【作者】 黄金山；

【导师】 胡志红；

【作者基本信息】 中国科学院研究生院（武汉病毒研究所） ， 微生物学， 2007， 博士

【摘要】 本论文构建了家蚕核多角体病毒（Bombyx mori nucleopolyhedrovirus，BmNPV）的Bac-to-Bac系统，在此基础上构建失活chitinase与v-cathepsin的Bac-to-Bac系统；并运用bacmid系统，对BmNPV的orf21进行功能研究，论文包括四个章节：第一章以综述的形式扼要介绍了杆状病毒的分类特征、基因组结构、病毒复制周期、致病的分子机制及杆状病毒的应用，重点介绍了杆状病毒表达载体的研究现状及应用，详细阐述了BmNPV的分子生物学研究及家蚕杆状病毒表达载体的研究与应用。第二章详细介绍了BmNPV的Bac-to-Bac系统的构建过程。成功地将含有一个Mini-F复制子、Kanamycin抗性基因、lacZ基因以及attTn7转座插入位点的8.6 kb的DNA片段置换到BmNPV基因组中的多角体基因位点，构建了能在大肠杆菌中稳定遗传的BmNPV bacmids，获得了17株不同的基因型，分别命名为BmBacJS1至BmBacJS17。将polyhedrin基因回复到与野生型BmNPV具有相似的限制性内切酶图谱的BmBacJS13中并进行了体内和体外感染性实验。实验结果表明BmBacJS13是一株具有与野生型病毒相同感染特性的bacmid。为了进一步分析BmBacJS13表达外源蛋白的性能，将egfp基因转座到BmBacJS13的Tn7转座插入位点，重组病毒感染细胞和幼虫后在细胞水平和虫体水平都观察到了强烈的绿色荧光。表明我们已成功构建了一套能在体内和体外表达外源基因的BmNPV Bac-to-Bac系统。第三章对己构建的BmNPV Bac-to-Bac系统进行改良，以期提高此系统的外源基因的表达效率。利用DNA同源重组技术在大肠杆菌中用氯霉素抗性基因替代BmBacJS13中的chitinase和v-cathepsin编码区域，获得了两基因同时失活的重组bacmid—BmBacJS13ACC。将AcMNPV几丁质酶信号肽与报告基因luciferase融合，转座到BmBacJS13和BmBacJS13ACC，构建重组病毒BmBacJS13ACC-luc与BmBacJS13-luc，比较两者对外源基因的表达量的影响。结果显示：无论是分泌到胞外的还是整个细胞的Luciferase的产量，BmBacJS13ACC-luc都显著高于对照病毒，表明改造后的BmBacJS13ACC是一个更有效的表达载体。第四章利用所构建的Bac-to-Bac系统对BmNPV orf21（Bm21）的功能进行分析。Bm21预测编码55.8kDa的蛋白，RT-PCR分析显示Bm21基因的转录产物在感染后12小时检测到，并持续到感染末期，而BM21的表达在感染后36小时可以检测到；将BM21的C端与EGFP融合瞬时转染BmN细胞，显示其定位于细胞核中，病毒超感染后其定位没有发生改变。为了进一步分析其功能，运用九噬菌体Red重组系统构建了缺失Bm21的重组病毒。结果表明对照病毒与缺失病毒之间BV的生长曲线和LC₅₀无明显差异，而缺失病毒的ST₅₀比对照病毒有8小时的延长，且差异达到了显著程度。因此Bm21是病毒复制的非必需基因，但可以加速感染虫体的死亡时间。更多还原

【Abstract】 Baculovirus expression vector system has been widely used for production ofrecombinant proteins. In this thesis, the Bac-to-Bac expression system of Bombyxmori nucleopolyhedrovirus （BmNPV） was constructed. The Bac-to-Bac system wasthen improved by deleting the chitinase and v-cathepsin genes of the bacmid toenhance the expression level of heterologous gene. The function of the orf21 ofBmNPV （Bm21） was also analyzed using the Bac-toBac system. The thesis containsfour chapters:Chapter One gave an overview of baculoviruses and the baculovirus expressionvector system（BEVS）. The molecular biology of BmNPV and advances of BmNPVBEVS were summarized.Chapter Two described the details of the construction of the BmNPVBac-to-Bac expression system. To construct the bacmid of BmNPV, a transfer vectorwas constructed which contained an Escherichia coli （E. coli） mini-F repticon,kanamycin resistance and a lacZ: attTN7: lacZ cassette within the upstream anddownstream regions of BmNPV polyhedrin gene. Through homologousrecombination in vivo, 17 different genotypes of BmNPV bacmids were screenedand named BmBacJS 1 to BmBacJS 17. One of bacmid colonies, BmBacJS13, whichhad similar restriction enzyme digestion profiles to that of wild-type BmNPV, wasselected for further research. The polyhedrin gene （ph） was inserted into BmBacJS13and the infectivity of BmBacJS13-ph was investigated in vivo or in vitro. The resultsindicated BmBacJS13-ph had the similar infectivities to that of wild-type BmNPV.Furthermore, the egfp gene was introduced into BmBacJS 13 to test the foreign gene expression both in vitro and in vivo. The green fluorescence could be detected inboth infected BmN cells and the tissues of infected B. mori larva. The above dataindicated that a Bac-to-Bac expression system of BmNPV was constructedsuccessfully.In Chapter Three the BmNPV Bac-to-Bac expression system was modified to enhance theexpression of foreign protein. The chitinase and v-cathepsin genes of BmBacJS13 were deleted togenerate BmBacJS 13ACC. The luciferase under signal peptide of AcMNPV chitinase was used asa marker gene and inserted into BmBacJS13 and BmBacJS13ACC, The intracellular and thesecreted Lusiferase activities of both viruses were compared. The results showed the expressionlevels of total and secreted recombinant protein of BmBacJS13ACC-Iuc were significantly higherthan that of BmBacJS13-luc. Therefore, the modified bacmid provides a better tool forrecombinant protein expression.In Chapter Four the function of orf21（Bm21） BmNPV was characterized.Bm21 was predicted to encode a protein of 55.8 kDa. RT-PCR analysis indicated thatthe transcriptional product of Bm21 was first detected at 12 h post infection. Westernblot analysis showed the protein could be first detected from the 36 h post infection.Transient-expression of BM21-EGFP with and without viral infection indicated thatBM21 was localized in the nuclei of the transfected BmN cells. A Bm21 knockoutbacmid BmBacJS13A21 was made and the recombinant virus was examined in BmNcells or insect larvae. The results showed that there no difference in BV growth curveand LC₅₀ between knockout virus and control virus, but the ST₅₀ of the knockoutvirus was about 8 h longer than the parental virus （significant difference）. Theseresults indicated that Bm21 was not essential for viral replication but couldaccelerate viral killing in the host insect.更多还原