[Construction of a lentiviral vector carrying human bcl-2 gene and its expression in human ovarian granulosa cells].

OBJECTIVE

To construct a lentiviral vector carrying human bcl-2 gene and investigate its expression in human ovarian granulosa cells (GCs).

METHODS

Human bcl-2 gene was amplified from the plasmid pCMV-SPORT6 using PCR and subcloned into the lentiviral vector pGC-FU to construct the lentiviral expression vector pGC-FU- bcl-2. The bcl-2 gene insert was confirmed by restriction enzyme digestion and sequencing. The recombinant lentiviruses generated by 293T cells co-transfected with pGC-FU-bcl-2 and the packaging plasmids pHelper1.0 and pHelper2.0. The resulting recombinant lentivirus GC-FU-bcl-2 carrying bcl-2 and EGFP genes were then used to infect human ovarian granulosa cells. EGFP and bcl-2 protein expressions in 293T and human ovarian GCs were detected by fluorescent microscope and Western blotting.

RESULTS

The plasmid pGC-FU-bcl-2 carrying the correct bcl-2 gene sequence could be expressed in human ovarian GC cells, and the recombinant lentivirus GC-FU-bcl-2 was generated by the packaging 293T cells. Stable expression of EGFP and bcl-2 proteins were detected by fluorescent microscope and Western blotting in 293T and human ovarian GCs after the infection. The recombinant lentivirus efficiently delivered bcl-2 gene into human ovarian GCs, in which bcl-2 expression was expressed efficiently and stably.

CONCLUSION

The recombinant lentivirus GC-FU-bcl-2 has been successfully constructed, which is capable of delivering the target gene bcl-2 into human ovarian GCs for its stable expression.