本文選題：L1-ORF2串聯(lián)片段 + GFP　； 參考：《河北醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的:人類基因組約有40-50%的序列屬于重復(fù)DNA序列,長散在核元件(long interspersed nuclear elements,LINEs)是其中重要的一種類型。LINEs包括LINE-1(L1)和LINE-2(L2),前者占據(jù)了人類基因組的16.9%�；蚪M中的L1多數(shù)為不完整序列,且大多數(shù)L1重復(fù)序列在人類基因中以反序形式存在。IL-2基因,T細(xì)胞受體基因側(cè)翼含有豐富的L1序列。因為L1在基因組中多數(shù)呈不完整片斷分布,所以有必要研究L1片段對基因表達(dá)的影響。L1-ORF2(L1重復(fù)序列第2讀碼框)按正序方向插入pEGFP-C1(C1)質(zhì)粒的GFP基因下游,強(qiáng)烈抑制GFP基因表達(dá),反序方向插入則導(dǎo)致GFP轉(zhuǎn)錄提前終止,但并不清楚L1-ORF2的這種特性是其整體特征還是由個別片段造成。為了研究L1-ORF2不同片段對上游基因的影響,將來自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串聯(lián)體,分別按正、反序方向插入C1質(zhì)粒,瞬時轉(zhuǎn)染HeLa細(xì)胞,Northern雜交和熒光顯微鏡檢測下游序列對GFP基因表達(dá)的影響。富含“A”堿基是L1-ORF2的序列特點,“A”堿基含量為40.89%,為了研究富含“A”堿基的簡單重復(fù)序列是否有正、反序列影響GFP基因表達(dá)不同的特征,將736bp的(AAACAAA)n或(AG)n按正、反方向插入C1質(zhì)粒,觀察簡單重復(fù)序列對GFP基因表達(dá)的影響。 方法:1重組質(zhì)粒構(gòu)建設(shè)計5對帶合適酶切位點的引物(Table 1),用RP11-29107克隆作模板,分別擴(kuò)增5段L1-ORF2片段(各280bp)。PCR擴(kuò)增片段與C1質(zhì)粒經(jīng)酶切和T4 DNA連接酶連接,獲得5種插入一個片段的重組質(zhì)粒。XbaⅠ和NheⅠ的粘性末端可以用T4 DNA連接酶連接,但是連接后不被XbaⅠ和/或NheⅠ切斷,利用該特性反復(fù)同向串聯(lián)構(gòu)建出含8串聯(lián)片段的重組質(zhì)粒。將8串聯(lián)重組質(zhì)粒插入片段反向插入,篩選出反序重組質(zhì)粒。構(gòu)建缺失3’端序列的ORF2(3212bp)片段反向插入表達(dá)載體,命名為p280-1~8as。合成上游帶有EcoRⅠ和XbaⅠ酶切位點,下游帶有NheⅠ和KpnⅠ酶切位點,中間分別帶有AAACAAA或AG簡單重復(fù)序列的寡核苷酸,引物擴(kuò)增使其成雙鏈,擴(kuò)增片段與C1質(zhì)粒經(jīng)酶切后連接,串聯(lián)重復(fù),獲得含有正、反序簡單重復(fù)序列( 736bp )的重組質(zhì)粒,命名為p(AAACAAA)Rep , p(AAACAAA)Repas , p(AG)Rep ,p(AG)Repas。 2細(xì)胞轉(zhuǎn)染將構(gòu)建成功的重組質(zhì)粒和C1質(zhì)粒分別以脂質(zhì)體法轉(zhuǎn)染HeLa細(xì)胞,37℃、5% CO2環(huán)境培養(yǎng)36小時,12孔板細(xì)胞用于提取RNA,24孔板細(xì)胞用于熒光觀察。 3 Northern雜交用Trizol提取質(zhì)粒轉(zhuǎn)染的HeLa細(xì)胞總RNA,甲醛變性瓊脂糖凝膠電泳,毛細(xì)法將RNA轉(zhuǎn)移到尼龍膜。α-32P標(biāo)記的GFP探針雜交、沖洗后放射自顯影。尼龍膜用剝離液處理,沖洗后用檢測Neo (neomycin resistance cassette) RNA的探針再次雜交,作為對照。 4熒光陽性細(xì)胞計數(shù)轉(zhuǎn)染質(zhì)粒的HeLa細(xì)胞經(jīng)4%多聚甲醛固定,×100倍視野白光下計數(shù)細(xì)胞總數(shù),同樣視野紫蘭光下計數(shù)熒光陽性細(xì)胞數(shù),計算熒光細(xì)胞陽性率。 結(jié)果:1重組質(zhì)粒 1.1重組質(zhì)粒構(gòu)建構(gòu)建以下18種重組質(zhì)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。經(jīng)酶切(Fig.2,Fig.4),PCR(Fig.3,Fig.4),測序(Fig.5,Fig.6,Fig.7)鑒定正確。 1.2其它重組質(zhì)粒p280-1*8、p280-4*8、pAlu*8、pORF2及pORF2as為本研究室其它工作構(gòu)建。本研究所使用的全部質(zhì)粒及說明見Table 2。 2 L1-ORF2不同串聯(lián)片段和Alu串聯(lián)序列對GFP報告基因表達(dá)的影響 2.1 Northern雜交分別用L1-ORF2串聯(lián)片段和Alu串聯(lián)正、反序重組質(zhì)粒轉(zhuǎn)染HeLa細(xì)胞,提取總RNA,Northern雜交,結(jié)果(Fig.8)顯示所有L1-ORF2串聯(lián)片段中反序GFP基因的表達(dá)均高于正序, Alu與L1-ORF2片段不同,正序的GFP基因表達(dá)高于反序;不同串聯(lián)片段轉(zhuǎn)錄終止位置不同,p280-1*8、p280-5*8、p280-9*8、p280-1*8as及p280-9*8as發(fā)生轉(zhuǎn)錄提前終止。 2.2熒光陽性細(xì)胞計數(shù)選擇p280-1*8、p280-5*8、p280-9*8及相應(yīng)的反序分別轉(zhuǎn)染HeLa細(xì)胞做熒光顯微鏡觀察(Fig.9),熒光細(xì)胞陽性率均值(±SD)分別為: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發(fā)生GFP基因轉(zhuǎn)錄提前終止 3.1 Northern雜交L1-ORF2反序插入C1質(zhì)粒(pORF2as)出現(xiàn)轉(zhuǎn)錄提前終止。去除L1-ORF2 3’端序列的反序重組質(zhì)粒(p280-1~8as)與pORF2as不同,不發(fā)生轉(zhuǎn)錄提前終止,而是出現(xiàn)轉(zhuǎn)錄延伸條帶的量高于提前終止性條帶(Fig.10)。 3.2熒光陽性細(xì)胞計數(shù)pORF2, pORF2-1~8as及pORF2as分別轉(zhuǎn)染HeLa細(xì)胞做熒光顯微鏡觀察(Fig.11),熒光細(xì)胞陽性率分別為: pORF2(0.16±0.03)% , pORF2-1~8as (3.93±0.25)%,pORFas(5.45±1.00)%。 4簡單重復(fù)序列對GFP報告基因表達(dá)的影響 4.1 Northern雜交插入AAACAAA或AG簡單重復(fù)序列反序的GFP基因轉(zhuǎn)錄強(qiáng)度分別高于其正序,且AAACAAA正、反序插入均發(fā)生GFP基因轉(zhuǎn)錄提前終止(Fig.12)。 4.2熒光陽性細(xì)胞計數(shù)p(AAACAAA)Rep, p(AAACAAA)Repas, p(AG)Rep,p(AG)Repas及C1質(zhì)粒分別轉(zhuǎn)染到HeLa細(xì)胞做熒光顯微鏡觀察(Fig.13),熒光細(xì)胞陽性率分別為: 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)%。 結(jié)論: 1所有pEGFP-C1下游插入的序列均抑制GFP基因表達(dá),但是抑制程度和轉(zhuǎn)錄終止位置不同。 2 L1-ORF2反序出現(xiàn)提前終止,去除L1-ORF2的3’端序列的反序片段不再出現(xiàn)轉(zhuǎn)錄提前終止,說明L1-ORF2反序?qū)е碌腉FP基因轉(zhuǎn)錄提前終止是由L1-ORF2的3’端部分序列決定的。 3插入AAACAAA、AG簡單重復(fù)序列反序的GFP基因轉(zhuǎn)錄強(qiáng)度高于其正序,說明簡單序列在ORF2正、反序影響GFP基因表達(dá)特征上起作用。AAACAAA正、反序均引起GFP轉(zhuǎn)錄提前終止,說明短的簡單重復(fù)序列也能調(diào)節(jié)GFP基因轉(zhuǎn)錄終止。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R394

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