The Cloning And Expression Of Human Nerve Growth Factor Beta Gene In Escherichia Coli And Its Identification

Never growth factor (NGF) is an important neurotrophic factor to immune system, hematogeneous system, genitourinary system, endocrine system and particularly to nervous system. It is known that NGF plays a critical role in growth, development, differentiation, survival, repair and regeneration of the neuron. The purified NGF from human placenta has been reported, but the tissue from which can be isolated the NGF is very limited and expensive, which can not meet the people's needs. So it is critical for primary research and clinic application to expression hNGF by genetic engineering.Objective: To further lay the foundation for the clinical application throughβ-NGF is highly expressed in Escherichia coli by constructing prokaryotic expression vector, and getting a biological activity rhβ-NGF after purification and renaturation.Methods: By polymerase chain reaction, gene fragment encoding the mature peptid part ofβ-NGF was amplified using the DNA of human peripheral blood as template, and was inserted the pMD18-T, which is a cloning vector. The cloned pMD18-T-β-NGF vector were digested by restriction enzyme NdeⅠ, HindⅢand the DNA fragment ofβ-NGF was gained. Then the fragment was inserted into expression vector pET28a+. After the recombination expression vector pET28a-β-NGF was identified by digestion and sequenced, it was transformed into E. coli BL21 (DE3). After inducing with IPTG theβ-NGF was expressed and washed by different buffer and purified by gel filtering chromatography. The reducingβ-NGF was oxidized slowly in buffer with 10μmol/L Cu2+. Biological activity of renaturedβ-NGF was identified by the test of never fiber growth of chicken embryo dorsal root ganglion and PC12 cell line. Results: Purity of genetic DNA extracting from human peripheral blood was rather high and OD260/OD280 ratio has been measured by ultraviolet spectrophotometer was between 1.8 and 2.0. 373bp gene fragment ofβ-NGF was amplified by polymerase chain reaction, which was coincidence with expectant length. Through digestion of restriction enzymes and amplification of PCR verified construction of recombinant cloning vector pMD18-T-β-NGF. The recombinant expression vector pET28a-β-NGF was confirmed by digestions of NdeⅠand HindⅢand sequencing of DNA and was transformed into E. coli BL21 (DE3). After inducing with IPTG theβ-NGF was highly expressed up to 23.5% of the total bacteria proteins. SDS-PAGE revealed theβ-NGF expression product had a Mr 16kDa and was mainly inclusion body. Through washing of different buffer and purification of gel filtering chromatography, the purity of rhβ-NGF was higher than 90%. After renaturation, yield of rhβ-NGF was 4.8mg/L expressing bacteria. The test of biological activity identification of chicken embryo DRG and PC12 cell line to rhβ-NGF displayed: never fiber growth of DRG was obvious, PC12 cells neural differentiation, cell body change, sprouted processes in rhβ-NGF concentration of 100μg/L or higher.Conclusion:β-NGF was rather high expressed in prokaryocyte expression system and had good biological activity after renaturation.