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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
史英力
学位类型博士
导师相建海 ; Yuanan Lu
2016-05-21
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词凡纳滨对虾β-actin启动子 启动子活性 Promoter Activity Litopenaeus Vannamei Shrimp 调控元件 Regulatory Elements Β-actin Promoter 重组昆虫杆状病毒 Recombinant Baculovirus 转导系统 Transduction System
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(actinT1)的基础上,首次扩增得到其β-actin 5’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,iPCR)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SbaP (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CArG motif),但是全长序列SbaP具有比5’侧翼序列更强的启动活性。为了评估SbaP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(TbaP)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,SbaP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HeLa (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,SbaP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TbaP启动子活性相近。而在sf21昆虫细胞系中,SbaP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.        SbaP调控元件结构及功能,以及优化后启动子活性研究
SbaP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SbaP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SbaPΔ-222/+1Δ-1325/-925 (命名为SbaP(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,SbaP(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SbaP(ENX)是否在其他细胞系中也具有较SbaP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SbaP相比,SbaP(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SbaP启动子的5%,HeLa细胞系中,也仅有16%。
3.        SbaP(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SbaP(ENX)为启动元件,以RFP为报告基因的重组昆虫杆状病毒Bac-SbaP(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-SbaP(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SbaP(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。
其他摘要The Pacific white shrimp, Litopenaeus vannamei, is an important economic aquaculture species found worldwide that accounted for over 79% of total shrimp aquaculture production and generated the highest commercial value (USD 16.5 billion) in 2013. However, over the past 20 years, shrimp farming has suffered enormous losses with an estimated amount of 1 billion USD per year due to infectious diseases. In present, most research has focused on the disease control, genetic breeding and aquaculture. Among these fields, current development of shrimp functional gene over-expression research has been hampered due to the lack of the suitable expression system. An expression system is consisted of expression vector and host system, and an effective expression vector should possess an efficient promoter.
Till now, few research are focused on the development of shrimp homologous promoter. β-actin promoters have been reported to be efficient ubiquitous regulatory elements and are widely used in transgenic mammalians and fish. In this thesis, in continuation of our previous description of the isolation and identification of genomic sequence actinT1 from the pacific white shrimp, we obtained the 5’-flanking sequence of actinT1, and further analyze its activity, application scope and cis-regulatory elements, meanwhile, we conducted a trial for the application of SbaP(ENX) in a baculovirus delivery system in vitro and in vivo. The objectives were to develop an efficient shrimp homologous promoter and effective expression system.
Three major research results are as follows:
1.        Molecular cloning, functional and application scope analysis of 5’upstream sequence of L. vannamei β-actin gene
A 1,642 bp sequence, containing 5’-flanking sequence, exon 1, intron 1 and partial exon 2, which are responsible for transcriptional initiation of shrimp β-actin (actinT1), was isolated from L. vannamei by inverse PCR method, and the sequence was designated as SbaP (Shrimp beta-actin Promoter).
To determine SbaP’s function, the structure domains were examined by constitutive expression of the luciferase reporter gene. We identified 5’ flanking region that played a central role in the expression of the β-actin gene, which contained two highly conserved transcriptional sites, CCAAT box and CArG motif. However, SbaP exhibited stronger promoter activity than 5’ flanking sequences. In order to evaluate and characterize the promoting function of SbaP, a systematical test was performed to compare this putative shrimp promoter with Cytomegalovirus (CMV), Simian vacuolating virus 40 (SV40), Polyhedrin (Polh) and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters commonly used and one β-actin promoter (TbaP) isolated from tilapia fish, using eight cell lines derived from different animal species. Transfection of these promoter-luciferase constructs followed by luciferase quantitation assays revealed that SbaP was able to drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green turtle), MS-1 (monk seal), 293T (human) and HeLa (human), but at different levels. Compared to other promoters tested, the promoting activity of SbaP was 10-fold lower than CMV promoter but much higher than Polh promoter in most of these cell lines (except for CHSE-214 cells). On the contrary, SbaP mediated luciferase expression in sf21 cells was over one order of magnitude higher than that ofCMV, SV40, Polh, and TbaP promoters.
2.        The structural and functional analysis of SbaP cis-regulatory elements, and the promoting activity examination of compact SbaP
Structural domain functional assay indicated that there might be cis-regulatory elements in 1st intron of SbaP. To characterize the function of regulatory element in 1st intron, a serial deletion promoter-luciferase constructs were generated on the basis of pGL-SbaP, and two negative (-1140/-925, -222/-21) and one positive (-810/-223) regulatory elements were identified in 1st intron according to dual-luciferase assay. Transient transfection assay with a construct containing proximal promoter and enhancer regions of the shrimp β-actin (SbaPΔ-222/+1Δ-1325/-925 which were named as SbaP(ENX)) coupled with luciferase and egfp (enhanced green fluorescent protein) showed its promoter activity was more than 8-fold higher than a viral-origin promoter (WSSV ie1) in sf21 cells. Particularly, SbaP(ENX) also drove a successful expression of luciferase in vivo and also showed higher promoter activity than the ie1 promoter. In addition, compared to the SbaP, SbaP(ENX) exhibited a relatively stronger promoter activity in EPC and PAC2 cells, but a 5% and 16% lower promoting effect in 293T and HeLa, respectively.
3.        The establishment of recombinant baculovirus system under the control of SbaP(ENX) and its potential application
For further application of SbaP(ENX) and establishment of an effective foreign gene expression system, we constructed a recombinant baculovirus system under the control of SbaP(ENX). Bac-SbaP(ENX)-RFP was confirmed that could successfully drive RFP gene expression in sf21 cells, and the stability tested indicated that there was no significantly decreased in titre in seawater and seawater mixture environment within 12 hours. Moreover, Bac-SbaP(ENX)-RFP was tested in both in vitro of transduced primary shrimp cells and in vivo of injected and immersed indicator shrimp. The results suggested that the expression of RFP could be detected in muscle and hepatopancreas by injected method.
In this study, we obtained the 5’ flanking sequence of L. vannamei β-actin gene, and further analyzed the function of its structural domain and cis-regulatory elements, which will benefit the understanding of shrimp homologous promoter; furthermore, these results warrant the potential value of the newly isolated shrimp promoter SbaP, particularly its derivative (SbaP(ENX)) in ectopic gene expression, which will facilitate the development of foreign gene expression system in shrimp functional gene over-expression research in future.
学科领域分子生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/112538
专题实验海洋生物学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
3.夏威夷大学
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史英力. 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[D]. 北京. 中国科学院大学,2016.
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