中试规模纯化重组鼠疫rF1-V融合蛋白及其免疫原性分析  

Pilot-scale purification of r F1-V fusion protein of Yersinia pestis and characterization of its immunogenicity

作　　者：房婷[1] 任军[1] 张金龙[1] 尹可欣[1] 杨秀旭[1] 于蕊[1] 张晓鹏[1] 于长明[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物工程学报》2016年第1期95-104,共10页Chinese Journal of Biotechnology

基　　金：国家传染病重大专项(No.2013ZX10004001)资助~~

摘　　要：重组F1-V融合蛋白(rF1-V)是目前在进行临床研究的鼠疫亚单位疫苗的主要成分。本研究摸索了rF1-V的可溶表达条件,并对条件进行了优化和放大,确定的中试发酵工艺为:在重组菌对数生长期中期加入50μmol/LIPTG,25℃诱导表达5h。通过硫酸铵分级沉淀、离子交换、疏水相互作用层析和凝胶过滤四步纯化,最终得到纯度为99%、回收率大于20%且各项检测指标合格的蛋白。在此基础上,将蛋白使用氢氧化铝佐剂进行吸附,在小鼠体内进行了免疫原性研究。ELISA测定两次皮下免疫后血清的抗体滴度。比较融合蛋白免疫组(rF1-V)与单一抗原免疫组(rF1、rV)以及联合抗原免疫组(rF1+rV)之间体液免疫反应的差异。结果显示:20μgrF1-V免疫剂量组诱导的抗F1抗体滴度明显高于其他组,抗V抗体滴度与其他组相比没有显著差异。表明本工艺制备的rF1-V抗原有望作为鼠疫亚单位疫苗的主要组分。Recombinant F1-V（r F1-V） fusion protein is the main ingredient of the current candidate vaccine against Yersinia pestis infection, which has been under investigation in clinical trial in USA. We investigated the soluble expression conditions of r F1-V in Escherichia coli BL21（DE3） that we constructed before. After scale-up and optimization of fermentation processes, we got the optimized fermentation process parameters： the culture was induced at the middle exponential phase with 50 μmol/L of IPTG at 25 °C for 5 h. Soluble r F1-V protein was isolated to 99% purity by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and gel filter chromatography. The protein recovery was above 20%. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing. Results of purity, quality and western blotting analysis indicated that the target protein is a consistent and properly folded product. Furthermore, the immunogenicity of various antigens formulated with aluminum hydroxide adjuvant was evaluated in mice. Serum antibody titers of 4 groups including 20 μg r F1, r V and r F1-V and 10 μg r F1＋10 μg r V, were assayed by ELISA after 2 doses. The antibody titers of anti-F1 with 20 μg r F1-V were obviously higher than titers with other groups; meanwhile there were no significant difference of anti-V antibody titers among them. These findings confirm that r F1-V would be the active pharmaceutical ingredient of the plague subunit vaccine.

关 键 词：鼠疫耶尔森菌 重组F1-V融合蛋白 发酵 分离纯化 免疫原性 亚单位疫苗 

分 类 号：R392[医药卫生—免疫学]