Expression, Purification, and Refolding of Chikungunya
Virus Full-Length Envelope E2 Protein along with B‑Cell and
T‑Cell Epitope Analyses Using Immuno-Informatics Approaches
posted on 2022-01-14, 16:06authored byManisha Shukla, Pankaj Chandley, Suman Tapryal, Narendra Kumar, Sulakshana P. Mukherjee, Soma Rohatgi
Chikungunya virus
(CHIKV) is a mosquito-transmitted alphavirus,
which causes severe illness in humans and is responsible for epidemic
outbreaks in Africa, Asia, North and South America, and Europe. Despite
its increased global prevalence, no licensed vaccines are available
to date for treating or preventing CHIKV infection. The envelope E2
protein is one of the promising subunit vaccine candidates against
CHIKV. In this study, we describe successful cloning, expression,
and purification of CHIKV E2 full-length (E2-FL) and truncated (E2-ΔC
and E2-ΔNC) proteins in the Escherichia coli expression system. The recombinant E2 proteins were purified from
inclusion bodies using Ni-NTA chromatography. Further, we describe
a detailed refolding procedure for obtaining the CHIKV E2-FL protein
in native conformation, which was confirmed using circular dichroism
and Fourier transform infrared spectroscopy. BALB/c mice immunized
with the three different E2 proteins exhibited increased E2-specific
antibody titers compared to sham-immunized controls, suggesting induction
of strong humoral immune response. On analyzing the E2-specific antibody
response generated in immunized mice, the CHIKV E2-FL protein was
observed to be the most immunogenic among the three different CHIKV
E2 antigens used in the study. Our B-cell and T-cell epitope mapping
results indicate that the presence of specific immunogenic peptides
located in the N-terminal and C-terminal regions of the CHIKV E2-FL
protein may contribute to its increased immunogenicity, compared to
truncated CHIKV E2 proteins. In summary, our study provides a detailed
protocol for expressing, purifying, and refolding of the CHIKV E2-FL
protein and provides an understanding of its immunogenic epitopes,
which can be exploited for the development of novel multiepitope-based
anti-CHIKV vaccine strategies.