以凡纳滨对虾β-actin基因启动子为元件的重组昆虫杆状病毒表达系统的建立

发布时间：2017-12-27 17:21

本文关键词：以凡纳滨对虾β-actin基因启动子为元件的重组昆虫杆状病毒表达系统的建立　出处：《中国科学院研究生院(海洋研究所)》2016年博士论文　论文类型：学位论文

【摘要】：凡纳滨对虾(Litopenaeus vannamei)隶属于甲壳亚纲十足目,是目前全世界对虾养殖产业中产量和产值最高的经济物种。由于病害侵袭,对虾养殖业每年面临严重的损失。目前大量的研究集中于病害防治、遗传育种和水产养殖等多方面,其中,在病害防治和基因功能研究中,基因过表达研究受制颇多,缺乏有效的表达系统便是其中之一。表达系统包括表达载体和宿主细胞,其中有效的表达载体需要高效的启动元件。目前有关对虾高效同源启动子的开发极少。β-actin启动子是目前在其他物种中常用的高效的同源启动子,本文以凡纳滨对虾为研究对象,在前期研究工作已获得凡纳滨对虾β-actin基因序列(actin T1)的基础上,首次扩增得到其β-actin5’上游序列,并对其活性、应用范围和调控元件功能进行深入研究,同时尝试将其与昆虫杆状病毒系统相结合,开发有效的表达系统。本研究的主要目的是:开发高效的对虾同源启动子和尝试建立有效的外源表达系统。本研究获得的主要进展如下:1.凡纳滨对虾β-actin基因5’上游序列的分子克隆、功能分析及应用范围研究通过反向PCR(inverse PCR,i PCR)等方法,我们获得了凡纳滨对虾β-actin基因5’上游序列,并将其命名为Sba P(Shrimp beta-actin promoter)。该序列全长1,642 bp,分别包含5’侧翼序列、第一外显子、第一内含子和第二外显子部分区域。通过生物信息学分析和启动活性检测发现,尽管5’侧翼序列对于β-actin基因表达起到非常重要的作用(该区域含有非常保守的CCAAT-box和CAr G motif),但是全长序列Sba P具有比5’侧翼序列更强的启动活性。为了评估Sba P启动活性强弱,将其与4种常用的病毒来源的组成型启动子,即巨细胞病毒(Cytomegalovirus,CMV)启动子,猴猿空泡病毒40(Simian vacuolating virus 40,SV40)启动子,昆虫杆状病毒多角体蛋白(Polyhedrin,Polh)启动子和白斑综合征病毒极早期基因1(white spot syndrome virus immediate early gene 1,WSSV ie1)启动子以及一种罗非鱼来源的β-actin启动子(Tba P)进行活性比较。由于启动子在不同细胞系中启动强度不同,活性分析在8种不同物种来源的细胞系中展开,通过检测构建的启动子-荧光素酶基因表达载体在转染的不同细胞系中的相对荧光素酶表达量,间接分析启动元件活性强弱。结果表明,在测试的8种细胞系中,Sba P能够全部启动报告基因的表达,它们分别是,sf21(昆虫),PAC2(斑马鱼),EPC(鲤鱼),CHSE-214(鲑鱼),GSTEF(绿龟),MS-1(僧海豹),293T(人类)和He La(人类)的细胞系,但是各自的表达模式不同。在除sf21昆虫细胞系外的其他细胞系中,Sba P介导的报告基因相对表达量均弱于CMV启动子10倍以上,但是显著性地高于Polh启动子(除CHSE-214细胞系),在某些细胞系与SV40,ie1和Tba P启动子活性相近。而在sf21昆虫细胞系中,Sba P介导的荧光素酶表达量显著性地高于除ie1外所有启动子,并且其荧光素酶相对表达量较其它组均高10倍以上。2.Sba P调控元件结构及功能,以及优化后启动子活性研究Sba P启动活性分析检测表明,其第一内含子区域可能存在调控元件,为检测调控元件结构及功能,我们构建了Sba P缺失突变-荧光素酶表达载体。通过检测缺失突变体启动活性发现,在第一内含子区域中存在有两个抑制子(-1140/-925,-222/-21)和至少一个增强子(-810/-223)区域。而缺少抑制子的突变体Sba PΔ-222/+1Δ-1325/-925(命名为Sba P(ENX)),在昆虫细胞中表现出了更强的启动活性,其报告基因相对表达量显著性高于ie1启动子组8倍以上。DNA注射实验进一步证实,Sba P(ENX)在凡纳滨对虾体内也具有显著性高于ie1启动子的启动活性。为检测Sba P(ENX)是否在其他细胞系中也具有较Sba P更强的启动活性,我们检测了其在其他几种不同物种细胞系中的启动活性。有趣的是,与Sba P相比,Sba P(ENX)在EPC和PAC2细胞系中也表现出较强的活性,但是在哺乳动物细胞系中其启动活性却显著性降低,介导的荧光素酶相对表达量在293T细胞系中仅为Sba P启动子的5%,He La细胞系中,也仅有16%。3.以Sba P(ENX)为启动元件的重组昆虫杆状病毒表达系统的建立及应用研究为进一步建立有效的表达系统,我们尝试构建以Sba P(ENX)为启动元件,以RFP为报告基因的重组昆虫杆状病毒Bac-Sba P(ENX)-RFP。结果表明,在表达能力和稳定性方面,构建的重组杆状病毒能够高效地在sf21昆虫细胞中启动报告基因RFP的表达,同时,在海水及半海水的条件下,在12 h内具有很好的稳定性。随后,通过将该重组杆状病毒体外转导原代血细胞和体内注射转导对虾组织以及浸泡感染的结果表明,Bac-Sba P(ENX)-RFP能够通过注射的方式在肝胰脏和肌肉中启动报告基因RFP的过表达。本研究获得了凡纳滨对虾β-actin基因5’上游序列,并对其结构、活性和调控元件功能进行了较为深入的研究,这有助于加深对对虾同源启动子的结构和功能的了解;此外,我们还开发了高效的对虾同源启动元件Sba P(ENX),并且尝试了将其与昆虫杆状病毒系统相结合应用于对虾外源基因表达系统的研究,为开发高效对虾外源基因过表达系统提供了一种新的可能。
[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.
【学位授予单位】：中国科学院研究生院(海洋研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78;S917.4

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