Expression, purification and serological analysis of hepatocellular carcinoma associated antigen HCA587 in insect cells.
OBJECTIVE
In order to assess hepatocellular carcinoma associated antigen HCA587 as a potential target for immunotherapy, the Bac-to-Bac expression system was used to express recombinant protein HCA587 in insect cells.


METHODS
The cDNA encoding HCA587 gene was cloned into donor vector pFasBacHtb and recombinant pFasBac Htb-587 was transformed into competent cells DH10Bac. Recombinant Bacmid-587 was transfected into Sf9 insect cells using CELLFECTIN, Recombinant HCA587 protein was produced in Sf9 insect cells after infection with recombinant baculovirus, and was purified using Ni-NTA resin. Sera from HCC patients were also screened using recombinant protein HCA587.


RESULTS
The molecular weight of the recombinant protein HCA587 expressed in insect cells was approximately 43kd. Western blot results proved the recombinant protein HCA587 had the similar antigenicity with its native counterpart. Serological analysis told that the rate of seroreactivity to HCA587 was not high in HCC patients.


CONCLUSIONS
The recombinant protein HCA587 was successfully expressed and purified using Bac-to-Bac expression system. It paved the way for generation of specific antibody and investigation of immunohistochemical analysis and immune responses of HCC in the future.
Li
Bing
B
Immunology Department of Peking University Health Science Center, 38 Xueyuan Road, Beijing, 100083, China. wfchen@public.bta.net.cn
Wu
Hong-Yan
HY
Qian
Xiao-Ping
XP
Li
Yan
Y
Chen
Wei-Feng
WF
D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera; D014162:Transfection
D000951:Antigens, Neoplasm; D017931:DNA Primers; C405429:MAGEC2 protein, human; D009363:Neoplasm Proteins; D011994:Recombinant Proteins