Cloning, prokaryotic expression and polyclonal antibody preparation of HCCR: a new biomarker for hepatocellular carcinoma.
- Author:
Jian-bo XIA
1
;
Zhen-hua ZHANG
;
Yong-jun TIAN
;
Zhong-ji MENG
;
Zhi-qun YU
;
Xi-ping ZHAO
;
Dong-liang YANG
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies, Monoclonal; biosynthesis; genetics; Biomarkers, Tumor; biosynthesis; genetics; Carcinoma, Hepatocellular; pathology; Cloning, Molecular; Escherichia coli; metabolism; Humans; Liver Neoplasms; pathology; Mice; Mice, Inbred BALB C; Prokaryotic Cells; metabolism; Proto-Oncogene Proteins; biosynthesis; genetics; immunology; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured
- From: Chinese Journal of Hepatology 2006;14(2):101-104
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVESTo construct a prokaryotic plasmid expressing truncated human cervical cancer oncogene (HCCR-1(167-360)), to express and purify the recombinant protein, and to develop the polyclonal antibody against HCCR.
METHODSHCCR-1(167-360) was amplified by RT-PCR from HepG2 cells and cloned into vector pRSET-B, then expressed in E.coli BL21(DE3) pLysS, which was induced by IPTG. The recombinant protein was purified using Ni-NTA spin column and acrylamide gel electrophoresis. A polyclonal antibody was developed by immunizing BALB/c mice with the purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immunosorbent assay, immunohistochemical staining and Western blot analysis.
RESULTSRecombinant plasmid expressing truncated HCCR-1167-360 was constructed. A protein of 2.70 x 10(4) was successfully expressed and purified. High titer polyclonal antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein.
CONCLUSIONSThe truncated recombinant HCCR-1(167-360) developed in this study is highly purified and shows strong antigenecity; the polyclonal antibody against this HCCR protein was generated by regular immunization method, showing both high sensitivity and specificity. The protein and the antibody can be used for further clinical examination and research of HCCR.
