摘要
目的确认重组人胰岛素类似物DesB30的结构,分析其生物效价。方法在毕赤酵母中构建表达含有短C肽AAK并去掉B链第30位氨基酸(T)的人胰岛素原类似物HMPIDesB30,经胰蛋白酶酶切纯化获得DesB30。利用N末端氨基酸序列测定,质量肽谱分析,圆二色谱等方法进行结构确认及质量分析。利用RP-HPLC法,小鼠血糖法,脂肪细胞法分析其生物效价。结果 DesB30的N端氨基酸序列、二硫键配对、圆二色谱值与理论值一致,其生物效价与人胰岛素基本一致。结论毕赤酵母分泌表达的胰岛素类似物DesB30结构正确,胰岛素去掉B链第30位的苏氨酸,不影响其生物活性。
Objective To confirm the structure of recombinant insulin analog DesB30 and analyze its biological potency. Methods Human Mini-proinsulin DesB30 （HMPIDesB30）, which contains short C peptide AAK and lacks the threonine （T） at site 30 of B chain, was constructed and expressed in Pichia pastoris. The purified DesB30 was obtained after trypsin digestion, and was confirmed and analyzed by N-terminal amino acid sequence determination, mass peptide mapping analysis and circular dichroism spectroscopy determination. RP-HPLC assay, mouse blood glucose bioassay and the fat cell assay were used for the biological potency determination of DesB30. Results The amino acid sequence, disulphide bridge and circular dichroism spectroscopy of insulin analog DesB30 were consistent with the theoretical value, and its biological potency was consistent with the that of human insulin. Conclusions The structure of insulin analog DesB30 is correct, and deletion ofthreonine Thr at site 30 of B chain does not affect its biological activity.
出处
《中国医药生物技术》
2013年第1期19-23,共5页
Chinese Medicinal Biotechnology
基金
"重大新药创制"国家科技重大专项(2009ZX09103-683)
