Production, purification and characterization of recombinant dengue multiepitope protein.
Dengue is an acute mosquito-borne viral disease of humankind. Dengue fever, dengue haemorrhagic fever and dengue shock syndrome have become global public health problems in recent years. rDME-G (recombinant dengue multiepitope protein that can specifically detect IgG) was produced in a 5-litre fermenter in Escherichia coli for use in diagnosis. The culture was induced with 1 mM isopropyl beta-D-thiogalactoside and cells were further grown for 4 h before harvesting. After fermentation, dry cell weight resulted in approx. 16.2 g/l. The rDME-G protein was purified from inclusion bodies using affinity chromatography. The final yield of purified rDME-G protein from fermentation resulted in approx. 168 mg/l of pure biologically active rDME-G protein. The purity of rDME-G protein was checked by SDS/PAGE analysis and the reactivity of this protein was further determined by Western blotting. The purified protein was used to develop an in-house dipstick ELISA and tested using a panel of 60 patient sera characterized using the commercially available tests for detection of dengue antibody. We compared our results with IgG-capture ELISA (Pan-Bio, Windsor, QLD, Australia) and rapid IC (immuno-chromatography) test (Pan-Bio). By using rDME-G protein as an antigen, in the dipstick ELISA, the results were in excellent agreement with commercial rapid IC test and IgG capture ELISA. These results show that the product has a promising potential to be used for diagnosis of dengue in both laboratory- and field-based detection systems with minimum cost and a high degree of sensitivity and specificity.
Tripathi
Nagesh K
NK
Division of Virology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India. tripathink@gmail.com
Shrivastva
Ambuj
A
Pattnaik
Priyabrata
P
Parida
Manmohan
M
Dash
Paban K
PK
Jana
Asha M
AM
Rao
P V Lakshmana
PV
D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*
D000939:Epitopes; D011994:Recombinant Proteins; D014764:Viral Proteins