Construction And Immune Activity Study Of A DNA Vaccine Encoding Folate Receptor α

OBJECTIVE:Folate receptor α, a 38 k D cell surface glycoprotein, is anchored to cell membranes through a glycosylphosphatidylinositol moiety. FRa exhibits limited normal tissue distribution, but is highly expressed in ovarian carcinoma. It is considered as an attractive biomarker for ovarian cancer, both in diagnosis and in treatment. In this project, a eukaryotic expression vector encoding human FRα gene was constructed and evaluated for its immune inducing activity.METHODS:Total RNA was isolated from human ovarian cancer cell line SKOV3. The DNA fragment encoding folate receptor α was amplified using RT-PCR and the gene was cloned into the eukaryotic vector pc DNA3.1. The recombinant plasmid was identified by colony PCR and DNA sequencing. HEK293 cells was transfected with the FRα-pc DNA3.1 plasmid using lipofectamine super FectinⅡ. The expression of the FRα in HEK293 cells was tested by RT-PCR, Western blot and immunofluorescence staining. The purified FRα-pc DNA3.1 DNA plasmid was injected intramuscularly in BALB/c mice at 14-day intervals, after 10 days of the third injection, the antibody titers was evaluated by ELISA, and lymphocyte proliferation and cytotoxic T lymphocy- tes experiments was performed to evaluated the induced cellularimmunity.RESULTS:A 750 bp DNA fragment was amplified by RT-PCR from SKOV3 cells.The eukaryotic expression vector FRα-pc DNA3.1 was constructed successfully and transfected in HEK293 cells. The expression of FRα in HEK293 cells was confirmed by RT-PCR, Western blot and immunofluorescence staining. Injection of plasmid increased antibody titers and lymphocyte proliferation. But for cytotoxic T lymphocytes activity, positive result was not observed.CONCLUSION:The recombinant eukaryotic expression plasmid of FRα-pc DNA3.1 was constructed successfully and FRα expression in eukaryotic cells was confirmed. The constructed FRα-pc DNA3.1 recombinant vector can induce both humoral immunity and cellular immunity and could be further explored for ovarian cancer immunotherapeutic study.