Alu串聯(lián)序列抑制GFP報告基因表達
發(fā)布時間：2018-08-02 21:40
【摘要】： 目的:基因組學的研究表明,98%的基因組DNA屬于非編碼DNA,在非編碼DNA中重復序列的含量最高,約占50%,對于這樣的事實一直不能很好的解釋。近年來的研究表明重復序列與多種生物學現(xiàn)象有關:基因轉座,基因突變,腫瘤發(fā)生,自身免疫病,生物進化等。Alu元件屬于重復序列中的短散布核元件(Short Interspersed Nuclear Elements),約占人類基因組的10%,在人類基因組中約有1,000,000個拷貝。近年來越來越多的證據(jù)表明Alu序列對人類基因組的結構、功能以及進化等方面有重要的影響。雖然對重復序列的研究取得了一定的進展但是對于其功能及機理性的研究還需要進一步的探討。本實驗是在pEGFP-C1質粒的報告基因GFP的下游依次裝入1, 2, 4,8,14個首尾串聯(lián)的Alu序列,然后在GFP基因和Alu14(14個串聯(lián)Alu)之間加入SV40早期mRNA加尾信號SV40-polyA反序(240bp),共構建出6個重組質粒。將這些質粒轉染入HeLa細胞中,觀察GFP綠色熒光蛋白的表達,研究基因的下游不同長度的Alu序列對上游基因表達的影響,為短散布核元件的功能研究積累實驗資料。 方法:1串聯(lián)表達載體構建 用基因分析軟件找出pEGFP-C1(pEGFP)質粒多克隆位點具有(Fig.1 Fig.2)但是待插入序列不具有的酶切位點。經分析Alu序列不含有EcoRⅠ, HindⅢ, NheⅠ, KpnⅠ和XbaⅠ酶切位點。XbaⅠ和NheⅠ兩種核酸內切酶切出的粘性末端可以用T4DNA連接酶連接,但是連接以后則對XbaⅠ和NheⅠ兩種核酸內切酶均不敏感。利用這種特性可以制備插有不同個數(shù)Alu串聯(lián)重復序列的質粒。設計引物上游帶有EcoRⅠ, XbaⅠ酶切位點,下游帶有KpnⅠ,NheⅠ酶切位點,用RP11-29107克隆作為模板,擴增Alu序列。EcoRⅠ和KpnⅠ作為Alu插入的位點,制備pEGFP-Alu1(以下簡稱p-Alu1)。pEGFP-Alu2的制備過程為:用HindⅢ和XbaⅠ酶切構建好的pEGFP-Alu1質粒,膠回收大片段,HindⅢ和NheⅠ酶切,膠回收小片段(Fig.3),再將大、小片斷用T4DNA連接酶連接,轉化DH5a感受態(tài)菌,PCR篩選含有目的序列的陽性菌,進一步用酶切和測序鑒定。反復重復上述步驟制備pEGFP-Alu4、pEGFP-Alu8。酶切和連接pEGFP-Alu4(以下簡稱p-Alu4)和pEGFP-Alu2(以下簡稱p-Alu2)質粒獲得pEGFP-Alu6(以下簡稱p-Alu6),再用pEGFP-Alu6和pEGFP-Alu8(以下簡稱p-Alu8)制備pEGFP-Alu14(以下簡稱p-Alu14)質粒。 2 pEGFP-polyAas-Alu14(以下簡稱p-polyAas-Alu14)構建在p-Alu14質粒GFP基因下游插入SV40早期mRNA加尾信號SV40-polyA反序(240bp),稱為p-polyAas-Alu14。 3細胞轉染和熒光計數(shù) 將構建好的六種質粒和pEGFP-C1以質脂體法分別轉入HeLa細胞,培養(yǎng)24小時后在熒光顯微鏡下觀察。在×100倍視野白光下計數(shù)細胞總數(shù),同樣視野紫蘭光下計數(shù)熒光陽性細胞數(shù)并在熒光和白光下拍下細胞照片。計數(shù)細胞總數(shù)至少500個,按以下公式計算熒光細胞陽性率:熒光細胞陽性率=熒光陽性細胞數(shù)/同樣視野細胞總數(shù)×100%。實驗數(shù)據(jù)用均數(shù)±標準差( x±SD)表示。 結果:1構建出的p-Alu1,p-Alu2,p-Alu4,p-Alu8,p-Alu14質粒及p-polyAas-Alu14的鑒定。 (1) p-Alu1,p-Alu2,p-Alu4,p-Alu8,p-Alu14質粒酶切鑒定圖譜(Fig.4 Fig.5)。 (2) p-polyAas-Alu14鑒定的序列和測序(Fig.6Fig.7)。 2 p-Alu1、p-Alu2、p-Alu4、p-Alu8、p-Alu14、p-polyAas-Alu14和pEGFP-C1七種質粒瞬時轉染HeLa細胞,24小時后熒光照片及熒光計數(shù)結果(Table 1 Fig.8 Fig.9)各質粒轉染后熒光細胞陽性率均值分別為: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%。 結論:1成功的在pEGFP-C1質粒中插入了不同串聯(lián)數(shù)目(1、2、4、8、14個)Alu序列。 2在pEGFP基因的下游插入不同長度的序列可以抑制熒光報告基因的表達,并且隨著長度的增加這種抑制作用增強。 3上述的這種抑制作用不是由于pEGFP基因下游插入基因的增多而導致的質粒增大引起的。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R346
本文編號：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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R346
【參考文獻】
相關期刊論文 前1條
1 韓葦,顏真,王俊樓,趙永同,石繼紅,張英起;EPO模擬肽基因4串聯(lián)體的構建和表達[J];第四軍醫(yī)大學學報;2001年04期
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