摘要
We prepared rabbit polyclonal antibodies against Kaposi＇s sarcoma-associated herpesvirus （KSHV）-encoded v- cyclin （ORF 72） and detected the natural viral protein using these polyclonal antibodies. Three antigenic polypep- tides 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 anti- bodies 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-v- cyclin 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 in- cluding ELISA and Western blotting assays.
We prepared rabbit polyclonal antibodies against Kaposi＇s sarcoma-associated herpesvirus （KSHV）-encoded v- cyclin （ORF 72） and detected the natural viral protein using these polyclonal antibodies. Three antigenic polypep- tides 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 anti- bodies 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-v- cyclin 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 in- cluding ELISA and Western blotting assays.
