RESUMEN
BACKGROUND: Allergen immunotherapy (AIT) still plays a minor role in the treatment of allergic diseases. To improve the acceptance of AIT by allergic patients, the treatment has to become more convenient and efficacious. One possibility is the oral application of allergens or derivatives thereof. Therefore, we sought to produce a recombinant allergen in the green alga Chlamydomonas reinhardtii as a novel production platform. METHODS: The major birch pollen allergen Bet v 1 was selected as candidate molecule, and a codon-optimized gene was synthesized and stably integrated into the microalga C. reinhardtii FUD50. Positive transformants were identified by PCR, cultured, and thereafter cells were disrupted by sonication. Bet v 1 was purified from algal total soluble protein (TSP) by affinity chromatography and characterized physicochemically as well as immunologically. RESULTS: All transformants showed expression of the allergen with yields between 0.01 and 0.04% of TSP. Algal-derived Bet v 1 displayed similar secondary structure elements as the Escherichia coli-produced reference allergen. Moreover, Bet v 1 produced in C. reinhardtii showed binding comparable to human IgE as well as murine Bet v 1-specific IgG. CONCLUSION: We could successfully produce recombinant Bet v 1 in C. reinhardtii. As microalgae are classified as GRAS (generally recognized as safe), the pilot study supports the development of novel allergy treatment concepts such as the oral administration of allergen-containing algal extracts for therapy.
Asunto(s)
Alérgenos/genética , Antígenos de Plantas/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Alérgenos/inmunología , Alérgenos/aislamiento & purificación , Antígenos de Plantas/inmunología , Antígenos de Plantas/aislamiento & purificación , Humanos , Inmunoglobulina E/inmunología , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
We have shown that melanoma cells produce viral particles that contain sequences which are homologous to human endogenous retroviruses. In this study particles derived from different melanoma cell lines and from melanoma cells of a lymph node metastasis were characterized. We determined the density and the reverse transcriptase (RT) activity of viral particles. Furthermore, we analyzed the sequence variability of multiple clones of each particle preparation. The particles were found to package sequences, which vary for each of the analyzed cell lines. Moreover, even particles derived from the same cell line contain heterologous sequences.
Asunto(s)
Retrovirus Endógenos/genética , Variación Genética , Genoma Viral , Melanoma/virología , Anciano , Secuencia de Bases , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Ganglios Linfáticos/virología , Metástasis Linfática , Melanoma/secundario , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
We show that human melanoma cells produce retrovirus-like particles that exhibit reverse transcriptase activity, package sequences homologous to human endogenous retrovirus K (HERV-K), and contain mature forms of the Gag and Env proteins. We also demonstrate expression of the pol gene and of Gag, Env, and Rec proteins in human melanomas and metastases but not in melanocytes or normal lymph nodes. The data suggest that expression of retroviral genes and production of retroviral particles is activated during development of melanoma.