Expression And Purification Of Human Des-γ-carboxy Prothrombin And Its Monoclonal Antibody Preparation And Identification

ObjectionHepatocellular carcinoma (HCC) is one of the most frequent malignant tumors and is the second most common cause of cancer death in China. The incidence and mortality of HCC is found increasing year by year. The prognosis should be significantly improved by early diagnosis and treatment. HCC conventional screening and diagnosis methods include physical examinations, imaging technologies and blood serum Alpha fetoprotein (AFP) examination.Nowadays, the diagnosis of HCC mainly due to AFP. It shows a sensitivity of50%to70%, but only40%for early HCC. Recent studies revealed that the sensitivity and specificity of DCP in sera are superior to AFP in the diagnosis of HCC. DCP is expected to be a potential biomarker for early diagnosis of HCC. So, the aim of this research is to prepare the monoclonal antibody (mAb) against DCP and identify the biological characteristics of the mAb.Methods1. The expression vector pCold II-DCP was constructed and transformed into E.coli. BL21(DE3) for expression by IPTG. The fusion protein was identification by SDS-PAGE of Proteins and purified with their fusion partner by His TrapFF affinity chromatography.2. The purified His-DCP fusion protein was used to immunize the BALB/c mice. Then, the mouse myeloma cells (Sp2/0) were fused with spleen cells from BALB/c immunized by the purified protein. Subsequently, three times limited dilution method was used to screen hybridoma cell lines.3. The titer of the mAb was detected by ELISA and its specificity was analyzed by Western blot. The subclasses of mAb were identified by the kit for Mouse Monoclonal subclasses.Results1. The expression vector pCold â…¡-DCP was constructed successfully.2. The DCP fusion protein was successfully expressed and purified. The concentration and purity of the protein is1.5mg/mL and more than80%respectively.3. Two hybridoma cell lines against DCP were obtained, which named3B5,6H4. The heavy chain of immunoglobulin subclasses was IgG2a, IgG2a respectively. And the titration is above1:243,000. Western blot showed that DCPmAb can identify the nature DCP protein in the lysate of cells.ConclusionsIn our research, the expression vector pCold â…¡-DCP was constructed successfully. DCP fusion protein was used as antigen and we successfully prepare a-DCP mAb. The successful preparation of DCP mAb may lays solid foundation for the research of the molecular mechanism of DCP in early diagnosis of HCC.