RESUMO
The Rift Valley fever virus (RVFV), transmitted through mosquito bites, leads to severe illness in humans and livestock throughout Africa and the Arabian Peninsula, causing significant morbidity and mortality. As of now, there are no verified and efficacious drugs or licensed vaccines accessible for the prevention or treatment of RVFV infections in both humans and livestock. The mature RVFV virion has two envelope proteins on its surface: glycoprotein N (GN) and glycoprotein C (GC). These proteins play a significant role in facilitating the virus's entry into the host cell, making them prominent targets for entry mechanism research as well as targets for drugs and vaccine development. The initial stage in obtaining atomic-resolution structural and mechanistic information on viral entry as well as developing biochemical and biophysical research tools involves recombinant protein production. In this chapter, we describe a simplified and scalable protocol facilitating the generation of high-quality, high-titer baculovirus virus for expression and purification of RVFV GC, utilizing the baculovirus-mediated expression system in insect cells.
Assuntos
Baculoviridae , Proteínas Recombinantes , Vírus da Febre do Vale do Rift , Proteínas do Envelope Viral , Baculoviridae/genética , Animais , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vírus da Febre do Vale do Rift/genética , Células Sf9 , Expressão Gênica , Humanos , Vetores Genéticos/genética , Clonagem Molecular/métodosRESUMO
Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.
Assuntos
Animais , Humanos , Camundongos , Técnicas de Transferência de Genes , Genoma Helmíntico/genética , Schistosoma japonicum/genética , Schistosoma mansoni/genética , Animais Geneticamente Modificados , Cromossomos/genética , Cromossomos/virologia , Elementos de DNA Transponíveis , DNA de Helmintos/genética , DNA Viral/genética , DNA Viral/isolamento & purificação , Vetores Genéticos , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/isolamento & purificação , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Interferência de RNA , Schistosoma japonicum/virologia , Schistosoma mansoni/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
The BVDV glycoproteins gp48 and gp53 were expressed in the baculovirus eukaryotic system. Both recombinant proteins were recognized in western blot analysis by monoclonal antibodies and polyclonal serum. Immunofluorescent test demonstrated that gp53 was localized on the cell surface whereas gp48 was in the cytoplasm. The expressed proteins were extracted by non-denaturing detergent treatment. Rabbit antiserum raised against gp53 recombinant protein efficiently neutralized the virus. These results demonstrate that the recombinant proteins have immunological properties similar to those of the native viral protein and that they can be useful as diagnostic reagents.