Clone And Expression Of Human GDNF Gene Segments And Protein Identification

Glial cell line-derived neurotrophic factor (GDNF) was firstly found in the culture supernatant of rat glial cell lines B49 as a neurotrophic factor in 1993 by Lin et al. It can enhance the growth and differentiation of mesencephal dopaminergic neuron, reinforce biological activity for intaking dopamine. It has been proved that GDNF can facilitate the survival of dynamoneure in vitro culturing when peripheral nerve was damaged. GDNF can restrain motor neuron retrograde degeneration induced by peripheral nerve damage and enhanceperipheral nerve regenerating after damage. So that, it has become a hot spots to study the function on prevention and treatment of the Parkinson's disease (PD).PD is a kind of frequent neurological retrogressive disease, often occurs in middle and old age people. GDNF is a more specific and effective factor for mesencephal dopaminergic neuron. The binding of TH and GDNF increases the synthesis of mesencephal DA and protects mesencephal dopaminergic neuron, which is the best choice for PD therapy. There are no different protein sustaining time obviously in vivo between prokaryotic expression system and eukarya expression system. But in contrast to eukarya expression system, prokaryotic expression system is more easily manipulated and it can contain bigger gene fragment. And it can get ready more easily and the cost is lower. Meanwhile, DNA intergration and mutation seldom occur in the system.We design and construct prokaryon recombinant plasmid and transform it to karyogamy expression system. It provides favourable foundation for the chronergy, duration, toxicity of GDNF expression. It is a key factor to produce much more G DNF with high purity and biological activity by the simpler method and the lower cost. Some experiments have proved that GDNF secreted by prokaryotic expression system,evenlacking glycosylation,but it has the same biological activity to that from eukaryotic system. We construct recombinant plasmid with GDNF gene and transfer it to the prokaryotic expression system, and obtain the extractable protein induced by IPTG, and affect to chickern embryonerve ganglion. The result showed it can inhance the growth of nerveaxon of chicken embryo root ganglion. It is proved that GDNF secreted by prokaryotic expression system has the same biological function to eukaryotic system. This experiment has six parts as followings:①to obtain the objective gene GDNF;②to construct expression vector;③to identify expression vector;④expression of the objective protein;⑤to extract the objective protein;⑥to detect the activity of objective protein. We obtain the gene fragment which has 405bp. It is the same to the sequence in GeneBank through DNA sequencing after connecting to T clone vector. By SDS-PAGE we have found the recombinant plasmid has the function of expressing specific protein whose molecular weight is about 32K It is the same to protein GDNF molecular weight. It is demonstrated that we construct expression vector succsessfully. The objective protein extracted promotes axon growth of chicken embryo root ganglion. It is proved that the protein expressed by recombinant vector has the biological activity of enhancing nerve cell growth. The advantage of the expression system is that they can grow steadily in vitro and operate easily. We can easily obtain the engineering cell line which ca n express exogenous gene stably by pharmacon screening. The study has excellent application perspective, and it provides fundamental basis for further study of GDNF on clinical application.