title: [Construction of Eukaryotic Expression Vector Containing ROP18-ROP12 of Toxoplasma gondii RH Strain].
abstract: OBJECTIVE
  To construct a recombinant eukaryotic expression plasmid containing ROP18-ROP12 (encoding rhoptry protein 18 and 12) complex gene of Toxoplasma gondii, and examine its expression in eukaryotic cells.
  
  
  METHODS
  Recombinant plasmids pVAX1-ROP18 and pVAX1-ROP12 were digested by restriction enzymes BamH I and Xba I . ROP12 gene was cloned into pVAX1-ROP18 to construct the eukaryotic expression plasmid pVAX1-ROP18- ROP12. After colony PCR, enzyme digestion and sequencing, the correct recombinant plasmid pVAX1-ROP18-ROP12 was transfected into HeLa cells. Along with it were groups of empty plasmid, pVAX1-ROP18 and pVAX1-ROP12. Total RNA was extracted from HeLa cells and reverse-transcribed to cDNA. RT-PCR was performed to evaluate mRNA expression of the housekeeping gene β-actin and ROP18-ROP12 complex gene. Immunofluorescence assay and Western blotting were performed to determine the protein levels of ROP18-ROP12 fusion protein.
  
  
  RESULTS
  Colony PCR in recombinant plasmid pVAX1-ROP18-ROP12 showed a specific band at about 2 373 bp, consistent with expectation. The extracted recombinant plasmids were confirmed by Hind III, BamH I and Xba I digestion. Sequencing results showed that the sequence of pVAX1-ROP18-ROP12 was 100% identical to that of T. gondii RH strain ROP18 gene (Accession No. AM075204.1) and 99% identical to that of T. gondii RH strain ROP12 gene (Accession No. DQ096559.1). Further, RT-PCR showed amplification products at 613 bp for β-actin in all the groups, while only the pVAX1-ROP18-ROP12 transfection group showed amplification products for the ROP18-ROP12 complex at 2,373 bp. In addition, the indirect immunofluorescence assay showed yellow-green fluorescence in HeLa cells transfected with pVAX1-ROP18-ROP12, but not in control cells. Western blotting showed that the ROP18-ROP12 fusion protein was expressed in HeLa cells transfected with recombinant plasmid pVAX1-ROP18-ROP12.
  
  
  CONCLUSIONS
  The recombinant eukaryotic plasmid pVAX1-ROP18-ROP-2 is constructed and can be expressed in eukaryotic system.
authors.forename: Ling-ling
authors.initials: LL
authors.lastname: Guo
authors.forename: Xiao-lei
authors.initials: XL
authors.lastname: Zhang
authors.forename: Jin-shun
authors.initials: JS
authors.lastname: Zhang
authors.forename: Xiao-hui
authors.initials: XH
authors.lastname: Jia
authors.forename: Chun-miao
authors.initials: CM
authors.lastname: Wang
authors.forename: Wen-jing
authors.initials: WJ
authors.lastname: Jiang
authors.forename: Xiao-bo
authors.initials: XB
authors.lastname: Zhu
authors.forename: Tian-jun
authors.initials: TJ
authors.lastname: Jia
mesh_terms: D000199:Actins; D015153:Blotting, Western; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D014122:Toxoplasma; D014162:Transfection
chemical_list: D000199:Actins; D011994:Recombinant Proteins