Expression of human NGF? gene segment encoding mature peptide in E.coli and determination of its bioactivity
- VernacularTitle:人神经生长因子成熟蛋白基因片段在大肠杆菌中的表达和生物活性鉴定
- Author:
Wei MA
;
Miao LIU
;
Guangxiao YANG
;
Quanying WANG
- Publication Type:Journal Article
- Keywords:
hNGF?gene segment encoding mature peptide;
molecular subcloning;
prokaryotic expression;
bioactivity
- From:Journal of Xi'an Jiaotong University(Medical Sciences)
1982;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express the hNGF? gene segment encoding mature peptide (hNGF? ) in E.coli and determine its bioactivity. Methods The resulting gene of hNGF? was subclonedinto the hNGF? site of the expression vector plasmid pBV220. The ligation products were used to transform the competent E.coli DH 5?. The proteins of hNGF? were expressed by temperature induction. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the expressed hNGF? tests of neurite growth of dorsal root knot of chicken embryo and tests of Brdu incorporation into PC12 cells was a biologically active protein. Results The recombinant plasmid pBV220/NGF? was successfully constructed. The NGF? was inserted to pBV220 plasma, a prokaryotic expression vector. Expression of NGF? in E.coli was induced by raising temperature to 42℃. SDS-PAGE electrophoresis showed that NGF? protein existed in inclusion. The solubility protein of NGF? was obtained through purification of inclusion by centrifugation and technique of protein repatriation. Recombinant NGF? protein was purified by affinity chromatography of heparin SepharoseCL-6B. The purity of NGF? was higher than 90% and yield of NGF? was 1.8～2.0mg/L expressing bacteria. The bioactivity of NGF? expressing prokaryotic cell was 1?10 5BU/g according to rule concerning examination of biological products in China. Conclusion The hNGF?gene with bioactivity can be expressed in E.coli.
