Cloning, Fusion-expression And Immunoadjuvant Effect Of The Extracellular Domain Of Canine CTLA-4

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a surface glycoprotein involved in cytotoxic T cell activation. Only activated T cells express CTLA-4 on their surface, which can compete with CD28 for binding the same B7 ligand to down-regulate T cell activation. The whole molecule of CTLA-4 consists of an extra-cellular domain, transmembrane region and a cytoplasmic tail. It has been shown that the extracellualr domain of mammalian CTLA-4 can be used to target the fused antigens to antigen presenting cells and thus can serve as a new-type molecular adjuvant.To study the immunoadjuvant effect of the extracellular domain of canine CTLA-4, total RNA was extracted from canine 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 to be 99.2% identical to the previously published sequence with no mutation in the hexapeptide motif (MYPPPY) for B7 binding. The DNA fragment was then subcloned into prokaryotic expression vector pQE-31 for expression study. The hydrophilic and antigenic region from 293 to 520 amino acids of the VP2 protein of canine parvovirus, called VP2S, was amplified by high fidelity PCR and subcloned into prokaryotic expression vector pQE-31 or pQE-31-CTLA4. After ITPG induction, two His-fusion proteins with molecular weight of 29kD and 42kD, 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 CPV antiserum. BALB/c mice were immunized 3 times with the same dose of the two proteins without adjuvant. Indirect ELISA showed that a quicker and much higher antibody response (1:100000) were induced in the CTLA-4-VP2S-immunized mice than that in VP2-immunized mice. The dymanics of HI antibody was the same as ELISA titer. These data demonstrate that the extracellular domain of canine CTLA-4 has high immunoadjuvant effect on its fused protein.