【作者】 陈寅；

【导师】 张志芳； 沈桂芳；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2006， 博士

【摘要】 利用半补齐法构建了BmNPV基因组随机文库。将BmNPV DNA用Sau3A Ⅰ部分酶切,用Sal Ⅰ完全消化质粒载体pUC19,分别以Klenow酶半补齐,连接、转化后得到覆盖率大于99.9%的文库。 通过与文库质粒DNA逐个共转染,鉴定了作用于早期基因helicase启动子的反式激活因子。筛选出的质粒均含有完整的ie-1基因。PCR克隆出ie-1基因共转染即可产生反式激活作用。为证实文库筛选的可靠性,分别克隆了其它主要早期基因并证实产物均不能对helicase启动子产生反式激活作用。瞬时表达分析表明,IE-1还可以对其它不同来源的启动子产生强烈的反式激活。 BmNPV hr3增强子的质粒不能激活另一报告质粒上的luc基因表达,但当细胞受到病毒感染时,便会强烈增强报告基因的表达。利用分别含有hr3的质粒、helicase启动子与luc基因的报告质粒和每个BmNPV随机文库质粒的DNA共转染家蚕细胞,结果显示杆状病毒的必需因子IE-1介导了hr3增强子在Bm细胞中的反式激活作用。进一步研究还证明IE-1因子作为蛋白质桥梁能使增强子对不同来源的启动子发挥广谱性的反式作用。这种增强子的反式作用一股能使转录活性提高40-100多倍。30bp回文序列是hr增强子反式激活中起作用的基本单元。 利用文库共转染筛选时发现一个较小的质粒能反式激活转录几十倍,其不包含任何病毒早期基因,但包含ie-1基因的5’侧翼非编码序列直至起始密码子上游19bp处。瞬时表达证实378bp的ie-1启动子序列能反式增强启动子转录40-100倍,说明此378 bp ie-1启动子中存在编码某个因子或存在某未知调控方式的一段序列。将此378 bp ie-1启动子分为不同长度片段以及定点突变证明并非编码因子造成这种反式激活。推测这种转录激活现象是增强序列的反式作用。而介导这种反式作用的蛋白是来源于宿主的细胞因子。将一段238 bp序列分别顺式连接于报告质粒的启动子上游或报告基因的下游后发现其可以不分正反,不分上下游并能远距离的增强目的启动子转录10,000倍以上。为了检测此非同源重复区增强子(non-homologous region enhancer,NHE)对不同启动子的增强活性,将其分别顺式连接至杆状病毒晚期启动子和哺乳动物病毒来源的启动子,发现NHE对它们都能发挥很强的增强作用。另外,通过构建同时含有同源重复和非同源重复这两种类型增强子的报告质粒转染后证实两种类型增强子能够协同作用于报告基因的转录。 通过将杆状病毒转移载体中的polyhedrin启动子替换为ie-1启动子,利用opa为报告基因,再将hr3增强子克隆于opd基因下游构建表达载体。共转染获得包含上述表达盒的重组病毒,通过对表达的OPD活性分析证明,该表达盒具有良好的表达效果。 将Pfu DNA聚合酶基因(PfuPol)克隆入转移载体使其处于多角体启动子控制之下。共转染获得重组病毒用于感染家蚕。SDS-PAGE分析显示表达的重组蛋白约90kD,将产物经一步法热变性后即可直接用于高保真PCR中。每毫升血淋巴中Pfu DNA聚合酶活性为2×10~5U。更多还原

【Abstract】 A genomic library of BmNPV was constructed by "partial filling-in" method. The genomic DNA of wildtype BmNPV was partially digested by Sau3A I, and the plasmid vector pUC19 was fully digested by Sal I , and subsequently filled in by the Klenow fragment. After ligation and transformation, the BmNPV genomic library with the representation of more than 99.9% was obtained.The viral factor involved in transactivation of the early helicase promoter was identified by co-transfection using the generated BmNPV genomic library. All of the screened plasmids contained intact ie-\ coding region. The ie-\ gene was cloned by PCR to further confirm that only IE-1 but not other factors encoded by flanking sequences functioned as transactivator. Other major baculovirus early genes exhibited no transactivation to the helicase promoter. Transient expression also revealed that IE-1 could give strong transactivation to other promoters from various origins.A plasmid hr3 enhancer failed to stimulate the expression of another plasmid containing luc gene under control of a promoter, but strong stimulation occurred when cells were infected by BmNPV. Co-transfections of hr3 enhancer, report plasmid, which containing luciferase gene driven by BmNPV helicase promoter, and the BmNPV genomic library revealed that IE-1, the essential viral factor, contributed to the hr3 enhancer function in trans. Further investigations proved that IE-1 could mediate hr3 enhancer function in trans as a broad-spectrum protein bridge to stimulate promoters from various origins. The stimulating effects ranged from 40 to more than 100 folds. Functional dissections of the hr3 enhancer clarified that the 30-bp imperfect palindrome was essential for the enhancer function in trans.During the screening of BmNPV transactivator for the helicase promoter, a relatively small library DNA was found to transactivate the target promoter distinctly but more slightly than the screened viral factor IE-1. No coding region of any viral early factor but the 5’ non-coding region until 19 bp upstream of ie-1 gene ATG was found by sequencing analyses. Further transient assays confirmed that the 378 bp ie-\ promoter can stimulate transcription of promoters 40-100 folds via co-transfection. Functional dissection analyses of four overlapping sub-fragments and site-directed mutagenesis revealed that the transactivation was not contributed by the potential viral factor. To further confirm the suppose that it is an enhancer acting in trans via a cellular factor, a 238 bp fragment was inserted into reporter plasmids to verify if this fragment could stimulate transcription from a linked promoter in a distance- and orientation-independent manner. After transfection, the fragment was proved to have prominent stimulation effects of more than 10,000 folds and meet all the classical definitions of an enhancer. This non-homologous region enhancer (NHE) was inserted into reporter plasmids (downstream of luc gene) containing baculovirus late promoter and mammalian virus originated CMV promoter and exhibited stimulation on both these two promoters. The NHE was subsequently proved to be function simultaneously with the homologous region enhancer hr3.In the transfer vector BacPAK8, using ie-\ promoter substituted for polh promoter, the opd gene was inserted into the transfer vector under the control of ie-\ promoter. The homologous region enhancer hr3 was cloned downstream of the opd gene to construct a new expression cassette for the expression via BEVS. After co-transfection with linearized parent baculovirus DNA the recombinant baculovirus was obtained and further plaque screened. The results revealed that this expression cassette had advantages in improving expression level.The PfuPol gene was cloned into the baculovirai transfer vector pVL1393 under the control of the polyhedrin promoter. After co-transfection with Bsu36 I linearized BmBacPAK6 genomic DNA, the recombinant baculovirus harboring PfuPol gene was obtained and plaque screening was further conducted. The recombinant virus was used to infect silkworm larvae. SDS-PAGE analysis revealed that the recombinant enzyme was approximately 90 kDa in size and could be purified in a single heat-treatment step. The heat-treated enzyme was sufficient and retained good performance for amplification in PCR directly. The Pfu DNA polymerase was overexpressed up to 2X105 U per ml hemolymph.更多还原