本文關(guān)鍵詞：以凡納濱對(duì)蝦β-actin基因啟動(dòng)子為元件的重組昆蟲(chóng)桿狀病毒表達(dá)系統(tǒng)的建立　出處：《中國(guó)科學(xué)院研究生院(海洋研究所)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 凡納濱對(duì)蝦β-actin啟動(dòng)子 啟動(dòng)子活性 調(diào)控元件 重組昆蟲(chóng)桿狀病毒 轉(zhuǎn)導(dǎo)系統(tǒng)

【摘要】：凡納濱對(duì)蝦(Litopenaeus vannamei)隸屬于甲殼亞綱十足目,是目前全世界對(duì)蝦養(yǎng)殖產(chǎn)業(yè)中產(chǎn)量和產(chǎn)值最高的經(jīng)濟(jì)物種。由于病害侵襲,對(duì)蝦養(yǎng)殖業(yè)每年面臨嚴(yán)重的損失。目前大量的研究集中于病害防治、遺傳育種和水產(chǎn)養(yǎng)殖等多方面,其中,在病害防治和基因功能研究中,基因過(guò)表達(dá)研究受制頗多,缺乏有效的表達(dá)系統(tǒng)便是其中之一。表達(dá)系統(tǒng)包括表達(dá)載體和宿主細(xì)胞,其中有效的表達(dá)載體需要高效的啟動(dòng)元件。目前有關(guān)對(duì)蝦高效同源啟動(dòng)子的開(kāi)發(fā)極少。β-actin啟動(dòng)子是目前在其他物種中常用的高效的同源啟動(dòng)子,本文以凡納濱對(duì)蝦為研究對(duì)象,在前期研究工作已獲得凡納濱對(duì)蝦β-actin基因序列(actin T1)的基礎(chǔ)上,首次擴(kuò)增得到其β-actin5’上游序列,并對(duì)其活性、應(yīng)用范圍和調(diào)控元件功能進(jìn)行深入研究,同時(shí)嘗試將其與昆蟲(chóng)桿狀病毒系統(tǒng)相結(jié)合,開(kāi)發(fā)有效的表達(dá)系統(tǒng)。本研究的主要目的是:開(kāi)發(fā)高效的對(duì)蝦同源啟動(dòng)子和嘗試建立有效的外源表達(dá)系統(tǒng)。本研究獲得的主要進(jìn)展如下:1.凡納濱對(duì)蝦β-actin基因5’上游序列的分子克隆、功能分析及應(yīng)用范圍研究通過(guò)反向PCR(inverse PCR,i PCR)等方法,我們獲得了凡納濱對(duì)蝦β-actin基因5’上游序列,并將其命名為Sba P(Shrimp beta-actin promoter)。該序列全長(zhǎng)1,642 bp,分別包含5’側(cè)翼序列、第一外顯子、第一內(nèi)含子和第二外顯子部分區(qū)域。通過(guò)生物信息學(xué)分析和啟動(dòng)活性檢測(cè)發(fā)現(xiàn),盡管5’側(cè)翼序列對(duì)于β-actin基因表達(dá)起到非常重要的作用(該區(qū)域含有非常保守的CCAAT-box和CAr G motif),但是全長(zhǎng)序列Sba P具有比5’側(cè)翼序列更強(qiáng)的啟動(dòng)活性。為了評(píng)估Sba P啟動(dòng)活性強(qiáng)弱,將其與4種常用的病毒來(lái)源的組成型啟動(dòng)子,即巨細(xì)胞病毒(Cytomegalovirus,CMV)啟動(dòng)子,猴猿空泡病毒40(Simian vacuolating virus 40,SV40)啟動(dòng)子,昆蟲(chóng)桿狀病毒多角體蛋白(Polyhedrin,Polh)啟動(dòng)子和白斑綜合征病毒極早期基因1(white spot syndrome virus immediate early gene 1,WSSV ie1)啟動(dòng)子以及一種羅非魚(yú)來(lái)源的β-actin啟動(dòng)子(Tba P)進(jìn)行活性比較。由于啟動(dòng)子在不同細(xì)胞系中啟動(dòng)強(qiáng)度不同,活性分析在8種不同物種來(lái)源的細(xì)胞系中展開(kāi),通過(guò)檢測(cè)構(gòu)建的啟動(dòng)子-熒光素酶基因表達(dá)載體在轉(zhuǎn)染的不同細(xì)胞系中的相對(duì)熒光素酶表達(dá)量,間接分析啟動(dòng)元件活性強(qiáng)弱。結(jié)果表明,在測(cè)試的8種細(xì)胞系中,Sba P能夠全部啟動(dòng)報(bào)告基因的表達(dá),它們分別是,sf21(昆蟲(chóng)),PAC2(斑馬魚(yú)),EPC(鯉魚(yú)),CHSE-214(鮭魚(yú)),GSTEF(綠龜),MS-1(僧海豹),293T(人類)和He La(人類)的細(xì)胞系,但是各自的表達(dá)模式不同。在除sf21昆蟲(chóng)細(xì)胞系外的其他細(xì)胞系中,Sba P介導(dǎo)的報(bào)告基因相對(duì)表達(dá)量均弱于CMV啟動(dòng)子10倍以上,但是顯著性地高于Polh啟動(dòng)子(除CHSE-214細(xì)胞系),在某些細(xì)胞系與SV40,ie1和Tba P啟動(dòng)子活性相近。而在sf21昆蟲(chóng)細(xì)胞系中,Sba P介導(dǎo)的熒光素酶表達(dá)量顯著性地高于除ie1外所有啟動(dòng)子,并且其熒光素酶相對(duì)表達(dá)量較其它組均高10倍以上。2.Sba P調(diào)控元件結(jié)構(gòu)及功能,以及優(yōu)化后啟動(dòng)子活性研究Sba P啟動(dòng)活性分析檢測(cè)表明,其第一內(nèi)含子區(qū)域可能存在調(diào)控元件,為檢測(cè)調(diào)控元件結(jié)構(gòu)及功能,我們構(gòu)建了Sba P缺失突變-熒光素酶表達(dá)載體。通過(guò)檢測(cè)缺失突變體啟動(dòng)活性發(fā)現(xiàn),在第一內(nèi)含子區(qū)域中存在有兩個(gè)抑制子(-1140/-925,-222/-21)和至少一個(gè)增強(qiáng)子(-810/-223)區(qū)域。而缺少抑制子的突變體Sba PΔ-222/+1Δ-1325/-925(命名為Sba P(ENX)),在昆蟲(chóng)細(xì)胞中表現(xiàn)出了更強(qiáng)的啟動(dòng)活性,其報(bào)告基因相對(duì)表達(dá)量顯著性高于ie1啟動(dòng)子組8倍以上。DNA注射實(shí)驗(yàn)進(jìn)一步證實(shí),Sba P(ENX)在凡納濱對(duì)蝦體內(nèi)也具有顯著性高于ie1啟動(dòng)子的啟動(dòng)活性。為檢測(cè)Sba P(ENX)是否在其他細(xì)胞系中也具有較Sba P更強(qiáng)的啟動(dòng)活性,我們檢測(cè)了其在其他幾種不同物種細(xì)胞系中的啟動(dòng)活性。有趣的是,與Sba P相比,Sba P(ENX)在EPC和PAC2細(xì)胞系中也表現(xiàn)出較強(qiáng)的活性,但是在哺乳動(dòng)物細(xì)胞系中其啟動(dòng)活性卻顯著性降低,介導(dǎo)的熒光素酶相對(duì)表達(dá)量在293T細(xì)胞系中僅為Sba P啟動(dòng)子的5%,He La細(xì)胞系中,也僅有16%。3.以Sba P(ENX)為啟動(dòng)元件的重組昆蟲(chóng)桿狀病毒表達(dá)系統(tǒng)的建立及應(yīng)用研究為進(jìn)一步建立有效的表達(dá)系統(tǒng),我們嘗試構(gòu)建以Sba P(ENX)為啟動(dòng)元件,以RFP為報(bào)告基因的重組昆蟲(chóng)桿狀病毒Bac-Sba P(ENX)-RFP。結(jié)果表明,在表達(dá)能力和穩(wěn)定性方面,構(gòu)建的重組桿狀病毒能夠高效地在sf21昆蟲(chóng)細(xì)胞中啟動(dòng)報(bào)告基因RFP的表達(dá),同時(shí),在海水及半海水的條件下,在12 h內(nèi)具有很好的穩(wěn)定性。隨后,通過(guò)將該重組桿狀病毒體外轉(zhuǎn)導(dǎo)原代血細(xì)胞和體內(nèi)注射轉(zhuǎn)導(dǎo)對(duì)蝦組織以及浸泡感染的結(jié)果表明,Bac-Sba P(ENX)-RFP能夠通過(guò)注射的方式在肝胰臟和肌肉中啟動(dòng)報(bào)告基因RFP的過(guò)表達(dá)。本研究獲得了凡納濱對(duì)蝦β-actin基因5’上游序列,并對(duì)其結(jié)構(gòu)、活性和調(diào)控元件功能進(jìn)行了較為深入的研究,這有助于加深對(duì)對(duì)蝦同源啟動(dòng)子的結(jié)構(gòu)和功能的了解;此外,我們還開(kāi)發(fā)了高效的對(duì)蝦同源啟動(dòng)元件Sba P(ENX),并且嘗試了將其與昆蟲(chóng)桿狀病毒系統(tǒng)相結(jié)合應(yīng)用于對(duì)蝦外源基因表達(dá)系統(tǒng)的研究,為開(kāi)發(fā)高效對(duì)蝦外源基因過(guò)表達(dá)系統(tǒng)提供了一種新的可能。
[Abstract]:Litopenaeus vannamei, which belongs to the order of the subclass crustaceans, is the economic species with the highest yield and output value in the shrimp culture industry all over the world. As a result of the disease, the shrimp aquaculture industry faces serious losses every year. At present, a large number of studies focus on disease control, genetic breeding and aquaculture. Many researches on gene overexpression in disease control and gene function research are quite numerous. Lack of effective expression system is one of them. The expression system includes the expression vector and the host cell, in which the effective expression vector needs the efficient starting element. At present, there are few development of high efficient homologous promoters in prawns. Beta -actin promoter is present in other species commonly used in high homologous promoter in Litopenaeus vannamei as the research object, in the previous research work has obtained the Litopenaeus vannamei beta -actin gene sequence (actin T1) on the basis of the first amplified beta -actin5 upstream sequence, and further study of its activity, application scope and function of regulatory elements, and try to combine it with the baculovirus expression system, the development of an effective system. The main purpose of this study is to develop efficient prawns homologous promoter and attempt to establish an effective exogenous expression system. Following results are obtained in this study: 1. Litopenaeus vannamei beta -actin gene 5 'upstream sequence of molecular cloning, function analysis and application study by reverse PCR (inverse PCR, I PCR) and other methods, we obtain the Litopenaeus vannamei beta -actin gene 5' upstream sequence, and named it Sba (P Shrimp beta-actin promoter). The total length of the sequence is 1642 BP, including the 5 'flanking sequence, the first exon, the first intron and the part of the second exon. Through bioinformatics analysis and detection of promoter activity, although the 5 'flanking sequences plays an important role in the expression of beta -actin gene (the region containing the conserved CCAAT-box and CAr G motif), but the full-length sequence of Sba P compared with the starting activity of 5' flanking sequences of stronger. In order to evaluate the Sba P promoter activity, and the 4 kinds of virus derived constitutive promoter, namely the cytomegalovirus promoter (Cytomegalovirus, CMV), Monkey (Simian vacuolating vacuolating virus 40 virus 40, SV40) promoter baculovirus polyhedrin protein (Polyhedrin, Polh) to start and the white spot syndrome virus early gene 1 (white spot syndrome virus immediate early gene 1, WSSV IE1) promoter and a source of beta tilapia -actin promoter (Tba P) were more active. The promoter strength promoter in different cell lines of different activity studies have been carried out on the 8 sources of different species of cell lines, the expression of luciferase expression vector in different cell lines transfected by detecting the amount of the promoter luciferase gene, promoter activity of indirect analysis. The results show that in the 8 cell lines tested, Sba P can initiate the expression of reporter genes, they are Sf21 (insects), PAC2 (zebrafish), EPC (carp), CHSE-214 (salmon), GSTEF (green turtle), MS-1 (Seng Haibao), 293T (human) and He La (Human) cell lines, but their different expression patterns. In the other cell lines except Sf21 insect cell lines, the relative expression of Sba P mediated reporter gene was weaker than that of CMV promoter, more than 10 times, but significantly higher than that of Polh promoter (except CHSE-214 cell line). In some cell lines, the activity of promoter was similar to SV40, IE1 and Tba P. In Sf21 insect cell lines, Sba P mediated luciferase expression was significantly higher than all promoters except IE1, and its luciferase expression was 10 times higher than that in other groups. The structure and function of 2.Sba P regulatory elements, and the optimized promoter activity analysis showed that the promoter activity of Sba P, the first intron region of possible regulatory elements for the detection of regulatory elements in structure and function, we constructed a Sba deletion mutation of P luciferase expression vector. By detecting the activation activity of deletion mutants, we found that there are two Suppressors (-1140/-925, -222/-21) and at least one enhancer (-810/-223) region in the first intron region. However, the mutant Sba P Delta -222/+1 -1325/-925 (Sba P (ENX)) showed a stronger activity in insect cells, and the relative expression level of its reporter gene was significantly higher than that of IE1 promoter group. It was more than 8 times higher than that in IE1 promoter group. The DNA injection test further confirmed that Sba P (ENX) was also significantly higher in the body of Penaeus vannamei than the IE1 promoter. In order to detect whether Sba P (ENX) has stronger Sba P activity in other cell lines, we detected its promoter activity in several other cell lines. Interestingly, compared with Sba P, Sba P (ENX) in EPC and PAC2 cell lines also showed strong activity in mammalian cell lines but its promoter activity was significantly decreased, the relative expression in 293T cell line is only Sba P 5% promoter mediated luciferase, He La cell line, only 16%. 3., the establishment and application of recombinant insect baculovirus expression system based on Sba P (ENX) as starting element. In order to further establish effective expression system, we attempt to build recombinant insect baculovirus Bac-Sba P (ENX) -RFP using Sba P (ENX) as starting element and RFP as a reporter gene. The results showed that the recombinant baculovirus could efficiently activate the expression of RFP in Sf21 insect cells, and had good stability in 12 h under seawater and semi seawater conditions. Subsequently, the recombinant baculovirus was transferred into the primary blood cells and injected into the shrimp tissues in vitro, and the results of immersion infection showed that Bac-Sba P (ENX) -RFP could be injected by injection.
【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(海洋研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78;S917.4

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