L1-ORF2不同位置串联片段及简单重复序列调节GFP报告基因表达

发布时间：2018-06-20 06:15

本文选题：L1-ORF2串联片段 + GFP　；参考：《河北医科大学》2008年硕士论文

【摘要】： 目的:人类基因组约有40-50%的序列属于重复DNA序列,长散在核元件(long interspersed nuclear elements,LINEs)是其中重要的一种类型。LINEs包括LINE-1(L1)和LINE-2(L2),前者占据了人类基因组的16.9%。基因组中的L1多数为不完整序列,且大多数L1重复序列在人类基因中以反序形式存在。IL-2基因,T细胞受体基因侧翼含有丰富的L1序列。因为L1在基因组中多数呈不完整片断分布,所以有必要研究L1片段对基因表达的影响。L1-ORF2(L1重复序列第2读码框)按正序方向插入pEGFP-C1(C1)质粒的GFP基因下游,强烈抑制GFP基因表达,反序方向插入则导致GFP转录提前终止,但并不清楚L1-ORF2的这种特性是其整体特征还是由个别片段造成。为了研究L1-ORF2不同片段对上游基因的影响,将来自Xq13.1位置的L1-ORF2(L1PA3家族)不同位置片段(各280bp)及Alu元件(283bp)的8串联体,分别按正、反序方向插入C1质粒,瞬时转染HeLa细胞,Northern杂交和荧光显微镜检测下游序列对GFP基因表达的影响。富含“A”碱基是L1-ORF2的序列特点,“A”碱基含量为40.89%,为了研究富含“A”碱基的简单重复序列是否有正、反序列影响GFP基因表达不同的特征,将736bp的(AAACAAA)n或(AG)n按正、反方向插入C1质粒,观察简单重复序列对GFP基因表达的影响。方法:1重组质粒构建设计5对带合适酶切位点的引物(Table 1),用RP11-29107克隆作模板,分别扩增5段L1-ORF2片段(各280bp)。PCR扩增片段与C1质粒经酶切和T4 DNA连接酶连接,获得5种插入一个片段的重组质粒。XbaⅠ和NheⅠ的粘性末端可以用T4 DNA连接酶连接,但是连接后不被XbaⅠ和/或NheⅠ切断,利用该特性反复同向串联构建出含8串联片段的重组质粒。将8串联重组质粒插入片段反向插入,筛选出反序重组质粒。构建缺失3’端序列的ORF2(3212bp)片段反向插入表达载体,命名为p280-1~8as。合成上游带有EcoRⅠ和XbaⅠ酶切位点,下游带有NheⅠ和KpnⅠ酶切位点,中间分别带有AAACAAA或AG简单重复序列的寡核苷酸,引物扩增使其成双链,扩增片段与C1质粒经酶切后连接,串联重复,获得含有正、反序简单重复序列( 736bp )的重组质粒,命名为p(AAACAAA)Rep , p(AAACAAA)Repas , p(AG)Rep ,p(AG)Repas。 2细胞转染将构建成功的重组质粒和C1质粒分别以脂质体法转染HeLa细胞,37℃、5% CO2环境培养36小时,12孔板细胞用于提取RNA,24孔板细胞用于荧光观察。 3 Northern杂交用Trizol提取质粒转染的HeLa细胞总RNA,甲醛变性琼脂糖凝胶电泳,毛细法将RNA转移到尼龙膜。α-32P标记的GFP探针杂交、冲洗后放射自显影。尼龙膜用剥离液处理,冲洗后用检测Neo (neomycin resistance cassette) RNA的探针再次杂交,作为对照。 4荧光阳性细胞计数转染质粒的HeLa细胞经4%多聚甲醛固定,×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,计算荧光细胞阳性率。结果:1重组质粒 1.1重组质粒构建构建以下18种重组质粒: p280-1*8as,p280-2*8,p280-2*8as,p280-4*8as,p280-5*8,p280-5*8as,p280-7*8,p280-7*8as,p280-8*8,p280-8*8as, p280-9*8 , p280-9*8as , pAlu*8as , p280-1~8as ,p(AAACAAA)Rep , p(AAACAAA)Repas , p(AG)Rep , p(AG)Repas。经酶切(Fig.2,Fig.4),PCR(Fig.3,Fig.4),测序(Fig.5,Fig.6,Fig.7)鉴定正确。 1.2其它重组质粒p280-1*8、p280-4*8、pAlu*8、pORF2及pORF2as为本研究室其它工作构建。本研究所使用的全部质粒及说明见Table 2。 2 L1-ORF2不同串联片段和Alu串联序列对GFP报告基因表达的影响 2.1 Northern杂交分别用L1-ORF2串联片段和Alu串联正、反序重组质粒转染HeLa细胞,提取总RNA,Northern杂交,结果(Fig.8)显示所有L1-ORF2串联片段中反序GFP基因的表达均高于正序, Alu与L1-ORF2片段不同,正序的GFP基因表达高于反序;不同串联片段转录终止位置不同,p280-1*8、p280-5*8、p280-9*8、p280-1*8as及p280-9*8as发生转录提前终止。 2.2荧光阳性细胞计数选择p280-1*8、p280-5*8、p280-9*8及相应的反序分别转染HeLa细胞做荧光显微镜观察(Fig.9),荧光细胞阳性率均值(±SD)分别为:p280-1*8(1.81±0.87)% , p280-1*8as(14±4.63)% ,p280-5*8(0.51±0.17)% , p280-5*8as(12.5±3.04)% ,p280-9*8(2.54±0.59)%,p280-9*8as(11.96±4.54)%。 3 L1-ORF2去除3’端序列后反向插入不发生GFP基因转录提前终止 3.1 Northern杂交L1-ORF2反序插入C1质粒(pORF2as)出现转录提前终止。去除L1-ORF2 3’端序列的反序重组质粒(p280-1~8as)与pORF2as不同,不发生转录提前终止,而是出现转录延伸条带的量高于提前终止性条带(Fig.10)。 3.2荧光阳性细胞计数pORF2, pORF2-1~8as及pORF2as分别转染HeLa细胞做荧光显微镜观察(Fig.11),荧光细胞阳性率分别为: pORF2(0.16±0.03)% , pORF2-1~8as (3.93±0.25)%,pORFas(5.45±1.00)%。 4简单重复序列对GFP报告基因表达的影响 4.1 Northern杂交插入AAACAAA或AG简单重复序列反序的GFP基因转录强度分别高于其正序,且AAACAAA正、反序插入均发生GFP基因转录提前终止(Fig.12)。 4.2荧光阳性细胞计数p(AAACAAA)Rep, p(AAACAAA)Repas, p(AG)Rep,p(AG)Repas及C1质粒分别转染到HeLa细胞做荧光显微镜观察(Fig.13),荧光细胞阳性率分别为: p(AAACAAA)Rep(2.86±0.25)% ,p(AAACAAA)Repas(5.71±0.41)%,p(AG)Rep(2.23±0.47)%,p(AG)Repas(2.86±0.34)%,pEGFP-C1(39±3.67)%。结论: 1所有pEGFP-C1下游插入的序列均抑制GFP基因表达,但是抑制程度和转录终止位置不同。 2 L1-ORF2反序出现提前终止,去除L1-ORF2的3’端序列的反序片段不再出现转录提前终止,说明L1-ORF2反序导致的GFP基因转录提前终止是由L1-ORF2的3’端部分序列决定的。 3插入AAACAAA、AG简单重复序列反序的GFP基因转录强度高于其正序,说明简单序列在ORF2正、反序影响GFP基因表达特征上起作用。AAACAAA正、反序均引起GFP转录提前终止,说明短的简单重复序列也能调节GFP基因转录终止。
[Abstract]:Objective: the sequence of 40-50% in the human genome belongs to the repetitive DNA sequence, and the long interspersed nuclear elements (LINEs) is one of the most important types of.LINEs including LINE-1 (L1) and LINE-2 (L2). The former occupies the incomplete sequence in the human genome, and most of the repeated sequences are repeated. The.IL-2 gene exists in the human gene in reverse order, and the flanking of the T cell receptor gene contains a rich L1 sequence. Because most of the L1 has incomplete fragment distribution in the genome, it is necessary to study the effect of L1 fragment on the gene expression (.L1-ORF2 (the second reading code frame of the L1 repeat sequence) under the GFP gene of the pEGFP-C1 (C1) plasmid in the positive direction. Swim, strongly inhibit the expression of GFP gene, and the reverse direction insertion leads to the early termination of GFP transcription, but it is not clear that this characteristic of L1-ORF2 is or is caused by individual fragments. In order to study the effect of different L1-ORF2 fragments on the upstream genes, the L1-ORF2 (L1PA3 family) from the Xq13.1 position (280bp) and Alu The 8 series of elements (283bp) were inserted into C1 plasmids in positive and reverse direction respectively, transient transfection of HeLa cells, Northern hybridization and fluorescence microscopy were used to detect the influence of downstream sequences on the expression of GFP gene. The rich "A" base was the sequence characteristic of L1-ORF2 and the content of "A" base was 40.89%, in order to study the simple repeat sequence rich in the "A" base. There is no positive, reverse sequence affects the characteristics of GFP gene expression, and 736bp (AAACAAA) n or (AG) n is inserted into the C1 plasmid in the opposite direction, and the effect of simple repeat sequence on the expression of GFP gene is observed.
Methods: 1 the 1 recombinant plasmids were designed to design 5 pairs of primers with appropriate enzyme cutting sites (Table 1), and RP11-29107 cloned as templates were used to amplify 5 segments of L1-ORF2 fragments (each 280bp) and C1 plasmids were linked with the enzyme cut and T4 DNA ligase. The sticky ends of the heavy regroup plasmids,.Xba I and Nhe I, could be used for T4 DNA. The connection was connected, but the connection was not cut off by Xba I and / or Nhe I, and the recombinant plasmid containing 8 series fragments was constructed in tandem with this characteristic repeatedly. The 8 series recombinant plasmid was inserted into the fragment and inserted to screen the reverse sequence recombinant plasmid. The ORF2 (3212bp) fragment of the missing 3 'terminal sequence was inserted into the reverse insert expression vector, named p280-1~8as. The upstream with EcoR I and Xba I enzyme tangent site, the downstream with Nhe I and Kpn I enzyme cut site, with AAACAAA or AG simple repeat sequence of oligonucleotides, primer amplification to make it double chain, amplified fragment and C1 plasmid after enzyme digestion, series repeat, to obtain a recombinant plasmid containing a positive, reverse sequence simple repeat sequence (736bp). It is named P (AAACAAA) Rep, P (AAACAAA) Repas, P (AG) Rep, P (AG) P.
The transfection of 2 cells transfected the successful recombinant plasmids and C1 plasmids to HeLa cells by liposome method, 37 C and 5% CO2 environment for 36 hours. 12 orifice cells were used to extract RNA, and 24 orifice cells were used for fluorescence observation.
3 Northern hybrids used Trizol to extract the total RNA of HeLa cells transfected by plasmids, formaldehyde denatured agarose gel electrophoresis, capillary method to transfer RNA to nylon membrane. The GFP probe labeled with alpha -32P was hybridized with autoradiography after flushing. The nylon membrane was treated with stripping solution, and after washing, the probe of Neo (neomycin resistance cassette) RNA was used as a second hybridization. Contrast.
The number of HeLa cells transfected with 4 fluorescent positive cells was fixed by 4% polyformaldehyde, the total number of cells was counted under 100 times of the white light, and the number of fluorescent positive cells was counted under the same visual field in the purple orchid. The positive rate of fluorescent cells was calculated.
Results: 1 recombinant plasmids
1.1 recombinant plasmids construct the following 18 recombinant plasmids: p280-1*8as, p280-2*8, p280-2*8as, p280-4*8as, p280-5*8, p280-5*8as, p280-7*8, p280-7*8as, p280-8*8, p280-8*8as, p280-9*8, p280-9*8as. The sequencing (Fig.5, Fig.6, Fig.7) was correctly identified.
1.2 other recombinant plasmids p280-1*8, p280-4*8, pAlu*8, pORF2 and pORF2as were constructed for other work in this laboratory. All the plasmids and instructions used in this study are Table 2..
Effects of 2 L1-ORF2 tandem fragments and Alu tandem sequences on GFP reporter gene expression
2.1 Northern hybrids were transfected with L1-ORF2 series fragments and Alu series respectively. The reverse sequence recombinant plasmid transfected into HeLa cells and extracted total RNA, Northern hybridization. Results (Fig.8) showed that the expression of reverse sequence GFP genes in all L1-ORF2 series fragments were higher than positive sequence. Alu was different from L1-ORF2 fragment, and the positive sequence of GFP gene expression was higher than reverse sequence; different series fragments were transferred. Transcription of p280-1*8, p280-5*8, p280-9*8, p280-1*8as and p280-9*8as terminated early.
2.2 p280-1*8, p280-5*8, p280-9*8 and corresponding reverse sequence were transfected to HeLa cells for fluorescence microscopy (Fig.9). The mean positive rate of fluorescent cells (+ SD) was p280-1*8 (1.81 + 0.87)%, p280-1*8as (14 + 4.63)%, p280-5 *8 (0.51 + 0.17)%, p280-5*8as (12.5 + 3.04)%, p280-9*8 (2.54 + 0.59)%, respectively. 8As (11.96 + 4.54)%.
3 L1-ORF2 removal of 3 'end sequence followed by reverse insertion without premature termination of GFP gene transcription.
The reverse sequence insertion of C1 plasmid (pORF2as) in 3.1 Northern hybrid L1-ORF2 appears to be terminated in advance. The reverse sequence recombinant plasmid (p280-1~8as) that removes the L1-ORF2 3 'end sequence is different from pORF2as, and does not occur in advance to terminate the transcription, but the amount of the transcriptional strip is higher than that of the premature termination band (Fig.10).
3.2 fluorescent positive cells were counted pORF2, pORF2-1~8as and pORF2as were transfected to HeLa cells to do fluorescence microscopy (Fig.11). The positive rates of fluorescent cells were pORF2 (0.16 + 0.03)%, pORF2-1~8as (3.93 + 0.25)%, pORFas (5.45 + 1)%, respectively.
4 Effect of simple repetitive sequences on GFP reporter gene expression
The GFP gene transcriptional intensity of 4.1 Northern blot inserting AAACAAA or AG simple repeat sequence was higher than that of its positive sequence, and AAACAAA was positive, and the reverse sequence insertion occurred early termination of GFP gene transcription (Fig.12).
4.2 P (AAACAAA) Rep, P (AAACAAA) Repas, P (AG) Rep, P (AG) Repas and plasmids were transfected to the fluorescent cells to observe the fluorescence microscope respectively. The positive rates of fluorescent cells were respectively (2.86 + 0.25)%, 2.23 + 0.47%, 2.86 + 0.34%, 39 + 39 ).
Conclusion: 1. All downstream sequences of pEGFP-C1 inhibit GFP gene expression, but the degree of inhibition is different from that of transcription termination.
The reverse sequence of 2 L1-ORF2 terminates early, and the reverse sequence of the 3 'end sequence of the L1-ORF2 is no longer terminated, indicating that the early termination of the GFP gene transcription caused by the reverse sequence of L1-ORF2 is determined by the 3' end sequence of L1-ORF2.
3 inserting AAACAAA, the GFP gene transcriptional intensity of AG simple repeat sequence is higher than that of its positive sequence, indicating that the simple sequence is positive in ORF2, the reverse order affects the.AAACAAA positive of the GFP gene expression, and the reverse sequence causes the GFP transcription to terminate ahead of time, indicating that a short simple repeat sequence can also regulate the terminating of the transcription of the node GFP gene.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R394

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