一种乙肝表面抗原结合蛋白（SBP）在Pichia Pastoris中的表达，纯化工艺及功能研究

【作者】 庞云；

【导师】 朱乃硕；

【作者基本信息】 复旦大学 ， 微生物， 2009， 硕士

【摘要】 乙型肝炎(hepatitis B,HB)是世界范围内流行的一种严重危害人类健康的疾病,由乙型肝炎病毒(HBV hepatitis B virus)引起.我国是乙肝高流行区,大约有1.12亿人慢性感染HBV,占全世界感染者的30%以上.乙肝疫苗是预防感染乙型肝炎和降低乙肝病毒携带率的最有效措施.但是,5-10%的个体在按标准程序接种乙肝疫苗后不能产生足够的抗体以抵抗乙肝病毒的感染.HBsAg-BP(Hepatitis B surface antigen binding protein,SBP)是本室以血清来源的乙肝表面抗原(HBsAg)为探针,通过印迹免疫技术从人肝cDNA噬菌体表达库中筛选获得,其特征是可以与乙肝表面抗原HBsAg特异性的相互作用.在本室前期的研究工作中,我们构建了原核表达载体pBV-SBP,用Ecoli大量表达重组SBP,并证实其具有增强乙肝疫苗免疫效果的作用.本文将SBP表达框克隆到在毕赤酵母表达载体pPICZαA中,通过电击法转化毕赤酵母菌株GS115,获得能够分泌表达SBP的GS115转化菌株;对培养工艺进行了优化,发现SBP表达菌株在pH为6.0,诱导剂甲醇浓度为1.0-1.5%的BMMY中诱导96小时,达到最佳表达水平,可以加入终浓度为50μg/ml的Amp和15μg/ml的Kan以防止染菌及2mM的EDTA以抑制降解且不影响蛋白表达;在最优条件下诱导表达SBP并收集培养液,通过超滤和亲和层析的方法纯化获得了一定量的重组SBP蛋白,ELISA法证实重组SBP具有与HBsAg特异性结合的能力,并用多种方法计算了二者间亲和常数,在小鼠免疫实验中证实重组SBP具有乙肝疫苗免疫增效作用.更多还原

【Abstract】 The current HBV vaccine performed certain protective role in preventing the infection of HBV. However, still about 5-10% people are under no protection because no response is detected after inoculation.In our previous study, we have found a protein named HBsAg Binding Protein (SBP), which can specifically interact with HBsAg. Several experiments have proved its ability on enhancing the immuno-response of HBV vaccine using E.coli expressed SBP.In the present study, SBP gene was cloned into the eukaryotic expression vector pPICZαA and transformed into Pichia pastoris strain GS115. The protein gene was placed downstream of the methanol inducible AOX1 promoter and theα-mating Factor signal sequence so the protein was expressed into the culture media. We studied on several factors which play important parts on SBP expression level, and found that when SBP expression was induced 72 hours under MeOH concentration of 1.0-1.5%, high expression level can be acquired. The using of 50μg/ml Amp and 15μg/ml Kan to prevent contamination and 2mM EDTA to prevent degradation had no effect on expression level. Subsequently we purified SBP through ultra-filtration and affinity chromatography.After identification through Western Blot and mass spectrometry analyses, we demonstrated that SBP has the ability to bind specifically with HBsAg and calculated the affinity constant value using two different methods. The function of SBP in enhancing HBV vaccine performance was also testified in vivo.更多还原