Abstract:

BACKGROUND: Enhanced green fluorescent protein (EGFP) can express photoprotein in vital cells as a report gene. Kozak consensus sequences at translation initiation site should increase the translation efficiency in eukaryotic cells.
OBJECTIVE: To construct the recombinant retroviral expression vector which contains report gene EGFP and Kozak consensus sequences increasing the translation efficiency in eukaryotic cells, and to identify this recombinant vector.
METHODS: EGFP gene was high-fidelity cloned from expression vector pEGFP-N1 containing EGFP report gene using polymerase chain reaction (PCR). Target gene was inserted to retroviral expression vector pLXSN with molecular cloning techniques. Positive recombinants were screened via colonial rapid PCR. The primers were designed through insertion site in vector in order to identify the insert direction of target gene, and PCR and restrictive enzymes digestion was used to identify its correction. Furthermore, DNA sequences of pLXSN-Kozak-EGFP were examined.
RESULTS AND CONCLUSION: The EGFP gene with Kozak consensus sequences at its upper flank was cloned, with length of 750 bp. Recombinant pLXSN-Kozak-EGFP was screened by colonial rapid PCR, gained the specific product about 750 bp. Recombinant plasmid pLXSN-Kozak-EGFP was identified with PCR individually, gained the specific product about 750 bp; and insert direction was identified with PCR, gained the specific product about 350 bp. Restrictive enzymes double digested the recombinant plasmid, obtained the specific products about 750 bp and 6 000 bp. Sequences alignment indicates 100% identity between target DNA and Kozak-EGFP. The results indicate that the retroviral expression vector pLXSN-Kozak-EGFP containing EGFP report gene and Kozak sequences has been constructed successfully in this experiment, and target gene insert direction is correct.