[Cloning, prokaryotic expression and polyclonal antibody preparation of HCCR: a new biomarker for hepatocellular carcinoma].
OBJECTIVE
To 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.


METHODS
HCCR-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.


RESULTS
Recombinant 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.


CONCLUSIONS
The 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.
Xia
Jian-bo
JB
Division of Clinical Immunology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhang
Zhen-hua
ZH
Tian
Yong-jun
YJ
Meng
Zhong-ji
ZJ
Yu
Zhi-qun
ZQ
Zhao
Xi-ping
XP
Yang
Dong-liang
DL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D014408:Biomarkers, Tumor / Q000096:biosynthesis* / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D011387:Prokaryotic Cells / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; C476732:LETMD1 protein, human; D011518:Proto-Oncogene Proteins