Clong, Expression Of Human Neuritin In E.coil And Its Associated Biological Activity Analysis

Neurotrophic factors are polypeptides which can promote neuron survival,growth ,differentiation and mediate the development ,differentiation and repair the damage of nervous systems. they play an important role in supporting the injured nervous system and neuron degenerative disorders.Neuritin is a neurotrophic factor discovered recently which acts the plasticity on the nerve specifically.the studies showed that Neuritin has widely biological function in nerve system ,which can induce neurite growth and promote the outgrowth and arborization of neurite . It has important clinical significance for the treatment and defence of nervous system diseases characterized by the injured of neurite and axon.Because the concentiation Neuritin in the human body is very low and difficult to isolate andpurify enough Neuritin t to supply for the study of preclinical medicine and clinic. It is quite effective way to obtain the recombinant human Neuritin by the methods of gene engineering.in this article, we expressed and purified the Neuritin in the E.coil and verified the biological function ,The research set the foundation for the theraphy of the neuron nerve system disease by gene methods.The ORF of neuritin was amplified by PCR from full-length neuritin cDNA,and the fragment was inserted into pET32a by T4 ligase. The recombinant plasmid pET32a-neuritin was transformed into the E.coil BL21, which is digested by the restriction enzyme and sequenced correctly,Then the recombinant plasmid pET32a-neuritin was induced by IPTG. SDS-PAGE analysis showed that after inducing 4 hours ,The expression of the molecular weight about 30kD Neuritin is the most. And the expressed protein was proved to own immuno competence by Western blot and was purified on Ni2+-NTA column. Lastly,the biological activity of recombinant Neuritin from E.coli were identified with pc12 and dorsal root gangria of the chick embryo which can stimulate the growth of nerve neurite of them .This is the first research of gene neuritin by E.coli through recombinant DNA technique and the recombinant Neuritin stimulated the growth of nerve neurite of the pc12 and dorsal root gangria of the chick embryo in vivo.The result can provide a good foundation for the clinical application of the Neuritin.