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Title: Codon optimization of the prM-E coding region generates stable genome-length cDNA clone for a chimeric dengue 2/3 virus that can be propagated in Escherichia coli
Other Titles: การปรับแต่งโคดอนของยีนพรีเอ็มและอีทําให้ได้โคลนซีดีเอ็นเอทั้งจีโนม ของเชื้อไวรัสเดงกี่ของไวรัสลูกผสม 2/3 ที่คงตัวสามารถเพาะเลี้ยงได้ในอี โคไล
Authors: Poonsook Keelapang
Sakawdaurn Yasom
Chunya Puttikhunt
Chutithorn Ketloy
Nopporn Sittisombut
Issue Date: 2017
Publisher: Faculty of Medicine, Chiang Mai University
Abstract: In an effort to generate a chimeric dengue virus containing the prM-E sequence from DENV-3 and with the rest of the viral genome derived from a DENV-2 strain 16681-3pm by in vitro ligation of subgenomic fragments, it was found that the ligated chimeric genome could not be stably propagated as genome-length cDNA clone in Escherichia coli. Native DENV-3 prM-E coding region may contain certain nucleotide sequence that results in plasmid instability when amplifi ed in E. coli. In this study, we modifi ed codons in the DENV-3 viral sequence into those that are most frequently used in human genome. Employing the Gibson assembly method, the modifi ed prM-E fragment was introduced into the full-length cDNA clone of strain 16681-3pm to replace the corresponding sequence in a DENV-2 genome. The full-length plasmid cDNA clone intended for the generation of a chimeric virus, D3 prMEopt/16661-3pm, was successfully propagated in E. coli. In vitro transcription of this clone produced full-length RNA transcripts that generated infectious viruses when introduced into cultured mammalian cells. Based on kinetics experiments, the replication of D3 prMEopt/16661-3pm with modifi ed codons in the prM-E gene was comparable to the one generated by the in vitro ligation approach. By combining the use of modifi ed codons and the Gibson assembly method, a full-length cDNA clone of a chimeric virus containing the DENV-3 prM-E sequence can be stably propagated in E. coli, facilitating further manipulation of the viral sequences. Availability of such an infectious cDNA clone would provide a valuable tool for investigating the role of nucleotide/ amino acid variations in the DENV-3 prM-E gene in viral replication, immunogenicity, and pathogenicity as well as accelerating dengue vaccine development.
Description: Chiang Mai Medical Journal (Formerly Chiang Mai Medical Bulletin) is an official journal of the Faculty of Medicine, Chiang Mai University. It accepts original papers on clinical and experimental research that are pertinent in the biomedical sciences. The Journal is published 4 issues/year (i.e., Mar, Jun, Sep, and Dec).
ISSN: 0125-5983
Appears in Collections:CMUL: Journal Articles

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