Preparation Of GPC3 As The Target Of Nanobody And The Target Recognition Effect On Hepatoma Cells

Liver cancer is regarded as one of the most common malignant tumors all over the world.Its morbidity and mortality rate show an increasing trend.It has caused serious damage to the health of human body.The treatment of liver cancer mainly includes liver transplantation,tumor resection and non resectable local therapy,such as hepatic artery chemoembolization.Because in Liver cancer especially primary hepatocellular carcinoma（HCC）is easy to metastasize early and relapse after treatment.Therefore,finding an index that can accurately predict prognosis and effective therapeutic targets for liver cancer is of great significance.Early diagnosis and treatment are one of the key factors to improve the curative effect of primary hepatocellular carcinoma.Phosphatidylinositol proteoglycan 3（Glypican 3,GPC3）,a member of heparan sulfate proteoglycan family,is involved in regulating cell proliferation,regulation,adhesion and migration.Some studies have shown that the specific high expression of GPC3 in primary liver cancer a nd low expression or non expression in normal liver tissues in adults.It is suggested that GPC3 has significant sensitivity and specificity in the diagnosis of liver cancer,and can be used as a specific tumor marker for identifying primary hepatocellular cancer.The human GPC3 gene was amplified by PCR to the p ET28a prokaryotic expression vector.The expression of phosphonoyl inositol fusion protein was induced in the E.coli BL21 expression strain.Purification of his-fusion protein by affinity chromatography was measured by SDS-PAGE electrophoresis.Finally,the protein content was also measured.The biological information of the structure and function of GPC3 gene is predicted with the help of bioinformatics online software.The Bactrian camel is immuned by the purified GPC3,lead to phage display Phage display library is constructed.After three rounds of panning to obtain anti-GPC3 nanobody protein.The affinity of the nanobody to eukaryotic protein was verified by ELISA in vitro,and the target identification effect of flow cytometry on hepatoma cells was explored.The results showed that the prokaryotic expression vector of pET28a-GPC3 was successfully constructed and optimize its expression.About 70kDa GPC3 fusion protein was obtained;The expression level of fusion protein was high at 32℃,IPTG0.5mmol/L and 6h.Bioinformatics shows that GPC3 protein was the stable hydrophilic protein which had signal peptide,and was located in the plasma membrane.The main secondary structure elements were mainly a-helix andβ-fold.It had 18 serines,7 threonines and 23 tyrosines,which might become phosphorylation sites of the protein kinase.GPC3 protein interacted with Wnt signal family proteins.The phage display library was successfully constructed by immune camel,and the anti GPC3 nanobody was screened from it.we obtained that storage capacity of the antibody library was 1.608×10⁶cfu/ml.The titer of the phage library were6.5×10¹³pfu/ml.The statistics of the enrichment were calculated,Indicated that the output of the third round was least.With the decrease of antigen coating amount,the specificity of antibody was gradually increased.And the prokaryotic expression vector of p ET28a-VHH_GPC3 was successfully constructed,and a fusion protein with a single molecular weight of about 13k Da was purified.The VHH_GPC3 protein was not only able to specifically binding to commercial human GPC3,but it also identified the surface of Hep G2 and BEL-7404 cells.and the fluorescence intensity of FITC in experimental group was significantly different from that in negative control group.Compared with HepG2,The affinity of nanobody to BEL-7404 was higher.