实验基础

A considerable amount of work has been done during the last 20 yr to genetically enhance the efficacy of baculovirus insecticides. Following construction of a genetically altered baculovirus, laboratory bioassays are used to quantify various parameters of insecticidal activity s ...

Probing the baculovirus infection process is essential in optimizing the recombinant protein production. Typically, researchers monitor the infection process in the stirred tank reactor, which, however, contains a population of cells infected at different times after virus in ...

Through green fluorescent protein (GFP) strategy, facile and fast monitoring and visualization of baculovirus infection in insect cells is possible in vivo. This chapter describes two novel techniques for simple determination of virus titer in the baculovirus expression system u ...

This chapter describes a technique for synthesizing and transfecting double stranded RNA (dsRNA) for RNA interference in Sf-21 cell culture. Transfection with dsRNA only requires 1 h and the cells are usually recovered within 12 h. Suggestions for designing dsRNA are included in the method ...

Apoptosis is a physiological program of cell suicide conserved in invertebrates and vertebrates. Apoptosis is crucial to the normal development of organisms and in tissue homeostasis, by promoting elimination of unwanted cells including damaged- or virusinfected cells. Because ...

Genetically modified baculoviruses can efficiently deliver and express genes in mammalian cells. The major prerequisite for the expression of a gene transferred by baculovirus is its control by a promoter that is active in mammalian cells. This chapter describes methods for producing ...

A protocol for the development of cancer vaccines is presented. The protocol is based upon the long-term in vitro treatment of cancer cells with interferon (IFN)-α to create cancer vaccine cells. This protocol has been used to develop cancer vaccines in mice against B16 melanoma, RM-1 prostate ca ...

Recent studies have revealed that type I interferons (IFNs) are powerful inducers of the differentiation and activation of dendritic cells (DCs). These findings emphasize the importance of these cytokines in linking innate and adaptive immunity, suggesting that effects of type I IFN on D ...

Plasmacytoid dendritic cells (PDCs) are the natural interferon (IFN-α)-producing cells in human peripheral blood that produce vast quantities of IFN-α in response to viral infection and other stimuli. PDCs are a rare cell type, making up less than 0.5% of peripheral blood mononuclear cells. To ...

A defining hallmark of the type 1 interferons (IFNs) is their ability to interfere with virus replication. As such, viruses have evolved diverse mechanisms to subvert the antiviral effects of interferons. Herpes simplex virus type 1 (HSV-1) is a large deoxyribonucleic acid virus best known for ...

Delivery of type I interferon (IFN) subtypes by intramuscular inoculation of mice with a recombinant mammalian expression vector encoding IFN stimulates the immune response. Such immunomodulation drives towards a Th1-like response. The degree of stimulation of the immune respon ...

Gene transfer is a widely used experimental approach to determine the value of specific genes under a variety of conditions. This chapter focuses on the expression of two human antiviral genes, interferon (IFN)-α2 and IFN-β, driven by a cytomegalovirus immediate-early promoter in a human M�l ...

The cytokine family of interferons (IFNs) has multiple functions, including antiviral, antitumor, and immunomodulatory effects and regulation of cell differentiation. The multiple functions of the IFN system are thought to be an innate defense against microbes and foreign subst ...

Interferons are the antiviral early inflammatory proteins produced in the cells in response to the infectious agents. The characterization of the interferon genes, their expression, and their function was advanced with the development of novel techniques in molecular and cellular ...

The primary focus of this chapter is on providing an overview of how we use the tools of functional genomics to study virus infection, the interferon response, and the mechanisms by which viruses attenuate or evade this response to ensure successful replication. We provide examples of the types of ...

The clinical symptoms associated with rotavirus (RV) gastroenteritis are not sufficiently characteristic to distinguish between RV infection and other causes of gastroenteritis. Therefore, laboratory procedures, including electron microscopy (EM) (1), enzyme-linked ...

We provide procedures for the panning of fully humanized Fab antibodies using guided selection. Human heavy and light chain genes are amplified. A parental light chain is cloned into a phage display vector and combined with the heavy chain library. After several rounds of panning, positive clo ...

This chapter outlines a protocol for the selection by phage display of single-chain variable antibody fragments with dual properties-specificity for tumor cells and the ability to be internalized. The protocol is based on a direct incubation of living target cells with antibody phage di ...

Many phage display techniques drive selection toward the isolation of highly specific antibodies. However, the identification of monoclonal antibodies that are cross-reactive has implications for the development of diagnostics, therapeutics, and vaccines against patho ...

Conventionally, antibody phage display has been used to isolate recombinant antibodies that are monovalent in their interaction with target antigens. These antibodies can be reengineered for expression in mammalian cell culture as full-length, monospecific immunoglobuli ...