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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 establish a stable and efficient fermentation and purification process of recombinant Yersinia pestis LcrV antigen-engineered bacteria. Methods The growth and expression of pET-V / BL21 (DE3) in vitro and in shake flasks of LcrV engineered bacteria were studied. The seed medium, induction time, induction time and induction concentration of IPTG were optimized and amplified to 30 L fermentor Cultivation, the establishment of a stable fermentation process. The cells were harvested, frozen and thawed, and the protein solution was collected by centrifugation. After high-pressure homogenization treatment, the cells were separated by Q. Sepharose HP anion exchange chromatography, Phenyl Sepharose 6 FF (hs) hydrophobic chromatography and Superdex 75 pg gel filtration chromatography Step chromatography column purification, the establishment of a stable purification process, three batches of continuous purification, and according to “Chinese Pharmacopoeia” three (2010 version) of the relevant requirements for a comprehensive test. Results Under the optimal conditions, the engineered bacteria could reach a density of 34 (A600), 36% of LcrV antigen (1.6 g / L). The fermentation product was purified by column chromatography. The yield of LcrV was more than 140 mg, the purity was more than 95% and the total protein recovery was 8.5%. The test indicators are in line with the “Chinese Pharmacopoeia” three (2010 version) of the relevant requirements. Conclusion The stable and suitable LcrV preparation process for large-scale production has been established, which lays the foundation for the development of a new type of plague vaccine.