Cloning Of LPS-induced TNF-α Factor (LITAF) From Freshwater Pearl Mussel, Hyriopsis Schlegelii And Its Apoptotic Effect On Hela Cells

Lipopolysaccharide induced TNF-α factor (LITAF) is an important and noveltranscriptional factor mediating TNF-α and various inflammatory expression andplaying important roles in invertebrate innate immune responses. In this study, partialcDNA sequence of a LITAF (designated as HsLITAF) gene was cloned and identifiedfrom freshwater pearl mussel Hyriopsis schlegelii. The results showed it contained anopening reading frame of453nucleotides encoding a predicted protein of150aminoacids which shared22.1%-44.1%similar residues and34%-73.3%identity to otherknown LITAF sequences from oyster to mammals. The predicted amino acidHsLITAF polypeptide had a calculated molecular weight of16.08kDa, and thetheoretical isoelectric point of8.04. Multiple amino alignment showed that HsLITAFcontained a typical LITAF domain at C-terminal with two conserved CXXC motifsforming a compact Zn2+-binding structure.Furthermore，the forward and reverse primers used for amplification of thissequence were the same. Further analysis found that this primer can amplify twosequences in all examined tissues (including adductor muscle, foot, gonad,hepatopancreas, gills, mantle and haemocytes) and they only differ in24bases and9amino acids. Two kinds of cDNA was named after LITAF1and LITAF2. Using theprimer to check genome structure, we found the genome of HsLITAF does notpossess intron structure.The expression level of HsLITAF was the highest in gill, moderate inhepatopancreas, hemocytes, adductor muscle and gonad, the lowest in heart andmantle. The HsLITAF mRNA expression was down-regulated at6h,24h and48h,but significantly up-regulated at72h in haemocytes after LPS stimulation. Theexpression level of HsLITAF was39-fold than the control at this time point. Thesemay indicate that HsLITAF participated in the innate immunity.To further investigate its mechanism of biological functions, the recombinantplasmid pcDNA3.1（+）-HsLITAF was constructed and transfected into Hela cells. Flow cytometer and RT-PCR analysis showed that the higher apoptosis ratio of Helacells was occurred in experimental group, and had significant effects on theexpression of p53and Bax gene, which the relative expression level was2.99-foldand2.53-fold than the control group. Taken together, we proposed that themitochondrion may be involved in LITAF-induced apoptosis. This study providesnew insights into the function of HsLITAF and the apoptosis pathway induced byHsLITAF.