Establishment of a stable cell line replicating hepatitis B virus DNA carrying the reverse transcriptase region derived from a clinical isolate / 中国医学科学院学报
Acta Academiae Medicinae Sinicae
;
(6): 13-18, 2013.
Artículo
en Chino
| WPRIM
| ID: wpr-284312
ABSTRACT
<p><b>OBJECTIVE</b>To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate.</p><p><b>METHODS</b>Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis.</p><p><b>RESULTS</b>Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained.</p><p><b>CONCLUSIONS</b>A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.</p>
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Plásmidos
/
Virología
/
Replicación Viral
/
ADN Viral
/
Línea Celular
/
Virus de la Hepatitis B
/
ADN Polimerasa Dirigida por ARN
/
Clonación Molecular
/
Hepatocitos
/
Biología Celular
Límite:
Humanos
Idioma:
Chino
Revista:
Acta Academiae Medicinae Sinicae
Año:
2013
Tipo del documento:
Artículo
Similares
MEDLINE
...
LILACS
LIS