重组人神经生长因子在大肠埃希菌中的表达及活性测定  

Expression of recombinant human nerve growth factor in E. coli and determination of activity of expressed product

作　　者：沈丽[1] 黄志立[1] 蒋伟[1] 林钰琼 王妍[1] 

机构地区：[1]深圳职业技术学院应用化学与生物技术学院,广东深圳518055

出　　处：《中国生物制品学杂志》2018年第2期134-139,共6页Chinese Journal of Biologicals

基　　金：深圳市科技创新委项目(JCYJ20140718171752670;JCYJ20170306144523128;CXZZ20120619162250509)

摘　　要：目的在大肠埃希菌中表达重组人神经生长因子(recombinant human nerve growth factor,rhNGF),并检测其活性。方法通过密码子优化,设计表达rhNGF的基因,构建重组表达质粒pET-28a(+)-hNGF,转化大肠埃希菌BL21pLyS(DE3)中,IPTG诱导表达。表达的蛋白经层析纯化后,SDS-PAGE及HPLC法检测纯度,Lowry法检测蛋白浓度,TF-1细胞增殖法检测生物学活性。结果构建了融合表达rhNGF的大肠埃希菌表达系统,表达的3批外源蛋白纯化后纯度均达到100%,比活分别为5.5×10~5、6.8×10~5和7.4×10~5 IU/mg,均高于小鼠颌下腺提取的NGF标准品。结论成功表达了rhNGF,纯化后纯度高,比活强,为规模化生产hNGF提供了参考。Objective To express recombinant human nerve growth factor （rhNGF） in E.coli and determine its activity. Methods The gene for expression of rhNGF was designed by codon optimization, based on which recombinant plasmid pET-28a-hNGF was constructed and transformed to E. coli pLyS （DE3） for expression under induction of IFFG. The expressed protein was purified by chromatography, then determined for purity by SDS-PAGE and HPLC, for concentration by Lowry method, and for biological activity by TF-1 cell proliferation assay. Results E. coli fusion expression system for hNGF was constructed. The purities of three batches of expressed protein were 100%, while the specific activities were 5. 5 × 10^5, 6. 8 ×10^5 and 7. 4 × 10^5 IU/mg respectively, which were higher than those of NGF standard extracted from mouse salivmy gland. Conclusion The hNGF with high purity and specificity was expressed successfully, which provided a reference for large-scale production of hNGF.

关 键 词：神经生长因子 大肠埃希菌 表达 活性 

分 类 号：Q789[生物学—分子生物学]