title: [Construction and identification of nemo-like kinase gene recombinant adenovirus vector].
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.
  
  
  CONCLUSIONS
  The NLK gene recombinant adenovirus vector was successfully constructed and identified.
authors.affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
authors.forename: Xiao-Wen
authors.initials: XW
authors.lastname: Cheng
authors.forename: Jun-Bo
authors.initials: JB
authors.lastname: Liang
authors.forename: Shi-Ying
authors.initials: SY
authors.lastname: Miao
authors.forename: Lin-Fang
authors.initials: LF
authors.lastname: Wang
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; C470551:NLK protein, human; D017346:Protein Serine-Threonine Kinases