摘要
The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatitis B vaccine in future. A single copy URA3 gene in the yeast genome was employed as internal control to test the plasmid copies by real-time PCR. The yeast cells carrying plasmids encoding HBsAg were cultured in regular non-feeding process and fed-batch process in shaking flasks at laboratory level and industrial fermentor. The cells at different stages of cultivation were collected and broken. Then the total DNAs of the cells were extracted and the plasmid copies were detected by real-time PCR with the method of relative quantification. The method of the real-time relative quantitative PCR used in this study was reliable with good accuracy and quickness. The plasmid copies in non-feeding and fed-batch flasks were 39.22±12.00 and 43.06±5.70, while the copies in fermentor were 96.16±21.00 and 82.50±7.78, respectively. The data show that the variation of the result is very small and the data precision is quite good. To prolong the fermentation time may increase the cell density without influence on the stability of the plasmid, and the method of relative quantification can also be applied to test the copy number of interest target gene in any host cell with certain known copy gene.
The purpose of this study is to detect the plasmid copies encoding HBsAg in recombinant yeast Saccharomyces cerevisiae by real-time PCR, and provide the data for setting up an optimal process of manufacturing hepatitis B vaccine in future. A single copy URA3 gene in the yeast genome was employed as internal control to test the plasmid copies by real-time PCR. The yeast cells carrying plasmids encoding HBsAg were cultured in regular non-feeding process and fed-batch process in shaking flasks at laboratory level and industrial fermentor. The cells at different stages of cultivation were collected and broken. Then the total DNAs of the cells were extracted and the plasmid copies were detected by real-time PCR with the method of relative quantification. The method of the real-time relative quantitative PCR used in this study was reliable with good accuracy and quickness. The plasmid copies in non-feeding and fed-batch flasks were 39.22 ± 12.00 and 43.06 ± 5.70, while the copies in fermentor were 96.16 ± 21.00 and 82.50 _± 7.78, respectively. The data show that the variation of the result is very small and the data precision is quite good. To prolong the fermentation time may increase the cell density without influence on the stability of the plasmid, and the method of relative quantification can also be applied to test the copy number of interest target gene in any host cell with certain known copy gene.
作者简介
Corresponding author： Jin LI, Beijing Tiantan Biological Products Co., 12d, Beijing 100024, P. R. China （Tel： 86-10-6576-2911. Fax ： 86-10-6576-2507. Email ： leegene@126.com）.
