关键词: 增强型绿色荧光蛋白, pGEM-T easy, DH5α大肠杆菌

Abstract:

To construct a recombinant plasmid (pGT-EGFP), the fragment of EGFP (enhanced green fluorescent protein) gene was amplified by PCR from the cloning vector pEGFPpA-CAN, and then inserted into the pGEM-T easy vector. The expression of EGFP was regulated by the SP6 promoter in pGEM-T easy. Compared with the culture of DH5α E. coli, the culture of DH5α E. coli with pGT-EGFP was a little greener in color. Through fluorescence microscope, the morphological features of DH5α E. coli with green fluorescence could be clearly observed. Moreover, the extraction of total protein from the lysate of DH5α E. coli with pGT-EGFP derived from ultrasonic treatment was done. The SDS-PAGE analysis showed that EGFP was a soluble protein, with a molecular weight of approximately 30 kD, which was identical to the theoretical size.

Key words: enhanced green fluorescent protein, pGEM-T easy, DH5αE. coli