Construction Of Plasmid Of Wild-type And Mutant MYOC Gene And Study On Eukaryotic Expression Of Them

Objective 1.To clone the sequence of the cDNA of POAG related MYOC gene and construct eukaryotic expression plasmid of wild—type MYOC gene. 2. To construct eukaryotic expression plasmid inserted by Pro370Leu mutation of the MYOC gene. 3. To compare mMYOC expression with wMYOC expression in eukaryotic cell for further understanding the mechanism of POAG .Methods 1.POAG related MYOC gene cDNA was amplified from eye tissue (cornea limbus, ciliary body) by RT-PCR.The cDNA was cloned into plasmid pGEM-T,then digesting pGEM-T by restriction enzyme,and the products were cloned into eukaryotic expression plasmid pEGFP-N3 for constructing recombinant plasmid pEGFP-N3-MYOC. The accuracy of pEGFP-N3-MYOC was confirmed by restriction enzyme digestion and DNA sequencing.2.Take pGEM-T-MYOC plasmid as plate to get Pro370Leu mutation MYOC gene by PCR site-directed mutagenesis,which was subcloned into eukaryotic expression plasmid pDsRed2-Nl to construct recombinant plasmid pDsRed2-N1-mMYOC. The accuracy of pDsRed2-Nl-mMYOC was confirmed by restricting enzyme digestion analysis and DNA sequencing.3.Recombinant plasmid pEGFP-N3-wMY0C and recombinant plasmid pDsRed2-N1-mMYOC were transfected respectively and co-transfected into Hela cells by lipofectin.EGFP and DsRed2 gene in recombinant plasmid were the markers to monitor wMYOC and mMYOCgene expression respectively. The localization of wMYOC and mMYOC proteins was examined by fluorescence microscope and their secretory properties were evaluated by Western blot analysis the apoptosis was surveyed by FCM.Results 1.By DNA sequencing, the sequence of MYOC cDNA was correct. By restriction enzymes digesting and DNA sequencing, recombinant plasmid pERFP-N3-MYOC was constructed correctly.2. With restricting enzyme digestion analysis and DNA sequencing, recombinant plasmid pDsRed2-Nl- mMYOC was constructed correctly.3. Observed under fluorescence microscope, wMYOC and mMYOC protein expression was proved when green and red florescences were found out under fluorescence microscope.The localization of both proteins was in cell plasma, but wMYOC protein appeared to distribute evenly in the whole cell plasma, whereas mMYOC protein appeared to aggregate, and wMYOC protein also appeared to aggregate with mMYOC protein when both proteins were co-transfected. Analyzed by Western blot, wMYOC protein was secreted whereas none of mMYOC protein was secreted from their corresponding transfected Hela cells.Besides none of wMYOC protein was also secreted when both proteins were coexpressed. Surveyed by FCM, mMYOC Protein accelerated apoptosis, which is the same when both proteins were coexpressed.Conclusion 1.P0AG related MYOC gene was cloned successfully.2. Eukaryotic expression plasmid pEGFP-N3-MYOC and pDsRed2-Nl-mMY0C were constructed successfully.3.Both of wMYOC and mMYOC genes can express successfully in cell plasma,but mMYOC protein appear to aggregate, fail to secrete and accelerate apoptosis.it affected the expression and secretion of wMYOC gene.