Studies On Cloning And Over Expression Of Drosophila Melanogaster Selenoprotein G-rich In E.coli And The Cellular Localization Of Endocytoplasmic Reticulum Stress Protein

Selenium(Se) has been recognized as an essential trace element in human life. Selenium, if supplied properly, is useful for preventing and treating many diseases such as heart diseases, tumor and so on. Selenoproteins are a kind of proteins which Se joins the polypeptide chain as selenocysteine(Sec) form. The researches of selenoproteins are practically important because the physiological functions of selenium are closely correlated with those of selenoproteins. There are three selenoproteins in Drosophila melanogaster. G-rich is the only one whose cellular localization and functions have not been known. The cloning and expressing of Selenoprotein G-rich in prokaryocytic cells is an international new aspect.The insertion process of Sec into selenoprotein is controlled by UGA codon which is located in CD area and a stem-loop (SECIS) which is located in 3'end. So the construction of the Sec Insertion Sequence (SECIS) is the emphasis of the experiment.First, the primers were designed with biologic software, Primer3. In the process the third base of part of the 3′end codons in G-rich coding region was changed for the purpose of inleting SacⅡin the gene sequence which could be used to insert SECIS next step. The G-rich coding region was amplified by PCR with the vector pAc-G-rich-GFP as a template and the SECIS of FDH-H of E.coli was synthesised in vitro, and both of them were subcloned into vector pTNTTM to construct vector pTNT-G-rich-SECIS. Then, the G-rich-SECIS sequence of vector pTNT-G-rich-SECIS was subcloned into vector pGEX-6p-1 to construct vector pGEX-6p-1-G-rich-SECIS, and vector pGEX-6p-1-G-rich-SECIS was over expressed in the E.coli cell, BL21 and Rosetta-gami (DE3) pLysS. The over expression conditions were also studied.It was found that Selenoprotein G-rich could be expressed in vector pGEX-6p-1-G-rich-SECIS transformed Rosetta-gami (DE3) pLysS, which contain the rare codons and is helpful for the forming of disulfide bonds in the object proteins. The suitable inducing temprature for transformed cells is 30℃and the cells grow better in the medium with glucose than those without glucose.The localization of Selenoprotein G-rich in human cell was also studied for the purpose to supply evidence for the cellular localization of the homolog selenoprotein SelK in human. The G-rich gene sequence was subcloned into the vector pEGFP-N1 to construct vector pEG-rich-GFP, and the constructed vector was transfected into human cell line, BGC-823. The cellular localization of G-rich gene was observed with laser scanning confocal microscopy with the staining of the transformed live cells by ER fluorescent stain as control. It was indicated that most of the selenoprotein G-rich were located in the endocytoplasmic reticulum of BGC-823 cells. Whether selenoprotein G-rich is located in other organoids needs further studies.Cg10091,cg15745,GstD21 are one kind of stress proteins in Drosophila melanogaster which are over expressed under the stimulating of tunicamycin. Most of the tunicamycin stress proteins are located in ER. To verify the cellular localization of the above proteins, the gene sequences of them were amplified by RT-PCR, subcloned to vector pAcPA, and tranfected into the cell line of Drosophila melanogaster, SL2, with the fluorescente staining dye of Endocytoplasmic Reticulum as control, and observed by laser confocal microscopy. It was indicated that all of the above Drosophila melanogaster stress protein, cg10091,cg15745,GstD21, were located in the endocytoplasmic reticulum of SL2 cell. Whether they were located in other organoids needs to be studied next.To sum up, the selenoprotein G-rich of Drosophila melanogaster was cloned and expressed successfully. Selenoprotein G-rich which contain the CD area, Sec codon TGA and SECIS was successfully over expressed in E.coli for the first time in the world. And it was hopeful to get the over expressed, bioactive, and Sec correctively inserted selenoprotein G-rich and the technique could be used to produce single content selenoprotein. The gene sequence of selenoprotein G-rich was also succesifully subcloned into the vector pEGFP-N1, and the cellular localization of Selenoprotein G-rich in human cells was successfully definited for the first time, which could give some indications for the cellular localization of human selenoprotein SelK. The gene sequences of cg10091,cg15745,GstD21 were also subcloned into vector pAcPA successfully for the first time, and the cellular localization of them were studied. These works make a solid basis for the functional studies of them next.