Gene Cloning And Expression And Functional Identification Of Human-Derived Neurotrophin-6

Neurotrophin (NT) is a kind of proteins with small molecular weight derived from neural tissues or its target tissues. Since the nerve growth factor (NGF), the first member of the NT family, was identified in the 50th of 20th century, including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6) and neurotrophin-7 (NT-7), totally six members of NTs have been reported. Because of its important role in enhancing the neurons occurrence, differentiation, survivance, antagonizing the neurons apoptosis and protecting and renovating the injured neurons, researches on NTs have always been the highlight of the neurobiology. At present, most studies are focused on the NGF, BDNF, NT-3 and NT-4. There are few about NT-6, and even fewer about NT-6 derived from human being. Hereby, the present project was carried out to study the structure and function of human-derived NT-6.Total RNA was extracted from aborted antenatal cerebral cortex and cDNA fragment of NT-6 was amplified through RT-PCR. The obtained NT-6 gene was cloned into pBK-CMVplasmid to construct NT-6 gene expression vector and its expression in the procaryotic cell was observed. Using the complete gene having been cloned as templet, we constructed fusion expression vector of human-derived NT-6 gene that encoded mature peptide pGEX1-NT-6. After induced by IPTG, the fusion protein was expressed in Escherichia coli. The mature peptide of NT-6 was collected and purified. On the cultured hippocampal nuerons and the rats with facial nerve transected, we identified the function of mature peptide of NT-6. The NT-6 gene expression vector was successfully constructed and Escherichia coli with recombinant vector expressed distinctive protein. Sequence BLAST and tertiary structure comparison indicated that the human-derived NT-6 possessed the inherent characteristics of NTs family. Escherichia coli with recombinant vector pGEX1-NT-6 expressed fusion protein of NT-6 mature peptide after induced by IPTG. The purified mature peptide of NT-6 could enhance the survivance of the cultured hippocampal nuerons and antagonize the retrograde-degeneration induced from the nerve transection. This study could not only increase our comprehension on the neurobiological characteristics of NTs, but also provide the theoretic basis for prevention and treatment of disease in the nervous system.