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.
Conclusion: The lentiviral expression vector for FcγRIIB was successfully prepared and its expression in HT-1080 cells is controllable via the alterations of Dox concentration.