title: [Construction of a replicative anti-tumor DNA vaccine PSCK-2PFcGB and its expression in vivo and in vitro].
abstract: OBJECTIVE
  To construct a replicative anti-tumor DNA vaccine PSCK-2PFcGB based on Semliki Forest Virus (SFV) replicon vector and observe its expression in vivo and in vitro.
  
  
  METHODS
  The plasmid pVAX1-2PFcGB was digested with Nhe I, and the digestion product was blunted prior to further digestion with BssH II to obtain the fragment 2PFcGB, a fusion gene containing the multitarget complex antigen 2PAG encoding both the most cytotoxic T lymphocyte epitopes of human survivin and chorionic gonadotropin β chain-CTP37 of human and monkey. The 2PFcGB fragment was inserted into the PSCK vector digested by Sma I. The products with the expected size were extracted and ligated, and the positive clones were screened by kanamycin and amplified. The recombinant PSCK-2PFcGB, following identification by colony PCR and restriction endonuclease Nde I, was transfected into 293T cells via lipofectamine 2000 and its expression was detected. The recombinant plasmid was also transfected into mouse quadriceps femoris muscle to observe its expression in vivo by immunohistochemistry.
  
  
  RESULTS
  Nde I digestion resulted in a fragment of the expected size. Transfection with the recombinant plasmid PSCK-2PFcGB resulted in successful expression of the antigen and adjuvant molecular protein in 293T cells, with the positivity rates of 5.70% and 19.75%, respectively. The fusion tumor antigen survivin and hCGβ-CTP37 were also detected in the muscular tissues of the mice.
  
  
  CONCLUSIONS
  A novel replicative anti-tumor DNA vaccine PSCK-2PFcGB has been successfully constructed and can be expressed in 293T cells and in the muscular tissues of immunized mice, which provide a basis for further studies of the antitumor activity and immunological mechanism of the DNA vaccine.
authors.affiliation: Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. zhangliangrpg@126.com
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.forename: Jin-qi
authors.initials: JQ
authors.lastname: Yan
authors.forename: Yue
authors.initials: Y
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiao
authors.forename: Kun
authors.initials: K
authors.lastname: Gao
authors.forename: Jin-kai
authors.initials: JK
authors.lastname: Dong
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.forename: Ji-yun
authors.initials: JY
authors.lastname: Yu
mesh_terms: D000818:Animals; D000974:Antibodies, Antinuclear / Q000276:immunology; D019496:Cancer Vaccines / Q000096:biosynthesis* / Q000276:immunology; D015870:Gene Expression; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism; D010957:Plasmids; D012672:Semliki forest virus / Q000235:genetics; D019444:Vaccines, DNA / Q000096:biosynthesis* / Q000276:immunology
chemical_list: D000974:Antibodies, Antinuclear; D019496:Cancer Vaccines; D019444:Vaccines, DNA