摘要
目的建立适于生产应用的重组人睫状神经营养因子(recombinant human ciliary neurotrophic factor,rh CNTF)纯化工艺,并对纯化产物进行鉴定。方法将CNTFnew CC22突变体工程菌BL21(DE3)菌体破碎上清经25%饱和度硫酸铵沉淀后,沉淀上清先后经Butyl HP疏水层析和Q HP阴离子交换层析进行纯化。通过非还原SDS-PAGE和反相高效液相色谱分析(RP-HPLC)检测样品纯度;质谱法测定rh CNTF的相对分子质量;Edman化学降解法测定rh CNTF的N-端氨基酸序列;TF-1细胞/CCK-8比色法和对饮食诱导性肥胖(diet-induced obesity,DDIO)大鼠的减肥作用分别测定rh CNTF的体外和体内生物学活性。结果纯化后rh CNTF的纯度可达99.6%,纯化回收率为34.5%;相对分子质量为21 153;N-端15个氨基酸序列为Ala-Phe-Thr-Glu-His-Ser-Pro-Leu-Thr-Pro-His-Arg-Arg-Asp-Leu;生物学比活性高于2×106 IU/mg,并能有效降低DIO大鼠体重。结论经3步纯化成功获得了高纯度的rh CNTF,为进一步对其性质、功能和生产应用研究奠定了基础。
Objective To develop a purification procedure suitable for recombinant human ciliary neurotrophic factor（rh CNTF）, and identify the purified product. Methods The supernatant of broken bacteria of recombinant E. coli BL21（DE3）mutant CNTFnew CC22 was precipitated with 25% saturated ammonium sulfate, and the precipitation supernatant was purified by Butyl HP hydrophobic interaction chromatography and Q HP anion exchange chromatography. The purified rh CNTF protein was determined for purity by non-reduced SDS-PAGE and RP-HPLC, for relative molecular mass by mass spectrometry, and for N-terminal amino acid sequence by Edman chemical degradation method. The in vitro and in vivo biological activities of rh CNTF were evaluated by TF-1 cell / CCK-8 colorimetric method and antiobesity effect on rats with diet-induced obesity（DDIO） respectively. Results The purified rh CNTF reached a purity of 99. 6%, of which the recovery rate was 34. 5%, the relative molecular mass was 21 153, the amino acid sequence at N-terminus was Ala-PheThr-Glu-His-Ser-Pro-Leu-Thr-Pro-His-Arg-Arg-Asp-Leu, and the specific biological activity was more than 2 × 10^6 IU / mg.The purified protein decreased the bodyweight of rats effectively. Conclusion Highly purified rh CNTF was successfully obtained by three steps of purification procedure, which laid a foundation of further study on its property, function and application.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第12期1321-1327,共7页
Chinese Journal of Biologicals
基金
北京市科委"生物医药与生命科学创新培育研究一创新品种临床前研究专项"(z151100003915068)
国药集团新产品开发基金(2011SW02)
关键词
重组人睫状神经营养因子
纯化
大肠埃希菌
减肥
Recombinant human ciliary neurotrophic factor
Purification
E.coli
Lose weight
