[Antigenicity of hepatitis C virus F protein and serum prevalence of anti-F in HCV-infected patients].
OBJECTIVE
To examine the antigenicity of hepatitis C virus (HCV) F protein and investigate serum prevalence of anti-F in HCV-infected patients.


METHODS
Eleven pairs of overlapping primers were used to synthesize the full-length HCV f gene, from which the truncated HCV f65 gene fragment was amplified by PCR. HCV f65 gene was then cloned into pET32a(+), and transformed into E. coli strain Plyss (DE3). This recombinant E.coli was induced by IPTG for the production of HCV F65 protein. The expressed HCV F65 protein, purified by Ni-NTA agarose, was further used in ELISA to detect serum anti-F, and to immunize rabbits for making polyclonal anti-F. The rabbit polyclonal anti-F was purified by Staphylococcus aureus protein A agarose.


RESULTS
After recombinant pET32a(+)-f65 was constructed successfully, HCV F65 protein was expressed and purified. The purified HCV F65 protein was used as a capture antigen in ELISA to detect serum anti-F in HCV infected patients (n = 30). The result showed that the mean A450 value and the positive rate of serum anti-F were 0.125+/-0.061 and 63.3%, respectively. The rabbit-derived polyclonal anti-F reacted specifically with HCV F65 protein, of which the titer was 1:30,000.


CONCLUSIONS
Our expressed HCV F65 protein is of antigenicity, and can be used to determine serum anti-F. Anti-F IgG does exist in the sera of the HCV-infected patients. Moreover, the rabbit-derived polyclonal anti-F can be used to detect HCV F protein.
Shao
Sheng-Wen
SW
Department of Microbiology, PLA Key Laboratory for Medical Microbiology, Second Military Medical University, Shanghai 200433, China.
Wu
Wen-Bin
WB
Yu
Jian-Guo
JG
Zhao
Ping
P
Qi
Zhong-Tian
ZT
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology; D006526:Hepatitis C / Q000097:blood / Q000453:epidemiology / Q000276:immunology*; D018936:Hepatitis C Antigens / Q000097:blood / Q000276:immunology; D006801:Humans; D015995:Prevalence; D011817:Rabbits; D014758:Viral Core Proteins / Q000097:blood* / Q000276:immunology; D014759:Viral Envelope Proteins / Q000276:immunology
D000914:Antibodies, Viral; D018936:Hepatitis C Antigens; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; C433031:hepatitis C protein F, Hepatitis C virus