Preparation and detection of cytokines polyclonal antibodies of Cyprinus carpio

Inflammatory cytokines are often used as one of the indicators to detect host immune responses. The lack of cytokines antibodies for fish has hindered analysis of cytokines protein expression by ELISA. In this paper, the fragments of TNF-α, IL-1β, IL-6, IL-12, IL-10 and TGF-β genes of Cyprinus carpio, containing partial antigenic determinants were cloned by PCR. The target sections of cytokines were cloned into plasmid pET-32a/21a vectors which had restriction sites for BamH Ⅰ and Hind Ⅲ. The recombinant plasmids were transformed into E. coli BL21 (DE3). The fusion proteins were highly expressed in E. coli BL21 (DE3) after being induced with IPTG. The purified fusion proteins were used as antigen to immunize New Zealand Rabbits by ear margin veins injection combined with subcutaneous injection to obtain rabbit anti carp polyclonal antibodies of cytokines. Enzyme-Linked Immuno Sorbent Assay (ELISA) was used to evaluate the antibody titers, and the antibodies were used as a test tool to detect the expression of inflammatory cytokines in carp serum after infection with Aeromonas hydrophila. The results showed that the sizes of fusion proteins were about 31.8, 31.7, 35.3, 32.5, 18.0 and 33.6 ku, respectively. The titers of the antiserum were about 2.4×10⁶. The pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-12 and anti-inflammatory cytokines IL-10 and TGF-β showed different expression patterns at different stages after A. hydrophila infection. The results indicated that the prepared antibodies performed high titer, affinity and specificity and could be applied to study the expression of cytokines in C. carpio. The availability of these polyclonal antibodies laid the foundation for the systematic study of immune response and cytokines expression in C. carpio. Meanwhile, these polyclonal antibodies could also be used to exploratively study the cytokines protein expression of TNF-α, IL-1β, IL-6, IL-12, IL-10 and TGF-β in other fishes.