Cloning, Expression And Bioactivity Analysis Of Goat Interleukin-4and Interleukin-18

Cytokines are multifunctional pleiotropic proteins which regulate communication between antigen-presenting cells (APCs), lymphocytes and other host cells in the course of an immune response. Interleukin-4(IL-4) and Interleukin-18(IL-18) are important to the development of humoral mediated immunity responses, cell-mediated immune responses, defending the infection of microbial and resisting the tumor.Two pairs of specific primers were respectively designed according to the sequences of goat IL-4and IL-18published in GeneBank. Goat IL-4and IL-18cDNA (pGIL-4and pGIL-18) were amplified by RT-PCR, and subsequently cloned into the vector pMD18-T. Sequence analysis of pGIL-4demonstrated an open reading frame (ORF) of408base pairs encoding for a135amino acid precursor protein. The signal peptide was composed of24amino acids. The ORF of the pGIL-18gene was582base pairs encoding a peptide of193amino acids. The leader peptide was composed of36amino acids. Phylogenetic relationship of IL-4and IL-18amino acid sequences of several mammalian were analyzed with CLUSTALX and MEGA4.0software.The mature GIL-4(mGIL-4) and mature GIL-18(mGIL-18) were accordingly amplified from pMD18T-pGIL-4and pMD18T-pGIL-18by PCR, and then subcloned into pET-32a(+) and pET-28a(+) respectively. The two recombinant plasmids of pET-32a(+)-mGIL-4and pET-28a(+)-mGIL-18were obtained after sequencing confirmation. High levels of rmGIL-4and rmGIL-18proteins were successfully expressed in E.coli strain BL21transformed with pET-32a(+)-mGIL-4and pET-28a(+)-mGIL-18. The SDS-PAGE analysis indicated that the fusion proteins were31.3KDa and22.3KDa in molecular weight and existed in the inclusion body.MTT analysis indicated that purified rmGIL-4protein has good bioactivity which induced the proliferation of activated T lymphocytes in goat’s PBMC in a dose-dependent manner and typically demonstrated8μg/ml of activity in vitro. To explore the biological activities of rmGIL-18, the mice peritoneal macrophages were activated by rmGIL-18in vitro. The optical density of the solution was tested after lysing the cultural macrophages phagocytizing neutral red. Nitric oxide secreted by the activated macrophages was assayed by NO assay kit. The founding was that rmGIL-18significantly enhanced the neutral red phagocytosis at500ng/ml and the NO secretion of the activated macrophages at250ng/ml. Under a certain dose range, the augment of rmGIL-18was dose-dependent.