Construction and prokaryotic expression of recombinant gene EGFRvIIIª²HBcAg and immunogenicity analysis of the fusion protein

    DUAN Xiaoª²yi1, WANG Jianª²sheng1, GUO Youª²min2*, HAN Junª²li2, WANG Quanª²ying3, YANG Guangª²xiao3

    1Department of Nuclear Medicine, the First Affiliated Hospital of Xi¡¯an Jiaotong University, Xi¡¯an 710061; 2Department of Image Center, the Second Affiliated Hospital of Xi¡¯an Jiaotong University, Xi¡¯an 710004, China

    \[Abstract\] AIM: To construct recombinant prokaryotic expression plasmid pET28a(+)/cª²PEPª²3ª²c and evaluate the immunogenicity of the fusion protein. METHODS: cDNA fragment encoding PEPª²3 was obtained from pGEMª²T Easy/PEPª²3 and inserted into recombinant plasmid pGEMEX/HBcAg. Then it was subcloned in prokaryotic expression vector and transformed into E.coli BL21(DE3). The fusion protein was expressed by inducing IPTG and purified by Ni2+ª²NTA affinity chromatography. BALB/c mice were immunized with fusion protein and the antibody titre was determined by indirect ELISA. RESULTS: The recombinant gene was confirmed to be correct by restriction enzyme digestion and DNA sequencing. After prokaryotic expression, fusion protein existed in sediment and accounted for 56% of all bacterial lysate. The purified product accounted for 92% of all protein and its concentration was 8 g/L. The antibody titre in blood serum reached 1¡Ã16000 after the fourth immunization and reached 1¡Ã2.56¡Á105 after the sixth immunization. The titre of antiª²PEPª²3 antibody reached 1¡Ã1.28¡Á105 and the titre of antiª²HBcAg antibody was less than 1¡Ã4¡Á103. CONCLUSION: Fusion gene PEPª²3ª²HBcAg is highly expressed in E.coli BL21. The expressed fusion protein can induce neutralizing antibody with high titer and specificity, which lays a foundation for the study of genetically engineering vaccine for malignant tumors with the high expression of EGFRvIII. ......