微生物学技术

Nondigestible oligosaccharides (NDOs), namely prebiotic oligosaccharides, are functional food ingredients that possess properties that are beneficial to the health of consumers. These include noncariogenicity, a low calorific value, and the ability to stimulate the growth ...

Enzymes control and regulate all biochemical processes in the body. In a single second, several million enzymes mediate chemical reactions occurring in a living system. Enzymes are highly specific in their function because each enzyme is programmed to carry out one special task. The immense ...

The biosensor field has grown enormously since the first demonstration of the biosensor concept by Leland C. Clark Jr. in 1962. Today’s biosensor market is dominated by glucose biosensors, mass-produced enzyme-electrodes for the rapid self-diagnosis of blood glucose levels by diabetes ...

Molluscum contagiosum virus (MCV) lesions are limited to the human epidermis and can persist for months, showing only weak signs of inflammation. Because a culture system has not yet been created to replicate MCV, this chapter describes how the virus can be isolated from patient specimens. From ...

Biochemical and functional analysis of poxvirus genomes, genes, and proteins has entered a new era with the recent sequencing of more than 30 poxvirus genomes. The management and analysis of this volume of sequence data in an efficient and effective manner requires specialized computer so ...

The vaccinia virus-based expression of viral envelope glycoprotein genes—derived from enveloped viruses that infect their respective host cells through a pH-independent mechanism of membrane fusion—has been a powerful tool in helping to characterize these important attach ...

Concern regarding the use of variola and monkeypox viruses as bioterrorist agents has led to an increased study of orthopoxviruses to understand the molecular and cellular basis of pathogenesis and develop safe and effective antivirals and vaccines against smallpox. Crucial to these ...

Studies that involve antigen processing and presentation often require de novo biosynthesis of the antigen both in vitro and in vivo. Additionally, biosynthesis of the antigen or engineered variants within the antigen-presenting cells is usually simpler than providing purified r ...

For the last 30 yr, interest in vaccinia virus immune monitoring has focused on the use of the vaccinia virus as a recombinant vaccine vector and the potential detrimental effect of antivector immunity on subsequent vaccination with a recombinant vaccinia virus. However, interest in this ar ...

Surface plasmon resonance (SPR) biosensors have become an increasingly popular technology for characterizing the protein-protein interactions of virus-host interactions. Various studies have exploited the versatility of SPR to probe the interaction between virus and host ...

The large size of poxvirus virions (approx 250 � 350 nm) makes them ideal candidates for microscopic studies. Recombinant vaccinia viruses that express a viral envelope-specific, green fluorescent protein (GFP) chimera produce enveloped virions that fluoresce green. This fluores ...

This chapter describes the methods for the study of binding and entry of the two different forms of vaccinia virus (VV)—the intracellular mature virus (IMV) and extracellular enveloped virus (EEV)—using immunofluorescent staining and confocal microscopy. After binding to or penetr ...

Cytoplasmic replication of poxviruses dictates the encoding of most, if not all, of the trans-acting factors required for faithful genome duplication. Several of these proteins have been identified through genetic and biochemical evaluation, including the catalytic DNA polyme ...

The study and use of vaccinia virus-derived RNA-modifying proteins has made a significant contribution to molecular biology. Here, the purification and assay of two such proteins, comprising the vaccinia poly(A) polymerase/cap-specific mRNA 2′-O-methyltransferase, is descri ...

This chapter describes a protocol that allows accurate in vitro transcription of vaccinia virus late genes. In this method, extracts are made from vaccinia virus-infected cells and used as enzyme sources to produce mRNAs from plasmid templates containing late gene promoter sequences.

Transcription of the vaccinia virus early genes occurs within the confines of the virion core structure. Therefore, isolated virions are a particularly rich source of proteins that function in early mRNA biosynthesis. Methods are described here for the extraction of purified vaccinia ...

Biologic and antigenic properties are often useful for identifying and differentiating orthopoxviruses (OPV). However, polymerase chain reaction (PCR) amplification, with either restriction cleavage or sequencing of amplicons, has been gaining credibility as a more rapid, ...

This chapter describes the preparation of template DNA from poxvirus-infected cells, plaques, or crude virus stocks for polymerase chain reaction (PCR) amplification. The advantages of this technique are that it is rapid, inexpensive, and, most importantly, reliable, requiring only ...

Poxviruses are cell-associated viruses that can be grown in adherent- or suspension-cell cultures or chorioallantoic membranes of embryonated hen’s eggs. The main principle of isolating the virus is to mechanically lyse infected cells. The virus can then be semipurified by centrifug ...

Modified vaccinia virus Ankara (MVA) is a valuable tool for the expression of recombinant genes used for such purposes as the study of protein functions or characterization of cellular and humoral immune responses. A major advantage of MVA is its clear safety record, and it can be handled under bio ...