title: [Preparation of lentivirus containing human FcγRIIB gene and FcγRIIB expression in HT-1080 cells].
abstract: OBJECTIVE
  To construct a lentiviral expression vector for FcγRIIB and identify its expression in HT-1080 cells.
  
  
  METHODS
  FcγRIIB gene fragment was obtained using reverse transcription with human mRNA as the template, and then was cloned into TRE lentiviral expression plasmid to construct the lentiviral expression recombinant plasmid TRE-FcγRIIB. The recombinant plasmid TRE-FcγRIIB and lentiviral inducible plasmid Tet were transfected into HEK293T cells respectively with lentivirus packaging mix plasmids to pack the expression lentivirus and inducible lentivirus. The viral titers of the two lentiviruses were measured respectively. HT-1080 cells were coinfected with the expression lentivirus and the inducible lentivirus and induced by gradient-concentration doxycycline (Dox). The expression of FcγRIIB was detected by immunofluorescence technique. Real-time quantitative PCR (qRT-PCR) and Western blotting were used to detect FcγRIIB mRNA and protein expression levels, respectively.
  
  
  RESULTS
  The recombinant plasmid was identified using PCR assay and enzyme digestion analysis, and gene sequencing demonstrated that the nucleotide sequence of the inserted fragment had a homology of 100% with the FcγRIIB nucleotide sequence provided by GenBank. The virus titer of the expression lentivirus was 10(6) TU/mL and the inducible lentivirus was 10(5) TU/mL. The immunofluorescence technique showed that the HT-1080 cells co-infected by expression lentivirus and inducible lentivirus expressed FcγRIIB under the induction of Dox. The qRT-PCR and the Western blotting showed that FcγRIIB mRNA and protein expression levels were positively correlated with the concentration of Dox.
  
  
  CONCLUSIONS
  The lentiviral expression vector for FcγRIIB was successfully prepared and its expression in HT-1080 cells is controllable via the alterations of Dox concentration.
authors.affiliation: Department of Pathogenic Biology and Immunology, School of Preclinical Medicine, Ningxia Medical University, Yinchuan 750004, China.
authors.forename: Huiyu
authors.initials: H
authors.lastname: Liu
authors.affiliation: Key Laboratory of Fertility Preservation and Maintenase, Ministry of Education, Yinchuan 750004, China.
authors.forename: Xiuqin
authors.initials: X
authors.lastname: Cao
authors.affiliation: Department of Pathogenic Biology and Immunology, School of Preclinical Medicine, Ningxia Medical University, Yinchuan 750004, China.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Yang
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008856:Microscopy, Fluorescence; D017452:Receptors, IgG / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: C499173:FCGR2B protein, human; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004318:Doxycycline