【作者】 肖庆利；

【导师】 何家禄； 张志芳；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养学， 2002， 博士

【摘要】 DNA复制、基因表达和调控过程中，DNA解旋酶承担了重要任务，负责打开DNA双链，参与新生链的合成，并在DNA修复和重组过程中发挥着不可或缺的作用。杆状病毒在宿主昆虫细胞内复制与基因表达是在一种有秩序的级联事件中发生的。本文通过研究家蚕杆状病毒(BmNPV)解旋酶基因启动子的结构与功能，BmNPV同源区hr3的增强子功能，病毒因子对解旋酶基因启动子的反式激活作用，探讨AcMNPV和BmNPV宿主域决定的机理，讨论了杆状病毒解旋酶参与DNA复制和晚期基因转录，关闭宿主蛋白质合成及决定杆状病毒宿主域方面所起的重要作用。 本文在研究杆状病毒早期基因表达调控的同时，利用极晚期多角体蛋白基因启动子构建的表达载体，在家蚕体内联合表达了鸡贫血病毒(CAV)主要抗原蛋白VP1和VP2，为研制抗鸡CAV感染的基因工程亚单位疫苗提供了一条可行的途径。本论文主要研究内容如下： (1)昆虫in vitro／in vivo瞬时表达体系的建立 在基因表达调控研究中，DNA转染是常见的技术手段，其中脂质体转染法是将外源基因导入培养细胞和组织的有效方法。目前尚无一套可以利用的完整体系用于昆虫细胞转染，加上商品化的脂质体价格昂贵，因此我们利用快速乙醇注射法制备了一种含阳离子脂质DDAB和中性辅助脂类DOPE(摩尔比为1：2)的阳离子脂质体。凝胶阻滞分析表明，该脂质体与DNA可形成很好的复合体。研究确定了DDAB／DOPE脂质体介导表达荧光素酶基因的报告质粒转染昆虫细胞系和家蚕幼虫的最佳条件，脂质体与质粒DNA的最佳混合比例为10～15nmol的DDAB／DOPE：1 ug DNA。试验证明该脂质体可成功介导长达128 kb的BmNPV基因组DNA转移进入Bm-5细胞。与商品化的Lipofectin试剂相比，该脂质体可提高转染效率8～10倍。本章研究结果为后面的解旋酶基因启动子 的功能研究奠定了基础。 （2）BmNPV解旋酶基因启动子的结构与功能 为了探讨家蚕核型多角体病毒DNA解旋酶基因的表达调控，我们通过PCR 技术克隆了 B衅V镇江株（B栅V ZJS）解旋酶基因 ATG上游的 sic hp启 动子区，序列分析发现，该启动子与BmN’PV T3及AcNtN－－－－－V解旋酶基因启动 于的同源性分别为99％，95 WJ。且同时具有早期和晚期RNA转录起始位。15， 分别为 TGTGC和 GTAAA。在ATG上游约一70 hp处有一小顺 反于带有qr Kozak规律的起始密码于ATG，在转录起始位点上游有典型的真核启动子TATA 盒存在。为了测定解旋酶基因启动子的活性，将起始密码突变为ATT后，引入 萤火虫荧光素酶基因构建报告质粒pBmhelslo山c，作瞬时表达分析。用 pBmhelsloluc转染BmN，Bm巧和SfZI细胞，发现解旋酶基因启动子能被细 胞的RNA聚合酶识别，具有早期启动子的特性。为了确定对BInNPV解旋酶基 因启动子功能影响较大的区段，我们通过PCR对该启动子区域进行了一系列缺 失分析，表明解旋酶基因启动子的调控区主要在5”上游区。 0）BmNPV同源区hr3增强子功能分析 杆状病毒同源区（hr）序列是病毒DNA复制起始点，又具有增强子功能， 在体外试验中能提高部分杆状病毒基因的表达水平。我们将BlnN’PV hi3序列与 解旋酶基因启动子（hels 0）克隆到同一载体中，分别构建 hr3位于报告基因h。 的下游和解旋酶基因启动子上游的重组质粒＠mh＊引01讥十3d。一和 pB mhelslo山c人r3up，并进IT瞬时表达研究。结果表明，hr3对helslo启动－于具 有显著的增强作用，且这种增强作用具有位置效应，hr3位于报告基因下游比位 于启动子上游的增强作用大。当 hr3位于报告基因的下游时，在转染的 SfZI， Bln－5和Bm＊细胞中，hr3可分别增强helslo启动子的转录活性达7654倍，刀刀 倍禾 3632倍。我们将瞬时表达质粒 pBmhelslouc－hr3down转染 5龄第二天的 家蚕幼虫，测定血淋巴细胞中的荧光素酶活力，发现在体内转染试验中，Ilf3仍 具有很高的增强作用，可提高heIS 10启动子的基础活性达1320倍。另外，从 1。Bml。el＊clue－hr3down在SfZI 细胞中的表达时相可以看出，在转染后6 11，纠J －二二－ 一 ／ 可测到较高水平的荧光素酶表柱量，36～48 h达最高，说明解旋酶基因为早期 基因，同时其表达具有累积作用。 （4）病毒因子对BmNPV解旋酶基因启动子的反式激活 杆状病毒立即早期基因产物能够反式激活延迟早期基因的表达。将报告质更多还原

【Abstract】 Acting during DNA replication and gene expression regulation, DNA helicases convert the duplex DNA to single strands and thereby activate the DNA for nascent strand synthesis. Helicases also play essential roles during DNA repair and recombination During infection baculovirus genes are expressed in a coordinately regulated cascade fashion in which each successive phase is dependent on the expression of genes during the previous phase. In this study, we examined the structure and function of the helicase gene promoter of BmNPV, the function of homologous region 3 (hr3) enhancer in BmNPV, and viral factors transactivating the helicase gene promoter. We also investigated the mechanism of host range determination between AcMNPV and BmNPV, and discussed the importance of baculovirus helicase involved in DNA replication, stimulating late gene transcription, closing host protein synthesis, and changing baculovirus host range.On the basis of studying the regulation of baculovirus early gene expression, we then successfully co-expressed chicken anaemia virus proteins VP1 and VP2 in silkworm using BmNPV expressing vector. This will provide an efficient method for development of CAV genetic engineering subunit vaccine. This paper mainly includes following parts:(1) Establishment of in vitro and in vivo transient expression system in insect cellsIn gene expression and regulation studies, Cationic liposomes have been commonly used to delivery functional genes into cells. Several cationic liposomes generally prepared by evaporation and sonication, are now commercially available;however, their large-scale use is confined for the great expense. So we prepared a cationic liposome reagent, composed of dirnethyldioctadecylammonium bromide (DDAB) and dioleoyl phosphatidylethanolamine (DOPE) (1:2 mol ratio). Then we investigated and determined the optimum conditions of this liposome mediated a reporter plasmid expressing luciferase into insect cell lines (Sf-21 and Bm-N) and silkworm larvae. Together the data demonstrated that Bomhyx mori nuclear polyhedrosis virus (BmNPV) genomic DNA (128 kb) was successfully transfected into Bm-5 cells using this liposome These results suggest that DDAB/DOPH liposome will be useful as delivery agents for gene transfer to insect cells both in vitro and in vivo. It is significant to understand the function of the helicase gene promoter deeply.(2) Structure and function of BmNPV helicase gene promoterTo examine the expression and regulation of DNA helicase gene of BmNPV, the promoter of the helicase gene (BmNPV ZJ strain), including 510 bp upstream of ATG, was amplified by PCR. Sequence analysis showed that it was 99% and 95% identity, respectively, with the homologues in BmNPV T3 strain and AcMNPV This promoter has both early (TGTGC) and late (GTAAA) RNA initiation sites. It has a minicistron at about -70 bp upstream of ATG. It also has the TATA box at the upstream of transcriptional initiation sites. To detect the activity of the helicase gene promoter, the initiation codon ATG was deleted by using point mutation, and luciferase gene, as a reporter gene, was fused with the promoter region to construct the plasmid pBmhe15101uc. When pBmhe15101uc was transfected into Bm-N, Bm-5 and Sf-21 cell lines, the helicase gene promoter was recognized by cellular RNA polymerase and had the characteristics of early promoters. Deletion analysis of the helicase gene promoter by PCR showed that cis-acting elements were mainly present within 5’ region of the promoter(3) Functional analysis of BmNPV homologous region 3 enhancerBaculovirus homologous regions (hrs) act as viral DNA replication start sites, which also have been shown to augment the expression of a small number of baculovirus genes in vitro. In this study, BmNPV hr3 was cloned into upstream or downstream of he!510-reporter gene construct, to study the effect of this enhancer on hel510 promoter activity. The results showed that hr3 could significantly enhance the activity of he!510 promoter. The promoter activity was increa更多还原