Cloning, Expression and Purification of Gene Encoding Human ?-NGF in E.coli
- VernacularTitle:人神经生长因子基因在大肠杆菌中的克隆、表达及产物纯化
- Author:
Juan ZHAO
;
Bing HE
;
Han-Min JIANG
;
Xiu-Wei CHENG
;
Xin-Da YU
;
- Publication Type:Journal Article
- Keywords:
Human ?-nerve growth factor Gene cloning Expression Purification Biological activity
- From:
China Biotechnology
2006;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Nerve growth factor is one of the most important factors playing an important role in regulating the growth, development and survival of the neuron. The purified NGF from human placenta has been reported, the tissue from which can be isolated the NGF is very limited. It is important for basic research and clinic application to expression hNGF by genetic engineering. By polymerase chain reaction,gene fragment encoding the mature part of ?-NGF was amplified using the DNA of human placenta as template. The fragment was sequenced and inserted into expression vector pET-15b, and the recombinant expression vector pET15b-NGF was transformed into E.coli BL21(DE3)pLysS. After inducing with IPTG the NGF was higher expressed up to 25% of the total cell proteins. The expression product was purified with metal chelate affinity chromatography on Ni-IDA agarose under denaturing condition. The purity of rNGF was higher than 90% and yield of rNGF was 4.56mg/L expressing bacteria. SDS-PAGE revealed the NGF expression product had a Mr 16kDa. Western-blot displayed the recombinant product had strong immunological activity with rabbit anti-human ?-NGF polyclonic antibodies. The expression products were dealed with solubilizing inclusion bodies and refolding protein. The test of nerve fiber growth of chicken embryo DRG neurons displayed rNGF had biological activity.
