| Hepatitis B (HB) is a severe global infectious disease caused by Hepatitis B virus (HBV). Due to shortage of blood sources and potential infection risk, widely used PDV has been replaced by genetically engineered HBsAg vaccines. CHO-derived recombinant HBsAg has the similar properties as PDV from serum of hepatitis B patients. Primate results also showed that CHO-derived recombinant HBsAg possessed higher immune activity, and produced higher titers of HBsAg antibodies.In this research, CHO-K1 adherent cells were successfully transfected with plasmids expressing Hepatitis B surface antigen (HBsAg) S protein. A stable CHO cell line producing high level of HBsAg S protein was obtained. At the first step, HBsAg S protein expressing plasmids (pS-1 and pcDNA-S-1) were purified and were tested in transient transfection of COS-1 cell and CHO-K1 cell, respectively. HBsAg S protein expression level was monitored by HBsAg ELISA. Results showed that PEI method had good transfection efficacy, but Calcium Phosphate Co-Precipitation method was unstable and had low efficiency. The best PEI/DNA ratio is 6.7. PEI also had very low cytotoxicity to CHO-K1 cell line. Therefore, CHO-K1 cells were permanently transfected with pcDNA-S-1 plasmid by PEI method at PEI/DNA ratio 6.7, and selected with G418 (Geneticin, 600ug/ml based on pilot G418 killing curve). Secreted HBsAg in conditioned medium was detected by a commercial HBsAg ELISA kit. After eight passages in T25 flasks, a stably transfected high-producing CHO-K1 clone, 6H3, was identified. HBsAg S protein in 6H3 clone CM was also detected by Transmission Electron Microscope (TEM).
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