title: [Construction of a recombinant lentiviral vector of p38 MAPK and establishment of  a human prostatic carcinoma cell line stably expressing p38 MAPK].
abstract: OBJECTIVE
  To construct a recombinant lentiviral vector for p38 MAPK and establish a human prostatic carcinoma cell line that stably expresses p38 MAPK.
  
  
  METHODS
  EGFP/p38 fusion gene was subcloned into the lentiviral vector pTYF- EF1α-IRES-EGFP. The recombinant lentiviral vector pTYF-EF1α-EGFP/p38 was indentified by restriction enzyme digestion, and packaged in HEK 293T cells using lipofectamintm2000 with the packaging plasmid psPAX2 and envelope plasmid pMD2.G. The viral titer was tested according to the expression level of GFP. The resulting recombinant lentiviral vector was transduced into human prostatic carcinoma DU145 cells, and stably transduced cells were selected by limiting dilution analysis. The intracellular expression level of total p38 was detected by Western blotting and the cell growth curve was drawn.
  
  
  RESULTS
  DNA restriction enzyme digestion demonstrated that the recombinant lentiviral vector of the fusion gene EGFP/p38 (pTYF-EF1α-EGFP/p38) was constructed successfully. The recombinant lentiviral vector was packaged in 293T with a viral titer of 4.7×10(6) TU/ml. A stable cell line, EGFP/p38-DU145, was established, which stably expressed exogenous EGFP/p38 MAPK fusion protein as detected by Western blotting and showed a lowered growth rate compared to the control cells.
  
  
  CONCLUSIONS
  We have successfully constructed a recombinant lentiviral vector of the fusion gene EGFP/p38 and established a stable cell line EGFP/p38-DU145. Overexpression of p38 has a significant inhibitory effect on the proliferation of DU145 cells in vitro.
authors.affiliation: Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. jingyumingdoc@163.com
authors.forename: Yu-Ming
authors.initials: YM
authors.lastname: Jing
authors.forename: Jie
authors.initials: J
authors.lastname: Luo
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Zhang
authors.forename: San-San
authors.initials: SS
authors.lastname: Chen
authors.forename: Pei
authors.initials: P
authors.lastname: Wan
authors.forename: Ren-He
authors.initials: RH
authors.lastname: Yan
authors.forename: Hong-Chang
authors.initials: HC
authors.lastname: Wang
authors.forename: Bai-Hong
authors.initials: BH
authors.lastname: Chen
authors.forename: Wan-Long
authors.initials: WL
authors.lastname: Tan
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Li
mesh_terms: D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism*; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D048051:p38 Mitogen-Activated Protein Kinases