重组水蛭素Ⅲ的质量控制标准研究  

Quality Control of Recombinant Hirudin Ⅲ

作　　者：韦利军 郭昱 吴梧桐[3] 章良[4] 王耀方 

机构地区：[1]常州千红生化制药有限公司,江苏常州213032 [2]南京生命能科技开发有限公司,江苏南京210016 [3]中国药科大学生命科学与技术学院,江苏南京210009 [4]苏州大学药学院,江苏苏州215123

出　　处：《药物生物技术》2006年第5期343-346,共4页Pharmaceutical Biotechnology

摘　　要：通过测定重组水蛭素Ⅲ的质粒中目的基因序列、重组水蛭素Ⅲ分子质量、C末端序列以及菌体蛋白和DNA残留量,对重组水蛭素Ⅲ的质量进行研究。结果:重组质粒经NheⅠ、HindⅢ双酶切和基因序列测定,表明工程菌所携带的质粒上含有完整的信号肽基因、NheⅠ酶切位点、水蛭素HV3基因和HindⅢ酶切位点;还原性SDS PAGE结果表明在14 000 u处有单一条带,为重组水蛭素Ⅲ二聚体;测定样品分子C端序列为E D YA D E P I P E F,与重组水蛭素Ⅲ的理论值一致;重组水蛭素Ⅲ样品中的残留菌体蛋白和DNA含量分别是0.02%和每剂量样品中残留DNA含量低于2.8 ng。结论:从基因水平、蛋白水平和杂质的角度证明重组水蛭素Ⅲ产品符合质量控制标准。Quality control of recombinant hirudin 3 （r-HV3） was studied. By sequencing plasmid in engineering bacteria expressing r-HV3, the recombinant sequence was conformed to theoretical r-HV3 sequence which proved that r-HV3 product corresponds to quality control standard at gene level. 11 amino acids of the obtained r-HV3 were determined by the analysis of C-terminal amino acid sequence to be corresponding to theoretical value which proved that r-HV3 product corresponds to quality control standard at protein level. The tropina and residual tissues DNA were determined to be consistent with the quality standard of r-hirudin which proved that r-HV3 product corresponds to quality control standard at the point of view of foreign materials. Recombinant hirudin 3 corresponds to quality control standard.

关 键 词：重组水蛭素 质量控制 质粒 C端序列 残留DNA 菌体蛋白 

分 类 号：Q51[生物学—生物化学]