摘要
在培养基中添加二甲基亚砜(DMSO)可使基因工程中国仓鼠卵巢细胞(CHO)外源蛋白乙肝表面抗原(HBsAg)的比生产速率提高4倍以上.为了进一步研究DMSO的作用机理,采用双相凝胶电泳技术分离在培养基中添加1.5%的DMSO后CHO细胞的总蛋白,胶态考马斯亮蓝G-250染色,GS-800凝胶扫描仪获取凝胶图像,并用PDQUEST软件分析,测得每张图谱平均斑点数量为1852±123,以其中一块胶为参考胶进行4块胶间的斑点匹配,其平均匹配的点数达1326±100,平均匹配率为71.6%以上.通过分析得到18个蛋白质点在添加了DMSO后表达量有明显变化(其中12个蛋白表达量明显降低,6个蛋白表达量明显增加.用基质辅助激光解析电离飞行时间质谱测定其胶内酶解后的肽质指纹谱图和串级质谱图,用GPS软件查询NCBI数据库进行了鉴定.鉴定结果表明,这些差异蛋白中一些是与细胞周期有关的蛋白,一些是与细胞能量代谢相关的蛋白,还有一些是与蛋白翻译后修饰有关的酶,这些结果进一步深化了对DMSO提高CHO细胞表达HBsAg的机理的理解.
Expression of the recombinant Hepatitis B surface Antigen （HBsAg） in the Chinese Hamster Ovary （CHO）cell lines was enhanced more than 4-fold in case of adding dimethyl sulfoside （DMSO） into the culture medium. The mechanism was studied via comparative investigation of the differential expression of the proteins in CHO cells before and after adding 1.5 % DMSO to the culture by using two-dimensional gel electrophoresis（2-DE） with the software PDQUEST for image analysis. 1 852±123 protein spots were found in each gel plate, and four gel plates were compared with one gel plate as reference. 1 326±100 protein spots were matched （percentage of 71.6）. 18 proteins that were differentially expressed were found. This 18 protein spots were digested into peptides and then analysed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry. NCBI database was used for search for the mass spectral data and tandem mass spectral data using GPS software. Results showed that among these 18 differential proteins, two important enzymes, namely protein disulfide-isomerase and peptidyl-prolyl cis-trans isomerase, and four enzymes related to glycolysis, triosephosphate isomerase, glyceraldehyde 3-phophate dehydrogenase（GADPH）,aldolase and phosphoglycerate kinase, were down-regulated.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
2005年第4期607-613,共7页
Journal of Fudan University：Natural Science
基金
国家重点基础研究发展规划资助项目(001CB510202)
关键词
蛋白质组
双相凝胶电泳
中国仓鼠卵巢细胞
质谱
二甲基亚砜
乙肝表面抗原
proteome
two-Dimension Electrophoresis
Chinese Hamster Ovary ceils
Matrix-assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry
dimethyl sulfoxide
Hepatitis B surface Antigen
