title: [Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].
abstract: OBJECTIVE
  To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis.
  
  
  METHODS
  Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis.
  
  
  RESULTS
  The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells.
  
  
  CONCLUSIONS
  The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.
authors.affiliation: Department of Transfusion Medicine, School of Biotechnology, Southern Medical University, Guangzhou 510515, China.
authors.forename: Zuoping
authors.initials: Z
authors.lastname: He
authors.affiliation: Department of Transfusion Medicine, School of Biotechnology, Southern Medical University, Guangzhou 510515, China.
authors.forename: Peifang
authors.initials: P
authors.lastname: Luo
authors.affiliation: Department of Transfusion Medicine, School of Biotechnology, Southern Medical University, Guangzhou 510515, China.
authors.forename: Feihuan
authors.initials: F
authors.lastname: Hu
authors.affiliation: Department of Transfusion Medicine, School of Biotechnology, Southern Medical University, Guangzhou 510515, China.
authors.forename: Yunceng
authors.initials: Y
authors.lastname: Weng
authors.affiliation: Department of Transfusion Medicine, School of Biotechnology, Southern Medical University, Guangzhou 510515, China.*Corresponding author, E-mail: wenjingking77@126.com.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Transfusion Medicine, School of Biotechnology, Southern Medical University, Guangzhou 510515, China.
authors.forename: Chengyao
authors.initials: C
authors.lastname: Li
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D017347:Brucella melitensis / Q000235:genetics*; D045744:Cell Line, Tumor; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008074:Lipoproteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins; C097319:OMP19 protein, Brucella abortus