Expression Of Human β-NGF Gene In E.coli And In CHO Cells

Nerve growth factor(NGF) is an important neurotrophic factor to immune system, hematogeneous system, genitourinary system, endocrine system, particularly to nervous system. NGF has yield good results in the clinic to treat central nervous system(CNS)'s disease such as Alzheimer'disease, neurodegeneration and neuro-injury in the peripheral nervous system(PNS). Natural NGF protein can be purifed from some part in human body. But this method is too boring to be used of NGF production.Moreover, due to the natural NGF protein has high molecular weight, it could not permeate through the blood brain barrier(BBB). To look for a good method to producing NGF which has low molecular weight, we designed two projects which are carried out through gene cloning and cell engineering techniques to provide the problem.Part I Construction of the recombinant PBV220- β -NGF from human β -NGF gene and expression in E.coli DH5 aObjective To study a new method of expressing protein β -NGFthrough technique of gene recombined in vivo. Method A pair of specific primers for the full sequence encoding human β-NGF was designed and synthesized. A 380bp fragment was amplified from human blood genomic DNA by polymerase chain reaction(PCR), and cloned into PUCm-T vector. The identified insert fragment from the recombinant PBV220-β -NGF was directedly ligated with linearized PBV220 with the compatible termini. E.coli DH5a was transformed with the expression recombinant PBV220- β -NGF and induced by heat. Results The cloned DNA fragment was identified as the full-length sequence encoding human β -NGF by restriction analysis and DNA sequencing. SDS-PAGE revealed the cloned NGF gene expressed as a fusion protein (20.7%)in the cells transformed by PBV220- β -NGF. Results The clone containing the full-length sequence encoding human 1? -NGF is obtained and successfully expressed in E.coli to be of use for studying the biological functions of it.