实时PCR法检测重组酵母中编码HBsAg质粒的拷贝数

发布时间：2018-04-17 16:01

本文选题：荧光定量PCR + 质粒拷贝数　；参考：《北京生物制品研究所》2005年硕士论文

【摘要】：用于乙型肝炎疫苗生产的重组酿酒酵母工程菌2150-2-3(pHBS56-GAP347/33)含有游离质粒,可在宿主细胞中独立复制并表达乙型肝炎表面抗原(HBsAg)。目前在生产中,为了从基因水平监控抗原表达量,对发酵细胞进行了质粒保有率的检测。但该方法只能测定细胞中有无质粒,不能对质粒进行定量。由于细胞内质粒拷贝的多少直接影响最终HBsAg的表达量,所以在生产过程中有必要对质粒拷贝数进行实时定量测定。测定质粒拷贝数的方法有很多,如氯化铯-溴化乙锭平衡梯度离心法、高效液相色谱法、核酸杂交方法等等,但它们都有各自的缺点,有的准确性不高,有的耗时长不适于实时检测,本文主要介绍了应用实时荧光定量PCR这一新兴检测技术对质粒拷贝数目的测定方法,通过提取重组酿酒酵母基因组和质粒DNA,以酿酒酵母细胞基因组中单拷贝基因URA3为内标,利用质粒HBsAg基因片段和内标的相对数量的测定来确定每个细胞中的质粒拷贝数。该方法结果准确,90min内即可完成检测,通过对重组酿酒酵母800L罐发酵细胞进行测定,最终质粒拷贝数为71.5个/细胞。为了验证该结果的可信程度,同时用DNA斑点杂交的方法进行验证,实验结果与PCR方法测定结果基本一致,证明了实时荧光定量PCR测定的质粒拷贝数的正确性。文章进一步介绍了该检测方法在实际应用:对生产菌种进行了亚克隆优选,选择16个亚克隆在三角瓶进行连续培养同时检测质粒拷贝数和抗原表达水平,试验结果显示质粒拷贝数与抗原表达水平具有一致性,同时在不同亚克隆之间存在较大的差异,说明拷贝数测定可作为菌种优选的一种有效手段;对三批三角瓶模拟补料发酵和两批生产补料发酵细胞在发酵第40小时、52小时、64小时测定
[Abstract]:Recombinant Saccharomyces cerevisiae 2150-2-3 pHBS56-GAP347 / 33) containing free plasmids can replicate and express HBsAg in host cells independently.In order to monitor the expression of antigens at the gene level, the retention of plasmids was detected in fermentative cells.However, this method can only determine whether there are plasmids in cells, and can not quantify plasmids.Because the number of plasmid copies in the cell directly affects the final HBsAg expression, it is necessary to quantify the plasmid copy number in real time during the production process.There are many methods for determining copy number of plasmids, such as cesium chloride and ethidium bromide equilibrium gradient centrifugation, high performance liquid chromatography, nucleic acid hybridization, etc.Some time-consuming methods are not suitable for real-time detection. In this paper, a new method of detecting plasmid copy number by real-time fluorescence quantitative PCR is introduced.By extracting the recombinant Saccharomyces cerevisiae genome and plasmid DNA, the single copy gene URA3 in the genome of Saccharomyces cerevisiae was used as the internal standard. The plasmid copy number was determined by the determination of the relative number of plasmid HBsAg gene fragment and internal standard.The results of this method could be completed within 90 minutes, and the final copy number of plasmid was 71.5 / cell by determining the fermentation cells of recombinant Saccharomyces cerevisiae in 800L pot.In order to verify the reliability of the results, the DNA dot blot method was used to verify the results. The experimental results were in good agreement with those of the PCR method, which proved the correctness of the plasmid copy number measured by real-time fluorescence quantitative PCR.The article further introduces the practical application of this method: the subclone selection of the producing strain was carried out, and 16 subclones were selected for continuous culture in the triangle flask to detect the plasmid copy number and antigen expression level simultaneously.The results showed that the copy number of plasmid was consistent with the level of antigen expression, and there were great differences among different subclones, which indicated that copy number determination could be used as an effective method for the selection of bacterial species.Three batches of triangulated flasks and two batches of feedstock fermentation cells were determined at the 40th hour and the 52nd hour and the 64th hour after fermentation.
【学位授予单位】：北京生物制品研究所
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R37

【引证文献】