Expression of HCV Alternative Reading Frame Protein (Core+1/F) in Baculovirus Expression System and its Evaluation for Assessment of Specific Anti-core+1 Antibody in Iranian HCV Infected Patients.
BACKGROUND
Hepatitis C virus (HCV) genome contains an overlapping reading frame which results in alternative core protein (ARFP). Baculovirus expression system was used as a powerful eukaryotic vector system to express core+1/F protein for the first time. This recombinant core+1/F protein was used to assess the anti-core+1 antibody in anti-HCV drug resistant and sustained virologic response (SVR) patients.


METHODS
The core+1 coding sequence from HCV genotype 1 was designed and synthesized in pUC57 vector. It was subcloned into baculovirus donor plasmid pFastBacTM HTA and transposed into baculovirus shuttle vector (bacmid) to transfect Sf9 cells. Recombinant core+1 protein was purified using Ni-NTA agarose under native condition and verified using SDS-PAGE electrophoresis and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was developed using this purified protein to assess anti-core+1 antibody in 28 anti-HCV drug resistant patients and in 34 patients with sustained virologic response (SVR) in comparison with 31 healthy volunteers used as the negative control.


RESULTS
Expression of HCV core+1 protein in Sf9 cells was confirmed by using SDS-PAGE and Western blotting. Antibody titer against core+1 protein in anti-HCV drug resistant patients was significantly higher than that in both the healthy volunteers and SVR patients (p < 0.0001).


CONCLUSIONS
HCV core+1 protein was expressed successfully in a baculovirus expression system in high yield in order to develop an ELISA to assess the anti-core+1 antibody. Further studies are needed to reveal the potential application of core+1 protein in anti-HCV treatment prognosis.
Pooneh
P
Rahimi
Rouhollah
R
Vahabpour
Farideh Sadat Sajadian
FS
Fard
Fatemeh
F
Motevalli
Fatemeh Fotouhi
FF
Chahouki
Mohammad Reza
MR
Aghasadeghi
Azam
A
Bolhassani
Seyed Mehdi
SM
Sadat
Ehsan
E
Mostafavi
Nasir
N
Mohajel
Ali
A
Jahanian-Najafabadi
Mohammad Reza
MR
Amiran
D000328:Adult; D016367:Baculoviridae / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006526:Hepatitis C / Q000188:drug therapy*; D018937:Hepatitis C Antibodies / Q000097:blood*; D006801:Humans; D008297:Male; D008875:Middle Aged; D011994:Recombinant Proteins / Q000276:immunology; D014758:Viral Core Proteins / Q000235:genetics* / Q000276:immunology
D018937:Hepatitis C Antibodies; D011994:Recombinant Proteins; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus