Molecular cloning and characterization of macrophage migration inhibitory factor from small abalone Haliotis diversicolor supertexta.
ABSTRACT The macrophage migration inhibitory factor (mif) cDNA and its genome were cloned from small abalone Haliotis diversicolor supertexta. Small abalone mif (samif) was originally identified from an expressed sequence tag (EST) fragment from a normalized cDNA library. It's 5' untranslated region (UTR) was obtained by 5' rapid amplification of cDNA end (RACE) techniques and its genomic DNA was cloned by PCR. The full-length cDNA of samif was of 535 bp, consisting of a 5'-terminal UTR of 49 bp, an open reading frame of 384 bp and a 3'-terminal UTR of 102 bp. The deduced protein was composed of 128 amino acids, with an estimated molecular mass of 14.0 kDa and a predicted pI of 6.90. The full-length samif genomic DNA comprises 3238 bp, containing three exons and two introns. Real time quantitative PCR analysis revealed that samif gene is constitutively expressed in 6 selected tissues, and its expression level in hepatopancreas is higher than that in the other tissues (p < 0.01). Samif expression level in the hepatopancreas at 24 and 48 h after Vibrio parahaemolyticus injection was upregulated significantly (p < 0.01), but there was no significant change after exposure to tributyltin (TBT) (p > 0.05).
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ABSTRACT: 544 INTRODUCTION Macrophage migration inhibitory factor (MIF) is a mysterious cytokine and is reportedly released by antii gennactivated T lymphocytes with the function of inhibiting vertebrate macrophage migration [1, 2]. Since it was found in 1966, more studies on MIF have been undertaken, showing that it is produced not only by activated T cells but by other immune cells such as monocytes, macrophages, and some nonnimmune cells [3, 4]. MIFs have been isolated and characterized from many organisms like the bovine brain [5, 6] and rat epididymis . Since human MIF complementary cDNA was first cloned in 1989, many MIF genes have been cloned successfully from various species such as Mus musculus [8, 9], the Qingdao amphioxus Brann chiostoma belcheri tsingtaunese , and the sheep Ovis aries . Recently, MIF has been reported to play many important roles in the internal environn ment. It has been confirmed to act as a mediator durr ing the inflammatory response [12, 13, 14]. Many other studies have shown that MIF is also involved in the processes of regulating cell proliferation and tumor angiogenesis . Ito et al demonstrated that MIF also functions as a growth factor during the period of embryonic differentiation . In addition, Yan et al  suggested that MIF produced at superrphysiological levels in a murine neuroblastoma cell line (Neuroo2a) inhibited TTcell activation and induced the death of TT cells through an γIFN pathway. Besides, studies [4, 17, 18] have also implied that MIF could activate the ERK1–ERK2–MAPK pathway of the intracellular signal system. Oxana et al  suggested that at heat stress temperatures, when heat shock proteins are not abundant, MIF may act as a chaperone to bind other proteins of similar properties to counteract the early onset of stress. Mud crabs (Scylla) are mostly distributed throughh out the IndooPacific and Indian Ocean regions  as one of the most commercially valuable species in these areas. However huge economic losses are caused each year by the death of mud crabs from diseases associi ated with Vibrio parahaemolyticus, the most important infectious agent. To enhance the immunity of mud crabs, researchers have paid more attention to the mechanisms of action of immunogenic proteins. Although the cloning of MIF from many species has been reported, there is no report about the cloning of MIF from Scylla paramamosain (SpMIF). In this study, we characterized the fullllength cDNA of
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ABSTRACT: Macrophage migration inhibitory factor (MIF) is an evolutionarily ancient and highly conserved cytokine with multiple functions. In the present study, a MIF-like gene was cloned from Zhikong scallop Chlamys farreri (designated as CfMIF) based on expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of CfMIF was of 2296bp, consisting of a 5' untranslated region (UTR) of 60bp, a 3' UTR of 1903bp with a poly(A) tail and an open reading frame (ORF) of 333bp encoded 111 amino acid residues with a calculated molecular mass of 12.6kDa and a theoretical isoelectric point of 5.63. The deduced amino acid sequence of CfMIF shared 27-50.5% similarity with those of other known MIFs. A conserved MIF domain was identified in the deduced amino acid sequence of CfMIF, and conserved proline(2) and lysine(33) were also found to be present in CfMIF. Phylogenetic analysis revealed that CfMIF is one of MIF members. The tissue distribution and temporal expression of CfMIF in hemocytes of scallop after lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan stimulation were detected by real-time RT-PCR. CfMIF gene was ubiquitously expressed in six selected tissues of healthy scallops, with the higher expression levels in hepatopancreas, mantle and gill. In comparison with the control group, the expression of CfMIF mRNA in hemocytes was up-regulated significantly at 6h, 24h and 48h after LPS treatment, and at all time points after PGN and glucan treatment. The cDNA fragment encoding mature peptide of CfMIF was recombined and expressed in Escherichia coli BL21 (DE3) pLysS. The recombinant protein of CfMIF (rCfMIF) promoted sheep fibroblast migration into scraped spaces in vitro. These results generated from the present study encourage us to suggest that CfMIF was a novel member of MIF family, and it was involved in immune response and wound healing by promoting fibroblast migration.Developmental and comparative immunology 01/2011; 35(1):62-71. · 3.29 Impact Factor
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ABSTRACT: The widespread use of genome sequencing provided evidences for the high degree of conservation in innate immunity signalling pathways across animal phyla. However, the functioning and evolutionary history of immune-related genes remains unknown for most invertebrate species. A striking observation coming from the analysis of the pea aphid Acyrthosiphon pisum genome is the absence of important conserved genes known to be involved in the antimicrobial responses of other insects. This reduction in antibacterial immune defences is thought to be related to their long-term association with beneficial symbiotic bacteria and to facilitate symbiont maintenance. An additional possibility to avoid elimination of mutualistic symbionts is a fine-tuning of the host immune response. To explore this hypothesis we investigated the existence and potential involvement of immune regulators in aphid agonistic and antagonistic interactions.BMC genomics. 09/2014; 15(1):762.