Cloning, Fusion-Expression And Immunoadjuvant Effect Of The Extracellular Domain Of Porcine CTLA-4 On PPV VP1 Antigen

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a surface glycoprotein expressed mainly on activated T lymphocytes, which competes with CD28 molecule for binding to the B7 molecule on antigen presenting cells and thus play a key role in down-regulating T cell activation. CTLA-4 molecule consists of an extracellular domain, a transmembrane region and a cytoplasmic tail. It has been demonstrated that the extracellualr domain of mammalian CTLA-4 can promote the presentation of its fused antigens to antigen presenting cells and thus serves as a new-type of molecular adjuvant.To study the immunoadjuvant effect of the extracellular domain of porcine CTLA-4, total RNA was extracted from porcine peripheral blood lymphocytes and a pair of primers was designed according to the published sequence. After 30 cycles of amplification by RT-PCR, the amplification product of correct size was cloned into T vector and sequence analysis showed a 99.2% identity to the previously published sequence with atypical hexapeptide motif (LYPPPY) for B7 binding. The DNA fragment was then subcloned into prokaryotic expression vector pQE-31 for expression study. The hydrophilic and antigenic region of the VP2 protein of porcine parvovirus, named VP2s, was amplified by high fidelity PCR and subcloned into prokaryotic expression vector pGEX-6p-1, and CTLA-4 was also subcloned into prokaryotic expression vector pGEX-6p-1.After ITPG induction, two GST-fusion proteins with molecular weight of 60kD and 74kD, respectively, were detected in the lysates of the recombinant E.coli by SDS-PAGE. Western-blotting assay revealed that the recombinant proteins were recognized by PPV antiserum. BALB/c mice were immunized 2 times with the same dose of the two proteins without adjuvant. Indirect ELISA showed that quicker and higher antibody response (1∶2 560-1∶5 120) was induced in the GST-CTLA4IgV-VP2s immunized mice than that in GST-VP2s immunized mice. The tendency of HI antibody was similar to ELISA titer. These datas demonstrate that the extracellular domain of porcine CTLA-4 has high immunoadjuvant effect on its fused protein.