title: [Construction and expression of eukaryotic expression plasmid pcDNA3.1-smac].
abstract: OBJECTIVE
  To construct the eukaryotic expression vector of human gene Smac pcDNA3.1-Smac and express it in the lung adenocarcinoma A549 cells.
  
  
  METHODS
  The Smac was amplified from human testis tissue by reverse transcriptase polymerase chain reaction (RT-PCR). Then recombined eukaryotic expression vector pcDNA3.1-Smac was constructed. After the reconbinant plasmid was proved to be constructed correctly by endonucleases digesting and DNA sequencing, we trasfected it into lung adenocarcinama cells A549 through liposome inducing. The expression of Smac in transfectant A549 was detected by RT-PCR and Western blot. And the cell growth inhibition ratio after trasfection was detected by MTT.
  
  
  RESULTS
  The amplified fragment by PCR was coincident with the anticipated result, and its sequence was in concordance with that published on GenBank.Therefore, the gene Smac was cloned successfully, and the recombinant plasmid pcDNA3.1-Smac was also constructed successfully. Both on the mRNA level and the protein level, the expression of Smac gene was increased obviously in the transfected A549 detected by RT-PCR and Western blot respectively. The cell growth inhibition ratio in the group transfected pcDNA3.1-Smac was significantly higher compared with the pcDNA3.1 group after 72 hours.
  
  
  CONCLUSIONS
  The recombinant eukaryotic expression vector pcDNA3.1-Smac was constructed, and it could be obviously expressed in lung adenocarcinoma cells A549. It is also proven that Smac has the function of growth inhibition.
authors.affiliation: Oncology Center, First Affiliated Hospital, Xi'an Jiaotong University College of Medicine, Xi' an 710061, China. sida.qin@gmail.com
authors.forename: Si-da
authors.initials: SD
authors.lastname: Qin
authors.forename: Hong
authors.initials: H
authors.lastname: Ren
authors.forename: Xiao-Jun
authors.initials: XJ
authors.lastname: Li
authors.forename: Cheng-Cheng
authors.initials: CC
authors.lastname: Yang
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.forename: Xiang-Zhong
authors.initials: XZ
authors.lastname: Zhang
authors.forename: Shi-Sen
authors.initials: SS
authors.lastname: Li
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Hu
mesh_terms: D051017:Apoptosis Regulatory Proteins; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins