豆伞滑刃线虫Bd-MIF-1基因的克隆与分析OA

[Objective]The macrophage migration inhibitory factor(MIF)is an effector protein with an inhibitory role in host immune response.The MIF gene was cloned from Bursaphelenchus doui,and its expression characteristics were investigated to provide a reference for the function in the interaction process between B.doui and its hosts.[Method]The anterior-end specific cDNA library of B.doui was constructed by using the solid-phase hybridization method and using the anterior part,including esophageal gland,secretory pore and cerebral organ as the teaters and the posterior part,including intestine as the driver.The cDNA library was screened by reverse-northern blotting and sequencing technology.The full-length cDNA sequence of an MIF homologue was cloned by using RACE technology.The nucleotide sequence and amino acid sequence were analyzed by using the bioinformatics method.The gene copy number was identified by Southern blot method and the developmental expression pattern of MIF gene was detected by the real-time fluorescent quantitative PCR.[Result]The anterior-end specific cDNA library of B.doui was constructed successfully.The full-length cDNA sequence of Bd-MIF-1 gene(GenBank:PQ275932)was cloned from B.doui.The cDNA sequence of Bd-MIF-1 was 528 bp in length and contains 3 exons and 2 introns.The gene encoding had 114 amino acids.The relative molecular weight and theoretical isoelectric point were 12.16 kD and 6.22 respectively.The Bd-MIF-1 protein with a 4-Oxalocrotonate-Tautomerase superfamily structural domain had high homology with the amino acid sequence of Bursaphelenchus.The Southern blot analysis indicated that Bd-MIF-1 gene belonged to a single copy gene in B.doui genomes.The real-time fluorescent quantitative PCR result showed that the Bd-MIF-1 gene was expressed at all intart stages of Bursaphelenchus doui but there was no significant difference.[Conclusion]The Bd-MIF-1gene is successfully cloned from B.doui and its encoding protein contains a 4-Oxalocrotonate_Tautomerase superfamily domain.The encoding protein with a tauromerase activity may be involved in the parasitic process of B.doui.