RESUMO
CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv-apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv-apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.
RESUMO
An RNAi system based on T7 RNA polymerase (TRNAP) was designed and examined in Aspergillus fumigatus. This system consists of two elements; an inducible T7RNAP expressing cassette and an AMA1-based episomal RNAi plasmid. These constructs were transformed into the A. fumigatus protoplasts and the efficiency of this system was tested in downregulation of alb1 gene. Upon the induction of T7RNAP expression, the recombinant T7RNAP was able to recognize T7 promoters, which were located on the episomal plasmid and in opposite direction. As a result, the bidirectional transcription of alb1 fragment led to the silencing of the target gene. However, our results demonstrated that this silencing system is unstable and may not be applicable in preparation of RNAi libraries.
Assuntos
Aspergillus fumigatus/genética , Proteínas Cdc20/genética , RNA Polimerases Dirigidas por DNA/genética , Interferência de RNA , Proteínas Virais/genética , RNA Interferente Pequeno/genética , Transformação Genética/genéticaRESUMO
The large family of annexins is composed of more than a thousand members which are typically phospholipid-binding proteins. Annexins act in a number of signalling networks and membrane trafficking events which are fundamental to cell physiology. Annexins exert their functions mainly through their calcium-dependent membrane binding abilities; however, some calcium-independent interactions have been documented in the literature. Although mammalian and plant annexins have been well characterized, little is known about this family in fungi. This mini review summarizes the available data on fungal annexins.
