[Expression, purification and identification of the domain III of DENV II envelop protein in Escherichia coli].
OBJECTIVE
To express the domain III of DENV II envelop protein in Escherichia coli, obtain the purified recombinant protein and identify its immunoreactivity.


METHODS
Suckling mice were inoculated with live DENV II in the brain. The total RNA was extracted from the brain of the infected mice, and the envelope protein DNA fragment was amplified by RT-PCR and ligated into pMD 18-T to construct pMD 18-T-DV2-E. The domain III DNA fragment of the envelope protein was amplified by PCR with pMD 18-T-DV2-E as the template and cloned into pET-32a(+) to construct the expression plasmid pET-32a(+)-DV2-E-DIII. The recombinant plasmid was transformed into E.coli BL21(DE3) and induced by IPTG, and the expressed products were analyzed by SDS-PAGE and Western blotting.


RESULTS
After RT-PCR amplification, a specific DNA fragment of about 1.5 kb was obtained and ligated into pMD 18-T to construct pMD 18-T-DV2-E. With pMD 18-T-DV2-E as the template, the domain III DNA fragment about 320 bp in length was amplified and the expression plasmid pET-32a(+)-DV2-E-DIII was successfully constructed. After induction with IPTG, a specific soluble protein with a relative molecular mass of 29000 was obtained and the expression product accounted for 52.50 percent; of the total protein of the cell lysate. Western blotting demonstrated reactivity of the recombinant protein with His-Tag McAb and DENV (Type I-IV) McAb.


CONCLUSIONS
The recombinant plasmid can be highly expressed in E.coli BL21(DE3) in a soluble form and the recombinant protein can react with DENV (Type I-IV) McAb.
Lei
Zi-qing
ZQ
Department of Pathogenic Biology, College of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. g.rayzq@163.com
Su
Yu-xin
YX
Zheng
Xue-li
XL
D000818:Animals; D003716:Dengue Virus / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D051379:Mice; D008815:Mice, Inbred Strains; D010957:Plasmids; D054730:Protein Interaction Domains and Motifs; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification*
D014759:Viral Envelope Proteins