[Construction and identification of nemo-like kinase gene recombinant adenovirus vector].
Zhongguo Yi Xue Ke Xue Yuan Xue Bao
; 33(6): 632-7, 2011 Dec.
Article
en Zh
| MEDLINE
| ID: mdl-22509546
ABSTRACT
OBJECTIVE:
To construct the nemo-like kinase (NLK) gene recombinant adenovirus vector.METHODS:
The AdEasy system was used to construct the recombinant adenovirus vector. Using reverse transcriptase polymerase chain reaction (RT-PCR), the full-length gene of NLK and its mutants (K155M, T286V, and C425Y) were amplified from HEK293 cells. The FLAG tag was appended at the C-terminal of NLK. After ligation and transformation, the NLK gene and its mutants were cloned into the pAdTrack-CMV vector. It was detected by PCR, sequencing, and Western blot analysis. Using DNA recombination and homogenous recombination, the normally expressed plasmids were linearized by the restriction enzyme-PmeI and PacI, then the enzyme-digested products were recycled by using ethanol precipitation. The purified product was transfected to HEK293A packaging cells with FuGENE HD transfection reagent. After amplification of the recombinant adenovirus, Western blot analysis was performed to detect the expression of NLK gene and its mutants.RESULTS:
The successful construction of pAdtrack-CMV-NLK (and mutants) was confirmed by PCR and sequencing. Western blot analysis showed that the target genes and the recombinant adenovirus were obtained. This recombinant virus was able to express NLK protein and its mutants correctly in HCT 116 cells.CONCLUSION:
The NLK gene recombinant adenovirus vector was successfully constructed and identified.
Buscar en Google
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Adenoviridae
/
Proteínas Serina-Treonina Quinasas
/
Péptidos y Proteínas de Señalización Intracelular
/
Vectores Genéticos
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
Zh
Revista:
Zhongguo Yi Xue Ke Xue Yuan Xue Bao
Año:
2011
Tipo del documento:
Article
País de afiliación:
China