RESUMO
Human noroviruses (NoVs) are the most common cause of acute gastroenteritis worldwide. One major obstacle in developing NoV vaccines is the lack of robust cell culture for efficacy evaluation. In this study, we successfully developed a NoV virus-like particle (VLP) entry assay based on split NanoLuc luciferase (LgBiT and HiBiT) complementation. HiBiT-tagged NoV GII.4 VLP (VLP-HiBiT) can be efficiently produced in Pichia pastoris and retain binding activity towards NoV receptor histo-blood group antigens (HBGAs). A 293T-FUT2-LgBiT cell line was established and was shown to stably express cell surface HBGAs and intracellular LgBiT. GII.4 VLP-HiBiT can bind and enter into the 293-FUT2-LgBiT cells, producing strong luminescence signals in live cells. Anti-GII.4 sera can inhibit VLP-HiBiT entry into the 293-FUT2-LgBiT cells in a dose-dependent manner, and neutralizing titers well correlate with their blocking titers measured by HBGAs-binding blockade assay. Moreover, such a surrogate infection/neutralization assay can be applied to other NoV genotypes such as GI.1 and GII.17. Together, the VLP-HiBiT entry assay can mimic both NoV attachment and internalization in live cells and thus facilitate reliable and comprehensive evaluation of NoV vaccine and antibodies.
Assuntos
Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/farmacologia , Luciferases/genética , Norovirus/genética , Norovirus/imunologia , Internalização do Vírus , Anticorpos Antivirais/imunologia , Infecções por Caliciviridae/virologia , Teste de Complementação Genética/métodos , Teste de Complementação Genética/normas , Genótipo , Células HEK293 , Humanos , Luciferases/metabolismo , Medições Luminescentes , Saccharomycetales/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Ligação ViralRESUMO
Genetic alterations affecting nucleotide excision repair, the most versatile DNA-repair mechanism responsible for removal of bulky DNA adducts including ultraviolet (UV) light-induced DNA lesions, may result in the rare, recessively inherited autosomal syndromes xeroderma pigmentosum (XP), Cockayne syndrome (CS), or trichothiodystrophy (TTD). Classical approaches such as somatic cell fusions or microinjection assays have formalized the genetic complexity of these related but clinically distinct syndromes, and contributed to the determination of seven, five, and three complementation groups for XP, CS, and TTD, respectively. XP patients are highly susceptible to photoinduced cutaneous cancers of epidermal origin. To better study the responses to UV irradiation of XP keratinocytes, and to objectively determine the extent to which cutaneous gene therapy may be realized, we set up experimental procedures adapted to ex vivo genetic complementation of keratinocytes from XP patients. We provide here detailed rationales and procedures for these approaches.
Assuntos
Reparo do DNA/genética , Teste de Complementação Genética/métodos , Queratinócitos/efeitos da radiação , Dermatopatias/genética , Raios Ultravioleta , Xeroderma Pigmentoso/genética , DNA/efeitos da radiação , Replicação do DNA , Teste de Complementação Genética/normas , Terapia Genética , Genótipo , Humanos , Queratinócitos/patologia , Fenótipo , Retroviridae/genética , Dermatopatias/patologia , Dermatopatias/terapia , Transdução Genética , Xeroderma Pigmentoso/patologia , Xeroderma Pigmentoso/terapiaRESUMO
Several recent studies have used quantitative complementation tests to identify relatively short chromosome regions that contain genes that influence life span and to screen for candidate life-span genes in flies. The methodology and logic of quantitative complementation tests are described. Arguments are presented that suggest that these tests may be misleading because there is a substantial, but unknown, likelihood of false positive results. The arguments are supported by the published results of quantitative complementation tests.