Baculovirus phylogeny and evolution

The family Baculoviridae represents one of the largest and most diverse groups of viruses and a unique model for studying the forces driving the evolution and biodiversity of double-stranded DNA viruses with large genomes. With the advent of comparative genomics, the phylogenetic relationships of baculoviruses have been put on solid bases. This, as well as improved bioinformatic approaches, has provided a detailed picture of baculovirus phylogeny and evolution. According to the present knowledge, baculoviruses can be classified into at least four evolutionary lineages: the most ancestral dipteran nucleopolyhedroviruses, the hymenopteran nucleopolyhedroviruses and the lepidopteran nucleopolyhedroviruses and granuloviruses. Despite the growing understanding of baculovirus phylogeny and macro-evolution, our knowledge of the micro-evolutionary processes within baculovirus species and virus populations is still limited. Here we present the state of the art on baculovirus phylogeny and evolution.     

Baculovirus DNA replication and processing

In this report, factors involved in baculovirus DNA replication are reviewed. These include factors that are required for DNA synthesis, other factors that have been implicated in genome processing or packaging, and homologs of proteins that are involved in DNA replication or repair in other systems. Conservation of a number of these factors in all baculovirus genomes suggest that many of the observations for specific viral systems may apply to the most if not all members of the Baculoviridae.     

Functional genomics

We are in the midst of a genomics revolution. The first chapter of this revolution will end later this year with the completion of the first draft of the entire human genome; estimates for the exact number of genes in the human genome vary from 50,000 to 140,000. This endeavor has been a major catalyst for the genomics revolution and has moved science into uncharted territories, which has led to the need to establish both new disciplines and a new vocabulary. Thus we now have pharmacogenomics, genotyping, pharmacogenetics, microarrays, biochips, differential display, bioinformatics and cheminformatic. The meeting provided a taste of the wealth of information that is now being accumulated under the name of both genomics and proteomics. The challenge ahead will be turning this information into knowledge and then translating this knowledge into new therapies.     

Structural genomics: bridging functional genomics and structure-based drug design

Considerable advances in structural genomics have been witnessed in the last year. Several pilot studies have begun to report their initial results, and new centers have been funded to join the endeavor. The legacies of the genome sequencing efforts, namely high-throughput molecular biology and whole-organism genome sequences, have been integrated as front-end modules for structural genomics pipelines. Impressive advances have been made in NMR spectroscopy and X-ray crystallography. New methods in structural bioinformatics and computational chemistry have been published that provide the means to exploit the wealth of new information in drug discovery. Not surprisingly, the biopharmaceutical industry has been quick to recognize the benefits of these new developments and has begun to adopt them. This article reviews recent results from structural genomics initiatives and the potential applications of new information and technologies in the drug discovery process.     

Baculovirus expression vectors for insect and mammalian cells

Functional expression of recombinant proteins has become a routine, but critical tool in modern molecular biology. Since their introduction, the use of baculovirus vectors to produce proteins for purification has become one of the most widely-used viral gene delivery systems as expression levels obtained are difficult to match with any other eukaryotic expression system. Extensive engineering to simplify and accelerate the process of recombinant virus construction has made this system accessible to virtually any modern biological laboratory. The utility of baculoviruses has been broadened with the discovery that appropriately modified virus can mediate gene expression in a wide variety of mammalian cell lines, and thus can function as a flexible cell-based assay development tool. The wide range of applications and potential for commercialization of products leads to consideration of a number of aspects of the system.     

Functional genomics of osteoarthritis

Functional genomics is a challenging new way to address a complex disease like osteoarthritis on a molecular level. Despite osteoarthritis being ultimately a biochemical problem, mainly characterized by an imbalanced cartilage matrix turnover, a deeper understanding of molecular events within the tissue cells (i.e., the chondrocytes) will provide not only a better understanding of pathogenetic mechanisms but also new diagnostic markers and cellular targets for therapeutic intervention. This innovative technology represents a challenging approach complementing (not replacing) classical research in previously described and new disease-relevant genes: large-scale functional genomics will open up new areas of so far unrecognized molecular networks. This will include as yet unidentified players in the anabolic-catabolic balance of matrix turnover of articular cartilage as well as disease-relevant intracellular signaling cascades so far hardly investigated in articular chondrocytes. However, care must be taken not to over or misinterpret results and some major challenges must be overcome in order to properly utilize the potential of this technology in the field of osteoarthritis.     

小鼠穿孔素在杆状病毒表达系统中的克隆与表达

穿孔素是机体发挥细胞毒作用的重要效应物质,在抗肿瘤、抗病毒及寄生虫治疗中有着重要的意义,但它在动物体内的含量极少,不便于进一步研究及新药开发,用基因工程方法来表达生产穿孔素是将小鼠的穿孔素是将小鼠的穿孔素基因在杆状病毒表达系统中进行克隆与表达。表达产物经ＳＤＳ－ＰＡＧＥ检测,薄层扫描确定表达量为细胞总蛋白的２５％,表达量在第４ｄ达到最高,其发挥溶血红细胞活性的最佳Ｃａ＾２＋浓度为２ｍｍｏｌ／Ｌ。穿     

人穿孔素在杆状病毒中的克隆和表达

穿孔素是机体免疫系统中具有免疫杀伤作用的一种重要效应物质,由于昆虫杆状病毒表达系统和原核表达系统相比能对表达产物进行翻译后加工,所以选择昆虫细胞真核表达人穿孔素,交人穿孔素基因插入杆状病毒转移载体pAcSecG2T中和野生昆虫杆状病毒共转染 sf9细胞,筛选得重组病毒,在sf9细胞中分泌表达含凝血酶位点的GST－hp融合蛋白,产物呈现钙依赖的溶红细胞活性,并且经Dot-Blot检测表明具有免疫活性。     

基因组学与心律失常的研究进展

基因组学与心血管病关系研究的重要进展是阐明导致家族性心肌病和离子通道病的几种基因异常。基因组学研究提示,人类基因组30亿个碱基中,有300万以上的碱基决定人群间的差异,包括对疾病的抵抗性和易感性。因此单核苷酸多态性是阐明遗传性心血管疾病的重要因素。     

Bac-to-Bac杆状病毒表达系统在BHK细胞表达鼠IL-2蛋白的研究

目的:利用Bac-to-Bac杆状病毒表达系统在BHK细胞表达重组鼠白细胞介素(IL)-2蛋白。方法:用Flag标记鼠IL-2并加入真核细胞启动子CMV,构建重组质粒pFB-CMV-IL-2并予酶切和PCR鉴定;将鼠IL-2克隆入杆状病毒表达载体pFastBacDual中,将pFB-CMV-IL-2转化到含杆状病毒穿梭载体Bacmid的DH10Bac感受态菌中,筛选阳性克隆,获得重组杆状病毒载体Bacmid-pFB-CMV-IL-2,抽提质粒;用脂质体转染法将Bacmid-pFB-CMV-IL-2转染Sf9昆虫细胞包装病毒,收获重组杆状病毒,将构建病毒转导BHK细胞培养,用Western blot检测IL-2蛋白。结果:通过杆状病毒表达系统,在BHK细胞成功表达重组的鼠IL-2蛋白。结论:重组杆状病毒在真核细胞中成功表达重组鼠IL-2蛋白。     

乙型肝炎病毒核心抗原HBcAg基因重组杆状病毒的构建及鉴定

目的利用杆状病毒表达系统（BEVS）构建含乙型肝炎病毒核心抗原（HBcAg）基因的重组杆状病毒。方法采用PCR法从含乙型肝炎病毒全基因序列（adw）的质粒pHBV1中扩增出HBVC基因,亚克隆到pGEM-T载体,然后将HBVC基因插入载体pAcGHLT-A中,构建含有HBVC基因的重组质粒pAcGHLT-HBcAg,与苜蓿银纹夜蛾核型多角体病毒（AcNPV）联合转染秋粘虫细胞（Sf9）,获得含有HBVC基因的重组杆状病毒。采用限制性内切酶酶切和DNA序列分析对重组病毒进行鉴定。结果利用杆状病毒表达系统,成功地构建了含HBVc基因的重组杆状病毒,感染昆虫细胞Sf9后,PCR检测可以扩增出相应大小的片段。结论利用BEVS成功构建了含有HBVC基因的重组杆状病毒,为进一步的研究奠定了基础。