RESUMO
To create a tool for eukaryotic surface display, this approach is aimed at demonstrating a direct modification of the native envelope protein gp64 of Autographa californica NPV without disturbing viral infectivity. Short affinity-tag peptides, the biotin mimic streptagII, and the gp41 amino-acid motif ELDKWA of HIV-1, specific for the human monoclonal antibody 2F5, were engineered into the baculovirus major coat protein gp64 and presented on the viral surface. Two different streptag peptides were inserted at the naturally occurring NotI site at amino-acid 278 of gp64. Additionally, the ten-amino-acid peptide GG-ELDKWA-GG, containing the epitope of mAb 2F5, was introduced into gp64 envelope protein at the same position. In all cases we were able to propagate viable virus-achieving infectious titers in the range of wild-type AcMNPV. Streptag and ELDKWA-epitope surface localization on purified virus particles was demonstrated by flow cytometry and Western blot analysis. We could also show selective retention of mutant viruses by specific interaction between chimeric virions and their target counterparts, recognizing the epitope or the streptag peptide in the viral envelope. These data provide evidence that altering the surface properties of the baculovirus virion could be of value in improving baculovirus display technology and developing new applications.
Assuntos
Nucleopoliedrovírus/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Western Blotting , Epitopos , Dados de Sequência Molecular , Spodoptera , Proteínas do Envelope Viral/imunologia , Vírion/isolamento & purificação , Replicação ViralRESUMO
Extended electroretinographic (ERG) testing has been carried out in a series of patients with retinitis pigmentosa, dominantly inherited. In 36 of 57 cases only cone b waves were evoked. In 20 of these, psychophysical tests showed only cones mediated vision (Massof class I), while in 16 statis scotopic perimetry demonstrated residual rod function (class II). In the remaining cases, where rod ERGs were seen, the light intensities required to evoke responses were not greatly elevated. A computer model was constructed to relate psychophysical threshold measurements to ERG data. This analysis of the results suggests that in one subgroup of patients the scotopic ERG is smaller than expected from the losses of visual field and that in another the psychophysical elevation of rod visual threshold is greater than expected from ERG measurements.
Assuntos
Células Fotorreceptoras/fisiopatologia , Retinose Pigmentar/fisiopatologia , Adolescente , Adulto , Idoso , Computadores , Eletrorretinografia , Genes Dominantes , Humanos , Pessoa de Meia-Idade , Modelos Biológicos , Psicofísica , Retinose Pigmentar/genética , Testes de Campo Visual , Campos VisuaisRESUMO
An electroretinographic (ERG) technique is described in which the relationship between scotopic b wave amplitude and stimulus light intensity is determined. The relative amplitude of scotopic to photopic responses is assessed by means of red light and flicker. The method is applied to the detection of ERG abnormalities in heterozygotes for X-linked retinitis pigmentosa. These have been found in only a proportion of cases. The ERG results can be used to suggest the nature of the retinal abnormality.
