重组鼠疫耶尔森菌LcrV抗原制备工艺的建立

Development of a Procedure for Preparation of Recombinant Yersinia pestis LcrV Antigen

目的建立稳定、高效的重组鼠疫耶尔森菌LcrV抗原工程菌的发酵与纯化工艺。方法研究LcrV工程菌pET-V/BL21(DE3)在试管和三角摇瓶中的生长和表达规律,对种子培养基、IPTG诱导时机、诱导时间及诱导浓度进行优化,并放大至30 L发酵罐培养,建立稳定的发酵工艺。收获菌体,经冻融、离心收集蛋白溶液,高压匀浆处理后,分别采用Q.Sepharose HP阴离子交换层析、Phenyl Sepharose 6 FF(hs)疏水层析及Superdex 75 pg凝胶过滤层析三步柱层析纯化,建立稳定的纯化工艺,连续纯化3批,并按《中国药典》三部(2010版)的相关要求进行全面检定。结果经优化的条件培养及诱导表达,工程菌的收菌密度(A600值)可达34,LcrV抗原的表达量达36%,含量为1.6 g/L。发酵产物经柱层析纯化,LcrV产量高于140 mg,纯度大于95%,蛋白总回收率达8.5%以上。各项检定指标均符合《中国药典》三部(2010版)的相关要求。结论已建立了稳定的、适合规模化生产的LcrV制备工艺,为新型鼠疫组分疫苗的研制奠定了基础。

Objective To develop a stable and effective procedure for fermentation of recombinant Yersinia pestis and purification of LcrV antigen.Methods The regularity of growth of recombinant E.coli strain pET-V / BL21（DE3） in tube and in conical flask as well as expression of LcrV antigen were investigated,based on which the medium,time point and duration for induction with IPTG and IPTG concentration were optimized and scaled up to a 30 L fermenter to develop a stable fermentation procedure.The harvested bacteria were subjected to freeze-thawing,from which protein solution was collected by centrifugation and treated by high pressure homogenization,the purified by Q.Sepharose HP anion exchange chromatography,Phenyl Sepharose 6 FF（hs） hydrophobic chromatography and Superdex 75 pg gel filtration chromatography,based on which a stable purification procedure was developed.Three consecutive batches of test samples were purified by the developed procedure and subjected to overall control tests according to the requirements in Chinese Pharmacopoeia（VolumeⅢ,2010 edition）.Results After culture and induction under the optimized condition,the density of harvested bacteria（A600 value） reached 34,while the expression level and amount of LcrV antigen reached were more than 36% and 1.6 g / L respectively.After purification by column chromatography,the yield,purity and total recovery rate of LcrV antigen were more than 140 mg,more than 95% and more than 8.5% respectively.All the quality indexes of purified LcrV antigen met the requirements in Chinese Pharmacopoeia（VolumeⅢ,2010 edition）.Conclusion A stable procedure suitable for large-scale production of LcrV antigen was developed,which laid a foundation of preparation of novel Y.pestis component vaccine.