实验动物总则

Complementary cell lines have become an accepted tool for the functional analysis of viral genes (1,2) and proteins, as well as an essential component in strategies for the construction of mutant viruses. More recent applications include the propagation of replication-defective vir ...

Lytic infection by herpes simplex virus (HSV) efficiently inhibits the synthesis of most cellular proteins while a large number of viral proteins is produced, including a host shut off protein (1–3). The inhibitory effect of the protein obviously needs a certain period of time to be fully effici ...

The isolation of an individual polypeptide from a heterogeneous mix is an essential process in characterizing a protein of interest. In purified form a protein can be used to generate specific polyclonal and monoclonal antibodies for in vivo studies, in vitro the enzymic properties or inter ...

Herpes simplex virus (HSV) remains a major human pathogen worldwide (25 causing cold sores, eye and genital infections, blindness, encephalitis, and neonatal infections. Most adults have antibodies against the oral form of the virus HSV-1 (9), and a significant number are infected with the g ...

Proteomics has been widely applied to develop two-dimensional polyacrylamide gel electrophoresis maps and databases, evaluate gene expression profiles under different environmental conditions, assess global changes associated with specific mutations, and define dr ...

Two-dimensional electrophoresis results in an adequate resolution of the proteome of microorganisms to allow the detection and identification of specific antigens after blotting on membranes and overlaying the protein pattern with patient’s sera. The complement of all identi ...

The practical problems encountered when purifying and visualizing small amounts of antigens from complex cellular and protein mixtures are explored. Practical aspects and the relative advantages and disadvantages of immunoprecipitation and blotting, the two most commonly u ...

In this chapter I describe the PCR-coupled subtractive hybridization technique of representational difference analysis of cDNA (cDNA RDA). cDNA RDA is based on the representational difference analysis (RDA) method previously described by Lisitsyn et al. 3, and can be used to identify ge ...

DNA microarray is an innovative technology for obtaining information on gene function. Because it is a high-throughput method, computational tools are essential in data analysis and mining to extract the knowledge from experimental results. Filtering procedures and statistical ...

Expression cloning involves the selection of specific polypeptides, generated from a cDNA or genomic DNA library, based on certain characteristics of the expressed proteins, such as antibody or ligand binding, recognition by T-cells, function, or complementation of cell defects. He ...

Recently, various bacterial components have been suggested as initiating and modulating immune activation, thereby substantially affecting the complex and dynamic host/pathogen interactions. Herein, we present a valuable and simple methodology for determining the capa ...

As a response to an infection, the immune system produces antibodies. The determination of the antigenic structure recognized by the antibody through epitope mapping provides information about the interaction between antigen and antibody for the diagnosis of a disease on a molecular l ...

Here we describe a method for T-cell epitope identification using a modified ELISpot assay that is both simple and efficient. By using a carefully constructed array of pools of overlapping peptides spanning the entire antigen sequence to stimulate T-cell responses, we are able to detect anti ...

By using automation and affinity-tag technologies, analysis of the large number of ORFs generated by genome-sequencing projects is greatly accelerated. Protocols describing culture of E. coli in automation-compatible formats and subsequent microto large-scale automated p ...

Many of the problems related with mammalian gene expression, such as low translation efficiency and mRNA halflife, can be solved by means of a rational gene design, based on modern bioinformatics, followed by the de novo generation of a synthetic gene. Moreover, high expression rates and prolon ...

The use of recombinant antigens is essential for the construction of robust and sensitive diagnostic assays. A critical step in the preparation of recombinant antigens is protein purification. Purification problems may be very different for related structural proteins expressed ...

This chapter describes a straightforward protein purification strategy for the specific separation of insoluble recombinant proteins (so-called “inclusion bodies”) located in the E. coli cytoplasm and their subsequent recovery in form of soluble recombinant proteins. Opti ...

Genome sequencing projects have led to the identification of an enormous number of open reading frames that code for unknown proteins. Elucidation of the structure and function of these proteins makes it necessary to produce proteins fast, in high yields and at low cost. The recombinant expre ...

Small-molecule-protein conjugates are often required to act as immunogeneic complexes in the production of both monoclonal and polyclonal antibodies against small antigens. When antibodies have been obtained, they (and/or the small antigens) need to be labeled to facilitate their ...

The primary structure of proteins is nowadays determined by DNA sequencing, and a variety of genomes are already known. Nevertheless, protein sequencing/identification is still indispensable to analyze the proteins expressed in a cell, to identify specific proteins, and to determi ...