Production and characterization of recombinant dengue virus type 4 envelope domain III protein.
Dengue fever, a mosquito-borne viral disease has become a major worldwide public health problem with a dramatic expansion in recent years. Cultivation process for production of recombinant dengue virus type 4 envelope domain III (rDen 4 EDIII) protein in Escherichia coli was developed for its diagnostic use as well as for further studies in immunoprophylaxis. The dissolved oxygen level was maintained at 20-30% of air saturation. The culture was induced with 1mM of isopropyl beta-d-thiogalactoside when dry cell weight was 13.78 g l(-1) and cells were further grown for 4h to reach 17.31 g l(-1) of culture. The protein was overexpressed in the form of insoluble inclusion bodies. The rDen 4 EDIII protein was purified by affinity chromatography and analyzed by SDS-PAGE. The final yield of purified rDen 4 EDIII protein in this method was approximately 196 mg l(-1) of culture. The purified protein was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue infected human serum samples. These results show that the product has the potential to be used for the diagnosis of dengue infection or for further studies in vaccine development. This production system may also be suitable for the high yield of other recombinant dengue proteins.
Tripathi
Nagesh K
NK
Division of Virology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India. tripathink@gmail.com <tripathink@gmail.com>
Babu
J Pradeep
JP
Shrivastva
Ambuj
A
Parida
Manmohan
M
Jana
Asha M
AM
Rao
P V Lakshmana
PV
D019149:Bioreactors; D018929:Cell Culture Techniques; D002846:Chromatography, Affinity; D003470:Culture Media; D003715:Dengue / Q000276:immunology; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry; D007075:Immunoglobulin M / Q000097:blood / Q000737:chemistry; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D009500:Neutralization Tests; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D003470:Culture Media; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins