with Hind III and Xba I enzyme, the gel recovered large fragments, Hind III and Nhe I enzyme cut, the glue recovered small fragments (Fig.3), and then the large fragments were connected by T4DNA ligase and converted to the receptive bacteria. The positive bacteria of the target sequence were further identified by enzyme digestion and sequencing. Repeat the above steps to prepare pEGFP-Alu4, pEGFP-Alu8. enzyme cutting and connection pEGFP-Alu4 (hereinafter referred to as p-Alu4) and pEGFP-Alu2 (hereinafter referred to as p-Alu2) plasmid to obtain pEGFP-Alu6 (hereinafter referred to as p-Alu6), and then use pEGFP-Alu6 and pEGFP-Alu8 (hereinafter referred to as p-Alu8) to prepare pEGFP-Alu14 (hereinafter referred to as p-Alu8). The following abbreviated p-Alu14) plasmids.
2 pEGFP-polyAas-Alu14 (hereinafter referred to as p-polyAas-Alu14) is constructed downstream of the p-Alu14 plasmid GFP gene into the SV40 early mRNA plus tail signal SV40-polyA reverse sequence (240bp), called p-polyAas-Alu14.
3 cell transfection and fluorescence counting
Six plasmids and pEGFP-C1 were transferred into HeLa cells with fat body method respectively. After 24 hours culture, the total number of cells was counted under 100 times of the white light. The number of positive cells was counted under the same vision and the number of cells was taken under the fluorescence and white light. The count cell count was at least 500, according to the number of cells. The positive rate of fluorescent cells was calculated by the next Formula: the positive rate of fluorescent cells = the number of fluorescent cells / the total number of cells in the same field of visual field * 100%. experimental data were expressed as mean standard deviation (x + SD).
Results: 1 the identification of p-Alu1, p-Alu2, p-Alu4, p-Alu8, p-Alu14 plasmids and p-polyAas-Alu14 were constructed.
(1) p-Alu1, p-Alu2, p-Alu4, p-Alu8, p-Alu14 plasmid identification map (Fig.4 Fig.5).
(2) p-polyAas-Alu14 identification sequence and sequencing (Fig.6Fig.7).
2 p-Alu1, p-Alu2, p-Alu4, p-Alu8, p-Alu14, p-polyAas-Alu14 and pEGFP-C1 were transiently transfected to HeLa cells. After 24 hours, the fluorescent photo and fluorescence count results (Table 1 Fig.8 Fig.9) were respectively 17.4 + 0.7%, 13.7 + 1.31%, 10.2 + 0.41%, 5.6 + 0.27%, respectively. U14 0.07 + 0.16%, p-polyAasAlu14 10 + 0.26% and pEGFP-C1 35.3 + 2.66%.
Conclusion: 1 successful insertion of different serial numbers (1,2,4,8,14) Alu sequences in pEGFP-C1 plasmid.
The expression of fluorescent reporter gene was inhibited by inserting different length sequences downstream of pEGFP gene, and the inhibition increased with the increase of length.
3 the above inhibition is not caused by the increase of plasmids caused by the insertion of pEGFP genes.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
本文编号：2160835
[Abstract]:Objective: genomics studies have shown that 98% of the genomic DNA is a non coded DNA, and the repeat sequence in the non coded DNA is the highest, accounting for about 50%. The fact that this fact is not well explained. Recent studies have shown that the repeat sequences are related to a variety of biological phenomena: gene transposition, gene mutation, oncology, autoimmune disease, .Alu components, such as biological evolution, belong to the Short Interspersed Nuclear Elements in the repetitive sequence, accounting for about 10% of the human genome, and about 1000000 copies in the human genome. In recent years, more and more evidence shows that Alu sequences have important effects on the structure, function and evolution of the human genome. Although some progress has been made in the study of repeat sequences, the study of its function and rationality needs to be further explored. This experiment is in the lower reaches of the pEGFP-C1 plasmid reporting gene GFP in sequence of 1, 2, 4,8,14 first and tail series of Alu sequences, and then adding early mRN to SV40 between the GFP base and Alu14 (14 series Alu). 6 recombinant plasmids were constructed with A plus tail signal SV40-polyA reverse order (240bp). These plasmids were transfected into HeLa cells, and the expression of GFP green fluorescent protein was observed. The effect of Alu sequence on the upstream gene expression in the downstream length of the gene was studied, and the experimental data for the function of short scattered nuclear elements were accumulated.
Method: 1 construction of tandem expression vector
The pEGFP-C1 (pEGFP) plasmid polyclonal site was found to have (Fig.1 Fig.2) but the inserted sequence did not have the enzyme tangent site. After analysis, the Alu sequence did not contain EcoR I, Hind III, Nhe I, Kpn I and Xba I enzyme tangent site.Xba I and Nhe I two kinds of nucleic acid endonucleases can be linked with the ligase. It is not sensitive to two kinds of endonucleases of Xba I and Nhe I after connection. Using this characteristic, we can prepare plasmids with different number of Alu series repeats. The primers are designed to carry the EcoR I, Xba I enzyme cut site upstream, the downstream with Kpn I, Nhe I enzyme cut site, RP11-29107 clones as templates, Alu sequence.EcoR I and Kp N I as the insertion site of Alu, the preparation process of pEGFP-Alu1 (hereinafter referred to as p-Alu1).PEGFP-Alu2 was prepared by the pEGFP-Alu1 plasmids constructed with Hind III and Xba I enzyme, the gel recovered large fragments, Hind III and Nhe I enzyme cut, the glue recovered small fragments (Fig.3), and then the large fragments were connected by T4DNA ligase and converted to the receptive bacteria. The positive bacteria of the target sequence were further identified by enzyme digestion and sequencing. Repeat the above steps to prepare pEGFP-Alu4, pEGFP-Alu8. enzyme cutting and connection pEGFP-Alu4 (hereinafter referred to as p-Alu4) and pEGFP-Alu2 (hereinafter referred to as p-Alu2) plasmid to obtain pEGFP-Alu6 (hereinafter referred to as p-Alu6), and then use pEGFP-Alu6 and pEGFP-Alu8 (hereinafter referred to as p-Alu8) to prepare pEGFP-Alu14 (hereinafter referred to as p-Alu8). The following abbreviated p-Alu14) plasmids.
2 pEGFP-polyAas-Alu14 (hereinafter referred to as p-polyAas-Alu14) is constructed downstream of the p-Alu14 plasmid GFP gene into the SV40 early mRNA plus tail signal SV40-polyA reverse sequence (240bp), called p-polyAas-Alu14.
3 cell transfection and fluorescence counting
Six plasmids and pEGFP-C1 were transferred into HeLa cells with fat body method respectively. After 24 hours culture, the total number of cells was counted under 100 times of the white light. The number of positive cells was counted under the same vision and the number of cells was taken under the fluorescence and white light. The count cell count was at least 500, according to the number of cells. The positive rate of fluorescent cells was calculated by the next Formula: the positive rate of fluorescent cells = the number of fluorescent cells / the total number of cells in the same field of visual field * 100%. experimental data were expressed as mean standard deviation (x + SD).
Results: 1 the identification of p-Alu1, p-Alu2, p-Alu4, p-Alu8, p-Alu14 plasmids and p-polyAas-Alu14 were constructed.
(1) p-Alu1, p-Alu2, p-Alu4, p-Alu8, p-Alu14 plasmid identification map (Fig.4 Fig.5).
(2) p-polyAas-Alu14 identification sequence and sequencing (Fig.6Fig.7).
2 p-Alu1, p-Alu2, p-Alu4, p-Alu8, p-Alu14, p-polyAas-Alu14 and pEGFP-C1 were transiently transfected to HeLa cells. After 24 hours, the fluorescent photo and fluorescence count results (Table 1 Fig.8 Fig.9) were respectively 17.4 + 0.7%, 13.7 + 1.31%, 10.2 + 0.41%, 5.6 + 0.27%, respectively. U14 0.07 + 0.16%, p-polyAasAlu14 10 + 0.26% and pEGFP-C1 35.3 + 2.66%.
Conclusion: 1 successful insertion of different serial numbers (1,2,4,8,14) Alu sequences in pEGFP-C1 plasmid.
The expression of fluorescent reporter gene was inhibited by inserting different length sequences downstream of pEGFP gene, and the inhibition increased with the increase of length.
3 the above inhibition is not caused by the increase of plasmids caused by the insertion of pEGFP genes.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
【参考文献】
相关期刊论文 前1条
1 韩苇,颜真,王俊楼,赵永同,石继红,张英起;EPO模拟肽基因4串联体的构建和表达[J];第四军医大学学报;2001年04期
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