[Construction and expression of humanized anti-HBsAg scFv targeting interferon-alpha in escherichia coli].
OBJECTIVE
To develop a bacteria expression system to produce the fusion protein of humanized anti-HBsAg scFV and interferon-alpha.


METHODS
The expression vector was constructed after cleaving the plasmids harboring the humanized anti-HBsAg scFv and interferon alpha respectively and ligating to linearized pET22b subsequence. The expression of fusion protein in E.coli was analyzed by SDS-PAGE. The binding activity and antiviral activity of the fusion protein was characterized by competing inhibition test and cytopathic effect reduction.


RESULTS
The plasmid harboring the in frame arranged fusion gene was constructed and identified. After induction for 12h, a new band close to 4.5  10(4) was observed using SDS-PAGE. Results of competing ELISA and cytopathic effect reduction showed the fusion protein retained its specific binding activity and antiviral activities.


CONCLUSIONS
The construction and expression of the fusion gene of humanized anti-HBsAg scFv and interferon in E.coli are successful.
Xia
Xiaobing
X
Gene Therapy Research Centre, 302nd Hospital of PLA, Beijing 100039, China.
Cheng
Jun
J
Yang
Jizhen
J
Zhong
Yanwei
Y
Wang
Gang
G
Fang
Hongqing
H
Liu
Yan
Y
Li
Ke
K
Dong
Jing
J
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006510:Hepatitis B Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006515:Hepatitis B virus / Q000187:drug effects; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D007128:Immunoglobulin Fragments; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv