Cloning And Expression Of PMELK-catalytic Domain And Its Transfection With PBCL-G Affects On Apoptosis

The maternal embryonic leucine zipper kinase(MELK) also known as MPK38 in mouse, pEg3 in Xenopus, which is a member of the sucrose nonfermenting protein kinase(snf1)/AMPK serine/threonine kinase family. It is highly expressed in the organ specific stem cells and cancer cells. MELK participates in a wide range of cell cellular processes that are related to the cell cycle, cell proliferation, cell apoptosis, spliceosome assembly, gene expression, embryonic development, hematopoiesis and tumor formation. In breast carcinogenesis:the kinase phosphorylates BCL-GL, inhibiting the latter’s proapoptotic function. Our laboratory had cloned the pMELK gene and pBCL-G gene, and had certified that pMELK protein can interact with pBCL-G protein. And had played an important role of repressing the function of proapoptotic of pBCL-G. In order to investigate wheather the catalytic domain of MELK performed the similar role, we constructed the expression vetcor and by the flow cytometry technique, to study the effect on apoptosis of catalytic domain. Our results are as followings:1. In order to obtain stable expression pBCL-G gene cell lines, this experiment tested the cell lines which had already storaged more than two years. The results show that the pBCL-G gene expression steady, which had laid a foundation for subsequent gene function research.2. We inserted the catalytic domain of porcine MELK into eukaryotic expression vetor pCMV-HA, which were named as pCMV-HA-MELK-cd. The recombinant plasmid were transfected into SUVECs by TransLipid Transfection Reagent. Western blot analysis confirmed the expression of pCMV-HA-MELK-cd 48 h later.3. We transfected recombinant plasmid pCMV-HA、pCMV-HA-pMELK-cd and pCMV-HA-pMELK into SUVECs expressing GFP-pBCL-G protein stably induced 18 h by 500 nM STS. Flow cytometry analysis showed that pMELK-cd cannot significantly inhibit pBCL-G and reduce cell apoptosis.