Preparation and application of polyclonal antibodiesagainst KSHV v-cyclin

Abstract

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 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-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 including ELISA and Western blotting assays.

Keywords: Kaposi's sarcoma-associated herpesvirus; polyclonal antibody; synthesized peptides; v-cyclin.

Fig 1.. Amplification of the KSHV v-cyclin gene and construction of recombination plasmid pEF v-cyclin Flag-IRES/Puro.

A: Amplification of the v-cyclin gene by PCR. B and C: pEF v-cyclin and fragments of pEF v-cyclin restrictly digested by NheI and XhoI.

Fig 2.. The sequence of pEF-MCS-Flag-IRES/Puro was compared with KSHV ORF 72 by DNA sequencing.

A fragment corresponding to the v-cyclin gene was inserted in an expression vector, pEF-MCS-Flag-IRES/Puro, after cleavage with NheI and XhoI. The correct insertion, identity, and integrity of the v-cyclin gene were demonstrated by DNA sequencing, which showed an identical sequence to that in GenBank.

Fig 3.. Western blotting analyses using the antibody against KSHV v-cyclin-flag.

A: Expression of the v-cyclin protein in 293T cells transfected with pEF-v-cyclin was detected with the anti-flag antibody in Western blotting. B: Expression of the v-cyclin protein in EA.hy926 cells transfected with pEF-v-cyclin was detected with the anti-flag antibody in Western blotting assays.

Fig 4.. Protein profiles of v-cyclin analyzed using the SUMOplot software.

Fig 5.. Detection of the full-length v-cyclin protein by anti-v-cyclin in Western blotting assays.

A and B: The KSHV v-cyclin protein (predicted MW of 28 kDa) was detected with the anti-v-cyclin antibodies from RB1A, RB1B (rabbits immunized with polypeptide No. 1); C and D: The KSHV v-cyclin protein was also detected with the anti-v-cyclin antibodies from RB2A, RB2B (rabbits immunized with polypeptide No. 2); E and F: The KSHV v-cyclin protein was also detected with the anti-v-cyclin antibodies from RB3A and RB3B (rabbits immunized with polypeptide No. 3).

Fig 6.. Detection of natural viral protein in BCBL-1, BC-3, and JSC-1 cells in Western blotting assays.

Lysates of KSHV+ BC-3, BCBL-1 PEL, and KSHV+ EBV+ JSC-1 PEL cells with or without TPA treatment were subjected to SDS-PAGE. The anti-v-cyclin antibody came from rabbit RB2B, immunized with polypeptide No. 2.