[Correlaion between serum Golph2 protein and hepatocellular carcinoma].
OBJECTIVE
To study the correlation between hepatocellular carcinoma (HCC) and serum Golph2 protein.


METHODS
Golph2 gene was cloned by RT-PCR using RNA from WBF44 cell line as template, the point mutations of the cloned sequence were corrected by PCR, then the gene (1206 bp) was cloned into pET-21 plasmid, and the resulted plasmid was transformed into E.coli DH5a. The expression of 6xHis and Golph2 fusion protein was induced by isopropylthio-beta-D-galactoside (IPTG). The expression of fusion protein was detected by SDS-PAGE and Western blot, and was purified by Ni NTA chelating agarose. The rabbit antibody against Golph2 protein was obtained by immunizing 2 rabbits with the purified Golph2 protein. The specificity and titer of the antibody was determined by Western-blot and ELISA respectively. Sandwich ELISA was used to detect the level of serum Golph2 protein.


RESULTS
There were two replacement mutation and 1 deletion mutation in the cloned sequence contrasted to NM177937 in Genbank, including 644(T-->C, L-->P) , 970 (G-->A, V-->I) and 802 G deletion. The sequence was completely reversed by PCR. The sequence of Golph2 gene cloned into expression vector was confirmed by DNA sequencing. SDS-PAGE and Western blot analysis showed that Golph2 protein was expressed in E.coli DH5a. The antiserum could bind to the 52 kD recombinant protein and serum 73 kD protein specifically. The mean A450 value of ELISA for serum Golph2 protein were significantly higher in HCC and other liver diseases than that in control groups. The sensitivity and specificity for HCC were 44.5% and 82.0%, respectively, at the cut off value is more than or equal to 0.40.


CONCLUSIONS
The polyclonal antibody against Golph2 protein is specific. The level of serum Golph2 is significantly higher in patients with HCC and other liver diseases than that in healthy controls.
Tan
Long-yi
LY
Department of Clinical Laboratory, First People's Hospital Baoshan Branch, Shanghai 200940, China. tanlongyi@hotmail.com
Chen
Jie
J
Wang
Hao
H
Ye
Han-qing
HQ
Li
Qian
Q
Gu
Ji-an
JA
Han
Li-qin
LQ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000097:blood; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis / Q000097:blood / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; C410824:GOLM1 protein, human; D008565:Membrane Proteins