Recombinant Expression Of Hepatoma Associated Gene HTA And Preparation Of Anti-HTA Monoclonal Antibody

Objectives:To study the role of HTA in the development and progression of hepatocellualr carcinoma and to clone the gene of a new biomarker and potential immunotherapy target point for hepatocellualr carcinoma, this study will recombinant express the Hepatoma Associated Gene(HTA) and develop the anti-HTA monoclonal antibody, pre-test the function of HTA protein and analysis the expression profile of HTA protein.Methods:HTA338-616DNA was amplified from HepG2 cells and cloned into the prokaryotic expression vector pET21 a(+)-MBP. The proteins MBP and MBP-HTA were induced by IPTG, purified by His-tag magnetic bead purification kit and identified by ELISA. HepG2 cells were stimulated with MBP and MBP-HTA proteins respectively. MTT assay and colony formation assay were employed to examine the proliferation of these cells and the changes of cell cycle distribution were determined by flow cytometry(FCM). Anti-HTA monoclonal antibody was developed by immunizing BALB/c mice with purified recombinant protein MBP-HTA and hybridoma technique, and their sensitivity and specificity were tested by ELISA and Western blot. The immunohisto-chemistry staining was used to analysis expression profile of HTA protein.Results:1. The fragment about 300bp of HTA338-616DNA was obtained from HepG2 cells by RT-PCR. The prokaryotic expression plasmid pET21 a(+)-MBP-HTA was successfully constructed and confirmed by PCR, endonuclease digestion and direct sequencing.2. The recombinant protein MBP-HTA could be expressed in abundance in the form of inclusion bodies. We got the purified purpose protein of 52kDa and Western blot analysis showed that the recombinant protein MBP-HTA was expressed correctly. 3. The proliferation of HepG2 cells stimulated with MBP-HTA was significantly higher than HepG2 cells stimulated with MBP and negative controls, which was analysised by MTT assay and colony formation assay. FCM results suggest that HepG2 cells stimulated with MBP-HTA showed significantly decrease fraction in G1 phase and increase fraction in S phase, the activity of cell proliferation was enhanced.4. High titer anti-HTA monoclonal antibody with high specificity was obtained. Using ELISA and Western blot analysis, the titer of the monoclonal antibody was 1:12800 and 1:1600 respectively. By immunohistochemistry, HTA was detected in hepatic carcinoma tissues, hepatic cirrhosis tissues, hepatic fibrosis tissues, colon cancer tissues, rectal cancer tissues, gastric carcinoma tissues, lung cancer tissues, cervical cancer tissues, normal hepatic tissues and normal colon tissues, but not in other detected tissues. The experimental results demonstrated cytoplasmic and membrane staining. Compared with the hepatic cirrhosis tissues(P<0.05), hepatic fibrosis tissues(P<0.05) and normal hepatic tissues(P<0.05), the positive expression rate of HTA protein was obviously higher in hepatocellular carcinoma tissues.Conclusions:1. The prokaryotic expression plasmid pET21a(+)-MBP-HTA was successfully constructed, and can express recombinant protein MBP-HTA with the molecular weight of about 52kDa.2. The recombinant protein MBP-HTA developed in this study had strong immunogenicity.3. HTA protein could significantly promote the proliferation of HepG2 cells, which may be related to the promotion of G1 phase cells transit to S phase cells.4. The anti-HTA monoclonal antibody was successfully prepared. This monoclonal antibody had both high sensitivity and high specificity, and can be applied in immunohistochemistry staining. Compared with the hepatic cirrhosis tissues, hepatic fibrosis tissues and normal hepatic tissues, the positive expression rate of HTA protein was obviously higher in hepatocellular carcinoma tissues.