Abstract:

BACKGROUND: The construction of herpes simplex virus (HSV-II) latency-associated transcript (LAT) open reading frame 1 (ORF1) (pEGFP-C2/LAT ORF1) eukaryotic expression vector can lay a foundation for the study of the function of HSV-2 LAT ORF1 in the virus latency and recurrence.
OBJECTIVE: To construct pEGFP-C2/LAT ORF1 eukaryotic expression vector and to verify its expression in vitro through the transfection of African green monkey kidney cells.
METHODS: A PCR primer was designed according to DNA sequencing of pEGFP-C2/LAT ORF1. HSV-Ⅱ333 standard plant genome was used as the template and ORF1 gene was amplified by PCR method and subcloned into the pEGFP-C2 eucaryotic experssion vector. And then enzyme digestion and DNA sequencing were used for identifying the recombinant plasmid pEGFP-ORF1. Finally, the recombinant plasmid pEGFP-C2/LAT ORF1 was transfected into Vero cells in vitro by X-fect transfection kits. Fusion green fluorescent protein expression was observed by inverted fluorescence microscope and its mRNA expression levels were detected by reverse transcription PCR.
RESULTS AND CONCLUSION: In vitro amplified targeted DNA fragment was 741 bp in length and the size of pEGFP-C2/LAT ORF1 eucaryotic experssion vector constructed was conformed to the expectation, moreover, its sequencing results were conformed to those of ORF1 gene sequencing included by NCBI. Recombinant plasmid pEGFP-C2/LAT-ORF1 was verified by reverse transcription PCR after transfected into Vero cells, and green fluorescent protein expression in the transfected Vero cells was determined by fluorescence microscope. These results suggest that the pEGFP-C2/LAT ORF1 eucaryotic experssion Vector have successfully constructed and realized its expression in African green monkey kidney cells (Vero cells).