Molecular Cloning And Characterization Of A Putative LITAF Gene Homologue From Zhikong Scallop Chlamys Farreri

Whether cytokines-like molecules exist in the invertebrates is controversial for a long time. By using cellular biological and immunological approaches, several cytokines-like molecules, such as TNF-α, IL-1αand IL-6, have been verified to exist in mollusk, arthropod and echinoderm. Unfortunately, there are still no direct evidences of nucleotide or protein sequences about the presence of cytokine-like molecules in invertebrates. Transferring eyes on the transcription factor that regulates cytokine expression could be a new method to research the cytokine-like molecules in invertebrate.Recently, a novel transcriptional factor LPS-induced TNF-αfactor (LITAF) was cloned and characterized from human monocyte/macrophage cell line THP-1. Subsequently homologues of LITAF were cloned from mouse and chicken. Functional experiments indicates that this factor is necessary for TNF-αexpression. However, there were no reports about the homologues of LITAF in invertebrates.The first scallop LITAF (named as CfLITAF) was cloned from Zhikong scallop Chlamys farreri by Expressed Sequence Tag (EST) and Polymerase Chain Reaction (PCR) techniques. The cDNA of CfLITAF was of 1240 bp and consisted of a 5'untranslated region (UTR) of 112 bp, a 3'UTR of 678 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 16.08 kDa and theoretical isoelectric point of 6.77. A typical conserved LITAF-domain was identified in CfLITAF by SMART analysis. The mRNA expression of CfLITAF in different tissues including haemocytes, muscle, mantle, heart, gill and gonad was measured by quantitative real-time PCR. CfLITAF mRNA transcripts could be detected in all tissues examined. In the tissues of heart, mantle, gill and gonad, there was a significant 2.45-fold, 2.82-fold, 9.23-fold and 24.93-fold relative abundance compared with mRNA abundance in haemocytes, while there was not significant difference of CfLITAF expression between muscle and haemocytes. The temporal expression in haemocytes challenged by LPS or peptidoglycan (PGN) was also measured by quantitative real-time PCR. The results indicated that CfLITAF was up-regulated in haemocytes after LPS challenge. No significant changes were observed after PGN stimulation.Molecular cloning, characterization, and the temporal expression profile of CfLITAF in scallop C. farreri not only simply explicated the innate immune response of scallop to LPS stimulation, but importantly provided the clue for the existence of TNF-αor TNF-α-like molecules in invertebrates.