Cloning And Expression Of Swine Melk And Analysis Of Its Tissue-specific Expression

The maternal embryonic leucine zipper kinase,also known as pEg3in Xenopus, is an atypical member of the snfl/AMPK family of serine-threonine kinases.MELK is involved in diverse physiological and pathological processes,including the cell cycle,cell proliferation,a-poptosis and RNA processing.Recently,MELK has emerged as an important gene in the field of cancer research and showing high level expression in malignant epithelial tumor samples from colon,lung,ovarian,breast,brain and skin.In order to investigate the function of MELK in swine,we cloned swine MELK gene by in silico according to human MELK sequence,and then confirmed it by molecullar experiments.Our results are as followings:1.Swine gene MELK was cloned through homology searching human MELK sequence in swine EST database.The characteristics and evolutionary progression of swine MELK were analyzed by bioinformatics.Our results showed that swine MELK was2072bp with17exons encoding650amino acids with an S_TKc conserved domain,and the homology of MELK protein between sus scrofa and Ovis aries was as high as94.68%.2.We inserted porcine MELK into eukaryotic expression vetor pEGFP-Cl,which were named as pEGFP-C1-MELK.Recombinant plasmid containing a green fluorescence reporter gene was transfected into Swine Umbilical Vein Endothelial Cells (SUVECs) by lipofectami-ne2000.Fluorescence microscope showed that fusion protein pEGFP-C1-MELK was expres-sion24hours post transfection. Western blotting analysis confirmed the expression of MELK48h later.3.To determined the expression abundance of MELK in the11tissues (heart, liver, spleen, lung,kidney,tonsil,thymus,superficial inguinal lymph node,hilar lymph node,mesenteric lym-ph node and chin lymph node), Semi-quantitative PCR was applied. Results indicated that porcine MELK was expressed highest in superficial inguinal lymph node,followed by tonsil,liver,heart,hilar lymph node and kidney.Mesenteric lymph node, lung, spleen chin lymph node an-d thymus showed very weak expression of swine MELK.