Preparation and application of polyclonal antibodies against KSHV v-cyclin

摘要

We prepared rabbit polyclonal antibodies against Kaposi's sarcoma-associated herpesvirus(KSHV)-encoded vcyclin(ORF 72)and detected the natural viral protein using these polyclonal antibodies.Three antigenic polypeptides of v-cyclin were designed and synthesized.A fragment of the v-cyclin gene was cloned into a eukaryotic expression vector pEF-MCS-Flag-IRES/Puro to construct a recombinant vector,pEF v-cyclin.Then,pEF v-cyclin was transfected into 293T and EA.hy926 cells to obtain v-cyclin-Flag fusion proteins.Six New Zealand white rabbits were immunized with KLH-conjugated peptides to generate polyclonal antibodies against v-cyclin.The polyclonal antibodies were then characterized by ELISA and Western blotting assays.Finally,the polyclonal antibodies against v-cyclin were used to detect natural viral protein expressed in BCBL-1,BC-3,and JSC-1 cells.The results showed that using the Flag antibody,v-cyclin-Flag fusion protein was detected in 293T and EA.hy926 cells transfected with pEF-v-cyclin.Furthermore,ELISA showed that the titer of the induced polyclonal rabbit anti-vcyclin antibodies was higher than 1:8,000.In Western blotting assays,the antibodies reacted specifically with the v-cyclin-Flag fusion protein as well as the natural viral protein.The recombinant expression vector pEF-v-cyclin was constructed successfully,and the polyclonal antibodies prepared can be used for various biological tests including ELISA and Western blotting assays.