【作者】 邓军；

【导师】 张宇锋；

【作者基本信息】 天津大学 ， 药剂学， 2008， 硕士

【摘要】 乙型肝炎是由乙型肝炎病毒(HBV)引起的全球性传染病。HBV除了引起急性肝炎外,还可导致慢性肝炎和肝硬化,而且与肝癌的发生有着密切的关系。曾在国内外广泛用于乙型肝炎预防的血源性乙肝疫苗(PDV)因血源供应有限和潜在危险性,己经逐步被基因工程疫苗所代替。中国仓鼠卵巢癌细胞(Chinese hamster ovary cell,CHO cell)经基因转染后分泌的重组乙肝表面S抗原,和乙肝病人血清来源的抗原同样具有系统的抗原活性,而且在灵长类动物的实验中发现CHO细胞重组乙肝抗原比血清抗原和酵母来源的抗原具有更高的免疫活性,表现为产生更多的抗体和更高的血清滴度。本课题主要内容是建立CHO-K1细胞基因转染乙肝表面S抗原基因的方法,并筛选出稳定表达的CHO细胞株。首先采用分子生物学的方法制备了转染级别的包含乙肝表面S抗原基因的两种质粒pS-1和pcDNA-s-1。然后,用两种转染方法分别转染COS-1细胞和CHO-K1细胞,并用ELISA的方法对转染结果进行检测。经过实验得知,磷酸钙转染法本身受其它很多因素影响且很大,转染方法不稳定;PEI转染法可以有效的转染细胞,实验稳定结果较好,并且确定了最佳的PEI/DNA的质量比为6.7。本实验还对PEI的毒性进行了测定,发现它对CHO-K1细胞毒性不大,细胞存活最少的也有76.01%存活。最后,确定了G418的最佳筛选浓度为600ug/ml后,用PEI转染法转染CHO-K1细胞,经G418加压筛选后,用酶联和透射电镜检测均得到细胞可稳定表达乙肝表面抗原的结论。在本课题中,成功的构建了稳定表达乙肝表面S抗原的CHO细胞株6H3。更多还原

【Abstract】 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 Due to shortage of blood sources and potential infection risk, widely used PDV has been replaced by genetically engineered HBsAg vaccines.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).更多还原