Cloning & expression of chikungunya virus genes coding structural proteins in Escherichia coli.
DNA complementary to the single stranded RNA genome of Chikungunya (CHIK) virus with poly A tract was cloned into the plasmid pGEM-3Zf(-) and 5Zf(+) by blunt end ligation strategy. Clones containing the cDNA inserts were selected by X-gal, IPTG system. They were tested for the expression of structural protein(s) of CHIK virus by in situ enzyme immunoassay and Western blot. The former assay system showed the presence of expressed viral proteins. Analysis of Western blot shows that three structural proteins, E1, E2 and capsid (C) are expressed in Esch. coli. The molecular weights of envelope proteins E1 and E2 were 44-46 Kd and 42-44 Kd respectively, which are lesser than the actual molecular weights of virional proteins (50-52 Kd). This may be due to the absence of glycosylation of these proteins in Esch. coli. In clone no. 382, a high molecular weight protein (56-58 Kd) was observed, which was probably the unglycosylated form of P62 polyprotein coded by the virus during its multiplication. A small protein of MW 6-8 Kd was also expressed in clone nos. 382 and 504, and this appeared to be the unglycosylated form of E3 protein of CHIK virus.
Ranadive
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National Institute of Virology, Pune.
Banerjee
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K
D002646:Chikungunya virus / Q000235:genetics*; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D012367:RNA, Viral / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
D004279:DNA, Viral; D012367:RNA, Viral; D015678:Viral Structural Proteins