Expression Of Fusion Protein Of DCD-1L And Enhanced Green Fluorescent Protein In E.Coli Transetta

Green Fluorescent Protein (GFP) is a reporter gene in the field of biology. Themost commonly used GFP mutant is the Enhanced Green Fluorescent Protein (EGFP).Use the unique light-emitting mechanism of EGFP and the recombinant technology ofDNA, The target genes and the EGFP gene are made up of fusion gene, and the fusiongene is usually transfected or transformed in suited cells for expression, then the targetproteins are observed by the fluorescence microscope. This technique is applicatedwidely. Dermcidin (DCD) is a new antibacterial peptide identified most recently fromhuman sweat glands. And the DCD-1L is a derivant of DCD, which has a broadspectrum of activities. Because of the characteristics of stable physicochemicalproperties, broad spectrum of activities against germ, difficult to generate resistance ofDCD-1L, it will be the best alternative for antibiotics. In this experiment, EGFP hasbeen expressed in E.coli Transetta. DCD-1L and EGFP gene have been fusionexpressed in E.coli Transetta. The unique light-emitting mechanism of EGFP was usedto mark DCD-1L, in order to detece the expression of DCD-1L. The results were asfollows：1. The EGFP gene has been obtained from the vevtor pEGFP-N1by PCR.2. The recombinant plasmid PET-32a (+)-EGFP has been obtained.3. The recombinant plasmid PET-32a (+)-EGFP has been transformed into E.coliTransetta and expressed. The green fluorescent of the expression products have beenobserved by fluorescence microscope. The EGFP gene has been expressed in E.coliTransetta successfully.4. The DCD-1L gene has been obtained by overlap extention.5. The recombinant plasmid PET-32a (+)-EGFP-DCD-1L has been obtained.6. The recombinant plasmid PET-32a (+)-EGFP-DCD-1L has been sequenced. Itwas found that a T base in EGFP gene terminal was missed, then the bases followed theT base were frameshift mutations.7. The recombinant plasmid PET-32a (+)-EGFP-DCD-1L has been transformedinto E.coli Transetta and expressed. The green fluorescent of the expression productshave been observed by fluorescence microscope, but the target band has not beendetected by SDS-PAGE. By analyzing, the base A in3’ ends of P2’ primer was missed,and then the base T in the EGFP gene downstream was missed in the process of PCR.The P2’ primer has been synthesized again and the experiment is ongoing.In this experiment, EGFP and DCD-1L gene have been obtained. The recombinantplasmid PET-32a (+)-EGFP has been constructed and expressed in E.coli Transetta successfully. The recombinant plasmid PET-32a (+)-EGFP-DCD-1L has beenconstructed successfully and expressed in E.coli Transetta. It will provide a foundationto construct high expressing DCD-1L recombinant bacterial strain which is marked byEGFP.