人血小板生成素重组细胞的培养工艺研究

【作者】 苏冬梅；

【导师】 夏焕章； 娄竞；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2001， 硕士

【摘要】 本研究的内容主要是建立高密度、高表达培养TPO重组细胞的工艺。进行了包括构建重组细胞株、确立重组细胞培养方法、建立TPO体外活性测定方法、纯化和鉴定表达产物等一系列实验。 首先将带有二氢叶酸还原酶（DHFR）基因的TPO表达质粒pTcho以电穿孔法转入二氢叶酸还原酶缺陷的CHO（DHFR）细胞，通过条件培养基选择及氨甲碟呤加压筛选，获得能够高效、稳定地表达rhTPO的细胞株，命名为STPO-1。经细胞稳定性等多项指标的鉴定，证实该细胞可以作为生产细胞株。 为了实现高表达细胞培养，通过实验选择了合适的培养基、pH值和表达刺激物质等，确定了STPO-1细胞在瓶中的培养及表达方法。使细胞在瓶培养时的TPO表达量达到2000U/ml（相当于7mg/ml）。为了进一步实现重组细胞的高密度培养，利用生物反应器进行了大规模连续培养，摸索出了STPO-1细胞在生物反应器中培养时合适的pH值、溶解氧值、转数、通气流量、葡萄糖浓度和流加速度等，确定了STPO-1细胞在生物反应器中的生长及表达条件，在体积为5L的生物反应器中，利用无蛋白培养基培养，细胞密度达6×10~6个/ml，TPO表达量达12000U/ml（相当于40mg/ml），细胞密度和表达量均达到了瓶培养的6倍，表达时间达24天，收获量达100L，收获液经纯化后可得TPO蛋白650mg，并且在表达期间细胞代谢和表达量比较稳定。 为验证表达产物的结构，进一步对表达产物进行纯化，获得了均一的rhTPO组分。经鉴定，产品的纯度大于99％，分子量为70-120KD，N端17个氨基酸序列依次为Ser-Pro-Ala-Pro-Pro-Ala-Cys-Asp-Leu-Arg-Val-Leu-Ser-Lys-Leu-Leu-Arg，等电点小于5，免疫印迹为单一条带。 通过实验获得了能高效、稳定地表达重组TPO的细胞株，并建立起一套完整有效的细胞培养及产物纯化技术。对大规模产业化生产和广泛的临床应用打下了坚实的基础。 在研制过程中，确定了TPO在体外定量测定的方法，该方法简便、灵敏沈阳药科大学硕士论文 人血小板生成素重组细胞的培养工艺研究度高、重复性好，可做为常规方法推广使用。更多还原

【Abstract】 In the current study, a method of high-density cell culture for producing rhTPO was developed. These research included construction of a CHO cell line stably transfected with rhTPO eDNA, selection of the cell line that highly expressed rhTPO protein, development of an assay for measuring the in-vitro activity of rhTPO, development of a method of cell culture using bioreactor, and finally, the purification and characterization of rhTPO.In brief, a mammalian expression plasmid containing TPO eDNA and a selectable marker was first introduced into CHO cells. A cell line, which was named STP0-l, was obtained following the standard selection and amplification procedures. This STP0-l cell line could stably express rhTPO at a high level.The optimization of cell culture was then performed and the resulting level of expression reached 2,000 units/mi (7 mglml) of rhTPO when the ST1抩-l cells were cultured in flasks. The high-density cell culture was studied using method of packed-bed bioreactor with continuous perfusion culture. Following optimization of the bioreaction condition, such as medium, pH, dissolved oxygen (DO2), agitation speed, glucose concentration, and perfusion rate, the cell density could reach 6 X 106/ml and level of TPO expression could reach 12,000 units/mI (40 mg/mi) by using a specific protein-free medium. As a result, an average of 100 liters of conditioned medium could be harvested using a bioreactor with a 5-liter vessel. A total of 650 mg of rhTPO protein could be obtained following the purification process, which included a series of filtration and chromatography steps.The characterization of purified rhTPO showed 99% purity by High-Performance-Liquid Chromatography (HPLC) and SDS-Polyacrylamide GelElectrophoresis (PAGE), 70 -120 kD of the apparent molecular weight, multiple Iso-Electric Focusing (IEF) bands with p1 <5, and a single smeared band on a WesternBlot analysis. The N-terminal amino acid analysis showed correct sequence as3deduced from TPO genetic code.In summary, a cell line that could highly express rhTPO was constructed, an efficient in-vitro activity assay of rhTPO and a complete process for production and purification of rhTPO was developed. These results provided a solid ground for large-scale manufacture process development as well as eventual clinical application of rhTPO.更多还原